Patent Application
20250114454
This application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML, created on Dec. 13, 2024, is named 167741-046002US_SL.xml and is 49,457 bytes in size.
Immune checkpoint blockade (ICB) has had remarkable success in several cancer types, suggesting that enhancing anti-tumor immunity is a fundamental strategy to combat cancer. Currently-approved immune checkpoint inhibitors block inhibitory signals to cytotoxic T lymphocytes (CTLs), which can directly recognize and eliminate tumor cells in an adaptive immune response. However, many patients do not respond to immunotherapy with ICB, and there is a lack of understanding of the immune evasion mechanisms active in different cancer contexts. The limitations of existing technologies have thus far precluded the systematic discovery of immune resistance mechanisms active in the multicellular ecosystem of the tumor microenvironment. Further, immune resistance can hinder the effectiveness of treatment of a cancer patient using engineered immune effector cells (e.g., in chimeric antigen receptor (CAR) T-cell therapy). Accordingly, compositions and methods for overcoming tumor immune resistance (e.g., in the context of a CAR T-cell therapy) are urgently required.
Therefore, there remains a need for improved immunotherapies for treating cancers, particularly therapies able to overcome tumor immune evasion.
As described below, the present disclosure features compositions and methods for the treatment of cancers that are able to evade the immune system.
In embodiments, the disclosure provides chimeric antigen receptor (CAR) expressing immune cells (e.g., CAR T cells) that have been modified to reduce and/or eliminate expression or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide, and methods for use of such cells to treat a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer).
In one aspect, the disclosure features a modified immune cell containing a chimeric antigen receptor polypeptide. The modified immune cell contains one or more genetic alterations that reduces or eliminates expression and/or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide.
In another aspect, the disclosure features a method for increasing the anti-tumor activity of an immune cell. The method involves introducing into the genome of the immune cell one or more genetic alterations that reduces or eliminates expression and/or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide.
In another aspect, the disclosure features a method for treating a neoplasia in a subject. The method involves administering to the subject a modified immune cell containing one or more genetic alterations that reduces or eliminates expression and/or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide.
In another aspect, the disclosure features a pharmaceutical composition containing the modified immune cell of any of the above embodiments and a pharmaceutically acceptable excipient.
In another aspect, the disclosure features a method for characterizing immune checkpoint blockade sensitivity in a neoplasia. The method involves detecting interferon-stimulated gene (ISG) expression and 6p21.3 copy number in the neoplasia. Loss of 6p21.3 and/or reduced ISG expression levels relative to a reference characterizes the neoplasia as sensitive to immune checkpoint blockade. Presence of intact 6p21.3 and increased ISG expression levels relative to a reference characterizes the neoplasia as resistant to immune checkpoint blockade.
In another aspect, the disclosure features a method for treating a selected patient having a neoplasia. The method involves administering to the selected patient an immune checkpoint blockade. The patient is selected by characterizing loss of 6p21.3 and/or reduced ISG expression levels relative to a reference.
In another aspect, the disclosure features a method for inducing expression of NKG2A and/or CD94 in a T cell. The method involves contacting the cell with an anti-CD3 monoclonal antibody, an anti-CD28 monoclonal antibody, and an IL-12 polypeptide. In embodiments, the method further involves contacting the cell with the anti-CD3 monoclonal antibody, the anti-CD28 monoclonal antibody, and the IL-12 polypeptide a second time. In embodiments, the first contacting and the second contacting each further involve contacting the cell with IL-2, IL-7, and IL-15. In embodiments, the IL-12 polypeptide is an IL-12p70 polypeptide. In embodiments, the second contacting is between about 7 and 14 days after the first contacting.
In any aspect provided herein, or embodiments thereof, the cell is a T cell, a natural killer (NK) cell, a gammadelta T cell, or a natural killer T cell.
In any aspect provided herein, or embodiments thereof, the chimeric antigen receptor specifically binds an antigen present on a neoplastic cell. In embodiments, the antigen is selected from one or more of CD19, BCMA, Mesothelin, MUC1, MUC16, GD2, CD79b19, April, EGFR, EGFRvIII, IL13Ra, HLA-G, and PSMA.
In any aspect provided herein, or embodiments thereof, the cell contains one or more genetic alterations that reduces or eliminates expression of the natural killer cell lectin A (NKG2A) polypeptide.
In any aspect provided herein, or embodiments thereof, the immune cell has reduced susceptibility to interferon-mediated immune inhibition on tumor cells.
In any aspect provided herein, or embodiments thereof, the cell is a T cell, a natural killer (NK) cell, a gammadelta T cell, a natural killer T cell.
In any aspect provided herein, or embodiments thereof, the immune cell further contains one or more genetic alterations that reduces or eliminates expression and/or activity of the natural killer cell lectin A (NKG2A) polypeptide.
In any aspect provided herein, or embodiments thereof, the immune cell is in vivo or in vitro. In any aspect provided herein, or embodiments thereof, the immune cell is a human immune cell. In any aspect provided herein, or embodiments thereof, the subject or patient is a mammal. In embodiments, the mammal is a human.
In any aspect provided herein, or embodiments thereof, the neoplasia is a cancer selected from one or more of skin, colon, pancreas, lung, and kidney cancer. In embodiments, the skin cancer is melanoma. In embodiments, the lung cancer is non-small cell lung cancer. In embodiments, the kidney cancer is renal clear cell carcinoma.
In any aspect provided herein, or embodiments thereof, the method further involves administering to the subject an immune checkpoint blockade therapy. In any aspect provided herein, or embodiments thereof, the immune checkpoint blockade is a PD1, PDL1, or CTLA4 inhibitor. In any aspect provided herein, or embodiments thereof, the immune checkpoint blockade contains an antibody. In embodiments, the antibody is selected from one or more of Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab, and ipilimumab.
In any aspect provided herein, or embodiments thereof, reference is the level of ISG expression present in a healthy cell or in a neoplasia containing intact 6p21.3.
In any aspect provided herein, or embodiments thereof, ISG expression is increased by at least about 20% relative to a reference.
In any aspect provided herein, or embodiments thereof, detecting ISG expression levels involves determining expression levels for one or more genes selected from one or more of ADAR, APOL6, ARID5B, ARL4A, AUTS2, B2M, BANK1, BATF2, BPGM, BST2, BTG1, C1R, C1S, CASP1, CASP3, CASP4, CASP7, CASP8, CCL2, CCL5, CCL7, CD274, CD38, CD40, CD69, CD74, CD86, CDKN1A, CFB, CFH, CIITA, CMKLR1, CMPK2, CMTR1, CSF2RB, CXCL10, CXCL11, CXCL9, DDX58, DDX60, DHX58, EIF2AK2, EIF4E3, EPSTI1, FAS, FCGR1A, FGL2, FPR1, GBP4, GBP6, GCH1, GPR18, GZMA, HELZ2, HERC6, HIF1A, HLA-A, HLA-B, HLA-DMA, HLA-DQA1, HLA-DRB1, HLA-G, ICAM1, IDO1, IFI27, IFI30, IF135, IF144, IF144L, IFIH1, IFIT1, IFIT2, IFIT3, IFITM2, IFITM3, IFNAR2, IL10RA, IL15, IL15RA, IL18BP, IL2RB, IL4R, IL6, IL7, IRF1, IRF2, IRF4, IRF5, IRF7, IRF8, IRF9, ISG15, ISG20, ISOC1, ITGB7, JAK2, KLRK1, LAP3, LATS2, LCP2, LGALS3BP, LY6E, LYSMD2, MARCHF1, METTL7B, MT2A, MTHFD2, MVP, MX1, MX2, MYD88, NAMPT, NCOA3, NFKB1, NFKBIA, NLRC5, NMI, NOD1, NUP93, OAS2, OAS3, OASL, OGFR, P2RY14, PARP12, PARP14, PDE4B, PELI1, PFKP, PIM1, PLA2G4A, PLSCR1, PML, PNP, PNPT1, PSMA2, PSMA3, PSMB10, PSMB2, PSMB8, PSMB9, PSME1, PSME2, PTGS2, PTPN1, PTPN2, PTPN6, RAPGEF6, RBCK1, RIPK1, RIPK2, RNF213, RNF31, RSAD2, RTP4, SAMD9L, SAMHD1, SECTM1, SELP, SERPING1, SLAMF7, SLC25A28, SOCS1, SOCS3, SOD2, SP110, SPPL2A, SRI, SSPN, ST3GAL5, ST8SIA4, STAT1, STAT2, STAT3, STAT4, TAP1, TAPBP, TDRD7, TNFAIP2, TNFAIP3, TNFAIP6, TNFSF10, TOR1B, TRAFD1, TRIM14, TRIM21, TRIM25, TRIM26, TXNIP, UBE2L6, UPP1, USP18, VAMP5, VAMP8, VCAM1, WARS1, XAF1, XCL1, ZBP1, and ZNFX1.
In any aspect provided herein, or embodiments thereof, detecting 6p21.3 copy number involves detecting a gene selected from one or more of TAP1, TAP2, TAPBP, PSMB8, and PSMB9. In embodiments, failure to detect one or more of the genes identifies a loss of 6p21.3.
In any aspect provided herein, or embodiments thereof, ISG expression level is detected by determining the expression levels for one or more genes selected from one or more of ADAR, APOL6, ARID5B, ARL4A, AUTS2, B2M, BANK1, BATF2, BPGM, BST2, BTG1, C1R, C1S, CASP1, CASP3, CASP4, CASP7, CASP8, CCL2, CCL5, CCL7, CD274, CD38, CD40, CD69, CD74, CD86, CDKN1A, CFB, CFH, CIITA, CMKLR1, CMPK2, CMTR1, CSF2RB, CXCL10, CXCL11, CXCL9, DDX58, DDX60, DHX58, EIF2AK2, EIF4E3, EPSTI1, FAS, FCGR1A, FGL2, FPR1, GBP4, GBP6, GCH1, GPR18, GZMA, HELZ2, HERC6, HIF1A, HLA-A, HLA-B, HLA-DMA, HLA-DQA1, HLA-DRB1, HLA-G, ICAM1, IDO1, IFI27, IFI30, IFI35, IFI44, IFI44L, IFIH1, IFIT1, IFIT2, IFIT3, IFITM2, IFITM3, IFNAR2, IL10RA, IL15, IL15RA, IL18BP, IL2RB, IL4R, IL6, IL7, IRF1, IRF2, IRF4, IRF5, IRF7, IRF8, IRF9, ISG15, ISG20, ISOC1, ITGB7, JAK2, KLRK1, LAP3, LATS2, LCP2, LGALS3BP, LY6E, LYSMD2, MARCHF1, METTL7B, MT2A, MTHFD2, MVP, MX1, MX2, MYD88, NAMPT, NCOA3, NFKB1, NFKBIA, NLRC5, NMI, NOD1, NUP93, OAS2, OAS3, OASL, OGFR, P2RYT4, PARP12, PARP14, PDE4B, PELI1, PFKP, PIM1, PLA2G4A, PLSCR1, PML, PNP, PNPT1, PSMA2, PSMA3, PSMB10, PSMB2, PSMB8, PSMB9, PSME1, PSME2, PTGS2, PTPN1, PTPN2, PTPN6, RAPGEF6, RBCK1, RIPK1, RIPK2, RNF213, RNF31, RSAD2, RTP4, SAMD9L, SAMHD1, SECTM1, SELP, SERPING1, SLAMF7, SLC25A28, SOCS1, SOCS3, SOD2, SP110, SPPL2A, SRI, SSPN, ST3GAL5, ST8SIA4, STAT1, STAT2, STAT3, STAT4, TAP1, TAPBP, TDRD7, TNFAIP2, TNFAIP3, TNFAIP6, TNFSF10, TOR1B, TRAFD1, TRIM14, TRIM21, TRIM25, TRIM26, TXNIP, UBE2L6, UPP1, USP18, VAMP5, VAMP8, VCAM1, WARS1, XAF1, XCL1, ZBP1, and ZNFX1.
In any aspect provided herein, or embodiments thereof, the method further involves administering to the selected patient a modified immune cell containing a chimeric antigen receptor polypeptide. The modified immune cell contains one or more genetic alterations that reduces or eliminates expression and/or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide. In aspect of the disclosure, or embodiments thereoof, the method further involves administering to the selected patient a modified immune cell containing a chimeric antigen receptor polypeptide if intact 6p21.3 is present in the neoplasia, ISG expression levels are increased in the neoplasia relative to a reference, and/or HLA-E levels are increased in the neoplasia relative to the reference, where the modified immune cell contains one or more genetic alterations that reduces or eliminates expression and/or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide.
In any aspect provided herein, or embodiments thereof, the modified immune cell is a T cell, a natural killer (NK) cell, a gammadelta T cell, a natural killer T cell.
In any aspect provided herein, or embodiments thereof, ISG expression level is detected by determining the expression levels for one or more genes selected from one or more of those genes listed in
The invention provides compositions containing chimeric antigen receptor (CAR) immune cells that have been modified to reduce and/or eliminate expression or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide, and methods for use thereof to treat a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “6p21.3” is meant locus 21.3 of the short arm (p) of chromosome 6. In embodiments, a cell lacking all or a fragment of a gene encoding TAP1, TAP2, TAPBP, PSMB8, and/or PSMB9 polynucleotide and/or failing to express the same is characterized as having a loss of 6p21.3.
By “cluster of differentiation 19 (CD19) polypeptide” is meant a CD19 protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to Genbank Accession No. AAB60697.1 An exemplary CD19 amino acid sequence from Homo sapiens is provided below (GenBank Accession No. AAB60697.1):
By “cluster of differentiation 19 (CD19) polynucleotide” is meant a nucleic acid PGP21,DNA molecule encoding a CD19 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD19 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for CD19 expression. An exemplary CD19 nucleotide sequence from Homo sapiens is provided below (GenBank Accession No. AH005421.2):
By “cluster of differentiation 94 (CD94) polypeptide” is meant a CD94 protein or fragment thereof, capable of dimerizing with an NKG2A polypeptide and having immunomodulatory activity and having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001107868.2. An exemplary CD94 amino acid sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. NP_001107868.2):
By “cluster of differentiation 94 (CD94) polynucleotide” is meant a nucleic acid molecule encoding a CD94 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a CD94 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for CD94 expression. An exemplary CD94 nucleotide sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No.: NM_001114396.3):
By “human leukocyte antigen E (HLA-E) polypeptide” is meant a HLA-E protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. ARB08449.1 An exemplary HLA-E amino acid sequence from Homo sapiens is provided below (GenBank Accession No. ARB08449.1):
By “human leukocyte antigen E (HLA-E) polynucleotide” is meant a nucleic acid molecule encoding a HLA-E polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a HLA-E polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for HLA-E expression. An exemplary HLA-E nucleotide sequence from Homo sapiens is provided below (GenBank Accession No.: KY497359.1):
Homo sapiens HLA-E (HLA-E) gene, complete cds
By “interferon gamma (IFNγ) polypeptide” is meant a IFNγ protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. AAB59534.1. An exemplary IFNγ amino acid sequence from Homo sapiens is provided below (GenBank: AAB59534.1):
By “interferon gamma (IFNγ) polynucleotide” is meant a nucleic acid molecule encoding a IFNγ polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a IFNγ polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for IFNγ expression. An exemplary IFNγ nucleotide sequence from Homo sapiens is provided below (GenBank Accession No. J00219.1):
Homo sapiens interferon-gamma (IFNG) gene,
By “natural killer cell lectin A (NKG2A) polypeptide” is meant a NKG2A protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to NCBI Ref. Seq. Accession No. NP_998823.1. An exemplary NKG2A amino acid sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. NP_998823.1):
By “natural killer cell lectin A (NKG2A) polynucleotide” is meant a nucleic acid molecule encoding a NKG2A polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a NKG2A polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for NKG2A expression. An exemplary NKG2A nucleotide sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. NM_213658.2):
By “proteasome 20S subunit beta 8 (PSMB8) polypeptide” is meant a PSMB8 protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to NCBI Ref. Seq. Accession No. NP_004150.1. An exemplary PSMB8 amino acid sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. NP_004150.1):
By “proteasome 20S subunit beta 8 (PSMB8) polynucleotide” is meant a nucleic acid molecule encoding a PSMB8 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a PSMB8 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for PSMB8 expression. An exemplary PSMB8 nucleotide sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. NM_004159.5):
By “proteasome 20S subunit beta 9 (PSMB9) polypeptide” is meant a PSMB9 protein or fragment thereof, having immunomodulatory activity and having at least about 95% amino acid sequence identity to NCBI Ref. Seq. Accession No. AQY77063.1. An exemplary PSMB9 amino acid sequence from Homo sapiens is provided below (NCBI Ref. Seq. Accession No. AQY77063.1):
By “proteasome 20S subunit beta 9 (PSMB9) polynucleotide” is meant a nucleic acid molecule encoding a PSMB9 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a PSMB9 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for PSMB9 expression. An exemplary PSMB9 nucleotide sequence from Homo sapiens is provided below (GenBank Accession No. KY500591.2):
By “transporter 1, ATP binding cassette subfamily B member (TAP1) polypeptide” is meant a TAP1 protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. AAS55412.1. An exemplary TAP1 amino acid sequence from Homo sapiens is provided below (GenBank Accession No. AAS55412.1).
