methods to identify a target to prepare an inhibitory chimeric antigen receptor or a protective chim

专利摘要:
The present invention provides a method for identifying a target for preparing an inhibitory chimeric antigen receptor (iCAR) or a protective chimeric antigen receptor (pCAR) capable of preventing or attenuating unwanted activation of an effector immune cell. Also provided is a list of iCAR targets, as well as vectors and transduced effector immune cells comprising the nucleic acid molecule and methods for treating cancer comprising administering the transduced effector immune cells.
公开号:BR112020006106A2
申请号:R112020006106-9
申请日:2018-09-28
公开日:2020-11-17
发明作者:Gideon Gross；Will Gibson；Dvir Dahary；Merav Beiman
申请人:Immpact-Bio Ltd.；Gavish-Galilee Bio Applications Ltd.；
IPC主号:

专利说明:

[001] [001] This order claims priority for US Provisional Order 62 / 564,454, filed on September 28, 2017, and US Provisional Order 62 / 649,429, filed on March 28, 2018, each of which is incorporated by reference. SEQUENCE LISTING
[002] [002] The present application contains a Sequence Listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 27, 2018, is called 120575-5003_ST25.txt. ASCII TABLE
[003] [003] The provisional patent application to which the current application claims priority contains a long table section. A copy of the table has been presented to the US Patent and Trademark Office on a compact disk in ASCII format with Provisional Priority Order 62 / 649,429, filed on March 28, 2018, and is hereby incorporated by reference and may be used in the practice of the invention. . The referred ASCII table, created on March 28, 2018, is as follows: 120575-5003-PR allCandExt1167Genes_5003_PR.txt, 272,719,870 bytes. FIELD OF THE INVENTION
[004] [004] The invention relates to the field of cancer immunotherapy by adoptive cell transfer, employing chimeric activation antigen receptors (aCARs), recognizing antigens expressed on the surface of tumor cells, CAR inhibitors (iCARs) and protective CARs (pCARs) )
[005] [005] The identification of targetable antigens that are expressed exclusively by tumor cells, but not by healthy tissues, is undoubtedly the biggest challenge of immunotherapy against cancer today. Clinical evidence that T cells are capable of eradicating tumor cells comes from numerous studies evaluating highly diverse approaches to harnessing T cells to treat cancer (Rosenberg and Restifo, 2015). These approaches employ bone marrow transplantation with infusion of donor lymphocytes, adoptive transfer of tumor-infiltrating lymphocytes (TILs), treatment with genetically redirected T cells in pre-selected antigens via CARs (Gross and Eshhar, 2016a) or T cell receptors ( TCRs), the use of immune checkpoint inhibitors or active vaccination. Of these, the use of genetically modified T cells and different strategies for active immunization involve pre-existing information on candidate antigens that are likely to have a durable clinical response, but minimal adverse effects. However, as stated in the title of a recent review by S. Rosenberg, “Finding suitable targets is the major obstacle to cancer gene therapy” (Rosenberg, 2014).
[006] [006] The concept of using chimeric antigen receptors (or CARs) to genetically redirect T cells (or other killer cells of the immune system, such as natural killer cells (NK) and cytokine-induced killer cells) against antigens of choice. independently of MHC it was first introduced by Gross and Eshhar in the late 1980s (Gross et al., 1989). They are produced synthetically from chimeric genes encoding a variable fragment of extracellular single chain antibody (scFv) fused through a flexible hinge and motif
[007] [007] Most other TAAs currently evaluated in preclinical and clinical studies are overexpressed by tumors, but are also present, usually at a lower level, in essential normal tissue.
[008] [008] The broad spectrum of strategies designed to combat autoimmunity in T cell CAR therapy can be divided into those that seek to eliminate or suppress transferred T cells when the damage is already evident (reactive measures) and those that aim to prevent potential damage in the first place. place (proactive measures) (Gross and Eshhar, 2016a). Reactive approaches often use suicide genes, such as herpes simplex virus thymidine kinase (HSV-tk) and iC9, a fusion polypeptide comprising a truncated human caspase 9 and a mutated FK506 binding protein. Other approaches use antibodies to selectively remove engineered cells that wreak havoc or, as recently demonstrated, a small molecule heterodimerizing agent that governs the coupling of the CAR recognition fraction to the intracellular signaling domain (Wu et al., 2015). Although some proactive measures are designed to limit the persistence or function of T CAR cells in vivo (for example, the use of
[009] [009] Although undoubtedly intriguing, this approach still faces the need for meticulous titration of the magnitude of both activation and co-stimulation signals, in order to achieve the ideal balance that would allow only an effective T-cell reactivity in the target tumor. Whether such a balance can be achieved routinely in the clinical setting is still
[0010] [0010] An entirely new approach to limiting the T cell response to only target cells that express a unique combination of two antigens has recently been published (Roybal et al., 2016a). Its core element functions as a 'genetic switch' that explores the mode of action of various cell surface receptors, including Notch. After binding such a receptor to its ligand, it undergoes double cleavage, resulting in the release of its intracellular domain, which translocates to the cell nucleus, where it functions as a transcription factor. The implementation of this principle implies the co-introduction of two genes in effector T cells. The first is expressed constitutively and encodes that chimeric cleavable receptor equipped with a recognition fraction directed to the first antigen. Involvement with this antigen on the surface of a target cell will activate the expression of the second gene that encodes a conventional CAR that is directed to the second antigen. The target cell will be killed only if it coexpresses this second antigen as well.
[0011] [0011] Inhibitory CARs. Reactivity outside the tumor occurs when the target antigen of killer cells redirected to CAR is shared with normal tissue. If this normal tissue expresses another surface antigen not present in the tumor, then coexpression in cells modified by genes from an additional CAR targeting this unshared antigen, which houses a fraction of inhibitory signaling, may prevent T cell activation by the tissue. normal.
[0012] [0012] Instead of an activation domain (such as FcRy or CD3-ζ), an iCAR has a signaling domain derived from an inhibitory receptor that can antagonize T cell activation, such as CTLA-4, PD-1 or an NK inhibitory receptor. If the normal tissue that shares the candidate aCAR antigen with the tumor expresses another surface antigen not shared with the tumor, an iCAR expressed by the same T cell
[0013] [0013] Unlike T cells, each of which expresses a single, two-stranded TCR encoded by somatic rearranged gene segments, NK cells do not express specific antigen receptors. Instead, NK cells express an array of germline-encoded activation and inhibition receptors that recognize, respectively, multiple activation and inhibition ligands on the cell surface of infected and healthy cells. The protective ability of an iCAR based on NK inhibitory receptors, such as KIR3DL1, has been described (US 9,745,368). KIR3DL1 and other NK-inhibitory receptors work by dismantling the immune synapse in a quick and comprehensive manner. There is compelling evidence that a single NK cell can spare a resistant cell that expresses both inhibition and activation ligands, while still killing a susceptible cell that it involves simultaneously, which expresses only the activation ligands (Abeyweera et al., 2011; Eriksson et al., 1999; Treanor et al., 2006; Vyas et al., 2001). This exquisite skill is governed by the different spatial organization of signal transduction molecules formed in each of the respective immune synapses, which consequently affects the exocytosis of cytolytic granules (see (Huse et al., 2013) for review). More recently, Fedorov et al. (Fedorov et al., 2013a; WO 2015/142314) successfully used the intracellular domains of PD-1 and CTLA-4 for this purpose. Unlike NK-inhibitory receptors, the regulatory effects of these iCARs affected the entire cell. However, these effects were temporary, allowing full activation of T cells upon subsequent encounter with target cells that express only the aCAR antigen.
[0014] [0014] The tissue distribution of the antigens targeted by iCAR and aCAR determines the ideal mode of action of the iCAR needed to provide maximum safety without compromising clinical effectiveness. For example, if
[0015] [0015] Next generation sequencing (NGS) allows the determination of the DNA sequence of all protein coding genes (~ 1% of the entire genome) in a given tumor biopsy and the comparison of the cancer 'exome' with that healthy tissue (usually white blood cells) from the same patient. Exome sequencing can be completed within several days after biopsy removal and at a relatively low cost. In parallel, the analysis of the transcriptome (RNA-seq) can provide complementary information about the genes that are actually expressed by the same cell sample.
[0016] [0016] It is becoming increasingly clear that the mutational scenario for each individual tumor is unique (Lawrence et al., 2013; Vogelstein et al., 2013). As a result of non-synonymous mutations, the tumor cell can potentially present a particular set of neopeptides to the patient's immune system in one or more of its HLA products. In fact, enormous efforts have been made in recent years to identify specific tumor neoepitopes that can be recognized by the patient's own CD8 or CD4 T cell repertoire and serve as targets for immunotherapy (for review, see (Blankenstein et al., 2015 ; Van Buuren et al., 2014; Heemskerk et al., 2013; Overwijk et al., 2013; Schumacher and Schreiber, 2015)). However, cumulative conclusions suggest that neoantigen-based T cell immunotherapies are more likely to be effective in
[0017] [0017] In short, the urgent need to identify suitable targets for cancer immunotherapy through the adoptive transfer of genetically redirected killer cells is still largely unmet. BRIEF SUMMARY OF THE INVENTION
[0018] [0018] In some respects, the present invention provides a method for identifying a target for preparing a chimeric antigen receptor inhibitor (iCAR) or a protective chimeric antigen receptor (pCAR) capable of preventing or attenuating unwanted activation of an immune cell effector, in which the target is identified by a method characterized by the fact that it comprises: (i) identifying a gene with at least two expressed alleles that encode a protein comprising an extracellular polymorphic epitope; (ii) determining that at least one of the expressed alleles exhibits a change in the amino acid sequence in the extracellular polymorphic epitope sequence with respect to an extracellular polymorphic epitope reference sequence; (iii) determining that the gene is located in a chromosomal region that suffers loss of heterozygosity (LOH) in a type of tumor; and
[0019] [0019] In some modalities, the LOH position is selected from the group consisting of substitution, deletion and insertion. In some modalities the position of LOH is a SNP. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA gene.
[0020] [0020] In some embodiments, the gene comprising the extracellular polymorphic epitope is an HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L gene, HLA-DM, HLA-DO, HLA-DP, HLA_DQ or HLA-DR In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-A gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-B gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-C gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-G gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-E gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-F gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-K gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-L gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-DM gene. In some embodiments, the gene that comprises the extracellular polymorphic epitope is an HLA-DO gene. In some embodiments, the extracellular polymorphic epitope is an HLA-DP gene. In some embodiments, the extracellular polymorphic epitope is an HLA_DQ gene. In some embodiments, the extracellular polymorphic epitope is an HLA-DR gene.