By “transporter 1, ATP binding cassette subfamily B member (TAP1) polynucleotide” is meant a nucleic acid molecule encoding a TAP1 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a TAP1 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for TAP1 expression. An exemplary TAP1 polynucleotide sequence from Homo sapiens is provided below (GenBank Accession No. AY523971.2):
By “transporter 2, ATP binding cassette subfamily B member (TAP2) polypeptide” is meant a TAP2 protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. AHW47975.1. An exemplary TAP2 amino acid sequence from Homo sapiens is provided below (GenBank Accession No. AHW47975.1):
By “transporter 2, ATP binding cassette subfamily B member (TAP2) polynucleotide” is meant a nucleic acid molecule encoding a TAP2 polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a TAP2 polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for TAP2 expression. An exemplary TAP2 nucleotide sequence from Homo sapiens is provided below (GenBank Accession No. KJ657697.1):
Homo sapiens clone HIP1009 major
By “TAP binding protein (TAPBP) polypeptide” is meant a TAPBP protein or fragment thereof, having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. AQY77142.1. An exemplary TAPBP amino acid sequence from Homo sapiens is provided below (GenBank: AQY77142.1):
By “TAP binding protein (TAPBP) polynucleotide” is meant a nucleic acid molecule encoding a TAPBP polypeptide, as well as the introns, exons, 3′ untranslated regions, 5′ untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, a TAPBP polynucleotide is the genomic sequence, cDNA, mRNA, or gene associated with and/or required for TAPBP expression. An exemplary TAPBP nucleotide sequence from Homo sapiens is provided below (GenBank Accession No. KY500670.2):
Homo sapiens isolate COX TAPBP gene, complete cds
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change in the structure, expression levels or activity of a polynucleotide or polypeptide as detected by standard art known methods such as those described herein. The alteration can be an increase or a decrease. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, a 50% or a greater change in expression levels. In embodiments, an alteration in structure is a genetic alteration. In embodiments, the genetic alteration is a missense mutation, deletion, or insertion that results in a loss of function.
By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
As used herein, the term “antibody” or “antigen-binding domain” refers to an immunoglobulin molecule or a fragment thereof that specifically binds to, or is immunologically reactive with, a particular antigen. Non-limiting examples of antibodies or antigen-binding domains include polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including e.g., Fab′, F(ab′)2, Fab, Fv, rlgG, and scFv fragments, as well as engineered antibodies, which include CrossMabs (e.g., CrossMabFabs, CrossMabCH1-CL and CrossMabVH-VL formats), or fragments thereof. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab′)2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (see Wahl et al., J. Nucl. Med. 24:316, 1983; incorporated herein by reference).
By “antigen” is meant an agent to which an antibody or other polypeptide capture molecule specifically binds. In an embodiment, the antigen is a tumor antigen. Exemplary antigens include small molecules, carbohydrates, proteins, and polynucleotides.
By “Chimeric Antigen Receptor” or alternatively a “CAR” is meant a polypeptide capable of providing an immune effector cell with specificity for a target cell. In embodiments, the target cell is a cancer cell. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule. In embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
By “chemotherapeutic agent” is meant an agent that inhibits cancer cell proliferation, inhibits cancer cell survival, increases cancer cell death, inhibits and/or stabilizes tumor growth, or that is otherwise useful in the treatment of cancer. In embodiments, chemotherapeutic agents provided herein are used as part of an immunotherapy. In embodiments, chemotherapeutic agents provided herein contain an immune checkpoint blockade (ICB). In embodiments, the ICB contains a PD-1/PD-L1 checkpoint inhibitor (e.g., atezolizumab, avelumab, BMS-936559, MDX-1105, cemiplimab, durvalumab, nivolumab, and/or pembrolizumab). In embodiments, an PD-1/PD-L1 checkpoint inhibitor contains an anti-CTLA-4 and/or anti-PD-1 antibody. In embodiments, the chemotherapeutic agents provided herein contain a CAR-T that has been modified to reduce or eliminate expression or activity of an NKG2A and/or CD94 polypeptide.
One of skill in the art can readily identify a chemotherapeutic agent of use in a method for treating a cancer described herein (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments of any of the aspects, the combination of agents provided herein decrease cancer cell proliferation or survival by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death (apoptosis) in a cell or cells within a cell mass.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. Any embodiments specified as “comprising” a particular component(s) or element(s) are also contemplated as “consisting of” or “consisting essentially of” the particular component(s) or element(s) in some embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In embodiments, the disease is a neoplasia. Exemplary neoplasias include, but are not limited to, cancers of the skin (e.g., melanoma), colon, pancreas, lung (non-small cell lung cancer), and kidney.
By “effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “immunomodulatory activity” is meant increasing, decreasing, or participating in an immune response.
By “immunotherapy” is meant a treatment that involves supplementing or stimulating the immune system. Non-limiting examples of immunotherapies include treatments involving administration of immune checkpoint blockades and/or CAR T cells.
By “immune checkpoint blockade” is meant an agent that blocks a checkpoint protein from binding it's partner. In embodiments, the agent is an antibody. In some cases, the polynucleotide and/or pathway functions in inhibiting an immune response. In some instances, an immune checkpoint inhibitor inhibits PD-1/PD-L1, CTLA-4, NKG2A, and/or CD94. In embodiments, an immune checkpoint blockade inhibits the interaction of a receptor (e.g., PD-1) with its respective ligand (e.g., PD-L1).
By “increase” is meant to alter positively by at least 5% relative to a reference. An increase may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
By “interferon-stimulated gene” is meant a gene with expression levels that increase when a cell containing the gene is contacted with an interferon. In embodiments, the interferon is IFNγ. Exemplary ISGs include, but are not limited to, ADAR, APOL6, ARID5B, ARL4A, AUTS2, B2M, BANK1, BATF2, BPGM, BST2, BTG1, C1R, C1S, CASP1, CASP3, CASP4, CASP7, CASP8, CCL2, CCL5, CCL7, CD274, CD38, CD40, CD69, CD74, CD86, CDKN1A, CFB, CFH, CIITA, CMKLR1, CMPK2, CMTR1, CSF2RB, CXCL10, CXCL11, CXCL9, DDX58, DDX60, DHX58, EIF2AK2, EIF4E3, EPSTI1, FAS, FCGR1A, FGL2, FPR1, GBP4, GBP6, GCH1, GPR18, GZMA, HELZ2, HERC6, HIF1A, HLA-A, HLA-B, HLA-DMA, HLA-DQA1, HLA-DRB1, HLA-G, ICAM1, IDO1, IF127, IF130, IF135, IF144, IF144L, IFIH1, IFIT1, IFIT2, IFIT3, IFITM2, IFITM3, IFNAR2, IL10RA, IL15, IL15RA, IL18BP, IL2RB, IL4R, IL6, IL7, IRF1, IRF2, IRF4, IRF5, IRF7, IRF8, IRF9, ISG15, ISG20, ISOC1, ITGB7, JAK2, KLRK1, LAP3, LATS2, LCP2, LGALS3BP, LY6E, LYSMD2, MARCHF1, METTL7B, MT2A, MTHFD2, MVP, MX1, MX2, MYD88, NAMPT, NCOA3, NFKB1, NFKBIA, NLRC5, NMI, NOD1, NUP93, OAS2, OAS3, OASL, OGFR, P2RY14, PARP12, PARP14, PDE4B, PELI1, PFKP, PIM1, PLA2G4A, PLSCR1, PML, PNP, PNPT1, PSMA2, PSMA3, PSMB10, PSMB2, PSMB8, PSMB9, PSME1, PSME2, PTGS2, PTPN1, PTPN2, PTPN6, RAPGEF6, RBCK1, RIPK1, RIPK2, RNF213, RNF31, RSAD2, RTP4, SAMD9L, SAMHD1, SECTM1, SELP, SERPING1, SLAMF7, SLC25A28, SOCS1, SOCS3, SOD2, SP110, SPPL2A, SRI, SSPN, ST3GAL5, ST8SIA4, STAT1, STAT2, STAT3, STAT4, TAP1, TAPBP, TDRD7, TNFAIP2, TNFAIP3, TNFAIP6, TNFSF10, TOR1B, TRAFD1, TRIM14, TRIM21, TRIM25, TRIM26, TXNIP, UBE2L6, UPP1, USP18, VAMP5, VAMP8, VCAM1, WARS1, XAF1, XCL1, ZBP1, and ZNFX1. In various embodiments, expression of an ISG increases by at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent relative to a reference. In other embodiments, the expression of an ISG increases by at least about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 2×, 3×, 4×, or 5×. In embodiments, ISGs level are considered as being “high” in a subject if the hallmark IFN gene expression signature for the subject is within the top 25% of that observed in a population (e.g., a patient cohort or a cohort of healthy subjects). In embodiments, expression is measured using RNAseq. In some instances, the increase is statistically significant (e.g., a p value cutoff of p<0.1, p<0.05, or p<0.01)
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a developmental state, condition, disease, or disorder.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
By “neoplasia” is meant a disease or disorder characterized by excess proliferation or reduced apoptosis. In embodiments, a neoplasia is a cancer or tumor. Illustrative neoplasms include breast cancer, esophageal cancer, head-and-neck cancer, pancreatic cancer, skin cancer, colorectal cancer, hepatocellular cancer, bladder cancer, bile duct cancer, luminal and non-luminal bladder cancer, basal bladder cancer, muscle-invasive bladder cancer, and non-muscle-invasive bladder cancer, pancreatic cancer, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, liver cancer, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In embodiments, the neoplasia may be colon adenocarcinoma (COAD), stomach adenocarcinoma (STAD), stomach cancer, and uterine corpus endometrial carcinoma (UCEC). In embodiments, the neoplasia may be a liquid tumor such as, for example, leukemia or lymphoma. In embodiments, the cancer is a colon, kidney, lung, pancreatic, renal (e.g., renal cell carcinoma or clear renal cell carcinoma), or skin cancer (e.g., a melanoma).
By “polypeptide” or “amino acid sequence” is meant any chain of amino acids, regardless of length or post-translational modification. In various embodiments, the post-translational modification is glycosylation or phosphorylation. In various embodiments, conservative amino acid substitutions may be made to a polypeptide to provide functionally equivalent variants, or homologs of the polypeptide. In some aspects the invention embraces sequence alterations that result in conservative amino acid substitutions. In some embodiments, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the conservative amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references that compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Non-limiting examples of conservative substitutions of amino acids include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In various embodiments, conservative amino acid substitutions can be made to the amino acid sequence of the proteins and polypeptides disclosed herein.
By “reduce” is meant to alter negatively by at least 5% relative to a reference. A reduction may be by 5%, 10%, 25%, 30%, 50%, 75%, or even by 100%.
By “reference” is meant a standard or control condition. Non-limiting examples of references include a healthy subject, a subject prior to a change in treatment or administration of an agent, and an unmodified cell. In some instances, a reference is a cell (e.g., an immune cell, such as a CAR T cell) that expresses a functional NKG2A and/or CD94 polypeptide.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant an animal. The animal can be a mammal. The mammal can be a human or non-human mammal, such as a bovine, equine, canine, ovine, rodent, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
The disclosure features compositions containing chimeric antigen receptor (CAR) immune cells that have been modified to reduce and/or eliminate expression or activity of a natural killer cell lectin (NKG2) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide, and methods for use thereof to treat a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer).
The invention is based, at least in part, upon the discovery that loss of IFNγ signaling by tumor cells sensitizes most (i.e., 6 of 8) cancer models to Immune Checkpoint Blockade (ICB). Using in vivo screening data, transcriptional profiling, and genetic interaction studies, it was revealed that the immune-inhibitory effects of tumor IFN sensing are the direct result of tumor upregulation of classical and nonclassical MHC-I genes. The interferon-MHC-I axis can inhibit anti-tumor immunity through two mechanisms: first, upregulation of classical MHC-I inhibits the cytotoxicity of natural killer cells, which are activated by ICB. Second, IFN-mediated upregulation of Qa-1b directly inhibits cytotoxicity by effector CD8+ T cells via the NKG2A/CD94 receptor, which is induced on CD8+ T cells by ICB. Finally, it was shown that high interferon-stimulated gene expression in is associated with decreased survival or poor response in ICB-treated ccRCC and advanced melanoma patients. The studies described in the Examples below reveal the underlying mechanism to explain the inhibitory role of tumor IFN sensing, demonstrating that IFN-mediated upregulation of classical and non-classical MHC-I inhibitory checkpoints can facilitate immune escape.
The findings described herein resulted from studies designed to comprehensively assess immune evasion strategies active in the tumor microenvironment in tumors with distinct genetic drivers and tissues of origin. To identify immune-related dependencies across preclinical tumor models, genome-scale and sub-genome-scale in vivo loss-of-function CRISPR screens were conducted across eight transplantable mouse tumor models representing a diversity of cancer types. These screens identified many new immunotherapy targets and resistance mechanisms, displaying both tumor specific and common patterns of dependency. Strikingly, loss of tumor-intrinsic interferon sensing sensitized most tumors to immune checkpoint blockade (ICB), a surprising finding given the importance of IFNγ for immune surveillance of cancer, and the association of loss-of-function mutations in Jak1 and Jak2 with immune checkpoint blockade (ICB) resistance.
Given the importance of IFNγ to anti-tumor immunity, screening data, transcriptional profiling, and in vivo mouse models were leveraged to determine the mechanism of IFN-mediated resistance to anti-tumor immunity and ICB. It was demonstrates that tumor IFN sensing inhibits NK cells through the upregulation of classical MHC-I and CD8+ T cells via the non-classical MHC-I Qa-1b. Using patient data, it was shown that the prognostic effect of IFN inflammation on survival is dependent on the nature of the immune response to the tumor, with interferon-stimulated gene (ISG) expression predicting response in T cell-dominant tumors and resistance in tumors with transcriptional profiling, and in vivo mouse models to determine the mechanism of IFN-mediated resistance to anti-tumor immunity and ICB. We demonstrate that tumor IFN sensing inhibits NK cytotoxicity. It was shown that tumor IFN sensing inhibits higher NK cell surveillance. Serum proteomics analysis of anti-PD-1-treated advanced melanoma patients revealed that IFN-mediated resistance is pronounced in on-treatment samples and high IFN signaling is predictive of disease progression. This study reveals that resistance to immune checkpoint blockade (ICB) mediated by tumor interferon (IFN) sensing and upregulation of MHC-I and Qa-1b (HLA-E) was a conserved adaptive resistance mechanism at play in most tumor microenvironments and that blocking these inhibitory axes can overcome resistance to immune checkpoint blockade. In particular, it was found that immune cells modified to reduce or eliminate expression or activity of a natural killer cell lectin (NKG2) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide are less susceptible to inhibition by neoplastic cells.
The invention is also based, at least in part, upon the discovery that loss of 6p21.3 and/or low ISG expression levels relative to a reference predicted that a neoplasia would be responsive to immunotherapy, and presence of 6p21.3 and high ISG expression levels relative to a reference neoplasia predicted that the neoplasia would be resistant to immunotherapy. Also, interferon-stimulated gene (ISG) expression predicted response in T cell-dominant tumors and resistance in tumors with higher NK cell surveillance.
Accordingly, the present disclosure provides CAR T cells that have improved resistance to inhibition by neoplastic cells, where the CAR T cells have been modified to reduce or eliminate expression or activity of a natural killer cell lectin A (NKG2A) polypeptide and/or a cluster of differentiation 94 (CD94) polypeptide.
In various aspects, the present disclosure provides CAR T cells that have been modified to reduce and/or eliminate expression and/or activity of NKG2A and/or CD94. NKG2A/CD94 is an inhibitory receptor that has HLA-E (Qa-1 in mice) as its ligand. NKG2A is a transmembrane protein type II that dimerizes with CD94 to form a functional heterodimeric receptor. CD94 contains a short cytoplasmic domain and is responsible for signal transduction. When the NKG2A/CD94 receptor on the surface of an immune cell (e.g., an NK cell or a T cell) is bound by HLA-E, the immune cell becomes inhibited. It is demonstrated in the Examples provided herein that one way that a neoplasia can evade immune cells is by expressing HLA-E on the surface thereof to inactivate immune cells by way of the NKG2A/CD94 receptor. Therefore, in various aspects, the invention provides CAR T cells that have been modified to reduce or eliminate expression and/or activity of NKG2A and/or CD94 to improve the ability of the CAR T cells to resist inactivation by neoplastic cells.
In various embodiments, immune cells of the present disclosure are modified using genome editing. Immune cells can be modified by knocking out (e.g., by deletion) a target gene(s) (e.g., an NKG2A or CD94 gene). Gene editing tools provide the ability to manipulate the DNA sequence of a cell (e.g., to delete a target gene) at a specific chromosomal locus, without introducing mutations at other sites of the genome. This technology effectively enables the researcher to manipulate the genome of a subject's cells in vitro or in vivo.
In one embodiment, gene editing involves targeting an endonuclease (an enzyme that causes DNA breaks internally within a DNA molecule) to a specific site of the genome and thereby triggering formation of a chromosomal double strand break (DSB) at the chosen site. If, concomitant with the introduction of the chromosome breaks, a donor DNA molecule may be introduced (for example, by plasmid or oligonucleotide introduction), interactions between the broken chromosome and the introduced DNA can occur, especially if the two sequences share homology. In this instance, a process termed “gene targeting” can occur, in which the DNA ends of the chromosome invade homologous sequences of the donor DNA by homologous recombination (HR). By using the donor plasmid sequence as a template for HR, a seamless repair of the chromosomal DSB can be accomplished. In some embodiments, no donor DNA molecule is introduced and the double-stranded break is repaired by the error-prone non-homologous end joining NHEJ pathway leading to knock-out or deletion of the target gene (e.g., through the introduction of indels or nonsense mutations). In some embodiments, an endonuclease(s) can be targeted to at least two distinct chosen sites located within a gene sequence so that chromosomal double strand breaks at the distinct sites leads to excision and deletion of a nucleotide sequence flanked by the two distinct sites.
In some embodiments, the chosen site is associated with or disposed within a nucleotide sequence encoding a gene selected from Klrc1 (NKG2A) and (Klrd1) CD94.
Current genome editing tools use the induction of double strand breaks (DSBs) to enhance gene manipulation of cells, including the deletion or knockout of genes. Such methods include zinc finger nucleases (ZFNs; described for example in U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, and U.S. Pat. Publ. Nos. 20030232410 and US2009020314, which are incorporated herein by reference), Transcription Activator-Like Effector Nucleases (TALENs; described for example in U.S. Pat. Nos. 8,440,431, 8,440,432, 8,450,471, 8,586,363, and 8,697,853, and U.S. Pat. Publ. Nos. 20110145940, 20120178131, 20120178169, 20120214228, 20130122581, 20140335592, and 20140335618, which are incorporated herein by reference), and the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system (described for example in U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,871,445, 8,889,356, 8,906,616, 8,932,814, 8,945,839, 8,993,233, and 8,999,641, and U.S. Pat. Publ. Nos. 20140170753, 20140227787, 20140179006, 20140189896, 20140273231, 20140242664, 20140273232, 20150184139, 20150203872, 20150031134, 20150079681, 20150232882, and 20150247150, which are incorporated herein by reference). For example, ZFN DNA sequence recognition capabilities and specificity can be unpredictable. Similarly, TALENs and CRISPR/Cas9 cleave not only at the desired site, but often at other “off-target” sites, as well. These methods have significant issues connected with off-target double-stranded break induction and the potential for deleterious mutations, including indels, genomic rearrangements, and chromosomal rearrangements, associated with these off-target effects. ZFNs and TALENs entail use of modular sequence-specific DNA binding proteins to generate specificity for ˜18 bp sequences in the genome.
RNA-guided nucleases-mediated genome editing, based on Type 2 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR Associated) systems, offers a valuable approach to alter the genome. In brief, Cas9, a nuclease guided by single-guide RNA (sgRNA), binds to a targeted genomic locus next to the protospacer adjacent motif (PAM) and generates a double-strand break (DSB). The DSB is then repaired either by non-homologous end joining (NHEJ), which leads to insertion/deletion (indel) mutations, or by homology-directed repair (HDR), which requires an exogenous template and can generate a precise modification at a target locus (Mali et al., Science. 2013 Feb. 15; 339(6121):823-6). Genetic manipulation using engineered nucleases has been demonstrated in tissue culture cells and rodent models of rare diseases.
CRISPR has been used in a wide range of organisms including baker's yeast (S. cerevisiae), zebra fish, nematodes (C. elegans), plants, mice, and several other organisms. Additionally, CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. Libraries of tens of thousands of guide RNAs are now available.
Since 2012, the CRISPR/Cas system has been used for gene editing (silencing, enhancing or changing specific genes) that even works in eukaryotes like mice and primates. By inserting a plasmid containing cas genes and specifically designed CRISPRs, an organism's genome can be cut at any desired location.
CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote's genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.
CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (E. coli, Y. pest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.
Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (about 30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype E. coli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. See also U.S. Patent Publication 2014/0068797, which is incorporated by reference in its entirety.
Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9's ability to home located its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA into a “single-guide RNA” molecule that, mixed with Cas9, could find and cut the correct DNA targets. It has been proposed that such synthetic guide RNAs might be able to be used for gene editing (Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).
Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Coinjection of Cas9 mRNA and sgRNAs into the germline (zygotes) generated mice with mutations. Delivery of Cas9 DNA sequences also is contemplated.
Guide RNA (gRNA)
As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type 21 promoter U6). Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a portion which is targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.
The invention provides immune cells that express chimeric antigen receptors (CARs) and that have been modified to reduce or eliminate expression or activity of an NKG2A polypeptide and/or a CD94 polypeptide. Modification of immune cells to express a chimeric antigen receptor can enhance an immune cell's immunoreactive activity, wherein the chimeric antigen receptor has an affinity for an epitope on an antigen, wherein the antigen is associated with an altered fitness of an organism. For example, the chimeric antigen receptor can have an affinity for an epitope on a protein expressed in a neoplastic cell. Because the CAR-T cells can act independently of major histocompatibility complex (MHC), activated CAR-T cells can kill the neoplastic cell expressing the antigen.
Some embodiments comprise autologous immune cell immunotherapy, wherein immune cells are obtained from a subject having a disease or altered fitness characterized by cancerous or otherwise altered cells expressing a surface marker. The obtained immune cells are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. Thus, in some embodiments, immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, these autologous immune cells are cultured and modified shortly after they are obtained from the subject. In other embodiments, the autologous cells are obtained and then stored for future use. This practice may be advisable for individuals who may be undergoing parallel treatment that will diminish immune cell counts in the future. In allogeneic immune cell immunotherapy, immune cells can be obtained from a donor other than the subject who will be receiving treatment. In some embodiments, immune cells are obtained from a healthy subject or donor and are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. The immune cells, after modification to express a chimeric antigen receptor, are administered to a subject for treating a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). In some embodiments, immune cells to be modified to express a chimeric antigen receptor can be obtained from pre-existing stock cultures of immune cells.
Immune cells and/or immune effector cells can be isolated or purified from a sample collected from a subject or a donor using standard techniques known in the art. For example, immune effector cells can be isolated or purified from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood cells by centrifugation. The immune effector cells can be further isolated or purified using a selective purification method that isolates the immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD4+ is used as a marker to select T cells. In one embodiment, CD8+ is used as a marker to select T cells. In one embodiment, CD4+ and CD8+ are used as a marker to select regulatory T cells.
One technique for isolating or purifying immune effector cells is flow cytometry. In fluorescence activated cell sorting a fluorescently labelled antibody with affinity for an immune effector cell marker is used to label immune effector cells in a sample. A gating strategy appropriate for the cells expressing the marker is used to segregate the cells. For example, T lymphocytes can be separated from other cells in a sample by using, for example, a fluorescently labeled antibody specific for an immune effector cell marker (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategy. In one embodiment, a CD4 gating strategy is employed. In one embodiment, a CD8 gating strategy is employed. In one embodiment, a CD4 and CD8 gating strategy is employed. In some embodiments, a gating strategy for other markers specific to an immune effector cell is employed instead of, or in combination with, the CD4 and/or CD8 gating strategy.
The immune effector cells contemplated in the invention are effector T cells. In some embodiments, the effector T cell is a naïve CD8+ T cell, a cytotoxic T cell, a natural killer T (NKT) cell, a natural killer cell, a gammadelta T cell (γδ T cell), or a regulatory T (Treg) cell. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4+CD8+ T cell or a CD4− CD8− T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Th1), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell). In some embodiments, the immune effector cell is any other subset of T cells.
Chimeric antigen receptors as contemplated in the present invention comprise an extracellular binding domain, a transmembrane domain, and an intracellular domain. Binding of an antigen to the extracellular binding domain can activate the CAR-T cell and generate an effector response, which includes CAR-T cell proliferation, cytokine production, and other processes that lead to the death of the antigen expressing cell. In some embodiments of the present invention, the chimeric antigen receptor further comprises a linker.
In various embodiments, the CAR specifically binds CD19 or any other antigen that can be targeted by a chimeric antigen receptor (CAR), such as BCMA, Mesothelin, MUC1, MUC16, GD2, CD79b19, April, EGFR, EGFRvIII, IL13Ra, HLA-G, or PSMA. Further non-limiting examples of antigens that can be bound by a CAR of the present disclosure include those described in Xu, et al. “The development of CAR design for tumor CAR-T cell therapy,” Oncotarget, 9(17) doi: 10.18632/oncotarget.24179, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
Chimeric antigen receptors, or any polypeptide of the present disclosure, can be delivered to an immune cell using a polynucleotide encoding the chimeric antigen receptor or polypeptide. For example, immune cells obtained from a subject may be transformed with a nucleic acid vector encoding the chimeric antigen receptor. The vector may then be used to transform recipient immune cells so that these cells will then express the chimeric antigen receptor. Efficient means of transforming immune cells include transfection and transduction. Such methods are well known in the art. For example, applicable methods for delivery the nucleic acid molecule encoding the chimeric antigen receptor (and the nucleic acid(s) encoding the base editor) can be found in International Application No. PCT/US2009/040040 and U.S. Pat. Nos. 8,450,112; 9,132,153; and 9,669,058, each of which is incorporated herein in its entirety. Additionally, those methods and vectors described herein for delivering the nucleic acid encoding the base editor are applicable to delivering the nucleic acid encoding the chimeric antigen receptor.
The chimeric antigen receptors of the invention include an extracellular binding domain. The extracellular binding domain of a chimeric antigen receptor contemplated herein comprises an amino acid sequence of an antibody, or an antigen binding fragment thereof, that has an affinity for a specific antigen. In some embodiments, the antigen is CD19.
In some embodiments the chimeric antigen receptor comprises an amino acid sequence of an antibody. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of an antigen binding fragment of an antibody. The antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of an antigen. In some embodiments, the antibody fragment portion of a chimeric antigen receptor is a single chain variable fragment (scFv). An scFv comprises the light and variable fragments of a monoclonal antibody. In other embodiments, the antibody fragment portion of a chimeric antigen receptor is a multichain variable fragment, which can comprise more than one extracellular binding domains and therefore bind to more than one antigen simultaneously. In a multiple chain variable fragment embodiment, a hinge region may separate the different variable fragments, providing necessary spatial arrangement and flexibility.
In other embodiments, the antibody portion of a chimeric antigen receptor comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion of a chimeric antigen receptor comprises two heavy chains, joined by disulfide bridges and two light chains, wherein the light chains are each joined to one of the heavy chains by disulfide bridges. In some embodiments, the light chain comprises a constant region and a variable region. Complementarity determining regions residing in the variable region of an antibody are responsible for the antibody's affinity for a particular antigen. Thus, antibodies that recognize different antigens comprise different complementarity determining regions. Complementarity determining regions reside in the variable domains of the extracellular binding domain, and variable domains (i.e., the variable heavy and variable light) can be linked with a linker or, in some embodiments, with disulfide bridges.
In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. Antigens can be heterologous, such as those expressed in a pathogenic bacteria or virus. Antigens can also be synthetic; for example, some individuals have extreme allergies to synthetic latex and exposure to this antigen can result in an extreme immune reaction. In some embodiments, the antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the antigen is expressed in a neoplastic cell.
The chimeric antigen receptors of the invention include a transmembrane domain. The transmembrane domain of the chimeric antigen receptors described herein spans the CAR-T cell's lipid bilayer cellular membrane and separates the extracellular binding domain and the intracellular signaling domain. In some embodiments, this domain is derived from other receptors having a transmembrane domain, while in other embodiments, this domain is synthetic. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain and, in some embodiments, humanized. By “humanized” is meant having the sequence of the nucleic acid encoding the transmembrane domain optimized such that it is more reliably or efficiently expressed in a human subject. In some embodiments, the transmembrane domain is derived from another transmembrane protein expressed in a human immune effector cell.
The chimeric antigen receptors of the invention include an intracellular signaling domain. The intracellular signaling domain is the intracellular portion of a protein expressed in a T cell that transduces a T cell effector function signal (e.g., an activation signal) and directs the T cell to perform a specialized function. T cell activation can be induced by a number of factors, including binding of cognate antigen to the T cell receptor on the surface of T cells and binding of cognate ligand to costimulatory molecules on the surface of the T cell. A T cell co-stimulatory molecule is a cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule. Activation of a T cell leads to immune response, Such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art.
The intracellular signaling domain of the chimeric antigen receptor contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises the primary signaling domain and a secondary, or co-stimulatory, signaling domain.
In various aspects, the methods of the disclosure involve characterizing a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). In some instances, the characterization of a neoplasia involves determining whether or not the neoplasia has a loss of 6p21.3 and/or measuring expression levels of one or more interferon-stimulated genes (ISGs) in the neoplasia. Such characterization and measurements can be carried out using methods familiar to one of skill in the art, which include, but are not limited to, those described herein.
In some cases, the methods provided herein can be used to detect loss of expression of a polypeptide (e.g., NKG2A (Klrc1) and/or CD94 (Klrd1)) in a cell (e.g., a modified immune cells, such as a CAR T cell). The methods can also be used to detect expression of a heterologous polypeptide in a cell (e.g., a chimeric antigen receptor).
In some instances, measuring expression levels of one or more interferon-stimulated genes (ISGs) involves measuring expression levels for about, at least about, or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more of the following genes: ADAR, APOL6, ARID5B, ARL4A, AUTS2, B2M, BANK1, BATF2, BPGM, BST2, BTG1, C1R, C1S, CASP1, CASP3, CASP4, CASP7, CASP8, CCL2, CCL5, CCL7, CD274, CD38, CD40, CD69, CD74, CD86, CDKN1A, CFB, CFH, CIITA, CMKLR1, CMPK2, CMTR1, CSF2RB, CXCL10, CXCL11, CXCL9, DDX58, DDX60, DHX58, EIF2AK2, EIF4E3, EPSTI1, FAS, FCGR1A, FGL2, FPR1, GBP4, GBP6, GCH1, GPR18, GZMA, HELZ2, HERC6, HIF1A, HLA-A, HLA-B, HLA-DMA, HLA-DQA1, HLA-DRB1, HLA-G, ICAM1, IDO1, IFI27, IFI30, IFI35, IFI44, IFI44L, IFIH1, IFIT1, IFIT2, IFIT3, IFITM2, IFITM3, IFNAR2, IL10RA, IL15, IL15RA, IL18BP, IL2RB, IL4R, IL6, IL7, IRF1, IRF2, IRF4, IRF5, IRF7, IRF8, IRF9, ISG15, ISG20, ISOC1, ITGB7, JAK2, KLRK1, LAP3, LATS2, LCP2, LGALS3BP, LY6E, LYSMD2, MARCHF1, METTL7B, MT2A, MTHFD2, MVP, MX1, MX2, MYD88, NAMPT, NCOA3, NFKB1, NFKBIA, NLRC5, NMI, NOD1, NUP93, OAS2, OAS3, OASL, OGFR, P2RY14, PARP12, PARP14, PDE4B, PELI1, PFKP, PIM1, PLA2G4A, PLSCR1, PML, PNP, PNPT1, PSMA2, PSMA3, PSMB10, PSMB2, PSMB8, PSMB9, PSME1, PSME2, PTGS2, PTPN1, PTPN2, PTPN6, RAPGEF6, RBCK1, RIPK1, RIPK2, RNF213, RNF31, RSAD2, RTP4, SAMD9L, SAMHD1, SECTM1, SELP, SERPING1, SLAMF7, SLC25A28, SOCS1, SOCS3, SOD2, SP110, SPPL2A, SRI, SSPN, ST3GAL5, ST8SIA4, STAT1, STAT2, STAT3, STAT4, TAP1, TAPBP, TDRD7, TNFAIP2, TNFAIP3, TNFAIP6, TNFSF10, TOR1B, TRAFD1, TRIM14, TRIM21, TRIM25, TRIM26, TXNIP, UBE2L6, UPP1, USP18, VAMP5, VAMP8, VCAM1, WARS1, XAF1, XCL1, ZBP1, and ZNFX1. In some cases, measuring expression levels of one or more ISGs involves measuring expression levels for IFNγ and/or HLA-E. In embodiments, the methods of the disclosure involve determining an ISG score based upon measured levels of one or more ISG genes. In some cases, a high ISG score relative to a reference indicates that a neoplasia will be or has an increased probability of being resistant to immunotherapy. In embodiments, the ISG score is an IFNγ score calculated using ssGSEA (Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108-112 (2009)) using the Hallmark Interferon Gamma Response gene set (Liberzon, A. et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1, 417-425 (2015)). In embodiments, an alteration in expression of any of the aforementioned genes is by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100%. In another embodiment, the alteration is a significant increase or reduction in the level or activity of any of the aforementioned genes or their associated polypeptides.