[0021] [0021] In some embodiments, the gene comprising the epitope
[0022] [0022] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 2. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY, 75
[0023] [0023] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 3. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, EPY8B1, DCBLD2, DR3, G3, GPR128, GPR15, GPR27, GRM2, GRM7, HEG1, HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1, LRRC15, LRRAL1, LRRN1, OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC4A7, SL1, SLA, TMEM44, TMPRSS7, TNFSF10, UPK1B, VIPR1 and ZPLD1.
[0024] [0024] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 4. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TMR6, TLR6, TLR10 TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1 and UNC5C.
[0025] [0025] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 5. In some
[0026] [0026] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 6. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A2, BTN3A2, BTN3A2, BTN3A2 BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR-6, HPR, HPR, 6 B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR2J2, OR2J OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1 and TREML2.
[0027] [0027] In some embodiments, the gene comprising the epitope
[0028] [0028] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 8. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A and TNFRSF10A.
[0029] [0029] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 9. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2 and VLDLR.
[0030] [0030] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 10. In some
[0031] [0031] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 11. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRP5, LRP5 MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10 OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D14, , OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8, OR8, OR8, OR8, OR8 , OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12,
[0032] [0032] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 12. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRFSC, LRP1, LR1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ, PTPRR, SCNNLCA, S8, S8, S5, SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2, TM1, TM2, TM2, TM1, TM2, TM2, TM2, TM2, TM2, TM2, TM2, TM2, TM2
[0033] [0033] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 13. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[0034] [0034] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 14. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2,
[0035] [0035] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 15. In some embodiments the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2 , FAM174B, GLDN, IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4, OR4M2, OR4N4, PRTG, RHCG, SCAMP5, SEMA4B, SEMA6D, SLC24A1, SLC24A1, SLC24A5, and TYRO3.
[0036] [0036] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 16. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, SEK6, PKD1 SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219 and TMEM8A.
[0037] [0037] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 17. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, LRC, KGA3, K3 LRRC37B, MRC2, NGFR, OR1A2, OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5AEM, TR2, TM2, TM2, TM2, TS2
[0038] [0038] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 18. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15 and TNFRSF11A.
[0039] [0039] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 19. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFARY, FPR, FX5 GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LIL, LIL, LIL, LIL, LIL, LIL, LIL, LIL, LIL LILRB4, LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7 PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, 3L, TMC4, TMEM91, TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B and ZNRF4.
[0040] [0040] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 20. In some embodiments, the gene comprising the extracellular polymorphic epitope is
[0041] [0041] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 21. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2 and UMODL1.
[0042] [0042] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 22. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6 and TNFRSF13C.
[0043] [0043] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on the X chromosome. In some embodiments, the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR1 , GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4 and XG.
[0044] [0044] In some embodiments, the tumor is selected from the group consisting of a breast tumor, a prostate tumor, an ovarian tumor, a cervical tumor, a skin tumor, a pancreatic tumor, a colorectal tumor, a renal tumor , a liver tumor, a brain tumor, lymphoma, leukemia, a lung tumor and a glioma.
[0045] [0045] In some modalities, the tumor is selected from the group that
[0046] [0046] The present invention also provides safe effector cells. In some embodiments, the present invention provides a safe effector immune cell that expresses (i) an iCAR or pCAR of any of claims 1 to 46 and (ii) a chimeric activation antigen receptor (aCAR).
[0047] [0047] In some embodiments, the safe effector immune cell of claim 47, wherein the aCAR is directed against or specifically binds to a tumor-associated antigen or a non-polymorphic cell surface epitope. In some embodiments, due to the protective effects of iCAR or pCAR, aCAR can be directed against any surface protein expressed in a cancer cell.
[0048] [0048] In some embodiments, aCAR is directed against or specifically binds to a tumor-associated protein, a CAR target as listed in Table 1, any cell surface protein that is expressed in a tumor tissue in which iCAR it is also expressed.
[0049] [0049] In some embodiments, the non-polymorphic cell surface epitope is selected from the group consisting of CD19, CD20, CD22, CD10, CD7, CD49f, CD56, CD74, CAIX Igκ, ROR1, ROR2, CD30, LewisY, CD33, CD34, CD38, CD123, CD28, CD44v6, CD44, CD41, CD133, CD138, NKG2D-L, CD139, BCMA, GD2, GD3, hTERT, FBP, EGP-2, EGP- 40, FR-α, L1-CAM, ErbB2,3,4, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, kRas, MAGE-A1, MUC1, MUC16, PDL1, PSCA, EpCAM, FSHR, AFP, AXL, CD80, CD89, CDH17, CLD18, GPC3,
[0050] [0050] 51. Safe effector immune cell according to any of claims 47 to 50, characterized by the fact that the safe effector immune cell is an autologous effector cell or a universal (allogeneic) cell.
[0051] [0051] In some embodiments, the safe effector immune cell is selected from the group consisting of a T cell, a natural killer cell and a cytokine-induced killer cell.
[0052] [0052] In some modalities, the expression level of iCAR or pCAR is greater than or equal to the expression level of aCAR.
[0053] [0053] In some modalities, iCAR or pCAR is expressed by a first vector and aCAR is expressed by a second vector.
[0054] [0054] In some modalities, the safe effector immune cell, iCAR or pCAR and aCAR are both expressed by the same vector.
[0055] [0055] In some embodiments of the safe effector immune cell, the nucleotide sequence encoding aCAR is downstream of the nucleotide sequence encoding iCAR or pCAR.
[0056] [0056] In some embodiments of the safe effector immune cell, the nucleotide sequence comprises a viral autoclivable 2A peptide between the nucleotide sequence encoding for aCAR and the nucleotide sequence encoding for iCAR or pCAR.
[0057] [0057] In some modalities of the safe effector immune cell, the viral autoclivable peptide 2A is selected from the group consisting of T2A of Thosea asigna virus (TaV), F2A of the foot and mouth disease virus (FMDV), E2A of the Equine rhinitis virus A (ERAV) and P2A from Porcine teschovirus-1 (PTV1).
[0058] [0058] In some embodiments of the safe effector immune cell, the nucleotide sequence encoding aCAR is linked via a flexible linker to iCAR or pCAR.
[0059] [0059] In some modalities of the safe effector immune cell, the
[0060] [0060] In some embodiments of the safe effector immune cell, the at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM), for example, CD3ζ or FcRγ chains.
[0061] [0061] In some embodiments of the safe effector immune cell, the at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to an activation killer cell immunoglobulin-like receptor (KIR), such as KIR2DS and KIR3DS .
[0062] [0062] In some embodiments of the safe effector immune cell, the at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to or an adapter molecule, such as DAP12.
[0063] [0063] In some embodiments of the safe effector immune cell, the at least one signal transducing element that activates or co-stimulates an effector immune cell is homologous to or a signal co-stimulating element of CD27, CD28, ICOS, CD137 (4 -1BB), CD134 (OX40) or GITR.
[0064] [0064] The present invention also provides a method for treating cancer in a patient having a tumor with the characteristic of LOH, characterized by the fact that it comprises administering to the patient a safe effector immune cell expressing an iCAR as described here.
[0065] [0065] In some embodiments, the invention further provides a method for treating cancer in a patient having a tumor with the characteristic of LOH, characterized by the fact that it comprises administering to the patient a safe effector immune cell as described here.
[0066] [0066] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a
[0067] [0067] In a further aspect, the present invention provides a vector comprising a nucleic acid molecule of the invention, as defined herein, and at least one control element, such as a promoter, operably linked to the nucleic acid molecule.
[0068] [0068] In another aspect, the present invention provides a method for preparing an inhibitory chimeric antigen receptor (iCAR) capable of preventing or attenuating the unwanted activation of an effector immune cell, according to the present invention, as defined herein, the a method comprising: (i) retrieving a list of human genomic variants of genes encoding proteins from at least one database of known variants; (ii) filter the list of variants retrieved in (i) by: (a) selection of variants resulting in an amino acid sequence variation in the protein encoded by the respective gene compared to its corresponding reference allele, (b) selection of variants of genes in which the amino acid sequence variation is in an extracellular domain of the encoded protein, (c) selection of variants of genes that suffer loss of heterozygosity (LOH) in at least one tumor and (d) selection of variants
[0069] [0069] In yet another aspect, the present invention provides a method for preparing a safe effector immune cell comprising: (i) transfecting a TCR engineered effector immune cell targeting a tumor associated antigen with a nucleic acid molecule comprising a sequence nucleotide encoding an iCAR as defined herein or transducing cells with a vector defined herein; or (ii) transfecting a naïve effector immune cell with a nucleic acid molecule comprising a nucleotide sequence encoding an iCAR as defined herein and a nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined herein; or by transducing an effector immune cell with a vector as defined herein.
[0070] [0070] In yet another aspect, the present invention provides a safe effector immune cell obtained by the method of the present invention, as
[0071] [0071] In a further aspect, the present invention provides a method for selecting a personalized biomarker for a subject having a tumor characterized by LOH, the method comprising (i) obtaining a tumor biopsy of the subject; (ii) obtaining a sample of normal tissue from the subject, for example, peripheral blood mononuclear cells (PBMCs); and (iii) identifying a simple allelic variant of a polymorphic cell surface epitope that is not expressed by the tumor cells due to LOH, but that is expressed by the cells of normal tissue, thereby identifying a personalized biomarker for the subject.
[0072] [0072] In a further aspect, the present invention provides a method for treating cancer in a patient having a tumor characterized by LOH, comprising administering to the patient an effector immune cell as defined herein, wherein the iCAR is directed to an allelic variant simple encoding a polymorphic cell surface epitope absent from tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of the patient's normal related mammalian tissue.
[0073] [0073] In yet an additional aspect, the present invention is directed to a safe effector immune cell, as defined herein, for use in
[0074] [0074] In still a further aspect, the present invention is directed to a method for treating cancer in a patient having a tumor characterized by LOH comprising: (i) identifying or receiving information identifying a simple allelic variant of a polymorphic cell surface epitope which is not expressed by tumor cells due to LOH, but which is expressed by cells of normal tissue, (ii) identifying or receiving information identifying a non-polymorphic cell surface epitope of an antigen or a simple allelic variant of an epitope of polymorphic cell surface, wherein said epitope is a tumor-associated antigen or is shared by cells of at least relative tumor and normal tissue in said cancer patient; (iii) selecting or receiving at least one nucleic acid molecule defining an iCAR as defined herein and at least one nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined herein, or at least one vector as defined herein, wherein iCAR comprises an extracellular domain that specifically binds to a (i) cell surface epitope and aCAR comprises an extracellular domain that specifically binds to a (ii) cell surface epitope; (iv) prepare or receive at least one population of safe redirected effector immune cells by transfecting immune effector cells with the nucleic acid molecules of (iii) or
[0075] [0075] In a similar aspect, the present invention provides at least one population of safe redirected immune effector cells to treat cancer in a patient having a tumor characterized by LOH, in which the safe redirected immune cells are obtained by (i) identifying or receiving information identifying a simple allelic variant of a polymorphic cell surface epitope that is not expressed by tumor cells due to LOH, but is expressed by cells in normal tissue, (ii) identifying or receiving information identifying a cell surface epitope non-polymorphic antigen or a simple allelic variant of a polymorphic cell surface epitope, wherein said epitope is a tumor-associated antigen or is shared by cells of at least a relative tumor and normal tissue in said cancer patient; (iii) selecting or receiving at least one nucleic acid molecule defining an iCAR as defined herein and at least one nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined herein, or at least one vector as defined herein, wherein iCAR comprises an extracellular domain that specifically binds to a (i) cell surface epitope and aCAR comprises an extracellular domain that specifically binds to a (ii) cell surface epitope; (iv) prepare or receive at least one population of safe redirected effector immune cells by transfecting immune effector cells with the nucleic acid molecules of (iii) or transducing effector immune cells with the vectors of (iii).
[0076] [0076] In another aspect, the present invention is directed to a combination of two or more nucleic acid molecules, each comprising a nucleotide sequence encoding a member
[0077] [0077] Fig. 1 shows the concept of iCARs (taken from (Fedorov et al., 2013a).
[0078] [0078] Fig. 2 shows the molecular design and mode of action of aCAR / pCAR. The binding of pCAR to its antigen in normal cells, whether they express the aCAR antigen or not, is expected to result in rapid RIP and breakdown of the polypeptide into 3 separate fragments.
[0079] [0079] Figs. 3A-C show the percentage of tumor samples undergoing LOH in the chromosomal region coding for the HLA class I locus. A. HLA-G, B. HLA-A, C. ZNRD1, in the tumor types from the
[0080] [0080] Fig. 4 shows the expression of HLA-A in relation to all other genes encoding proteins in the genome. The value for each gene reflects the average median RPKM value of tissue obtained from GTEX (gtexportal.org)
[0081] [0081] Fig. 5 shows a proposed workflow for analyzing the loss of heterozygosity of the HLA protein through cancers in Example 5.
[0082] [0082] Fig. 6 shows LOH frequency in the pancan12 database using ABSOLUTE processed copy number data. The lines represent 95% binomial confidence intervals for frequency.
[0083] [0083] Fig. 7 shows the types of LOH observed in HLA-A. Of 588 episodes of HLA-A LOH, none involved a breakpoint within the HLA-A gene.
[0084] [0084] Fig. 8 shows the length distribution (in base pairs) of deletions spanning HLA-A. A large fraction of these deletions is greater than the length of chromosome 6p.
[0085] [0085] Fig. 9 shows the correlation between fraction of patients who have HLA-A LOH in relative copy number data and ABSOLUTE with a threshold of -0.1.
[0086] [0086] Fig. 10A-10C shows that comparing the LOH rate of HLA-A, HLA-B and HLA-C across 32 cancers reveals an almost identical LOH pattern.
[0087] [0087] Fig. 11 shows the screen capture of IGV of the profiles of numbers of AML copies classified for deletion of chromosome 6p. Blue indicates deletion, red indicates amplification. There is no denouncement of HLA-A.
[0088] [0088] Fig. 12 shows the proportion of uveal melanoma tumors undergoing LOH for all SNPs.
[0089] [0089] Fig. 13 provides TCGA Study Abbreviations (also available at https://gdc.cancer.gov/resources-tcga-users/tcga-code- tables / tcga-study-abbreviations).
[0090] [0090] Fig. 14 represents the loss of a chromosomal region adjacent to the tumor suppressor protein TP53 encoded on chromosome 17. The genes encoded on chromosome 17, which have been identified as iCAR targets can be used to treat a patient's RC001.
[0091] [0091] Fig. 15 provides a schematic diagram of iCAR and aCAR constructs.
[0092] [0092] Fig. 16 provides data regarding IL-2 secretion as measured by ELISA. iCAR specifically inhibits IL-2 secretion through interaction with target cells expressing iCAR target.
[0093] [0093] Fig. 17 shows that iCAR specifically inhibits IL-2 secretion through interaction with target cells expressing iCAR target as measured by CBA.
[0094] [0094] Fig. 18 shows the specific activation of CD19 aCAR Jurkat-NFAT by target cells expressing CD19.
[0095] [0095] Fig. 19 shows the specific inhibition of NFAT activation in CD19 aCAR / HLA-A2 iCAR Jurkat-NFAT
[0096] [0096] Fig. 20 shows specific inhibition of NFAT activation in different E / T ratios.
[0097] [0097] Fig. 21 provides the sequences for the iCAR and aCAR constructs in Fig. 15.
[0098] [0098] Fig. 22 provides the 1,167 potential targets for iCAR, pCAR and / or aCAR.
[0099] [0099] Fig. 23 provides the 3,288 SNPs of the 1,167 genes listed in Fig.
[00100] [00100] With reference to the revolutionary concept of tumor suppressor genes (TSGs) that was presented in 1971 by AG Knudson (Knudson Jr., 1971), Devilee, Cleton-Jansen and Cornelisse declared in the opening paragraph of their essay entitled 'Ever since Knudson '(Devilee et al., 2001): “Many publications have documented LOH on many different chromosomes in a wide variety of tumors, implying the existence of multiple TSGs. The Knudson two-hit hypothesis predicts that these LOH events are the second step in inactivating both alleles of a TSG ”. In their seminal review of genetic instabilities in human cancers (Lengauer et al., 1998), Lengauer, Kinzler and Vogelstein wrote: “Karyotype studies have shown that most cancers have lost or gained chromosomes and molecular studies indicate that
[00101] [00101] The initial LOH events can be detected in pre-malignant cells of the same tissue, but not in surrounding normal cells (Barrett et al., 1999). LOH is irreversible and events can only accumulate, so that the heterogeneity of the tumor reflects the accumulation of losses along the progression of the tumor. Although tumor subclones may develop that differ in later LOH events, a minimal LOH signature is expected to be shared by premalignant cells,
[00102] [00102] An inevitable result of crude LOH events is the concomitant loss of all other genes residing in the deleted chromosomal material and these naturally include many genes that encode transmembrane proteins. Regarding your identity, a catalog of
[00103] [00103] More recently, Bausch-Fluck et al. (Bausch-Fluck et al., 2015) applied their Chemoproteomic Cell Surface Capture technology to identify a combined set of 1,492 cell surface glycoproteins in 41 types of human cells. A large fraction of the surfaceoma is expected to be expressed by any given tumor, each displaying a distinctive profile. It was found that genes encoding cell surface proteins are slightly enriched for single nucleotide polymorphisms (SNPs) in their coding regions than all other genes (Da Cunha et al., 2009). Polymorphic insertions and deletions, which are rarer, contribute even more to the number of variants and are likely to have more robust structural effects on polypeptide products than SNPs that alter the peptide sequence (not synonymous). In total, a typical genome contains 10,000 to 12,000 sites with non-synonymous variants and 190–210
[00104] [00104] A significant fraction of allelic variations in surface proteins would affect the extracellular portion of the respective gene products, potentially creating epitopes restricted to distinct alleles that, in principle, can be recognized and distinguished from other variants by highly specific mAbs. It is well documented that mAbs can be isolated that discriminate between two variants of the same protein that differ only in a single amino acid (see, for example, an initial example of mAbs that recognizes Ras mutation products with exquisite specificity (Carney et al., 1986)). Interestingly, it has been shown that two specific mAbs for a single exchange of amino acids in a protein epitope can use variable regions that are structurally distinct from their heavy and light chain V gene groups (Stark and Caton, 1991). Recently, Skora et al. (Skora et al., 2015) reported the isolation of specific peptide scFvs that can distinguish between HLA-I-linked neopeptides derived from mutated KRAS and EGFR proteins and their wild-type counterparts, differing in both cases in an amino acid
[00105] [00105] Together, a unique antigenic signature of the
[00106] [00106] The logic presented above argues that a single molecular representation is inevitably modeled by LOH for almost all tumors, which is marked by the absence of numerous polymorphic surface structures that are present in normal cells. The conversion of this postulated signature of the individual tumor into a target set of antigenic epitopes implies a practicable immunological strategy to translate the recognition of a particular 'absence' into a suggestion of activation capable of triggering target cell killing. Importantly, the incorporation of a safety device to ensure that reactivity outside the tumor on the target is strictly avoided will be highly favorable in the future clinical implementation of this strategy
[00107] [00107] The present invention addresses this challenge by coexpressing in each therapeutic killer cell a single pair of genes. One partner in this pair encodes an activation CAR (aCAR) and the other encodes a protective CAR (pCAR) or an inhibiting CAR (iCAR) II. SELECTION SETTINGS
[00108] [00108] The term "nucleic acid molecule", as used herein, refers to a DNA or RNA molecule.
[00109] [00109] The term "coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA or an mRNA, to serve as a model for the synthesis of other polymers and macromolecules in biological processes having any of a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if the transcription and translation of mRNA corresponding to that gene produce the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcribing a gene or cDNA, can be termed as encoding the protein or other product of that gene or cDNA.
[00110] [00110] Unless otherwise specified, a "nucleotide sequence that encodes an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.
[00111] [00111] The term "endogenous" refers to any material from or produced within an organism, cell, tissue or system.
[00112] [00112] The term "exogenous" refers to any material from or produced outside an organism, cell, tissue or system.
[00113] [00113] The term "expression", as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence triggered by its promoter.
[00114] [00114] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be
[00115] [00115] The term "genomic variant", as used here, refers to a change of at least one nucleotide at the genomic level in a sequenced sample compared to the reference or consensus sequence at the same genomic position.
[00116] [00116] The term "corresponding reference allele", as used here with reference to a variant, means the reference or consensus sequence or nucleotide in the same genomic position as the variant.
[00117] [00117] The term "extracellular domain", as used here with reference to a protein, means a region of the protein that is outside the cell membrane.
[00118] [00118] The term "loss of heterozygosity" or "LOH", as used here, means the loss of chromosomal materials, such as a complete chromosome or part of it, in a copy of the two chromosomes in a somatic cell.
[00119] [00119] The term “sequence region”, as used herein, with reference to a variant or reference allele, means a sequence starting upstream and ending downstream of the variant's position, which can be translated into an “epitope peptide ”That can be recognized by an antibody.