In various cases, loss of 6p21.3 is detected as a loss of expression of and/or a gene encoding one or more of a TAP1, TAP2, TAPBP, PSMB8, and/or PSMB9 polypeptide. The presence or absence of a gene can be determined using methods familiar to one of skill in the art including, but not limited to, the gene sequencing methods described herein. In embodiments, loss of 6p21.3 indicates that a neoplasia is likely to be responsive to immunotherapy; whereas, presence of 6p21.3 indicates that a neoplasia is likely to be resistant to immunotherapy.
Gene expression levels can be detected using biomarkers (e.g., polynucleotides or polypeptides). In some cases a biomarker is a polynucleotide (e.g., mRNA, a portion of a genome, and/or a gene). The biomarkers of this invention can be detected by any suitable method. The methods described herein can be used individually or in combination for a more accurate detection of the biomarkers (e.g., biochip in combination with mass spectrometry, immunoassay in combination with mass spectrometry, and the like).
Detection paradigms that can be employed in the invention include, but are not limited to, optical methods, electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
These and additional methods are describe below.
Detection by Sequencing and/or Probes
In particular embodiments, the biomarkers of the invention are measured by a sequencing- and/or probe-based technique (e.g., RNA-seq).
RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling. In embodiments, to mitigate sequence-dependent bias resulting from amplification complications to allow truly digital RNA-Seq, a set of barcode sequences can be used to ensure that every cDNA molecule prepared from an mRNA sample is uniquely labeled by random attachment of barcode sequences to both ends (see, e.g., Shiroguchi K, et al. Proc Natl Acad Sci USA. 2012 Jan. 24; 109(4):1347-52). After PCR, paired-end deep sequencing can be applied to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance can be measured based on the number of unique barcode sequences observed for a given cDNA sequence. The barcodes may be optimized to be unambiguously identifiable. This method is a representative example of how to quantify a whole transcriptome from a sample.
Detecting a target polynucleotide sequence or fragment thereof associated with a biomarker that hybridizes to a probe sequence may involve sequencing, FACS, qPCR, RT-PCR, a genotyping array, and/or a NanoString assay (see, e.g., Malkov, et al. “Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System”, BMC Research Notes, 2: Article No: 80 (2009)), or any of various other techniques known to one of skill in the art. Various detection methods may be used and are described as follows.
Preparation of a library for sequencing may involve an amplification step. Amplification may involve thermocycling or isothermal amplification (such as through the methods RPA or LAMP). Cross-linking may involve overlap-extension PCR or use of ligase to associate multiple amplification products with each other. Amplification can refer to any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR. In particular, the isolated RNA can be subjected to a reverse transcription assay that is coupled with a quantitative polymerase chain reaction (RT-PCR) in order to quantify the expression level of a biomarker.
Detection of the expression level of a biomarker can be conducted in real time in an amplification assay (e.g., qPCR). In one aspect, the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems in the art. DNA-binding dyes suitable for this application include, as non-limiting examples, SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
Other fluorescent labels such as sequence specific probes can be employed in the amplification reaction to facilitate the detection and quantification of the amplified products. Probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product. It utilizes fluorescent, target-specific probes (e.g., TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are taught, for example, in U.S. Pat. No. 5,210,015.
Sequencing may be performed on any high-throughput platform. Methods of sequencing oligonucleotides and nucleic acids are well known in the art (see, e.g., WO93/23564, WO98/28440 and WO98/13523; U.S. Pat. App. Pub. No. 2019/0078232; U.S. Pat. Nos. 5,525,464; 5,202,231; 5,695,940; 4,971,903; 5,902,723; 5,795,782; 5,547,839 and 5,403,708; Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977); Drmanac et al., Genomics 4:114 (1989); Koster et al., Nature Biotechnology 14:1123 (1996); Hyman, Anal. Biochem. 174:423 (1988); Rosenthal, International Patent Application Publication 761107 (1989); Metzker et al., Nucl. Acids Res. 22:4259 (1994); Jones, Biotechniques 22:938 (1997); Ronaghi et al., Anal. Biochem. 242:84 (1996); Ronaghi et al., Science 281:363 (1998); Nyren et al., Anal. Biochem. 151:504 (1985); Canard and Arzumanov, Gene 11:1 (1994); Dyatkina and Arzumanov, Nucleic Acids Symp Ser 18:117 (1987); Johnson et al., Anal. Biochem. 136:192 (1984); and Elgen and Rigler, Proc. Natl. Acad. Sci. USA 91(13):5740 (1994), all of which are expressly incorporated by reference).
The sequencing of a polynucleotide can be carried out using any suitable commercially available sequencing technology. In embodiments, the sequencing of a polynucleotide is carried out using a chain termination method of DNA sequencing (e.g., Sanger sequencing). In some embodiments, commercially available sequencing technology is a next-generation sequencing technology, including as non-limiting examples combinatorial probe anchor synthesis (cPAS), DNA nanoball sequencing, droplet-based or digital microfluidics, heliscope single molecule sequencing, nanopore sequencing (e.g., Oxford Nanopore technologies), GeneGap sequencing, massively parallel signature sequencing (MPSS), microfluidic Sanger sequencing, microscopy-based techniques (e.g., transmission electronic microscopy DNA sequencing), RNA polymerase (RNAP) sequencing, single-molecule real-time (SMRT) sequencing, SOLiD sequencing, ion semiconductor sequencing, polony sequencing, Pyrosequencing (454), sequencing by hybridization, sequencing by synthesis (e.g., Illumina™ sequencing), sequencing with mass spectrometry, and tunneling currents DNA sequencing.
In embodiments, levels of biomarkers in a sample are quantified using targeted sequencing. Methods for targeted sequencing are well known in the art (see, e.g., Rehm, “Disease-targeted sequencing: a cornerstone in the clinic”, Nature Reviews Genetics, 14:295-300 (2013)).
In embodiments, a probe comprises a molecular identifier, such as a fluorescent or chemiluminescent label, a radioactive isotope label, an enzymatic ligand, or the like. The molecular identifier can be a fluorescent label or an enzyme tag, such as digoxigenin, β-galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.
Methods used to detect or quantify binding of a probe to a target biomarker will typically depend upon the molecular identifier. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. Enzymatic labels can be detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and colorimetric labels can be detected by visualizing a colored label.
Specific non-limiting examples of molecular identifiers include radioisotopes, such as 32P, 14C, 125I, 3H, and 131I, fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin, and ruthenium. In the case where biotin is employed as a molecular identifier, streptavidin bound to an enzyme (e.g., peroxidase) may further be added to facilitate detection of the biotin.
Examples of fluorescent molecular identifiers include, but are not limited to, Atto dyes, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinyl sulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives; eosin, eosin isothiocyanate, erythrosin and derivatives; erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine
A fluorescent molecular identifier may be a fluorescent protein, such as blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, red fluorescent protein, yellow fluorescent protein or any photoconvertible protein. Colorimetric molecular identifiers, bioluminescent molecular identifiers and/or chemiluminescent molecular identifiers may be used in embodiments of the invention.
Detection of a molecular identifier may involve detecting energy transfer between molecules in a hybridization complex by perturbation analysis, quenching, or electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes. The fluorescent molecular identifier may be a perylene or a terrylen. In the alternative, the fluorescent molecular identifier may be a fluorescent bar code.
The molecular identifier may be light sensitive, wherein the label is light-activated and/or light cleaves the one or more linkers to release the molecular cargo. The light-activated molecular cargo may be a major light-harvesting complex (LHCII). In another embodiment, the fluorescent molecular label may induce free radical formation.
In an advantageous embodiment, agents may be uniquely labeled in a dynamic manner (see, e.g., international patent application serial no. PCT/US2013/61182 filed Sep. 23, 2012). The unique labels are, at least in part, nucleic acid in nature, and may be generated by sequentially attaching two or more detectable oligonucleotide tags to each other and each unique label may be associated with a separate agent. A detectable oligonucleotide tag may be an oligonucleotide that may be detected by sequencing of its nucleotide sequence and/or by detecting non-nucleic acid detectable moieties to which it may be attached.
In embodiments, the molecular identifier is a microparticle, including, as non-limiting examples, quantum dots (Empodocles, et al., Nature 399:126-130, 1999), or gold nanoparticles (Reichert et al., Anal. Chem. 72:6025-6029, 2000).
In particular embodiments, the biomarkers of the invention are measured by immunoassay. Immunoassay typically utilizes an antibody (or other agent that specifically binds the marker) to detect the presence or level of a biomarker in a sample. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.
This invention contemplates traditional immunoassays including, for example, Western blot, sandwich immunoassays including ELISA and other enzyme immunoassays, fluorescence-based immunoassays (e.g., flow cytometry), and chemiluminescence. Nephelometry is an assay done in liquid phase, in which antibodies are in solution. Binding of the antigen to the antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR).
Immunoassays can be carried out on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other forms that supports binding of the antibody to the marker and subsequent detection. A single marker may be detected at a time or a multiplex format may be used. Multiplex immunoanalysis may involve planar microarrays (protein chips) and bead-based microarrays (suspension arrays).
In a SELDI-based immunoassay, a biospecific capture reagent for the biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry.
In embodiments, a sample is analyzed by means of a biochip (also known as a microarray). The polypeptides and nucleic acid molecules of the invention are useful as hybridizable array elements in a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.
The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.
In embodiments, a sample is analyzed by means of a protein biochip (also known as a protein microarray). Such biochips are useful in high-throughput low-cost screens to identify alterations in the expression or post-translation modification of a biomarker, or a fragment thereof. In embodiments, a protein biochip of the invention binds a biomarker present in a sample and detects an alteration in the level of the biomarker. Typically, a protein biochip features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the invention) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).
In embodiments, the protein biochip is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a patient, such as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.
Many protein biochips are described in the art. These include, for example, protein biochips produced by Ciphergen Biosystems, Inc. (Fremont, CA), Zyomyx (Hayward, CA), Packard BioScience Company (Meriden, CT), Phylos (Lexington, MA), Invitrogen (Carlsbad, CA), Biacore (Uppsala, Sweden) and Procognia (Berkshire, UK). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Pat. Nos. 6,225,047; 6,537,749; 6,329,209; and 5,242,828; PCT International Publication Nos. WO 00/56934; WO 03/048768; and WO 99/51773.
In aspects of the invention, a sample is analyzed by means of a nucleic acid biochip (also known as a nucleic acid microarray). To produce a nucleic acid biochip, oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.). Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, e.g., as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, ascites, cyst fluid, and the like); a homogenized tissue sample (e.g., a tissue sample obtained by biopsy); or a cell isolated from a patient sample. For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are well known in the art. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the biochip.
Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions include, as non-limiting examples, temperatures of at least about 30° C., of at least about 37° C., or of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In an embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In embodiments, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In other embodiments, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., of at least about 42° C., or of at least about 68° C. In embodiments, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In other embodiments, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
Detection system for measuring the absence, presence, and amount of hybridization for all of the distinct nucleic acid sequences are well known in the art. For example, simultaneous detection is described in Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997. In embodiments, a scanner is used to determine the levels and patterns of fluorescence.
In embodiments, the biomarkers of this invention are detected by mass spectrometry (MS). Mass spectrometry is a well-known tool for analyzing chemical compounds that employs a mass spectrometer to detect gas phase ions. Mass spectrometers are well known in the art and include, but are not limited to, time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The method may be performed in an automated (Villanueva, et al., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with the mass spectrometer operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC-MS/MS). Methods for performing mass spectrometry are well known and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979 and the references disclosed therein.
In embodiments, the mass spectrometer is a laser desorption/ionization mass spectrometer. In laser desorption/ionization mass spectrometry, the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer. The analysis of proteins by LDI can take the form of MALDI or of SELDI. The analysis of proteins by LDI can take the form of MALDI or of SELDI.
Laser desorption/ionization in a single time of flight instrument typically is performed in linear extraction mode. Tandem mass spectrometers can employ orthogonal extraction modes.
In embodiments, the mass spectrometric technique for use in the invention is matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). In related embodiments, the procedure is MALDI with time of flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on a membrane with an agent that absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV or IR laser light into the vapor phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass/charge (m/z) reading which is very accurate and sensitive. MALDI spectrometers are well known in the art and are commercially available from, for example, PerSeptive Biosystems, Inc. (Framingham, Mass., USA).
Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, in embodiments, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.
MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the invention to produce an array of spots on a collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using an server (e.g., ExPASy) to generate the data in a form suitable for computers.
Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on a collection membrane. These include, but are not limited to, the use of delayed ion extraction, energy reflectors, ion-trap modules, and the like. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap, TOF, single quadrupole, multi-quadrupole mass spectrometers, and the like. The use of such devices (other than a single quadrupole) allows MS-MS or MSn analysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.
Capillary infusion may be employed to introduce the biomarker to a desired mass spectrometer implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques including, but not limited to, gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with mass spectrometry. One variation of the technique is the coupling of high-performance liquid chromatography (HPLC) to a mass spectrometer for integrated sample separation/and mass spectrometer analysis.
Quadrupole mass analyzers may also be employed as needed to practice the invention. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers high resolution and the ability of tandem mass spectrometry experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.
In embodiments, the mass spectrometric technique for use in the invention is “Surface Enhanced Laser Desorption and Ionization” or “SELDI,” as described, for example, in U.S. Pat. Nos. 5,719,060 and 6,225,047, both to Hutchens and Yip. This refers to a method of desorption/ionization gas phase ion spectrometry (e.g., mass spectrometry) in which an analyte (here, one or more of the biomarkers) is captured on the surface of a SELDI mass spectrometry probe.
SELDI has also been called “affinity capture mass spectrometry.” It also is called “Surface-Enhanced Affinity Capture” or “SEAC”. This version involves the use of probes that have a material on the probe surface that captures analytes through a non-covalent affinity interaction (adsorption) between the material and the analyte. The material is variously called an “adsorbent,” a “capture reagent,” an “affinity reagent” or a “binding moiety.” Such probes can be referred to as “affinity capture probes” and as having an “adsorbent surface.” The capture reagent can be any material capable of binding an analyte. The capture reagent is attached to the probe surface by physisorption or chemisorption. In certain embodiments the probes have the capture reagent already attached to the surface. In other embodiments, the probes are pre-activated and include a reactive moiety that is capable of binding the capture reagent, e.g., through a reaction forming a covalent or coordinate covalent bond. Epoxide and acyl-imidizole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors. Nitrilotriacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact non-covalently with histidine containing peptides. Adsorbents are generally classified as chromatographic adsorbents and biospecific adsorbents.
“Chromatographic adsorbent” refers to an adsorbent material typically used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators (e.g., nitrilotriacetic acid or iminodiacetic acid), immobilized metal chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, simple biomolecules (e.g., nucleotides, amino acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g., hydrophobic attraction/electrostatic repulsion adsorbents).
A biospecific adsorbent is an adsorbent comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). In certain instances, the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Examples of biospecific adsorbents are antibodies, receptor proteins and nucleic acids. Biospecific adsorbents typically have higher specificity for a target analyte than chromatographic adsorbents. Further examples of adsorbents for use in SELDI can be found in U.S. Pat. No. 6,225,047. A “bioselective adsorbent” refers to an adsorbent that binds to an analyte with an affinity of at least 10−8 M.