[00120] [00120] The term "CAR", as that term is used here, refers to a chimeric polypeptide that shares structural and functional properties with an immune function receptor of a cell or molecule that adapts, for example,
[00121] [00121] The term "specific binding", as used here in the context of an extracellular domain, such as a scFv, which specifically binds to a simple allelic variant of a polymorphic cell surface epitope, refers to the relative binding of scFv to an allelic variant and its failure to bind to the corresponding different allelic variant of the same polymorphic cell surface epitope. As this depends on the avidity (number of copies of CAR in the T cell, number of antigen molecules on the surface of the target cells (or cells to be protected) and the affinity of the specific CARs used, a functional definition would be that the specific scFv would provide a significant signal in an ELISA against the simple allelic variant of a cell surface epitope for which it is specific or cells transfected with a CAR displaying scFv would be clearly marked with the simple allelic variant of a polymorphic cell surface epitope on a FACS assay, while the same assays using the corresponding different allelic variant of the same polymorphic cell surface epitope would not give any detectable signal.
[00122] [00122] The term "treatment", as used herein, refers to means to obtain a desired physiological effect. The effect can be therapeutic in terms of partially or totally curing an illness and / or symptoms attributed to the illness. The term refers to the inhibition of the disease, for example, preventing its development; or improving the disease, poor example, causing the disease to regress.
[00123] [00123] As used in this document, the terms "subject" or "individual" or "animal" or "patient" or "mammal" refer to any subject, particularly a mammal subject, for whom the diagnosis,
[00124] [00124] The phrase "safe effector immune cell" or "safe effector cell" includes those cells described by the invention that express at least one iCAR or pCAR as described here. In some embodiments, the "safe effector immune cell" or "safe effector cell" is capable of administration to a subject. In some embodiments, the "safe effector immune cell" or "safe effector cell" further expresses an aCAR as described here. In some embodiments, the "safe effector immune cell" or "safe effector cell" further expresses an iCAR or a pCAR as described herein. In some embodiments, the "safe effector immune cell" or "safe effector cell" further expresses an iCAR or a pCAR as described herein and an aCAR as described herein.
[00125] [00125] Pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The carrier (s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient.
[00126] [00126] The phrase "effective amount" or "therapeutically effective amount" is used here interchangeably and refers to an amount of a compound, formulation, material or composition, as described here, effective to achieve a particular biological result .
[00127] [00127] The following example of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which carriers, modes of administration, dosage forms, etc., can be selected for use with the present invention. Those skilled in the art will understand, however, that any formulation and mode of administration selected data must first be tested to determine that they achieve the desired results.
[00128] [00128] Methods of administration include, but are not limited to,
[00129] [00129] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition can comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polvidone or povidone), tragacanth gum, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, corn starch and the like; a lubricant or surfactant, such as magnesium stearate or sodium lauryl sulfate; and a glidant, such as colloidal silicon dioxide.
[00130] [00130] The term "peripheral blood mononuclear cell (PBMC)", as used herein, refers to any blood cell having a round nucleus, such as a lymphocyte, a monocyte or a macrophage. Methods for isolating PBMCs from blood are readily apparent to those skilled in the art. A non-limiting example is the extraction of these cells from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under a plasma layer or by leukapheresis, the preparation of leukocytes concentrates with the return of red cells and low leukocyte plasma for the donor.
[00131] [00131] The term "cancer", as used here, is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,
[00132] [00132] It should be emphasized that the present invention provides a new avenue allowing specific targeting of tumor cells, while keeping normal cells safe. The concept presented here provides the identification of new targets for iCARs (or pCARs or protective CARs), these targets defined as comprising simple allelic variants of polymorphic cell surface epitopes, which are lost from tumor cells due to the LOH of the chromosomal region in which they reside, although remaining expressed in normal tissue. Due to the polymorphic variation, it is possible to distinguish the two alleles and reach only the missing allele in the tumor cells. In addition, the target antigen may not necessarily be a tumor suppressor gene, or a gene predicted to be involved with cancer, since it is chosen because it is in a region lost by LOH and therefore could simply be linked to these genes. This is conceptually different from the methods used or suggested so far in cancer therapy, which target tumor-associated antigens or unregulated antigens in tumors, regardless of the polymorphism. The present methods also provide to expand the selection of aCAR in addition to tumor-associated antigens, providing protection of normal cells through the coexpression of iCAR and / or pCAR, as described here.
[00133] [00133] The distinction is crucial because LOH, being a genomic event, results in total loss of a specific tumor variant, with a very rare chance of winning back the lost allele. If the LOH event occurs too early in the development of tumors, it ensures a uniform target signature on all tumor cells derived from the initial premalignant tissue, including metastatic tumors. Additionally, the
[00134] [00134] According to a strategy, the two CARs in each given pair specifically recognize the product of a different allelic variant of the same target gene for which the patient is heterozygous. The basic principle is as follows: aCAR targets an allele variant of a selected cell surface protein that is expressed by the given tumor cells and is not affected by LOH, while pCAR or iCAR targets the product encoded by allele variant of the same gene that was lost from these tumor cells due to LOH. In other normal tissues of that individual patient that express the said gene, both alleles are present and are known to be equally functional, that is, the expression is biallelic in all tissues (in contrast to other genes that may exhibit random monoallelic expression ( Chess, 2012; Savova et al., 2016) .In one scenario, the two CARs target two related epitopes residing in the same location in the protein product, which differ in one or only a few amino acids. In another scenario, aCAR targets a non-polymorphic epitope on the same protein, while pCAR or iCAR is allele specific. In this case, the density of the aCAR epitope in normal cells would generally be twice as high as that of iCAR
[00135] [00135] Another strategy uses the protein products of hosekeeping genes as the targets of pCAR or iCAR. Since, by definition, these genes are expressed in every cell in the body, they are safe targets for pCAR or iCARs. That is, if the pCAR or iCAR targets a membrane product of a hosekeeping gene for which the given patient is heterozygous, all cells in the body, except tumor cells that have lost this allele due to LOH, will be protected. This strategy allows decoupling of the aCAR target gene product from pCAR or iCAR. In fact, the target of aCAR can then be any non-polymorphic epitope expressed by the tumor. A variation on this strategy would be to use a known aCAR targeting a non-polymorphic tumor-associated antigen, for example, an aCAR in clinical use or under examination in clinical experiments, in combination with an iCAR or pCAR targeting a gene product from a gene for which the given patient is heterozygous and which is expressed in at least the tissue of origin of the tumor and preferably in additional vital normal tissues in which the aCAR target antigen is expressed.
[00136] [00136] Following the same logic that allows the decoupling of the aCAR target antigen from iCAR / pCAR, the latter should not necessarily be the product of a hosekeeping gene. In some embodiments, iCAR and / or pCAR are the product of any gene whose expression pattern is broad enough to protect the vital normal tissues that express the aCAR target antigen in addition to the tumor. As a corollary, the aCAR antigen can be, as argued for housekeeping genes, any non-polymorphic epitope expressed by the tumor, not restricted to known 'tumor-associated antigens', a consideration that can broadly expand the candidate list of aCAR. In general, for both hosekeeping and non-hosekeeping genes, identification
[00137] [00137] Care must be taken to ensure that the inhibitory signal transmitted by iCAR is strictly and permanently dominant over the aCAR signal and that no recognition takes place between iCAR and aCAR. The dominance of iCAR ensures that activation of the killer cell upon encounter with normal cells that express the two alleles would be prevented. This standard brake, however, does not operate by coupling with tumor cells: in the absence of its target antigen, iCAR would not emit inhibitory signals, thus triggering anticipated aCAR-mediated cell activation and subsequent lysis of tumor cells
[00138] [00138] iCAR technology can be based on immune checkpoints. In this sense, the demonstration (Fedorov et al., 2013b; WO 2015/142314) that the regulatory elements of PD-1 and CTLA-4 have a potent inhibitory capacity for T cells when incorporated as iCAR signaling components is encouraging, but most of these observations have been questioned recently (Chicaybam and Bonamino, 2014, 2015). In addition, although the precise molecular pathways triggered by these checkpoint proteins are not fully understood, their involvement decreases T cell activation through proximal and distal mechanisms, making T cells unresponsive to concomitant activation stimuli (Nirschl and Drake, 2013). Therefore, although the state of inactivation ensured by iCARs PD-1 and CTLA-4 is really temporary and reversible (Fedorov et al., 2013b), it would not allow T cell activation in tissues that express both iCAR and aCAR targets. On the other hand, the dominance of NK inhibitory receptors over activation receptors ensures that healthy cells are spared the attack of NK cells by a spatial rather than a temporal mechanism. (Long et al., 2013). There is convincing evidence that a
[00139] [00139] The strategy based on the control affirmed by the iCARs depends on the dominance of the iCAR activity over the aCAR activity, as explained above. In some embodiments, the present invention provides this type of iCAR, here called a pCAR (for 'protection CAR, see Fig. 2), designed to operate on CAR T cells in a synapse selective manner and to ensure complete dominance over aCAR coexpressed. In some embodiments, the iCAR provided by the present invention is this particular type of iCAR referred to here as a protective CAR (pCAR).
[00140] [00140] In some embodiments, the pCAR of the present invention integrates two technological feats. First, pCAR allows to decouple the aCAR activation fraction (FcRγ / CD3-ζ) from the recognition unit and the co-stimulator element (for example, CD28, 4-1BB, CD134 (OX40, GITR, IL2Rβ and STAT3 binding motif (YXXQ)), placing them genetically in two different polypeptide products.The re-coupling of these elements, which is mandatory for the function of aCAR, will occur only by the addition of a heterodimerizing drug that can join the respective binding sites incorporated in each of the polypeptides separately (Fig. 2B) The reconstruction of a fully functional CAR combining similarly divided recognition and activation fractions due to a heterodimerizing drug was recently reported by Wu et al. (Wu et
[00141] [00141] Second, the graft from the pCAR recognition unit and the missing activation domain, respectively, on the two surfaces of the transmembrane domain of a RIP-controlled receptor that contains the two intramembrane cleavage sites (Fig. 2A). The binding of pCAR to its antigen will trigger double cleavage of the encoded polypeptide first by a member of the extracellular disintegrin and metalloproteinase (ADAM) family that removes the ectodomain and then by intracellular γ-secretase, which releases the intracellular domain of the pCAR. This first cleavage event is predicted to disrupt the ability of the truncated aCAR to gain access to a functional configuration, anchored in a membrane, of its activation element
[00142] [00142] The proposed mode of action described above is predicted to exert local effects, so that only aCARs residing on the same synapse are affected and are no longer able to bind their antigen productively and form an immunological synapse. As a result, even when multiple aCAR interactions with large numbers of non-tumor cells are likely to occur, they are expected to be transient and non-functional, so that the cells are fully capable of additional interactions.
[00143] [00143] The dominance of pCARs over their aCAR counterparts is inherent in this system, as the function of aCARs depends entirely on the presence of pCARs. The relative scarcity of pCARs in a given T cell would render aCARs non-functional due to the lack of an activation domain. In some modalities the relative scarcity of pCARs in a given T cell
[00144] [00144] It is critical that both the recognition and activation domains are located on the plasma membrane (Wu et al., 2015). Therefore, the second cleavage, which highlights the plasma membrane activation domain, would render this domain non-functional and prevent unwanted cell activation. In some modalities, the recognition and activation domains are located on the plasma membrane. In some embodiments, the second cleavage highlights the plasma membrane activation domain and makes this domain non-functional and prevents unwanted cell activation.
[00145] [00145] aCAR and pCAR were designed to work through mutually exclusive mechanisms. The ability of pCAR to undergo cleavage does not depend on the resistance of inhibitory signaling, so there will be no conclusion about the signaling result. While pCARs are cleaved, aCARs cannot function, regardless of the relative avidity of their interactions with their respective antigens, a scenario that ensures another crucial level of security.
[00146] [00146] In some embodiments, the mammalian tissue is human tissue and in other embodiments the related normal mammalian tissue is the normal tissue from which the tumor developed.
[00147] [00147] In some embodiments, the effector immune cell is a T cell, a natural killer cell or a killer cell induced by cytokines.
[00148] [00148] In some embodiments, the at least one signal transduction element capable of inhibiting an effector immune cell is homologous to a signal transduction element of an immune checkpoint protein, such as an immune checkpoint protein selected from the group consisting of PD1; CTLA4; BTLA; 2B4; CD160; CEACAM, such as CEACAM1; KIRs, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LIR1, LIR2,
[00149] [00149] In some embodiments, the immune checkpoint protein is an inhibitory receptor of natural killer cells, e.g., KIRs, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3; or a leukocyte Ig-like receptor, such as LIR1, LIR2, LIR3, LIR5, LIR8; and CD94-NKG2A, a type C lectin receptor that forms heterodimers with CD94 and contains 2 ITIMs.
[00150] [00150] Methods for preparing and using killer cell receptors in iCARs have been described in US 9,745,368, incorporated by reference as if it were fully disclosed herein.
[00151] [00151] In some modalities, the extracellular domain of any of the modalities is fused through a transmembrane flexible and motif canonical hinge to the said intracellular domain. i. TARGET IDENTIFICATION: aCAR, iCAR and pCAR
[00152] [00152] The present invention provides methods for identifying aCAR, iCAR and / or pCAR targets based on the identification of candidate genes having extracellular polymorphic epitopes. In some embodiments, aCAR can be targeted to any extracellular protein expressed in the tumor tissue. In some embodiments, the aCAR target is still expressed in non-tumor tissues and the iCAR target is also expressed in non-tumor tissues, but it is not expressed in tumor tissues.
[00153] [00153] In some modalities, the method of identifying genes
[00154] [00154] In some embodiments, the target for use in iCAR and / or pCAR is selected based on the identification of a gene having at least one extracellular polymorphic epitope and in which said gene has at least two expressed alleles. In some modalities, the target for use in iCAR and / or pCAR is selected based on the identification of a gene having located in a chromosomal region that suffers loss of heterozygosity. In some modalities, the target for use in iCAR and / or pCAR is selected based on the identification of a gene having located in a chromosomal region that suffers loss of heterozygosity in cancer. In some modalities, the score for a theoretical SNP is calculated and a threshold limit is determined. For example, if only 32% of SNPs had a gene
[00155] [00155] In some embodiments, the target for use in iCAR and / or pCAR is selected from a gene having at least one extracellular polymorphic epitope. In some embodiments, the target is a gene that is located on chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 6, chromosome 6, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, chromosome 17, chromosome 18, chromosome 19, chromosome 20, chromosome 21, chromosome 22 or chromosome X.
[00156] [00156] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 1. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101 , CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C, CD244, CD34, CD46,
[00157] [00157] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 2. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207 , CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP1B, LRPY , MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO and TRABD2A.
[00158] [00158] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 3. In some
[00159] [00159] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 4. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ANTXR2, BTC, CNGA1, CORIN, EGF , EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TL10 , TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1 and UNC5C.
[00160] [00160] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 5. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9 , C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2, ILQRA, IL6
[00161] [00161] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 6. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1 , BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR116, GPR126, GPR126 , HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA -F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1 , OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1 and TREML2. In some embodiments, the gene that comprises the extracellular polymorphic epitope is located on chromosome 6 and comprises an HLA target. In some embodiments, the target for use in iCAR and / or pCAR is HLA-A, HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1 , HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G. In some embodiments, the target for use in iCAR and / or pCAR is HLA-A2,
[00162] [00162] In some embodiments, the gene comprising the epitope
[00163] [00163] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 8. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9 , ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A10, TNF
[00164] [00164] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 9. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9 , CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 , OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2 and VLDLR.
[00165] [00165] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 10. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group
[00166] [00166] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 11. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24 , CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP4, LRP4 , MCAM, MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10 , OR10Q1, OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4, 51, OR4, 51, OR4, 51 , OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B, OR5, OR5, OR5, OR5, OR5 1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1, OR8, OR8, OR8, OR8, OR8, OR8, OR8, OR8 OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12, SLCO2B1, SORL1, ST14, TM4, TM4, TM4, TM4, TM4
[00167] [00167] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 12. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163 , CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KRRF1, KLRF1, KLRF, , OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ, SPRA, SLCA, SCR1, , SLC8B1, SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2, TM1, TM2, TM2, TM2, TM2, TM2, TM2 .
[00168] [00168] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 13. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A , KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[00169] [00169] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 14. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1 , FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4 and SYNDIG1L.
[00170] [00170] In some embodiments, the gene comprising the epitope
[00171] [00171] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 16. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15 , CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PDKN1, PD , SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219 and TMEM8A.
[00172] [00172] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 17. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2 , C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGA3, ITGA3, ITGA3, ITG3, IT3 , LRRC37A, LRRC37B, MRC2, NGFR, OR1A2, OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC26A11, , TTYH2 and TUSC5.
[00173] [00173] In some embodiments, the gene that comprises the extracellular polymorphic epitope is located on chromosome 18. In some
[00174] [00174] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 19. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59 , C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCPR, FF1 , FXYD5, GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KIL1R, LILY, LILY, LILY, LILY , LILRB3, LILRB4, LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7 , OR7G3, PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, TMF, M161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B and ZNRF4.
[00175] [00175] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 20. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN , CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC4A3, SLC4A3, SLC4A3, SLC4A3, SLC4A3, SLC4A3, SLC4A3, SLC24A3, SLC4A3
[00176] [00176] In some embodiments, the gene comprising the epitope
[00177] [00177] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on chromosome 22. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1 , GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6 and TNFRSF13C.
[00178] [00178] In some embodiments, the gene comprising the extracellular polymorphic epitope is located on the X chromosome. In some embodiments, the target for use in iCAR and / or pCAR is selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4 and XG.
[00179] [00179] In some embodiments, the one used to treat cancer is directed against or specifically binds to any membrane protein that is expressed in tumor tissue, provided that iCAR is expressed in all normal tissues in which the target aCAR protein is expressed. In some embodiments, aCAR can bind or target specifically to a tumor-associated protein, tumor-associated antigen and / or antigens in clinical experiments, a CAR target as listed in Table 1, as well as any cell surface protein that is expressed in a tumor tissue to which an iCAR can be combined or paired in relation to the target binding, according to the criteria listed in the application. In some embodiments, aCAR can be any protein expressed with an extracellular domain, as long as iCAR is expressed in the same tissues
[00180] [00180] The present invention also provides recognition fractions designed to provide specific binding to the target. The recognition fraction allows to target the specific link and the targeted link of aCAR, iCAR and / or pCAR. In some embodiments, the recognition fraction designed to provide specific binding to the target provides specific binding to an extracellular polymorphic epitope. In some
[00181] [00181] Generally, any relevant technology can be used to engineer a fraction of recognition that gives aCARs and pCAR or iCARs specific binding to their targets. For example, recognition fractions comprising this iCAR-aCAR Library can be derived from a master recognition fraction group, ideally selected from a combinatorial display library, so that: - Collectively, the selected recognition fractions target the products of cell surface of an array of genes that reside in each of the two arms of all 22 human autosomes. The shorter the distance between neighboring genes, the greater the coverage, hence, the greater the universality of use. - For each of the selected genes, a set of specific allele recognition fractions is isolated, each allowing for strict discrimination between different allelic variants that are prevalent in the human population. The greater the number of targeted variants, the greater the number of therapeutic gene pairs that can be offered to patients.
[00182] [00182] A given allelic product can become a potential pCAR or iCAR target in one patient and a useful aCAR target in another patient harboring the same allele, depending on the particular LOH pattern in each case. Hence, when suitable recognition fraction genes are identified, each will be grafted into either a pCAR or iCAR gene scaffold and aCAR gene scaffold. Therefore, it is desirable that all recognition fractions targeting allelic variants of the same gene have binding affinities in a similar range. Within this given set of recognition fractions, all possible combinations of pCAR-aCAR or iCAR-aCAR pairs can be pre-assembled, in order to ensure the highest coverage of potential allelic compositions of this gene in the entire population.
[00183] [00183] In some embodiments, the patient is heterozygous for the major and minor allele, whose products differ in a single position along the encoded polypeptide as a result of a non-synonymous SNP or, less frequently, an indel. In some other modalities, a patient is heterozygous for two smaller alleles that differ from the main one in two separate positions. Depending on the particular LOH event involving that gene in individual patients, a given variant epitope can serve as an iCAR target in one patient and an aCAR target in another. In some embodiments, the variant epitope that can serve as an iCAR target is not the primary allele variant. In some embodiments, the variant epitope that can serve as an iCAR target is a minor allele.
[00184] [00184] The identification of a variant-specific mAb (say, a mAb specific for the epitope encoded by the minor allele 'a') is well known in the art and is similar, in principle, to the identification of a mAb against any conventional antigenic determinant and can generally be best done via high throughput screening from an scFv library
[00185] [00185] By definition, the corresponding epitope (in the same position), which is encoded by the main allele ('A'), creates a unique antigenic determinant that differs from that created by 'a' in the identity of a single amino acid (SNP) or length (indel; for example, insertion or deletion). This determinant can, in principle, be recognized by a different set of mAbs identified by the same, or other, antibody display screening technology. The ability of distinct members in each of the two sets of identified mAbs to distinguish between the two epitopes or variants, for example, an antibody from the first set binds the allele product 'a', but not 'A', and an Ab of the second league set
[00186] [00186] In some embodiments, for example, with respect to the HLA class I HLA-A, HLA-B and HLA-C genes as the target genes, there are numerous allele-specific monoclonal antibodies available, for example, but not limited to, antibodies listed in Example 3.