Protein biochips produced by Ciphergen comprise surfaces having chromatographic or biospecific adsorbents attached thereto at addressable locations. Ciphergen's ProteinChip® arrays include NP20 (hydrophilic); H4 and H50 (hydrophobic); SAX-2, Q-10 and (anion exchange); WCX-2 and CM-10 (cation exchange); IMAC-3, IMAC-30 and IMAC-50 (metal chelate); and PS-10, PS-20 (reactive surface with acyl-imidazole, epoxide) and PG-20 (protein G coupled through acyl-imidazole). Hydrophobic ProteinChip arrays have isopropyl or nonylphenoxy-poly(ethylene glycol)methacrylate functionalities. Anion exchange ProteinChip arrays have quaternary ammonium functionalities. Cation exchange ProteinChip arrays have carboxylate functionalities. Immobilized metal chelate ProteinChip arrays have nitrilotriacetic acid functionalities (IMAC 3 and IMAC 30) or O-methacryloyl-N,N-bis-carboxymethyl tyrosine functionalities (IMAC 50) that adsorb transition metal ions, such as copper, nickel, zinc, and gallium, by chelation. Preactivated ProteinChip arrays have acyl-imidazole or epoxide functional groups that can react with groups on proteins for covalent binding.
Such biochips are further described in: U.S. Pat. No. 6,579,719 (Hutchens and Yip, “Retentate Chromatography,” Jun. 17, 2003); U.S. Pat. No. 6,897,072 (Rich et al., “Probes for a Gas Phase Ion Spectrometer,” May 24, 2005); U.S. Pat. No. 6,555,813 (Beecher et al., “Sample Holder with Hydrophobic Coating for Gas Phase Mass Spectrometer,” Apr. 29, 2003); U.S. Patent Publication No. U.S. 2003-0032043 A1 (Pohl and Papanu, “Latex Based Adsorbent Chip,” Jul. 16, 2002); and PCT International Publication No. WO 03/040700 (Um et al., “Hydrophobic Surface Chip,” May 15, 2003); U.S. Patent Application Publication No. US 2003/-0218130 A1 (Boschetti et al., “Biochips With Surfaces Coated With Polysaccharide-Based Hydrogels,” Apr. 14, 2003) and U.S. Pat. No. 7,045,366 (Huang et al., “Photocrosslinked Hydrogel Blend Surface Coatings” May 16, 2006).
In general, a probe with an adsorbent surface is contacted with the sample for a period of time sufficient to allow the biomarker or biomarkers that may be present in the sample to bind to the adsorbent. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; preferably, aqueous solutions are employed. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature. Unless the probe has both SEAC and SEND properties (as described herein), an energy absorbing molecule then is applied to the substrate with the bound biomarkers.
In yet another method, one can capture the biomarkers with a solid-phase bound immuno-adsorbent that has antibodies that bind the biomarkers. After washing the adsorbent to remove unbound material, the biomarkers are eluted from the solid phase and detected by applying to a SELDI biochip that binds the biomarkers and analyzing by SELDI.
The biomarkers bound to the substrates are detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer. The biomarkers are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a biomarker typically will involve detection of signal intensity. Thus, both the quantity and mass of the biomarker can be determined.
The methods provided herein can be used for treating a subject for a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer) and/or for selecting a subject an agent (e.g., using CAR T cells and/or an immune checkpoint blockade (ICB)) to administer to a subject to treat a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). In embodiments, the methods provided herein can be used for selecting a subject for treatment using an immunotherapy. In embodiments, a subject is administered, for example, checkpoint blockade therapy comprising a PD-1/PD-L1 checkpoint inhibitor (e.g., Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, Cemiplimab, Dostarlimab). Non-limiting examples of checkpoint inhibitors include anti-PD-1 and anti-CTLA-4 antibodies (e.g., ipilimumab). Thus, the methods provided herein include methods for the treatment of a neoplasia (e.g., cancer, such as skin, colon, pancreas, lung, and kidney cancer). Generally, the methods provided herein include administering a therapeutically effective amount of a treatment as provided herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. The treatments can be selected based upon interferon-stimulated gene expression levels (e.g., an IFNγ score). Treatments can be selected based upon loss of 6p21.3. In some cases, a high IFNγ score selects a patient for administration of an immune checkpoint blockade comprising an NKG2A/CD94 receptor inhibitor.
Non-limiting examples of NKG2A/CD94 receptor inhibitors include anti-NKG2A and/or anti-CD94 antibodies, such as Monalizumab.
In embodiments, the methods provided herein can be used for selecting a subject for inclusion in or exclusion from a clinical trial. In embodiments, the clinical trial is designed to test the efficacy of an immunotherapy. The methods provided herein can assist in selecting patients likely to respond to a particular agent for inclusion in a clinical trial for the study of patient response to the agent. In embodiments, the methods of the invention involve using interferon-stimulated gene expression levels and/or loss of 6p21.3 to separate subjects likely to respond to an agent from those likely not to respond to the agent.
The methods provided herein include selecting a subject for and/or administering to a subject having or having a propensity to develop a neoplasia a treatment that includes a therapeutically effective amount of an immunotherapeutic agent, such as a CAR T cell of the disclosure (e.g., CAR T cells modified to reduce or eliminate expression or activity of NKG2A and/or CD94) and/or an immune checkpoint blockade. In some cases, the immune checkpoint blockade agent comprises a PD-1/PD-L1 pathway inhibitor.
The protein, Programmed Death 1 (PD-1), is an inhibitory member of the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells. The structure and function of PD-1 is further described in e.g., Okazaki et al., Curr. Opin. Immunol., 14:779-782 (2002); and Bennett et al., J. Immunol., 170:711-718 (2003), the teachings of each of which are incorporated herein by reference in their entireties. Non-limiting examples of PC-1/PD-L1 pathway-inhibitors include anti-PD-1 antibodies and/or anti-CTLA-4 antibodies.
Two ligands for PD-1 include PD-L1 (B7-H1, also called CD274 molecule) and PD-L2 (b7-DC). The PD-L1 ligand is abundant in a variety of human cancers. The interaction of PD-L1 with PD-1 generally results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells. See, e.g., Dong et al., Nat. Med., 8:787-789 (2002); Blank et al., Cancer Immunol. Immunother., 54:307-314 (2005); and Konishi et al., Clin. Cancer Res., 10:5094-5100 (2004), the teachings of each of which have been incorporated herein by reference in their entireties.
Inhibition of the interaction of PD-1 with PD-L1 can restore immune cell activation, such as T-cell activity, to reduce tumorigenesis and metastasis, making PD-1 and PD-L1 advantageous cancer therapies. See, e.g., Yang J., et al., J Immunol. August 1: 187(3): 1113-9 (2011), the teachings of which has been incorporated herein by reference in its entirety.
Non-limiting examples of PD-1/PD-L1 inhibitors that can be administered to a subject in need of treatment include Atezolizumab (Tecentriq, MPDL3280A, RG7446); Avelumab (Bavencio, MSB0010718C); BMS-936559 (MDX-1105); Cemiplimab (Libtayo REGN-2810, REGN2810, cemiplimab-rwlc); Durvalumab (MED14736, MEDI-4736); Nivolumab (Opdivo ONO-4538, BMS-936558, MDX1106); and Pembrolizumab (Keytruda, MK-3475).
In some embodiments, the agent(s) provided herein (e.g., CAR T cells modified to reduce or eliminate expression or activity of NKG2A and/or CD94) is administered in combination with an additional chemotherapeutic agent. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time.
An effective amount of an agent can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound or agent (i.e., an effective dosage) depends on the therapeutic compounds or agents selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic agents provided herein can include a single treatment or a series of treatments.
Dosage, toxicity and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents which exhibit high therapeutic indices are preferred. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test agent which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration (e.g., oral administration, intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, intracranial, intraspinal, subcutaneous, intraarticular, intrasynovial, intrathecal, topical, or inhalation routes) is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles described in Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.
For in vivo administration of any of the agents of the present disclosure, normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of an individual's and/or subject's body weight or more per day, depending upon the route of administration. In some embodiments, the dose amount is about 1 mg/kg/day to 10 mg/kg/day. In some embodiments, the dose amount of a CAR T cell is about, at least about, and/or no more than about 1e5 cells, 1e6 cells, 1e7 cells, 1e8 cells, 1e9 cells, 1e10 cells, 1e11 cells, 1e12 cells, 1e13 cells, 1e14 cells, 1e15 cells, or 1e16 cells. For repeated administrations over several days or longer, depending on the severity of the disease, disorder, or condition to be treated, the treatment is sustained until a desired suppression of symptoms is achieved.
An effective amount of an agent of the instant disclosure may vary, e.g., from about 0.001 mg/kg to about 1000 mg/kg or more in one or more dose administrations for one or several days (depending on the mode of administration). In certain embodiments, the effective amount per dose varies from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150 mg/kg.
An exemplary dosing regimen may include administering an initial dose of an agent of the disclosure of about 200 μg/kg, followed by a weekly maintenance dose of about 100 μg/kg every other week. Other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is contemplated herein. In certain embodiments, dosing ranging from about 3 μg/kg to about 2 mg/kg (such as about 3 μg/kg, about 10 μg/kg, about 30 μg/kg. about 100 μg/kg, about 300 μg/kg, about 1 mg/kg. or about 2 mg/kg) may be used. In certain embodiments, dosing frequency is three times per day, twice per day, once per day, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. Progress of the therapy is easily monitored by conventional techniques and assays. The dosing regimen, including the agent(s) administered, can vary over time independently of the dose used.
Methods for characterizing the efficacy of a treatment for a neoplasia are well known in the art (e.g., computerized tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), position emission tomography (PET) scan, ultrasound X-ray, biopsy, etc.).
Provided also are pharmaceutical compositions for use in treating a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). In an embodiment, the compositions include CAR T cells modified to reduce or eliminate expression or activity of an NKG2A and/or CD94 polypeptide, as described herein and an acceptable carrier, excipient, or diluent.
The agents of the disclosure (e.g., chemotherapeutic agents, CAR T cells, and/or immune checkpoint blockades (ICBs)) may be contained in any appropriate amount in any suitable carrier substance, and is/are generally present in an amount of 0.01-95% by weight of the total weight of the composition. The pharmaceutical composition may be provided in a form that is suitable for a parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration route, such that the agent, such as a vector described herein, is systemically delivered.
The pharmaceutical compositions of the present invention can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed. 2005). In general, the immune cell, or population thereof is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.
Some nonlimiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
Pharmaceutical compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.
Pharmaceutical compositions can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.
The skilled artisan can readily determine the number of cells and amount of optional additives, vehicles, and/or carriers in compositions and to be administered in methods of the invention. Typically, additives are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and still more preferably about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent such as a mouse); and, the dosage of the composition(s), concentration of components therein, and the timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein, and the time for sequential administrations can be ascertained without undue experimentation.
Pharmaceutical compositions may be formulated to release an agent substantially immediately upon administration or at any predetermined time or time after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) compositions that create a substantially constant concentration of the agent within the body over an extended period of time; (ii) compositions that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) compositions that sustain action during a predetermined time period by maintaining a relatively constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) compositions that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with a target site or location, e.g., in a region of a tissue or organ; (v) compositions that allow for convenient dosing, such that doses are administered, for example, once every one, two, or several weeks; and (vi) compositions that target a specific tissue or cell type.
In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. The pharmaceutical composition may be administered systemically.
The pharmaceutical composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the agent (e.g., CAR T cells, immune checkpoint blockade (ICB), or other chemotherapeutic agent), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
In some embodiments, the pharmaceutical composition are formulated for intravenous delivery. As noted above, the compositions according to the described embodiments may be in a form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be employed include water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
The pharmaceutical composition described herein can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and/or conditions.
The present disclosure also relates to a computer system involved in carrying out the methods of the disclosure relating to both computations and sequencing. In the methods described herein, analyses (e.g., calculating expression levels and/or calculation of an ISG score and/or IFNγ score) can be performed on general-purpose or specially-programmed hardware or software. One can then record the results on tangible medium, for example, in computer-readable format such as a memory drive or disk or simply printed on paper, displayed on a monitor (e.g., a computer screen, a smart device, a tablet, a television screen, or the like), or displayed on any other visible medium. The results also could be reported on a computer screen.
In aspects, the analysis is performed by an algorithm. The analysis of sequences will generate results that are subject to data processing. Data processing can be performed by the algorithm. One of ordinary skill can readily select and use the appropriate software and/or hardware to analyze a sequence.
In aspects, the analysis is performed by a computer-readable medium. The computer-readable medium can be non-transitory and/or tangible. For example, the computer readable medium can be volatile memory (e.g., random access memory and the like) or non-volatile memory (e.g., read-only memory, hard disks, floppy discs, magnetic tape, optical discs, paper table, punch cards, and the like).
Data can be analyzed with the use of a programmable digital computer. The computer program analyzes the sequence data to indicate alterations (e.g., aneuploidy, translocations, and/or MM driver mutations) observed in the data. In aspects, software used to analyze the data can include code that applies an algorithm to the analysis of the results. The software also can also use input data (e.g., biomarker measurements) to determine an ISG score.
A computer system (or digital device) may be used to receive, transmit, display and/or store results, analyze the results, and/or produce a report of the results and analysis. A computer system may be understood as a logical apparatus that can read instructions from media (e.g. software) and/or network port (e.g. from the internet), which can optionally be connected to a server having fixed media. A computer system may comprise one or more of a CPU, disk drives, input devices such as keyboard and/or mouse, and a display (e.g. a monitor). Data communication, such as transmission of instructions or reports, can be achieved through a communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection, or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections (or any other suitable means for transmitting information, including but not limited to mailing a physical report, such as a print-out) for reception and/or for review by a receiver. The receiver can be but is not limited to an individual, or electronic system (e.g. one or more computers, and/or one or more servers).
In some embodiments, the computer system may comprise one or more processors. Processors may be associated with one or more controllers, calculation units, and/or other units of a computer system, or implanted in firmware as desired. If implemented in software, the routines may be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other suitable storage medium. Likewise, this software may be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the internet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc. The various steps may be implemented as various blocks, operations, tools, modules and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software. When implemented in hardware, some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc.
A client-server, relational database architecture can be used in embodiments of the disclosure. A client-server architecture is a network architecture in which each computer or process on the network is either a client or a server. Server computers are typically powerful computers dedicated to managing disk drives (file servers), printers (print servers), or network traffic (network servers). Client computers include PCs (personal computers) or workstations on which users run applications, as well as example output devices as disclosed herein. Client computers rely on server computers for resources, such as files, devices, and even processing power. In some embodiments of the disclosure, the server computer handles all of the database functionality. The client computer can have software that handles all the front-end data management and can also receive data input from users.
A machine readable medium which may comprise computer-executable code may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
The subject computer-executable code can be executed on any suitable device which may comprise a processor, including a server, a PC, or a mobile device such as a smartphone or tablet. Any controller or computer optionally includes a monitor, which can be a cathode ray tube (“CRT”) display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display, etc.), or others. Computer circuitry is often placed in a box, which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard, mouse, or touch-sensitive screen, optionally provide for input from a user. The computer can include appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
A computer can transform data into various formats for display. A graphical presentation of the results of a calculation (e.g., sequencing results) can be displayed on a monitor, display, or other visualizable medium (e.g., a printout). In some embodiments, data or the results of a calculation may be presented in an auditory form.
The disclosure also provides kits for use in characterizing and/or treating a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). Kits of the instant disclosure may include one or more containers comprising an agent for characterization of a neoplasia and/or for treatment of a neoplasia. In some embodiments, the kits further include instructions for use in accordance with the methods of this disclosure. In some embodiments, these instructions comprise a description of use of the agent to characterize a neoplasia and/or use of the agent (e.g., CAR T cells) for treatment of a neoplasia (e.g., skin, colon, pancreas, lung, and kidney cancer). The kit may further comprise a description of how to analyze and/or interpret data.
Instructions supplied in the kits of the instant disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for practicing any of the methods described herein.