[00187] [00187] In some embodiments, the target for use in generating a recognition fraction comprises at least one extracellular polymorphic epitope. In some embodiments, the target is the product of a gene that is located on chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, chromosome 17, chromosome 18, chromosome 19, chromosome 20, chromosome 21, chromosome 22 or X chromosome.
[00188] [00188] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00189] [00189] In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected gene from chromosome 1. In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a gene selected from the group consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C, CD1A, CD1C, CD24 , CD46, CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B, FCGRAB, FCGRA , FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33, GPR157,
[00190] [00190] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00191] [00191] In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome 2 gene. In some embodiments, the recognition fraction for
[00192] [00192] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00193] [00193] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one epitope
[00194] [00194] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00195] [00195] In some modalities, the fraction of recognition for use in iCAR or pCAR provides specificity for at least one epitope
[00196] [00196] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00197] [00197] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one epitope
[00198] [00198] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00199] [00199] In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected gene from chromosome 6. In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENP3 , ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B, HLA-C, HLA-DA, HLA-DA1 , HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2 , LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2, OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16 , RAET1E, R AET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1 and TREML2.
[00200] [00200] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product from a selected chromosome gene
[00201] [00201] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 7. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A2, OR2AE, P2, OR2, P1, OR2, P1 PODXL, PTPRN2, PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213, TTYH3, ZAN and ZP3.
[00202] [00202] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 8. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ADAM18, BTC, ADAM28, CORIN, EGF, EMCN, ENPEP, ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1 , CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A and TNFRSF10B.
[00203] [00203] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 8. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymer epitope in a gene product selected from the group consisting of ADAM18, BTC, ADAM28, CORIN, EGF, EMCN, ENPEP, ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17 , CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A and TNFRSF10A and TNFRSF10A and TNFRSF10B.
[00204] [00204] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 9. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, MIA9 , MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2, OR1K1, OR1L1, OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1, OR2, OR2, OR2, OR2 , SEMA4D, SLC31A1, TEK, TLR4, TMEM2 and VLDLR.
[00205] [00205] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 9. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least
[00206] [00206] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 10. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GRFR2, GRFR2 , IL15RA, IL2RA, ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR, SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TSP5, TS5
[00207] [00207] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 10. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FTP2 , GPR158, GRID1, IL15RA, IL2RA, ITGA8,
[00208] [00208] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 11. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP, MMP26, MPEG1, MPRX, MPRX, MPRX, MPRX MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1, OR2, OR2, OR2, OR2, OR2, OR2, OR2, OR2 OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, O R51E1, OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A1, OR5A2, OR5A2, OR5A2, OR5A OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8, OR2, OR8, OR8, OR8, OR8 OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12, SLCO2EM8, TM4, S2, TM4,
[00209] [00209] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 11. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP, MPRX, MMP26, MRG26, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR10G8, OR1, OR2, OR1, OR1, OR1, OR1, OR1, OR1, OR1 OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR5B, OR5B, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8, OR8, OR8, OR8, OR8H3, OR8I2, OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLCMA1, SLCMA1, SLC5A1, TMEM225, TMPRSS4, TMPRSS5, TRIM5, TRPM5, TSPAN18 and ZP1.
[00210] [00210] In some modalities, the fraction of recognition for
[00211] [00211] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 12. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC2A, CLEC2A, CLEC2A CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1, MANSC4, OLR1, OR6, OR1, OR6, OR6, OR6, OR6, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2,
[00212] [00212] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 13. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope on a gene product selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[00213] [00213] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 13. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK19 and TNFRS.
[00214] [00214] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 14. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of
[00215] [00215] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 14. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G , OR4K1, OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4 and SYNDIG1L.
[00216] [00216] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 15. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN, IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11 NO, , NRG4, OCA2, OR4F4, OR4M2, OR4N4, PRTG, RHCG, SCAMP5, SEMA4B, SEMA6D, SLC24A1, SLC24A5, SLC28A1, SPG11, STRA6, TRPM1 and TYRO3.
[00217] [00217] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 15. In some modalities, the fraction of
[00218] [00218] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 16. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, IT4F , ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B, SEZ6L2, SLC22A31, SLC5A11, SLC7A6, TM, TM, TM, TM
[00219] [00219] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 16. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT1 , IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT,
[00220] [00220] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 17. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CNTNAP1 CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, OR1, OR1, OR1, OR1, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E, TNFSF12, TRPV3, TTYH2 and TUSC5.
[00221] [00221] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 17. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A2, LRRC37A2 OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11,
[00222] [00222] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 18. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15 and TNFRSF11A.
[00223] [00223] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 18. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC11 and TNFRSF .
[00224] [00224] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 19. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3,
[00225] [00225] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 19. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5, GFY, GP6, GPR42, GRINFLB, ICAM, GRINFL IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1, LDLR, LILRA1, LILRA2, LILRA4, LILRA, LILR3, LILR3, LILR3, LILR3 MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10,
[00226] [00226] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 20. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPB , OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4 and THBD.
[00227] [00227] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 20. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymeric epitope in a gene product selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1 , LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4 and THBD.
[00228] [00228] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 21. In some modalities, the recognition fraction for
[00229] [00229] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 21. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymer epitope in a gene product selected from the group consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS2, TMPRSS2, TMPRSS2, TMPRSS2, TMPRSS2, and UMODL1.
[00230] [00230] In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 22. In some modalities, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, SCXF, PLX , SUSD2, TMPRSS6 and TNFRSF13C.
[00231] [00231] In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product of a gene selected from chromosome 22. In some modalities, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product
[00232] [00232] In some embodiments, the recognition fraction for use in aCAR provides specificity for at least one extracellular polymorphic epitope in a selected X-chromosome gene product. In some embodiments, the recognition fraction for use in aCAR provides specificity for hair minus an extracellular polymorphic epitope in a gene product selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4.
[00233] [00233] In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a selected X-chromosome gene product. In some embodiments, the recognition fraction for use in iCAR or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLX8, .
[00234] [00234] Sequences encoding the variable regions of these antibodies can be easily cloned from the relevant hybridoma and used to construct genes encoding scFvs against any desired target including, for example, scFvs against specific HLA allele epitope variants, and which would be suitable for incorporation into a CAR construct using widely available tools as disclosed, for example, in Molecular Cloning: A Laboratory Manual (Fourth Edition) Green and Sambrook, Cold Spring Harbor Laboratory Press; Antibodies: A Laboratory Manual (Second Edition), Edited by Edward A. Greenfield,
[00235] [00235] The present invention provides a database comprising DNA sequences of polymorphic variants lost in tumor cells due to LOH and encoding cell surface products, wherein the variation in the DNA sequence results in a variation in the sequence of amino acid in an extracellular domain of the encoded protein. The information was retrieved from various databases open to the general public, such as TCGA, available on the TCGA National Institute of Health data portal (https://gdc.cancer.gov/), which provides, among others, data that can be used to infer the relative copy number of the gene in a variety of tumor types and the cbio portal for TCGA data at http://www.cbioportal.org (Cerami et al., 2012, Gao et al., 2013 ); the Exome Aggregation Consortium (ExAC) database (exac.broadinstitute.org, Lek et al., 2016), providing, among others, allele frequencies of Genotype- Tissue Expression (GTEX) v6p database (dbGaP Accession phs000424.v6 .p1) (https://gtexportal.org/home, Consortium GT. Human genomics, 2015), which includes tissue expression data for genes; and databases providing structural protein information, such as the Human Protein Atlas (Uhlen et al., 2015); o Cell Surface Protein Atlas (Bausch-Fluck et al., 2015), a database based on mass spectrometry of N-glycosylated cell surface proteins and UniProt database (www.uniprot.org/downloads).
[00236] [00236] The present invention further provides a method for identifying the entire genome of genes encoding expressed cell surface proteins that undergo LOH. The identified genes must meet the following criteria: 1) The gene encodes a transmembrane protein - therefore, having a portion expressed on the cell surface to allow binding of iCAR or pCAR; 2) The gene has at least two
[00237] [00237] In principle, the genes described above, suitable for encoding targets for iCAR or pCAR binding, can be identified by any method known in the art, and not just by database mining. For example, the concept of LOH is not new and the LOH information for specific genes, chromosomes or genomic / chromosomal regions in specific tumors has already been published in the literature and candidate genes can therefore be derived from the publications available. Alternatively, this information can be found by entire genomic hybridizations with chromosomal markers, such as microsatellite probes (Medintz et al., 2000, Genome Res. 2000 Aug; 10 (8): 1211–1218) or by any other suitable method (Ramos and Amorim, 2015, J. Bras. Patol. Med. Lab. 51 (3): 198-196).
[00238] [00238] Likewise, information on allelic variants is publicly available in various databases and can also be easily obtained for a personalized case by genomic sequencing of a suspected region. In addition, information on protein structure and expression pattern is publicly available and easily accessible, as described above.
[00239] [00239] Consequently, as information on the various criteria for many genes and SNPs is publicly available and the techniques for retrieving them are generally known, the main novelty of the application is to use LOH as a criterion for choosing a target for iCAR recognition or pCAR, and the concept of personalizing the treatment
[00240] [00240] As a non-limiting example, it has been found, according to the present invention, that HLA genes, including LOH from non-classical HLA-I and HLA-II genes (for example, HLA-A, HLA-B HLA- C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR HLA-K and HLA-L), in varying frequencies, is a relatively frequent in many types of tumors (see Figs. 10A-C), which would make these genes good candidates for use as targets for iCAR / pCAR recognition for the purposes of the present invention.
[00241] [00241] Recognition of the aCAR target in normal cells in any healthy essential tissue in the absence of the pCAR or iCAR target would be harmful and is strictly prohibited. In this sense, the concept of pCAR-aCAR or iCAR-aCAR pairs, as proposed here, constitutes a fail-safe activation switch, since: i) cells that do not express the selected gene (in the case of aCAR and pCAR or iCAR target different products of the same gene) will not be targeted due to the absence of the target antigen aCAR; ii) normal cells that express this same gene will coexpress both alleles and will not be targeted due to the dominance of pCAR or iCAR; iii) in case the pCAR or iCAR reaches the product of a polymorphic housekeeping gene, all cells in the body will be protected; and iv) only tumor cells that express the aCAR target, but not pCAR or iCAR, will be attacked. In some embodiments, recognition of the aCAR target in normal cells in any healthy essential tissue in the absence of the pCAR or iCAR target would be detrimental. In some modalities, cells that do not express the selected gene (in the case that aCAR and pCAR or iCAR target different products from the same gene) will not be targeted due to the absence of the target antigen from aCAR. In some modalities, normal cells that express this same gene will coexpress both alleles and will not be targeted due to the dominance of pCAR or
[00242] [00242] As emphasized above, according to the invention, there must be permanent dominance of the inhibitory signal over the activation signal. Therefore, it is necessary to ensure that no aCAR gene is expressed in a given killer cell, at any time, in the absence of its iCAR partner. This can be implemented through the tandem set of these iCAR-aCAR gene pairs as single chain products or through an appropriate bicistronic modality, based, for example, on an internal ribosome entry site or on one of several 2A peptides of viral auto-cleavage. As suggested by the vast amount of data reported on bicistronic expression, the iCAR gene will always be positioned upstream from its partner aCAR to ensure favorable stoichiometry. Another option would be to engineer the killer cells to express both aCAR and iCAR or pCAR, transfecting or transducing the killer cell with two independent constructs, each coding for either aCAR or iCAR / pCAR. This is obviously not a problem when using a pCAR-aCAR gene pair. In some embodiments, the inhibitory signal is dominant over the activation signal. In some embodiments, aCAR and iCAR or pCAR are expressed simultaneously in the same cell.
[00243] [00243] Another attractive option to ensure the dominance of iCAR is to highlight the fraction of aCAR recognition of its activator / co-stimulation portion, so that both entities can be assembled only in a functional receptor in the presence of a small heterodimerizing molecule. The ability to tightly control the state
[00244] [00244] In addition, the expected dominance is likely to be intrinsic to the particular composition of the iCAR signaling elements incorporated in the intracellular portion in the selected iCAR project that must 'compete' with the signaling intensity of the chosen aCAR platform. This ability will also be influenced by the relative affinities of the two fractions of recognition for their respective target epitopes (which were discussed above) and by the general activities of their interactions. In relation to the latter, the proposed strategy ensures both a favorable stoichiometry of iCAR / aCAR and a balanced distribution of its respective target epitopes in normal cells. Again, this is not an issue when using a pCAR-aCAR gene pair.
[00245] [00245] To further ensure safety, other conventional means currently implemented in the field of CAR and TCR immunotherapy can be employed, such as the use of suicidal genes or the use of mRNA electroporation for transient expression.
[00246] [00246] Although LOH often leaves cells with only one allele of a given gene, it is often accompanied by duplication of the remaining chromosome, or part of the chromosome, resulting in a 'neutral copy number' LOH (Lo et al. , 2008; O'Keefe et al., 2010; Sathirapongsasuti et al., 2011). In these circumstances, the appearance of variants of epitope loss requires two independent events and is thus less likely. The expression of several pairs of pCAR-aCAR or iCAR-aCAR in different fractions of the cells modified by genes will prevent the appearance of mutational leaks, even in cases of 'copy number loss' LOH, in which only a single copy of the allele target has been retained. However, since single copy genes can become essential, their
[00247] [00247] In view of the above, in one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an inhibitory chimeric antigen (iCAR) receptor capable of preventing or attenuating unwanted activation of an effector immune cell, wherein the iCAR comprises an extracellular domain that specifically binds to a unique allelic variant of a polymorphic cell surface epitope absent in mammalian tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of normal tissue related mammal or vital organs in which aCAR is expressed; and an intracellular domain comprising at least one signal transducing element that inhibits an effector immune cell.
[00248] [00248] In some embodiments, the polymorphic cell surface epitope is part of an antigen encoded by a tumor suppressor gene or a gene genetically linked to a tumor suppressor gene, since these genes are likely to be lost due to LOH in tumors. In addition, the polymorphic cell surface epitope may be part of an antigen encoded by a gene normally residing on a chromosome or chromosomal arm that frequently undergoes LOH in cancer cells, such as, but not limited to, 3p, 6p chromosomal arms , 9p, 10q, 17p, 17q or 18q or chromosome 19. These epitopes can be easily identified in the relevant databases, as described here.
[00249] [00249] In some embodiments, the polymorphic cell surface epitope is from a housekeeping gene product, such as AP2S1, CD81, GPAA1, LGALS9, MGAT2, MGAT4B, VAMP3; the CTNNA1 NM_001903, CTNNB1, CTNNBIP1 NM_020248, CTNNBL1 NM_030877, CTNND1 NM_001085458 delta catenin cell adhesion proteins; ABCB10 NM_012089, ABCB7 NM_004299, ABCD3 channels and conveyors
[00250] [00250] Any relevant technology can be used to engineer
[00251] [00251] In some embodiments, aCAR comprising an extracellular domain that specifically binds to a non-polymorphic cell surface epitope of an antigen or to a simple allelic variant of a polymorphic cell surface epitope. In some embodiments, the extracellular domain of aCAR binds to an epitope that is a tumor-associated antigen epitope. In some embodiments, the extracellular domain of aCAR binds to an epitope that is a tumor-associated antigen and is shared by at least related tumor cells and normal tissue and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates an effector immune cell. In some embodiments, the aCAR used to treat cancer is directed against or specifically binds to any membrane protein that is expressed in tumor tissue, provided that the iCAR target is expressed in all normal tissues in which the target aCAR protein is expressed. In some embodiments, aCAR is directed against or specifically binds to a non-polymorphic cell surface epitope selected from, but not limited to, the following list of antigens: CD19, CD20, CD22, CD10, CD7, CD49f, CD56, CD74 , CAIX Igκ, ROR1, ROR2, CD30, LewisY, CD33,
[00252] [00252] In some embodiments, iCAR is directed against or specifically binds to a simple allelic variant of an antigen that does not include ephrin receptors (eg EPHA 7) and claudins. In some embodiments, iCAR is directed against or specifically binds to an epitope encoded by a simple allelic variant of an HLA gene (HLA-A gene, HLA-B gene or HLA-C gene. Iii. INTRACELLULAR DOMAINS: aCAR, iCAR and pCAR
[00253] [00253] The present invention also provides intracellular domains as part of aCAR, iCAR, and / or pCAR. In some embodiments, the intracellular domain comprises at least one signal transducing element. In some embodiments, the intracellular domain comprises at least one signal transducing element that inhibits an effector immune cell.
[00254] [00254] Generally, any relevant technology can be used to engineer a signal transducing element that gives aCARs and pCAR or iCARs the ability to induce cellular function including, for example, the ability to inhibit an effector immune cell or to activate or co-stimulate an effector immune cell.
[00255] [00255] In some embodiments, the at least one signal transducing element is capable of inhibiting an effector immune cell. In some
[00256] [00256] In some modalities, the signal transduction element is capable of activating or co-stimulating an effector immune cell. In some embodiments, the signal transduction element is an activation domain. In some embodiments, the signal transduction element is a co-stimulating domain. In some embodiments, the signal transduction element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM), an activator killer cell-type immunoglobulin receptor or an adapter molecule and / or an co-stimulatory signal transduction element. In some embodiments, the signal transduction element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, ITAM is a protein including, but not limited to, CD3ζ or FcRγ chains. In some embodiments, the signal transduction element that activates or
[00257] [00257] In some embodiments, the extracellular domain is fused through a transmembrane flexible and motif canonical hinge to the said intracellular domain.
[00258] [00258] In some modalities, the use of a pCAR allows decoupling to decouple the aCAR activation fraction (FcRγ / CD3-ζ) from the recognition unit and the co-stimulating element (for example, CD28, 4-1BB). In some embodiments, these elements are genetically placed in two different polypeptide products. In some embodiments, the re-coupling of these elements, which is mandatory for the function of aCAR, will occur only by the addition of a heterodimerization drug that can join the respective binding sites incorporated in each of the polypeptides separately.
[00259] [00259] Instead of an activation domain (such as FcRy or CD3-ζ), an iCAR has a signaling domain derived from an inhibitory receptor that can antagonize T cell activation. In some embodiments, iCAR has a domain signaling derived from a
[00260] [00260] In some embodiments, aCAR is encoded by a first nucleic acid vector and iCAR or pCAR is encoded by a second nucleic acid vector. In some embodiments, aCAR is encoded by a first nucleic acid vector and iCAR or pCAR is encoded by a second nucleic acid vector. In some embodiments, aCAR is encoded by a first nucleic acid vector and iCAR or pCAR is encoded by a second nucleic acid vector. In some embodiments, the nucleotide sequence that encodes for iCAR or pCAR is in a second vector.
[00261] [00261] In some embodiments, the present invention provides a vector comprising a nucleic acid molecule of the invention, as defined in any of the above embodiments, and at least one control element, such as a promoter, operably linked to the acid molecule nucleic.
[00262] [00262] In some modalities, the vector is a lentiviral vector (LV). In some embodiments, the LV vector is a commercially available LV vector. In some embodiments, the LV vector includes, but is not limited to, pLVX-Puro, pLVX-IRES-Puro / Neo / Hygro, pLVx-EF1a-IRES (TAKARA) and / or pcLV-EF1a (Sirion). In some embodiments, the LV vector is pLVX-Pure. In some modalities, the LV vector is pLVX-IRES- Pure / Neo / Hygro. In some modalities, the LV vector is pLVx-EF1a-IRES (TAKARA). In some embodiments, the LV vector is pcLV-EF1a (Sirion).
[00263] [00263] In some embodiments, the vector comprises an EF1 promoter. In some modalities, the vector comprises a promoter of
[00264] [00264] In some embodiments, the vector further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an aCAR comprising an extracellular domain that specifically binds a non-polymorphic cell surface epitope to an antigen or a simple allelic variant of a polymorphic cell surface epitope, wherein said epitope is a tumor-associated antigen or is shared by at least related tumor cells and normal tissue and an intracellular domain comprising at least one signal transduction element that activates and / or co-stimulates an effector immune cell.
[00265] [00265] In some embodiments, the extracellular domain of the aCAR encoded by the nucleic acid comprised in the vector specifically binds to a non-polymorphic cell surface epitope of an antigen and the extracellular domain of the iCAR specifically binds to a simple allelic variant of an epitope of polymorphic cell surface of an antigen other than that to which the extracellular domain of said aCAR binds.
[00266] [00266] In some embodiments, the extracellular domain of the iCAR encoded by the nucleic acid comprised in the vector is directed against or specifically binds to a single allele variant of HLA genes, including, for example, HLA-A gene, HLA-B gene or gene HLA-C; or against a single allelic variant of a gene listed in Table 8.