The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
An in vivo pooled CRISPR screening approach (
To identify sgRNAs with immune-dependent fitness effects, tumors were implanted subcutaneously into immunocompetent wild-type mice (WT) and immune checkpoint blockade (ICB)-treated WT mice, with immunodeficient NOD SCID I12rg−/− (NSG) mice as controls. For immunotherapy treatment, B16 melanoma was treated with an irradiated GM-CSF-expressing tumor vaccine (GVAX) and anti-PD-1, and MC38 colon adenocarcinoma was treated with anti-PD-1 alone. All other tumor models were treated with a combination of anti-PD-1 and anti-CTLA-4 (Table 1). Endogenous anti-tumor immunity or an immunotherapy-dependent inhibition of tumor growth was observed in each model (
Quality control analyses indicated good screen performance. Across all screens, the majority of sgRNAs were well represented in all experimental conditions (
All screens revealed many genes that, when deleted, sensitize tumors to immune-dependent selective pressure, including gene deletions that sensitized two or more models to immunotherapy (
In addition to identifying many genes previously proposed to cause immune checkpoint blockade (ICB) resistance when lost such as Nlrc5, Pten, and Casp8 (FDR<0.05), several novel potential immune checkpoint blockade (ICB) resistance mechanisms were identified such as loss of Ccar1, Ubr5, Dotl1, Smg9, and Pdcd10 in the genome-scale screens (
Gene targets that were depleted in the sub-genome and genome immune checkpoint blockade (ICB)-treated cohorts relative to the NSG cohorts revealed previously reported mechanisms of immunotherapy sensitization, including Ptpn2, TNF signaling genes Traf2 and Ripk1, granzyme inhibitor Serpinb9, phagocytosis inhibitor Cd47, and the autophagy pathway genes Atg5 and Atg7 (
Many of these novel targets were specific to a subset of the cancer models screened rather than universally depleted across screens (
H2-T23, the mouse ortholog of human HLA-E, scored in the top 15 genetic dependencies in 7 out of the 8 models and was the top depleted hit overall, suggesting that this molecule is a potent negative regulator of anti-tumor immunity across cancer types (
To more systematically identify common and context-specific mechanisms of immune evasion, gene set overrepresentation analysis of depleted sgRNAs from each genome-scale screen was performed and network and tensor decomposition methods were used to derive a core set of 52 gene sets representative of 21 pathways (
Markov clustering of STRING network annotations of a set of 37 genes depleted across multiple genome-scale screens yielded 6 modules with 3 singletons (
The observation of interferon (IFN)-sensing genes as depleted targets was surprising given the essential role for IFNγ in tumor control and previous reports of immunotherapy resistance mediated by loss of interferon (IFN) sensing. Interferon (IFN) signaling increases tumor expression of MHC-I antigen presentation molecules, which is required for specific recognition by CTLs. Indeed, in vitro CRISPR screens have identified that loss of interferon and antigen presentation in similar murine cancer models caused resistance to CD8+ T cell cytotoxicity (
Experiments were undertaken to validate this observation in the WT C57BL/6 mice (KPC) pancreatic cancer and CT26 carcinoma models, which showed a marked dependency on interferon sensing genes in the in vivo screens. Jak1-, Ifngr1-, and Ifnar1-deficient KPC and CT26 cells were created and it was demonstrated that loss of either type I or type II IFN sensing significantly enhanced the response to ICB. Notably, for both KPC and CT26, the effects of deleting Jak1, which ablates both type I and type II IFN sensing, were more pronounced than deleting either Ifnar1 or Ifngr1 alone, confirming that tumor-intrinsic loss of both type I and type II interferon signaling increased sensitivity to anti-tumor immunity elicited by immune checkpoint blockade (ICB) (
To find immune inhibitory mechanisms downstream of interferon sensing, RNAseq was used to profile the transcriptional response to IFN in vitro in all 8 cancer models and IFN-regulated genes were identified that were enriched or depleted in the genome-scale screens (
To test this hypothesis, an in vivo competition assay was designed to test for genetic epistasis between IFNγ sensing genes and the MHC-I pathway by deleting Tap1 or B2m, which reduces cell surface expression of MHC-I and scored as sensitizing to immune checkpoint blockade (ICB) in the in vivo screens (
To test this hypothesis, an in vivo competition assay was designed to test for genetic epistasis between IFNγ sensing genes and the MHC-I pathway by deleting Tap1 or B2m, which reduces cell surface expression of MHC-I and scored as sensitizing to ICB in our in vivo screens (1G and 4B). Using the KPC model, 1:1 mixes of control and Ifngr1 or Jak1 sgRNA-transduced cells were made on either a control, Tap1-null, or B2m-null background (
In order to examine the effect of disrupted MHC-I presentation on tumor interferon signaling in human cancers, a recurring deletion in the 6p21.3 genomic locus was identified across human cancers in TCGA (
Both innate and adaptive immune cells, including natural killer (NK) cells, CD4+ T cells, and CD8+ T cells, have roles in anti-tumor immunity. NK cells, in particular, are known to eliminate cells lacking self MHC-I expression, and their cytotoxicity is inhibited by expression of MHC-I on target cells. However, because PD-1 and CTLA-4 are not expressed on most NK cells, the role of NK cells in immune checkpoint blockade (ICB)-mediated tumor destruction is not clear. To identify the immune subsets responsible for preferential killing of interferon sensing-null tumors in vivo, Jak1-deficient or control KPC cells were injected into immune checkpoint blockade (ICB)-treated WT mice with or without depleting antibodies for CD8+ T cells, CD4+ T cells, or NK cells. Depletion of CD8+ T cells had no effect on the enhanced response of Jak1-null tumors to PD-1 blockade (
Having established the functional importance of both CD4+ T cells and NK cells in the response of interferon sensing-deficient tumors to immune checkpoint blockade (ICB), experiments were undertaken to determine how immune checkpoint blockade (ICB) impacts these immune subsets using single cell transcriptional profiling. CD45Y tumor-infiltrating lymphocytes (TILs) from untreated and immune checkpoint blockade (ICB)-treated KPC tumors (
Only low levels of cytotoxic gene expression in the activated CD4+ T cells could be detected (
Given the link between interferon (IFN) sensing and MHC-I-mediated evasion of NK killing described above, it was suspected that most of the interferon (IFN) and MHC-I pathway genes that had scored as sensitizing hits in the screens (
Based on the observation that guides targeting H2-T23, Tap1, Tap2, B2m, and H2-D1 were still strongly depleted in the absence of NK cells in vivo, experiments were undertaken to examine potential inhibitory effects of H2-T23 on other immune cell populations. H2-T23 encodes the non-classical MHC-I molecule Qa-1b (HLA-E in humans) that binds the inhibitory NKG2A/CD94 receptor. The inhibitory activity of Qa-1b requires presentation of a TAP-dependent peptide (Qdm) derived from the signal sequence of the H2-D1 heavy chain in B6 mice. Because Qa-1b was the strongest hit overall across all in vivo screens (
H2-T23, the mouse ortholog of human HLA-E, scored in the top 15 genetic dependencies in 7 out of the 8 models and was the top depleted hit overall, suggesting that this molecule was a potent negative regulator of anti-tumor immunity across cancer types (
To clarify which immune subsets were inhibited by Qa-1b expression in vivo, H2-T23-null KPC tumors were implanted into WT mice treated with anti-PD-1 alone or anti-PD-1 and antibodies to deplete either NK1.1+ cells or CD8+ T cells. While there was still strong efficacy of PD-1 blockade for H2-T23-deficient tumors in anti-NK1.1-treated mice, depletion of CD8+ T cells completely suppressed the efficacy of anti-PD-1 and tumors grew progressively (
Because CD8+ T cells were primarily responsible for control of H2-T23-deficient tumors, subsets of tumor-infiltrating CD8+ T cells were delineated and examined for their expression of NKG2-family receptors along with canonical markers of effector function, activation and exhaustion using the single-cell RNA sequencing data (
Experiments were next undertaken to determine whether expression of Qa-1b directly inhibited CD8+ T cell cytotoxicity. In contrast to tumor-infiltrating CD8+ T cells, splenic CD8+ T cells activated in vitro with anti-CD3/CD28 antibodies and IL-2 expressed only low levels of NKG2A/CD94 (
Because not only Qa-1b, but also the antigen presentation genes required for presentation of signal peptide on Qa-1b (Tap1, Tap2, B2m, and H2-D) are upregulated by IFNγ (
Experiments were next undertaken to determine whether tumor upregulation of Qa-1b could be a major mechanism of interferon-mediated inhibition of T cells. To directly examine the relationship between tumor interferon (IFN) sensing and upregulation of inhibitory ligands such as Qa-1b, CD19 CAR-T cells were co-cultured with a 1:1 mixture of fluorescently labeled and IFNγ-stimulated control and Jak1-null CD19+ KPC tumor cells (
To examine the effect of interferon (IFN) signaling on patient survival, data was reanalyzed from two immune checkpoint blockade (ICB)-treated patient cohorts with clear cell renal cell carcinoma (ccRCC) or advanced melanoma (Liu, D. et al. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat. Med. 25, 1916-1927 (2019); Braun, D. A. et al. Interplay of somatic alterations and immune infiltration modulates response to PD-1 blockade in advanced clear cell renal cell carcinoma. Nat. Med. 26, 909-918 (2020)). To examine the epistatic effect of disrupted MHC-I presentation on tumor interferon signaling in human cancers, a recurring deletion was identified in the 6p21.3 genomic locus across human cancers in The Cancer Genome Atlas (TCGA), including in renal cell carcinoma (RCC) and melanoma (
In the clear cell renal cell carcinoma (ccRCC) cohort, a high interferon-stimulated gene (ISG) signature was significantly associated with poor overall survival in tumors with intact 6p21.3 loci (
While the analyses of pre-treatment biopsies provided evidence for the context specificity of interferon (IFN)-mediated immune inhibition, the data from the mouse models suggested that interferon (IFN) signaling activated adaptive resistance to immune checkpoint blockade (ICB), and thus inhibitory effects of interferon would be more pronounced in on-treatment samples. To that end, serum protein expression was collected for 50 ISGs from 203 immune checkpoint blockade (ICB)-treated melanoma patients from a pre-treatment time point and 6-week and 6-month post-treatment time points (
To systematically discover tumor-intrinsic immune evasion pathways in the tumor microenvironment, genome-scale in vivo CRISPR screens were performed in a collection of transplantable mouse tumor models treated with immune checkpoint blockade. This dataset revealed many new genetic dependencies and resistance mechanisms for anti-tumor immunity that provided a deeper understanding of how tumors evolve to evade immunity. The dataset can enable the generation of new therapeutics that enhance immune checkpoint blockade (ICB) efficacy. Importantly, the approach also revealed critical mechanisms of immune resistance that were shared by most tumors, such as interferon (IFN)-mediated inhibition of NK cells and CD8+ T cells by upregulation of classical MHC-I and the non-classical MHC-I Qa-1b.
The finding of a dominant immune inhibitory effect from tumor interferon (IFN) sensing from in vivo screens for immune evasion genes was surprising because the production of IFNγ by CD8+ T cells and NK cells has long been appreciated as critical for control of tumors by the immune system, and mutations in the interferon-sensing pathway have been associated with clinical resistance to immune checkpoint blockade (ICB) or tumor progression. In the above Examples, it has been demonstrated that IFNγ-mediated upregulation of classical and non-classical MHC-I genes inhibited natural killer cells and CD8+ T cells, respectively, and was a central mechanism used by tumor cells to evade cytotoxic lymphocyte-mediated killing. The Examples represent the first report of adaptive resistance to immune checkpoint blockade (ICB) caused by IFNγ mediated upregulation of MHC-I family genes.
It is shown in the above Examples that a high interferon-stimulated gene (ISG) signature in a pre-treatment biopsy predicted resistance to immune checkpoint blockade (ICB) in clear cell renal cell carcinoma (ccRCC), while the same gene signature predicted response in patients with melanoma. The analysis revealed that the role of interferon (IFN) sensing can depend on the immune context of the tumor. In melanoma tumors with high expression of ligands that activate NK cell cytotoxicity such as MICA and MICB, high interferon-stimulated gene (ISG) expression predicts poor response, consistent with the mechanistic data showing that interferon (IFN)-driven upregulation of MHC-I inhibits NK cells. However, in melanoma tumors with highly clonally expanded T cell populations, it was observed that high interferon-stimulated gene (ISG) expression predicted favorable response to immune checkpoint blockade (ICB), consistent with interferon (IFN) signaling driving increased antigen presentation to T cells. Not intending to be bound by theory, in many cancers with a lower average number of mutations per megabase and fewer neoantigens, such as ccRCC, anti-tumor immune responses may not always be mediated by clonally-expanded CD8+ T cells recognizing high-affinity MHC-I epitopes, and instead may be dominated by NK cells or other effector populations. In these settings, where the presentation of high-affinity epitopes is not driving an immune response, the upregulation of MHC-I genes is immune-protective, as a large number of ITIM-containing inhibitory receptors (NKG2A, KIR family, LIR family) can recognize MHC-I and are expressed across several immune subsets including T cells, NK cells and macrophages56. Thus, the IFN-mediated upregulation of MHC-I serves to promote immunity via antigen presentation when immunogenic MHC-I epitopes are present but then dampen immunity by providing ligands for ITIM-containing immune receptors when immunogenic epitopes are absent. The classical MHC-I genes play a dual role in both antigen presentation to CD8+ T cells and inhibition of NK cell killing, thus highlighting the importance of understanding the nature of immunotherapy responses in different contexts.
However, certain members of the MHC-I family such as the non-classical MHC-I Qa-1b/HLA-E are primarily inhibitory ligands and present antigen only in limited contexts, positioning them as key inhibitory molecules induced by interferon (IFN) that are less context-dependent than classical MHC-I.
The Qa-1b/NKG2A inhibitory axis was identified as the top immune evasion mechanism across cancer types. Not intending to be bound by theory, the data suggested that the activation of CD8+ T cells by immune checkpoint blockade (ICB) upregulated the expression of NKG2A/CD94 as they differentiate into cytotoxic terminal effector and exhausted T cells. At the same time, the IFNγ inflammation induced by immune checkpoint blockade (ICB) drove the expression of Qa-1b, the ligand for the NKG2A/CD94 inhibitory receptor. This adaptive resistance mechanism reveals how immune checkpoint blockade (ICB)-induced CD8+ T cell cytotoxicity can be dampened in the setting of strong interferon (IFN) inflammation despite high tumor cell expression of MHC-I. The data also revealed the importance of understanding the context-dependent activity of immune checkpoints, as the inhibitory interaction between Qa-1b and NKG2A was difficult to model in vitro due to poor expression of NKG2A on CD8+ T cells under standard CD3/28+IL-2 activation conditions. It was found that IL-12 stimulation caused the upregulation of NKG2A on mouse CD8+ T cells in vitro, but further study was required to determine the exact signals that lead to NKG2A expression in vivo. Finally, the data on Qa-1b(HLA-E) also suggested that the other non-classical MHC-I genes HLA-G and HLA-F may play a role in interferon (IFN)-mediated inhibition of anti-tumor immunity in human cancer. HLA-G and HLA-F were not assessed in the in vivo screens because they have no clear mouse orthologs. These non-classical MHC-I molecules are expressed in many tumors and their expression has generally been associated with poor prognosis. Thus, IFNγ-mediated inhibition of anti-tumor immunity may also involve additional non-classical MHC-I genes beyond HLA-E in humans and these inhibitory effects could impact a broad range of host immune cells.
Experiments were undertaken to demonstrate that CD8+ tumor-infiltrating lymphocytes (TILs) express high levels of NKG2A and CD94 in vivo. The experiments were carried out as shown in
An experiment was undertaken to evaluate the impact of the tumor microenvironment on gene expression in tumor-infiltrating human chimeric antigen receptor expressing (CAR) T cells in vivo. At day 0 (input) NSG mice bearing A375 tumors were injected with CAR T cells targeting the tumors, and gene expression in CAR T cells within the tumors was evaluated after 7 and 14 days using RNAseq. RNAseq of the T cells from the tumors revealed that KLRC1 (encoding NKG2A) and KLRD1 (encoding CD94) were both upregulated on tumor-infiltrating human CD8+ CAR T cells in vivo (
These results demonstrate, among other things, that knockout of NKG2A and/or CD94 polypeptide expression in CAR T cells may reduce susceptibility to immune inhibition within the tumor microenvironment.
To facilitate the examination of the impact of NKG2A and/or CD94 up-regulation on T cell (e.g., CAR T cell) function, experiments were undertaken to develop a method for inducing expression of NKG2A and/or CD94 in T cells in vitro. On day zero CD8+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using a CD8+ T cell isolation kit (Miltenyi Biotec). Isolated CD8+ T cells were then subjected to a first stimulation using T Cell TransAct™ available from Miltenyi Biotec following the manufacturer's protocol and cultured in TexMACS™ Medium (with Penicillin/Streptomycin) available from Miltenyi Biotec and supplemented with human IL-2, IL-7, IL-15, and/or IL-12p70. In the first stimulation, 1e6 cells+10 μl TransAct™ were combined in 1 ml TexMACS™ medium (1:100) in a 48-well plate format (Cytokine usage: IL-2 50 U/ml (PeproTech); IL-7 10 ng/ml (PeproTech); IL-15 10 ng/ml (PeproTech); IL-12p70 10 ng/ml (PeproTech)) and allowed to expand. Fresh medium with all cytokines was added to the cell cultures every 1-2 days, and TransAct™ was removed on day 2 according to the manufacturer's protocol. On day 14, the cells were subjected to a second stimulation similar to the first stimulation using TransAct at a 1:500 dilution. On day 16-18, cells were harvested for use in further analysis (e.g., use in a co-culture experiment) or frozen down with 50% CryoStor® CS10 cryopreservation medium (StemCell).
It was determined that T cells stimulated twice using an TransAct™, with IL-2, IL-7, IL-15, and IL-12 showed induced expression of NKG2A and CD94 (
These results demonstrate the development of a new method for inducing NKG2A and CD94 expression in T cells in vitro.
Experiments were undertaken to demonstrate that the interaction of HLA-E expressed on the surface of tumor cells within the tumor microenvironment (TME) mediated the immune inhibition of T cells within the TME.
First, co-culture experiments were undertaken to demonstrate that deletion of HLA-E in tumor cells sensitized the cells to killing by NK cells. NK cells were used because a large frequency of NK cells surface-express CD94 and NKG2A without any need for induction. The tumor cells evaluated included HT-29 colon carcinoma cells, 786-0 renal carcinoma cells, SU.86.86 pancreatic carcinoma cells, and PANC-1 pancreatic carcinoma cells. Target cells were stained with either CellTraceViolet or CellTraceFarRed, seeded in culture flasks, and evaluated by flow cytometry to quantify cell input ratios (e.g., ratio of HLA-E knockout tumor cells to control tumor cells). Prior to co-culture, the tumor cells were stimulated with human IFNg for 24-48 hours (10 ng/ml for HT-29 tumor cells and 100 ng/ml for other tumor cells). On the day of coculture, human PBMC-derived NK cells were thawed, incubated and rested in culture medium (LGM-3 medium) for 3-4 hours and then counted. NK cells were added directly to the target cells without any media changes and then co-cultured for 24 hours with the tumor cells. By co-culturing the NK cells for 24 h at different effector-to-target cell ratios with tumor cells expressing or deficient in expression of HLA-E (i.e., control cells or HLA-E/B2M knockout (KO) cells, respectively), it was found that the deletion of HLA-E from the tumor cells sensitized them to killing by the NK cells (
In further support of this result, it was found that Monalizumab reversed preferential killing of HLA-E KO tumor cells by NK cells. Co-cultures containing HT-29 or SU.86.86 cells (target cells) and the NK cells line NK-92MI or primary NK cells (effector cells) were administered 10 μg/ml Monalizumab. It was found that the Monalizumab eliminated preferential killing of HLA-E knockout (KO) cells over the control cells that expressed HLA-E (
Experiments were undertaken to demonstrate that CD8+ T cells with expression of NKG2A induced as described in Example 7 above preferentially killed HLA-E knockout (KO) tumor cells in a 24-hour co-culture. Killing of HT-29 tumor cells and HT-29 tumor cells deficient in expression of HLA-E (i.e., HLA-E KO or B2M KO HT-29 tumor cells) by T cells was evaluated using a CD19/CAR system (i.e., a co-culture of anti-CD19 chimeric-antigen receptor (CAR) T cells and HT-29 target cells engineered to express truncated human CD19) and using an anti-CD3 redirected killing assay over different effector-to-target cell ratios (i.e., CD8+ T cell to tumor cell ratios). In the redirected killing assay, the T cells expressed both CD3 and CD28 and the target HT-29 tumor cells surface-expressed both a membrane-tethered anti-CD3 scFv and human CD80. In both the CD19/CAR system and the anti-CD3 redirected killing assay, it was found that NKG2A+ primary human CD8+ T cells (i.e., the CAR T cells or the T cells expressing both CD3 and CD28) preferentially killed HLA-E knockout tumor cells in vitro (
Experiments were then undertaken to demonstrate the preferential killing of HLA-E knockout tumor cells by T cells in vivo. In mice a pool of HLA-E knockout tumor cells (HT29 colorectal adenocarcinoma cells or A375 melanoma cells), CD274 (PD-L1) knockout tumor cells, and tumor cells expressing both HLA-E and PD-L1 (control cells), it was found that human T cells administered to the mice preferentially killed the HLA-E knockout tumor cells (
The following materials and methods were employed in the above examples.
The CT26.WT colon carcinoma (referred to as CT26), Renca renal cell carcinoma, and Lewis Lung Carcinoma (referred to as LLC) cell lines were purchased from ATCC. The YUMMER1.7 Braf/Pten melanoma cell line (referred to as YUMMER) was a gift from M. Bosenberg and S. Kaech. The KPC pancreatic cancer cell line was a gift from A. Maitra and S. Dougan. The Panc02 pancreatic ductal adenocarcinoma was a gift from S. Dougan. The B16-F10 (referred to as B16) melanoma and B16 GM-CSF secreting cells (GVAX) were a gift from S. Dougan and G. Dranoff The MC38 colon carcinoma was a gift from A. Sharpe. CT26 and Renca cells were cultured in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum (FBS) and antibiotics. B16, MC38, LLC, Panc02, and KPC were grown in DMEM (GIBCO) supplemented with 10% fetal bovine serum and antibiotics. YUMMER cells were grown in DMEM supplemented with non-essential amino acids (GIBCO), 10% FBS and antibiotics. All cell lines were tested and found negative for the Mouse Essential CLEAR Panel w/C. bovis (Charles River Research Animal Diagnostic Services). The cell lines CT26, KPC, B16, MC38, and Panc02 were subcloned prior to screening in order to reduce heterogeneity in the screening cell populations. The clones were assessed for in vivo growth and response to immunotherapy and selected based on their similarity to the parental cell lines. The cell lines LLC, Renca, and YUMMER were not subcloned and screened directly.
All mice were housed at the Broad Institute's specific-pathogen free facility. Seven to ten-week old wild-type (WT) Balb/C and C57BL/6J mice were obtained from Jackson laboratories. A colony of NOD.Cg-Prkdcscid I12rgtm1Wjl/SzJ (NSG) immunodeficient mice were bred on site. For all WT tumor challenges, age-matched female mice were used with the exception of tumor challenges with YUMMER cells where age-matched male mice were used. All animal studies were approved by the Broad Institute IACUC committee.
For validation experiments, 1e6 tumor cells of the indicated cell lines were resuspended in Hanks Balanced Salt Solution (HBSS; GIBCO) and subcutaneously injected into the right flank on day 0. Mice were treated with 200 μg of rat monoclonal anti-PD1 antibodies (Bio X Cell, clone: 29F.1A12) on days 6, 9, and 12 via intraperitoneal (i.p.) injection. For CD8+ depletion experiments, mice were treated with 200 μg of anti-CD8b monoclonal antibody (Bio X Cell, clone 53-5.8) via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation. For CD4+ depletion experiments, mice were treated with 200 μg of anti-CD4 monoclonal antibody (Bio X Cell, clone GK1.5) via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation. For C57BL/6J mouse NK depletion experiments, mice were treated with 200 μg anti-mouse NK1.1 (Bio X Cell, clone PK136) via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation unless otherwise indicated. For Balb/C mouse NK depletion experiments, mice were treated with 50 μg anti-asialo GM1 Polyclonal Antibody (Invitrogen) via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation. Each tumor was measured every 3-4 days beginning on day 6 after challenge until either the survival endpoint was reached or no palpable tumor remained. Measurements were assessed manually by measuring the longest dimension (length) and the longest perpendicular dimension (width). Tumor volume was estimated with the formula: (L x W2)/2. For all experiments, at least five mice were included in each group, based upon prior knowledge of the variability of experiments with immune checkpoint blockade. Animals were randomized before treatment and no blinding was performed.
For the sub-genome scale in vivo screens, a library of 9,672 sgRNAs targeting 2,368 genes with 4 sgRNAs per gene along with non-targeting control guides (Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 547, 413-418 (2017)) was cloned into either the pXPR_024_sgRNA vector, the pXPR_055_sgRNA vector, or the pSCAR_sgRNA_1 (pXPR_BRD060) (Addgene #162076) vector (Dubrot, J. et al. In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma. Immunity (2021) doi:10.1016/j.immuni.2021.01.001). Briefly, this library targeted genes that were enriched in the Gene Ontology (GO) term categories kinase, phosphatase, cell surface, plasma membrane, antigen processing and presentation, immune system process, and chromatin remodeling, and that were expressed in B16 melanoma. For the KPC and LLC cell lines, screening cells were engineered by transducing pLX311-Cas9-expressing (Addgene #96924) cells with the pXPR_024_sgRNA lentiviral library. For the MC38 cell line, screening cells were engineered by transducing pLX311-Cas9-expressing cells with the pXPR_055_sgRNA lentiviral library. For the CT26 cell line, screening cells were engineered by transducing CT26 cells expressing pSCAR_Cas9_BSD (Addgene #162074) with the pSCAR_sgRNA_1 (pXPR_BRD060) lentiviral library. After allowing ˜1 week for selection and editing, the pool was then transduced with IDLV_Cre (Addgene #162073) at >5000× coverage/sgRNA (described in7). Vector expression was monitored by fluorescent reporters until <10% of cells expressed GFP or mKate2.
For the genome-scale in vivo screens, a library of 19,280 genes, each targeted by 4 sgRNAs delivered in 4 separate cohorts (Brie Library was cloned into either the pXPR_024_sgRNA vector, the pXPR_055_sgRNA vector, or the pSCAR_sgRNA_1 (pXPR_BRD060) vector. For the B16 and LLC cell lines, screening cells were engineered by transducing pLX311-Cas9-expressing cells with the pXPR_024_sgRNA lentiviral library. For the MC38 cell line, screening cells were engineered by transducing pLX311-Cas9-expressing (Addgene #96924) cells with the pXPR_055_sgRNA lentiviral library.
For the CT26, KPC, Panc02, Renca, and YUMMER cell lines, screening cells were engineered by transducing cells expressing pSCAR_Cas9_BSD or pSCAR_Cas9_HBP (Addgene #162075) with the pSCAR_sgRNA_1 (pXPR_BRD060) lentiviral library. After allowing ˜1 week for selection and editing, the pool was then transduced with IDLV_Cre at >5000×coverage/sgRNA. Vector expression was monitored by fluorescent reporters until <10% of cells expressed GFP or mKate2. For the CT26 screen, cells were sorted via FACS for GFP− mKate2− cells prior to implantation.
All pool infections were conducted with infection rates of 15-30%, to yield libraries with >1000×coverage/sgRNA; transduced cells were selected in puromycin (Millipore; pXPR_024_sgRNA lentiviral library and pSCAR_sgRNA_1 (pXPR_BRD060) lentiviral library) or hygromycin (Gibco; pXPR_055_sgRNA lentiviral library) and were maintained in culture at >1000×library coverage at all times following infection.
For the tumor challenges, 4.0×106 cells per tumor in a 50/50 mix of growth-factor-reduced Matrigel (Corning) and HBSS were implanted subcutaneously into the right flank only or both the right and left flanks for bilateral tumor injections (genome-scale screens with LLC, Panc02, and YUMMER) of NOD SCID I12rg−/− (NSG) and WT mice. To estimate the number of tumors required to maintain sufficient coverage of the library, an engraftment rate of ˜2.5-5% was empirically determined by comparing the library complexity recovered from tumors to purposefully subsampled in vitro libraries. Based on the engraftment rate, 40-100 tumors were used per experimental arm (NSG, untreated WT, WT+immune checkpoint blockade (ICB)) to maintain ˜250× coverage of each pool of the library.
For the B16 genome-scale screen, treated mice received subcutaneous injections on the abdomen on days 1 and 4 with 1.0e6 GVAX cells that had received 35 Gy of irradiation prior to administration. These mice then received i.p. injections of 100 μg of rat monoclonal anti-PD1 antibodies on days 6, 9, and 12. For the CT26 and LLC sub-genome and genome-scale screens, treated mice received i.p. injections of 200 μg of rat monoclonal anti-PD1 antibodies and 200 μg of mouse anti-CTLA-4 antibodies (Bio X Cell, clone 9D9) on days 6, 9, and 12. For the NK depletion arm of the CT26 sub-genome screen, mice received i.p. injections of 200 μg of rat monoclonal anti-PD1 antibodies and 200 μg of mouse anti-CTLA-4 antibodies on days 6, 9, and 12 and 50 μg anti-asialo GM1 Polyclonal Antibody via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation. For the MC38 sub-genome and genome-scale screens, treated mice received i.p. injections of 50 μg of rat monoclonal anti-PD1 antibodies on days 12 and 15. For the KPC sub-genome screen, treated mice received i.p. injections of 200 μg of rat monoclonal anti-PD1 antibodies on days 6, 9, and 12; for the NK depletion arm, mice received i.p. injections of 200 μg of rat monoclonal anti-PDT antibodies on days 6, 9, and 12 and 200 μg anti-mouse NKT.1 via i.p. injection every four days for the duration of the experiment, starting 1 day prior to tumor implantation. For the KPC, Renca, Panc02, and YUMMER genome-scale screens, treated mice received i.p. injections of 100 μg of rat monoclonal anti-PD1 antibodies and 100 μg of mouse anti-CTLA-4 antibodies on days 6, 9, and 12.
Cells were also maintained in culture at >2000× coverage/sgRNA for the duration of the screen. Tumors and in vitro pools were harvested 12-18 days after implantation, minced with scissors, and pooled by equal tissue mass in groups of 5-10 tumors within each experimental arm. Pooled tissue was then digested with proteinase K (QIAGEN) and buffer ATL (QIAGEN) and genomic DNA extracted using the QIAGEN Blood Maxi Kit. From 40-240 μg of gDNA per pooled sample, the sgRNA region was PCR-amplified (using the P5 and P7 Illumina primers) and sequenced using an Illumina HiSeq.
Guide sequences were demultiplexed and quantified using PoolQ v2.2.0 (portals.broadinstitute.org/gpp/public/software/poolq). Read counts were library normalized per million reads and log 2-transformed with a pseudocount of one. Gene-targeting guides were z-normalized by the control sgRNA distribution. Guide fold changes were calculated as residuals fit to a natural cubic spline with 4 degrees of freedom (
gProfiler was used to perform pathway enrichment analysis of enriched and depleted genes with FDR<0.25 on GO Biological Pathways, GO Molecular Functions, and Reactome pathway annotations, excluding terms with >1000 genes and ordering genes by STARS Score (Raudvere, U. et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 47, W191-W198 (2019)). The odds ratio for overrepresentation was computed using the fisher_exact function from scipy v1.6.2. To group similar pathways, a graph was drawn by using k-nearest neighbors (k=10) on Jaccard similarity between pathway gene membership and used the Louvain algorithm on the resulting network to discover communities. Louvain communities were further refined by performing Tucker tensor decomposition on a sparse gene by pathway by screen tensor for each community using the TensorLy python package. The gap statistic was used with agglomerative clustering to determine the optimum number of subclusters for each community (Tibshirani, R., Walther, G. & Hastie, T. Estimating the Number of Clusters in a Data Set via the Gap Statistic. J. R. Stat. Soc. Series B Stat. Methodol. 63, 411-423 (2001)). Finally, similar subclusters were merged based on Jaccard similarity between genes that scored in screens.
Tumor cells were stimulated with 100 ng/mL of IFNγ (PeproTech) or 103 activity units/mL INFβ for 48 hours. RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. First-strand Illumina-barcoded libraries were generated using the NEB RNA Ultra II Directional kit according to the manufacturer's instructions using a 10-cycle PCR enrichment. Libraries were sequenced on an Illumina NextSeq 500 instrument using paired-end 37 bp reads. Data were trimmed for quality using Trimmomatic v0.36 with the following parameters: LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:37. Gene abundances were quantified by pseudoalignment to the mm10 reference transcriptome using Kallisto (Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525-527 (2016)). Differential expression was performed by importing gene abundances using tximport and analyzing with the DESeq2 R package (Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res. 4, 1521 (2015); Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)).
The interferon inducible promoter, pLX_311_Irf1, was created by arranging 6 tandem gamma-activated site (GAS) elements and 3 tandem interferon-stimulated response elements (ISRE) upstream of the promoter sequence of Irf1. For the overexpression vectors, pLX_311_mCD19_truncated, Hygro_PGK_Ova, pLX_311_hCD19, pLX_311_Irf1 (inducible Qa1 up-regulation), and pLX311_Qa1_mutPAM (constitutive Qa1 up-regulation), cDNA (Origene) was PCR-amplified using primers containing attB sites and cloned into the pLX_311-Gateway destination vector using Invitrogen BP Clonase II and LR Clonase II Gateway reactions according to the manufacturer's instructions.
For CRISPR knockout validation studies and epistasis experiments, cells were transiently transfected with pX459_Cas9_sgRNA (Addgene #62988) targeting control, Jak1, H2-T23, Tap1, B2m, or H2-K1 with the Lipofectamine transfection reagent (Thermo Fisher Scientific, L3000015). Transfected populations were selected in antibiotics for 2-4 days and bulk transfectant populations were used for subsequent experiments. For epistasis experiments, previously transfected cell lines were lentivirally transduced with pSCAR_Cas9_HBP, pSCAR_sgRNA_1 (pXPR_BRD060) vector or pSCAR_sgRNA_6 (pXPR_BRD065) (Addgene #162072) vectors targeting control, Jak1, or Ifngr1, and then IDLV-Cre (as described in7). All cell lines were validated by flow cytometry analysis for >95% reduction of expression of relevant surface protein expression.
For CAR-T cell killing and adoptive transfer experiments, single- or double-knockout KPC cells were lentivirally transduced with pLX_311_mCD19_truncated. For Qa-1b overexpression in MC38, cells were lentivirally transduced with overexpression constructs pLX_311_hCD19, pLX_311_Irf1 (inducible Qa1 up-regulation), and pLX_311_Qa1 mutPAM (constitutive Qa1 up-regulation). Cells were then transduced with pSCAR_sgRNA_1 (pXPR_BRD060) with different non-targeting controls. For OT-1 adoptive transfer experiments, the Qa-1b overexpressing MC38 cells were lentivirally transduced with Hygro_PGK_Ova.
For flow cytometry of cell lines, trypsin was added to the culture, cells were washed in PBS+2% FBS+5 mM EDTA. Where IFNγ stimulation is indicated, cells were cultured with 20-100 ng/mL IFNγ (PeproTech). Where IFNβ stimulation is indicated, cells were cultured with 103 activity units/mL IFNβ (PeproTech). Cells were stained for surface proteins for 15 min at 4° C. with indicated conjugated fluorescent monoclonal antibodies against H2-Db (clone HK95, BioLegend), H2-Kb (clone AF6-88.5, BioLegend),), H2-Dd (clone 34-2-12, BD Biosciences), H2-Kd (clone SF1-1.1, BioLegend), Qa-1b (clone 6A8.6F10.1A6, Miltenyi Biotec), PD-L1 (CD274) (MIH5, eBioscience), or NKG2A (clone 16A11, BioLegend). For validation of CAR-T cell transduction, cells were stained with conjugated fluorescent polyclonal F(ab′)2 fragment specific antibodies (112-545-006, Jackson ImmunoResearch).
For tumor infiltrate analyses, mice were injected subcutaneously with 1e6 of indicated tumor cells. Tumors were collected on day 12 post-inoculation, mechanically diced, and dissociated with the mouse Tumor Dissociation Kit (Miltenyi Biotec) as per manufacturer's instructions. After filtering through a 70-μm filter and washing, cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (L34975, Invitrogen) as per manufacturer's instructions. Cells were then stained with conjugated fluorescent monoclonal antibodies against CD45 (clone 30-F11, BioLegend), NK1.1 (clone PK136, BioLegend), TCRP (clone H57-597, BioLegend), and NKG2A (clone 16A11, BioLegend). All samples were acquired using a Beckman Coulter Cytoflex instrument and analyzed with FlowJo software (FlowJo, LLC).
Spleens of C57BL/6J mice were mechanically dissociated, filtered through a 70-μm filter, and incubated in 1 mL ACK lysing buffer/spleen (Life Technologies, Inc.) for 1-2 minutes. Cells were quenched in 10× the volume of PBS+2% FBS+5 mM EDTA. T cells were isolated with the mouse Pan T cell Isolation Kit II (Miltenyi Biotec) per manufacturer's instructions. T cells were cultured on a plate coated with purified NA/LE hamster anti-mouse CD3e antibody (BD Pharmingen) in T/NK cell media (RPMI+10% FBS+antibiotics+non-essential amino acids+10 mM HEPES+55 μM 2-Mercaptoethanol) supplemented with 1 μg/mL purified NA/LE hamster anti-mouse CD28 antibody (BD Pharmingen) and 100 U/mL recombinant mouse IL-2 (BioLegend). On day 2 (˜18 hours post-stimulation), T cells were transduced with 100× concentrated 1D3_CAR lentivirus during an hour-long centrifugation followed by a 4-6 hour incubation at 37° C. LentiBOOST lentivirus transduction enhancer solution (Mayflower Bioscience) was used to increase transduction efficiency. T cells were removed from viral media overnight and then transduced for a second time on day 3, following the same protocol. On day 4, transduction of T cells was confirmed via either flow cytometry or specific killing of mCD19-expressing tumor cells. CAR-T cells were expanded and stimulated with anti-CD3e and anti-CD28 antibodies through day 5 and with IL-2 for the entire duration of the culture. CAR-T cells were stimulated with 2 ng/mL IL-12 p70 (PeproTech) on day 5 (at least 48 hours prior to use in killing assay).
Spleens of OT-1 mice were mechanically dissociated, filtered through a 70-μm filter, and incubated in 1 mL ACK lysing buffer/spleen for 1-2 minutes. Cells were quenched in 10× the volume of PBS+2% FBS+5 mM EDTA. T cells were isolated with the mouse CD8a+ T Cell Isolation Kit (Miltenyi Biotec) per manufacturer's instructions. T cells were cultured on a plate coated with Purified NA/LE Hamster Anti-Mouse CD3e antibody and in T/NK cell media supplemented with 1 μg/mL Purified NA/LE Hamster Anti-Mouse CD28 antibody and 100 U/mL IL-2 for 24 hours. OT-1 T cells were cultured with 100 U/mL IL-2 and 2 ng/mL IL-12 p70 for 3 days prior to adoptive transfer.
For the in vivo competition experiments, 1:1 mixes of the indicated cell lines were grown in culture for one passage prior to implantation. On the day of tumor challenge, the mixes were mixed 1:1 with unmodified cells and 106 cells were implanted into NOD SCID I12rg−/− (NSG) and WT mice as described above. Cells were maintained in culture for the duration of the in vivo competition assay. Tumors and in vitro cultures were harvested 12-15 days after implantation, minced with scissors, and then digested with proteinase K and Buffer ATL.
For the CAR-T cell adoptive transfer in vivo competition assay, 1:1 mixes of the indicated mCD19+ tumor cells were grown in culture in 2 ng/mL IFNγ for 48 hours prior to tumor challenge. On the day of tumor challenge, the mixes were mixed 1:1 with unmodified cells and 2e6 of the indicated tumor cells were implanted subcutaneously into NOD SCID I12rg−/− (NSG) mice. On day 7 following tumor implantation, 1.5e6 activated CAR-T or untransduced cells were transferred to tumor-bearing NSG mice via tail vein injection. Tumor cells were maintained in culture for the duration of the in vivo competition assay. Tumors and in vitro cultures were harvested 7 days following T cell transfer, minced with scissors, and then digested with proteinase K and Buffer ATL. Matched spleens from tumor-bearing mice were also harvested, mechanically dissociated, filtered through a 70-μm filter, and incubated in 1 mL ACK lysing buffer/spleen for 1-2 minutes. Splenocytes were then analyzed by flow cytometry to confirm successful adoptive transfer and immune cell depletion. A cutoff of >5% CD8+TCRβ+ transferred T cells in the spleens was applied for downstream analysis.
For the OT-1 adoptive transfer in vivo competition assay, 1:1 mixes of the indicated OVA+ tumor cell lines were grown in culture for one passage prior to implantation. On the day of tumor challenge, the mixes were mixed 1:1 with unmodified cells and 2×106 of the indicated tumor cells were implanted subcutaneously into NSG mice. On day 6 following tumor implantation, 3×106 activated OT-1 cells were transferred to tumor-bearing NSG mice via tail vein injection. Tumor cells were maintained in culture for the duration of the in vivo competition assay. Tumors and in vitro cultures were harvested 7 days following T cell transfer, minced with scissors, and then digested with proteinase K and Buffer ATL.
For all in vivo competition experiments, genomic DNA was extracted using the QIAGEN Blood Maxi Kit. From 1-10 μg of gDNA per sample, the sgRNA region was PCR-amplified (using the P5 and P7 primers listed in the key resources table) and sequenced using an Illumina MiSeq. Base intensities were converted to fastqs and demultiplexed by sample using Illumina's bcl2fastq v2.20.0 program. Count and log (RPM+1) matrices of sgRNAs per sample were generated using PoolQ v2.2.0.
In Vitro NK Killing Assays Spleens of C57BL/6J mice were mechanically dissociated, filtered through a 70-μm filter, and incubated in 1 mL ACK lysing buffer/spleen for 1-2 minutes. Cells were quenched in 10× the volume of PBS+2% FBS+5 mM EDTA. NK cells were isolated with the mouse NK Cell Isolation Kit (Miltenyi Biotec) per manufacturer's instructions. Purified NK cells were cultured in T/NK cell media supplemented with 10 ng/mL mouse recombinant IL-15 (BioLegend) and 100 U/ml IL-2 for 6 days. On day 6, NK cells were activated with 10 ng/mL mouse IL-12 and 100 ng/mL mouse IL-18 (MBL International) in addition to IL-2 and IL-15 for 24 hours. Differentially-labeled epistasis tumor cells were mixed 1:1 and plated in 24-well plates with 20 ng/mL IFNγ 24 hours prior to co-culture. The following day, NK cells were added to the tumor cells at the indicated effector to target ratios and maintained in IL-12, IL-18, IL-2, and IL-15. The co-cultures were maintained for 24-72 hours and tumor cells were collected, stained for live cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, and analyzed by flow cytometry for changes in the ratio of differentially labeled tumor cells.
For in vitro CAR-T cell killing assays, indicated mCD19+ tumor cells were stimulated with 20 ng/mL IFNγ for 24 hours, differentially labeled using Life Technologies Cell Trace Proliferation Kits (Thermo Fisher Scientific), and then mixed 1:1 and plated in 24-well plates. Activated untransduced or CAR-T cells were added to the tumor cell cultures at the indicated effector to target ratios while maintaining in 100 U/mL IL-2 and 2 ng/mL IL-12 p70. When indicated, 5 μg/mL of anti-PD1 antibodies or anti-mouse NKG2A/C/E (Bio X Cell, clone: 20D5) were added to the co-cultures. After 72 hours of co-culture, tumor cells were collected, stained for live cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, and analyzed by flow cytometry for changes in the ratio of differentially-labeled tumor cells.
For in vitro OT-1 T cell killing assays, indicated OVA+ tumor cell lines were differentially labeled using Life Technologies Cell Trace Proliferation Kits and then mixed 1:1 and plated in 24-well plates. Activated OT-1 T cells were added to the tumor cell cultures at the indicated effector to target ratios while maintaining in 100 U/mL IL-2 and 2 ng/mL IL-12 p70. After 48 hours of co-culture, tumor cells were collected, stained for live cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, and analyzed by flow cytometry for changes in the ratio of differentially-labeled tumor cells.
The Cancer Genome Atlas (TCGA) copy number and patient annotation data were downloaded from the UCSC Xena platform (Goldman, M. J. et al. Visualizing and interpreting cancer genomics data via the Xena platform. Nat. Biotechnol. 38, 675-678 (2020)). For the expanded melanoma cohort, RNAseq expression was normalized across pre-treatment cohorts using ComBat (Johnson, W. E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118-127 (2007)). Loss of 6p21.3 was defined using thresholded GISTIC scores (Goldman, M. J. et al. Visualizing and interpreting cancer genomics data via the Xena platform. Nat. Biotechnol. 38, 675-678 (2020)). For the Braun ccRCC dataset, 6p21.3 loss was defined as deletion of peaks 7 or 8, spanning chromosomal locations chr6:26008431-26241216 and chr6:33053841-33136415, respectively; for the Liu melanoma dataset, loss was defined as copy number loss of any of the following genes in the 6p21.3 locus: TAP1, TAP2, TAPBP, PSMB8, PSMB9. IFNγ scores were calculated using ssGSEA (Barbie, D. A. et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462, 108-112 (2009)) using the Hallmark Interferon Gamma Response gene set (Liberzon, A. et al. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst 1, 417-425 (2015)). NK ligand score was calculated from ssGSEA of a list of the NK cell activating ligands: ULBP1, ULBP2, ULBP3, RAET1E, RAET1G, RAET1L, MICA, MICB, NCR3LG1, PVR, and NECTIN2. CD3 expression was calculated as the mean expression of CD3D, CD3E, and CD3G. Mean T cell clone size was calculated using MiXCR on RNAseq read data (Bolotin, D. A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12, 380-381 (2015)). The optimal cut point for survival stratification using IFNγ score was determined using 6p21.3 disomy patients using conditional inference procedures in the coin R package (Hothorn, T., Hornik, K. & Zeileis, A. Unbiased Recursive Partitioning: A Conditional Inference Framework. J. Comput. Graph. Stat. 15, 651-674 (2006)). Kaplan-Meier survival analysis was performed using the Lifelines Python package, and Cox proportional hazards interaction modeling was performed using the survival R package (Davidson-Pilon, C. et al. CamDavidsonPilon lifelines: v0.25.7. (2020). doi:10.5281/zenodo.4313838; Therneau, T. M. & Grambsch, P. M. The Cox Model. in Modeling Survival Data: Extending the Cox Model (eds. Themeau, T. M. & Grambsch, P. M.) 39-77 (Springer New York, 2000)). Statistical significance for survival stratification of 6p21.3 deletion and thresholded IFNγ score was assessed using log-rank tests.
Metastatic melanoma patients at MGH provided written informed consent for the collection of blood samples (DF/HCC IRB approved Protocol 11-181). Whole blood was collected in BD Vacutainer CPT tubes (BD362753) prior to treatment and 6 weeks on treatment with immune checkpoint blockade, and 6 months after initial treatment. Three mL of plasma was isolated after centrifuging tubes for 30 minutes at room temperature, and plasma was stored at −80° C. for further use. The Olink Proximity Extension Assay (PEA) was performed as described in Filbin, M. R. et al. Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions. Cell Rep Med 2, 100287 (2021). Briefly, the full OLINK® Explore1536 library consists of 1536 assays of 1472 proteins and 48 controls. Amongst these, a curated list of 50 ISGs were selected for analysis. Pairs of oligonucleotide-labeled monoclonal or polyclonal antibodies against distinct epitopes were used to bind target proteins, facilitating hybridization of oligonucleotides when they are in close proximity, followed by an extension step that generates a unique sequence used for digital identification of the analyte using Illumina sequencing. Final libraries were sequenced using an Illumina NovaSeq 6000 sequencer. Data was delivered as NPX, which is Olink's relative protein quantification unit on a log 2 scale. Data generation of NPX consists of normalization to the extension control (known standard), log 2-transformation, and level adjustment using the plate control (plasma sample). Fold change calculations were performed relative to baseline measurements and z-score normalized across patients. Serum ISG protein score at 6 weeks and 6 months was determined as mean normalized fold change of all 50 ISGs at the respective timepoints. Serum ISG protein score was used for patient ranking in GSEA enrichment of non-responders, performed using GSEA v4.1.059 (Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A 102, 15545-15550 (2005)), and used for Cox proportional hazards and Kaplan-Meier survival analyses. Cox proportional hazards models were controlled for patient age and sex.
Mice were injected subcutaneously with 2e6 KPC tumor cells and half of the animals were treated with 100 μg of anti-PD-1 and 100 μg anti-CTLA-4 at 6 and 9 days after inoculation. Tumors were collected on day 12 post-inoculation, mechanically diced, and dissociated with the mouse Tumor Dissociation Kit as per manufacturer's instructions. After filtering through a 70-μm filter, live cells were isolated using a gradient with Lympholyte-M separation media (Fisher Scientific) as per manufacturer's instructions. Tumor-infiltrating lymphocytes were enriched by CD45+ MACS positive selection (Miltenyi Biotec). Four representative samples each of untreated and PD-1/CTLA-4 blockade-treated samples were selected and droplet-based isolation of single cells was performed with the Chromium Controller (10× Genomics). Subsequent generation of 3′ sequencing libraries was performed as per manufacturer's instructions (10× Genomics). Characterization of the sequencing library was performed with TapeStation (Agilent) and Qubit (ThermoFisher) instruments. Pooled equimolar 3′ 10× libraries were sequenced with an Illumina NextSeq 500 instrument using paired-end 91 bp reads.
Count matrices were generated using the Cellranger v3.0.0 pipeline. Downstream analysis was performed using Scanpy v1.6.076 (Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018)). Cells with >10% mitochondrial gene content or <200 genes detected were filtered. Doublets were scored and removed using Scrublet (Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: Computational Identification of Cell Doublets in Single-Cell Transcriptomic Data. Cell Syst 8, 281-291.e9 (2019)). Gene abundances were library size normalized to 100,000 and log-transformed with a pseudocount of one. Cell types were identified on the basis of marker genes discovered by one-vs-rest differential expression using the Wilcoxon rank-sum test in Seurat 4.0.078 (Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cold Spring Harbor Laboratory 2020.10.12.335331 (2020) doi:10.1101/2020.10.12.335331) (
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application is a continuation of International Application No. PCT/2023/020680, filed on May 2, 2023, which claims priority to and the benefit of U.S. Provisional Application No. 63/337,930, filed May 3, 2022, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63337930 | May 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/020680 | May 2023 | WO |
| Child | 18931863 | US |