[00267] [00267] In some embodiments, the extracellular domain of the aCAR encoded by the nucleic acid comprised in the vector is directed against or specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in Table 1, such as CD19. In some
[00268] [00268] In some embodiments, the extracellular domain of the iCAR encoded by the nucleic acid comprised in the vector is directed against or specifically binds to a simple allelic variant of HLA genes, including, for example, HLA-A gene, HLA-B gene or gene HLA-C or against a single allelic variant of a gene listed in Table 8; and the extracellular domain of aCAR encoded by the nucleic acid comprised in the vector is directed against or specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in Table 1, such as CD19. In some embodiments, the target for aCAR is any target with an extracellular domain.
[00269] [00269] In some embodiments, the at least one aCAR signal transducing element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM), for example, CD3ζ or FcRγ chains; a transmembrane domain of a killer cell immunoglobulin-like receptor (KIR) comprising a positively charged amino acid residue or a positively charged side chain or a transmembrane KIR domain activating, for example, KIR2DS and KIR3DS, or an adapter molecule, such like DAP12; or a co-stimulating signal transducing element of, for example, CD27, CD28, ICOS, CD137 (4-1BB) or CD134 (OX40).
[00270] [00270] In some modalities, iCAR or pCAR is expressed by a first vector and aCAR is expressed by a second vector. In some modalities, iCAR or pCAR and aCAR are both expressed by the same vector.
[00271] [00271] In some embodiments, the nucleotide sequence of the vector comprises an internal ribosome entry site (IRES) between the nucleotide sequence encoding the aCAR and the sequence of
[00272] [00272] In some embodiments, the nucleotide sequences encoding aCAR and iCAR are encoded in a simple vector. In some embodiments, the vector comprises an internal ribosome entry site (IRES) between the nucleotide sequence that encodes aCAR and the nucleotide sequence that encodes iCAR. In some embodiments, the nucleotide sequence encoding aCAR is downstream of the nucleotide sequence encoding iCAR. In some embodiments, the nucleotide sequence comprises a viral autoclivable 2A peptide located between the nucleotide sequence encoding aCAR and the nucleotide sequence encoding iCAR. In some embodiments, the nucleotide sequence of the vector comprises a viral autoclivable 2A peptide between the nucleotide sequence encoding aCAR and the nucleotide sequence encoding iCAR. In some embodiments, the viral autoclivable peptide 2A includes, but is not limited to, T2A of Thosea asigna virus (TaV), F2A of the foot and mouth disease virus (FMDV), E2A of the Equine rhinitis A virus (ERAV) and / or P2A from Porcine teschovirus-1 (PTV1). In some embodiments, the viral self-cleavable 2A peptide is T2A from the Thosea asigna virus (TaV). In some embodiments, the viral self-cleavable 2A peptide is foot and mouth disease virus (FMVD) F2A. In some embodiments, the viral autoclivable 2A peptide is E2A from the Equine rhinitis A virus (ERAV). In some embodiments, the viral autoclivable peptide 2A is P2A from Porcine teschovirus 1 (PTV1).
[00273] [00273] In some embodiments, the vector comprises a nucleotide sequence that encodes the constitutive aCAR linked via a ligand
[00274] [00274] Immune cells can be transfected with the appropriate nucleic acid molecule described herein by, for example, RNA transfection or by incorporation into a plasmid suitable for replication and / or transcription in a eukaryotic cell or viral vector. In some embodiments, the vector is selected from a retroviral or lentiviral vector.
[00275] [00275] Combinations of retroviral vector and an appropriate packaging line can also be used, where the capsid proteins will be functional to infect human cells. Several amphotropic virus-producing cell lines are known, including PA12 (Miller, et al. (1985) Mol. Cell. BioI. 5: 431-437); PA317 (Miller, et al. (1986) Mol. Cell. Bioi. 6: 2895-2902); and CRIP (Danos, et al. (1988) Proc. Nati. Acad. Sci. USA 85: 6460-6464). Alternatively, non-amphotropic particles can be used, such as pseudotyped particles with VSVG, RD 114 or GAL V envelopes. The cells can further be transduced by direct co-culture with producer cells, for example, by the method of Bregni, et al. (1992) Blood 80: 1418-1422, or culture only with viral supernatant or stocks of concentrated vectors, for example, by the method of Xu, et al. (1994) Exp. Hemat. 22: 223-230; and Hughes, et al. (1992) J Clin. Invest. 89: 1817.
[00276] [00276] In another aspect, the present invention provides a method for preparing an inhibitory chimeric antigen receptor (iCAR) capable of preventing or attenuating the unwanted activation of an effector immune cell, according to the present invention, as defined above, a method comprising: (i) retrieving a list of human genomic variants of genes encoding proteins from at least one database of known variants; (ii) filter the list of variants retrieved in (i) by: (a) selection of variants resulting in an amino acid sequence variation in the protein encoded by the respective gene compared to its corresponding reference allele, (b) selection of variants of genes in which the variation
[00277] [00277] In some modalities, the candidate variants of genes that are selected suffer LOH in at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in a certain type of tumor.
[00278] [00278] In some modalities, the lowest allele frequency for each selected variant is equal to or greater than 1, 2, 3, 4 or 5% in at least one population.
[00279] [00279] In another aspect, the present invention is directed to a combination of two or more nucleic acid molecules, each comprising a nucleotide sequence encoding a different member of a controlled effector immune cell activation system, the
[00280] [00280] In some embodiments, the first member is selected from: (a) a constitutive aCAR further comprising an intracellular domain comprising at least one signal transducing element that activates and / or co-stimulates an effector immune cell; and (b) a conditional aCAR further comprising an intracellular domain comprising a first member of a binding site for a small heterodimerizing molecule and optionally at least one co-stimulatory signal transduction element, but lacking an activation signal transduction element; and the second member is: (c) an inhibitory chimeric antigen receptor (iCAR) further comprising an intracellular domain comprising at least one signal transducing element that inhibits
[00281] [00281] In some embodiments (i) the extracellular domain of iCAR or pCAR specifically binds to a simple allelic variant of a polymorphic cell surface epitope of an antigen, which is an antigen other than that to which the aCAR extracellular domain binds; (ii) the extracellular domain of said pCAR or iCAR specifically binds to a simple allelic variant of a polymorphic cell surface epitope other than the same antigen to which the extracellular domain of said aCAR binds; or (iii) the extracellular domain of said pCAR or iCAR specifically binds to a simple allelic variant other than the same polymorphic cell surface epitope to which the extracellular domain of said aCAR binds.
[00282] [00282] In some pCAR modalities, the substrate for a sheddase is a substrate for a disintegrin and metalloproteinase (ADAM) or a beta-secretase 1 (BACE1). In some embodiments, the substrate forms part of the extracellular domain and comprises Lin 12 / Notch repeats and an ADAM protease cleavage site.
[00283] [00283] It is generally accepted that there is no consistent sequence motif predicting ADAM cleavage, but Caescu et al. (Caescu et al., 2009) disclose in Table 3 a large number of ADAM10 and / or ADAM17 substrate sequences, which are hereby incorporated by reference as if fully disclosed herein and which can serve as a substrate for ADAM in the pCAR of the present invention. In some
[00284] [00284] It may be advantageous to use an ADAM10 cleavage sequence in the pCAR of the present invention, because ADAM 10 is constitutively present at comparatively high levels in, for example, lymphocytes. In contrast to ADAM10, the close relative TACE / ADAM17 is detected only at low levels in unstimulated cells. The surface expression of ADAM17 in T cell blasts is rapidly induced by stimulation (Ebsen et al., 2013).
[00285] [00285] Hemming et al. (Hemming et al., 2009) report that no consistent sequence motifs predicting BACE1 cleavage has been identified in substrates versus non-substrates, but discloses in Table 1 a large number of BACE1 substrates having BAC1 cleavage sequences, which are hereby incorporated by reference as if fully disclosed herein and which may serve as a substrate for BACE1 in the pCAR of the present invention.
[00286] [00286] In some pCAR modalities, the substrate for an intramembrane cleavage protease is a substrate for an SP2, a γ– secretase, a signal peptide peptidase (spp), a spp-like protease or a rhomboid protease.
[00287] [00287] Rawson et al. (Rawson, 2013) disclose that SP2 substrates have at least one propeller covering type 2 membrane and include a propeller destabilizer motif, such as an Asp-Pro motif on an SP2 substrate. This article describes in Table 1 a number of SP2 substrates having
[00288] [00288] Haapasalo and Kovacs (Haapasalo and Kovacs, 2011) teach that the precursor of amyloid protein β (AβPP) is a substrate for presenilin-dependent γ-secretase (PS) (PS / γ-secretase) and that at least 90 proteins additional factors were considered to undergo similar proteolysis by this enzyme complex. Γ-secretase substrates have some common characteristics: most substrate proteins are type I transmembrane proteins; y-cleavage mediated by PS / γ-secretase (corresponding to & - cleavage in AβPP, which releases AICD) occurs at or near the limit of the transmembrane and cytoplasmic domains. The & type cleavage site flanks a stretch of hydrophobic amino acid sequence rich in lysine and / or arginine residues. It appears that the cleavage of PS / y-secretase is not dependent on a specific amino acid target sequence at or adjacent to the cleavage site, but rather, perhaps, in the conformational state of the transmembrane domain. Haapasalo and Kovacs disclose in Table 1 a list of γ-secretase substrates, whose cleavage sequences are hereby incorporated by reference as if fully disclosed herein and which can serve as a substrate for γ-secretases in the pCAR of the present invention.
[00289] [00289] Voss et al. (Voss et al., 2013) teach that so far no consensus cleavage site based on primary sequence elements within the substrate has been described for GxGD aspartyl proteases (spps). The transmembrane domains of membrane proteins preferentially adopt α-helical confirmation in which their peptide bonds are difficult to access to proteases. In order to make the transmembrane domains susceptible to intramembrane proteolysis, it was therefore postulated that their α-helical content needs to be reduced by destabilizing helix amino acids. Consistent with this hypothesis, it was demonstrated
[00290] [00290] Bergbold et al. (Bergbold and Lemberg, 2013) teach that, for rhomboid proteases, two different models for substrate recognition have been suggested. In the first model, the conformational flexibility of the substrate peptide backbone combined with immersion of the membrane in the vicinity of the rhomboid active site is sufficient to provide specificity. For the well-characterized Drosophila substrate
[00291] [00291] In view of the above, since in most cases no consensus motive has yet been established for intramembrane cleavage proteases and since assays to identify intramembrane cleavage protease substrates are well known in the art, as described in the literature cited above, pCAR can comprise an amino acid sequence identified as such and can further comprise destabilizing residues of the transmembrane helix.
[00292] [00292] In some embodiments, the substrate forms part of the transmembrane motif canon and is homologous / derived from a Notch transmembrane domain, ErbB4, E-cadherin, N-cadherin, ephrin-B2, amyloid precursor protein or CD44.
[00293] [00293] In some embodiments, the comprises a nucleotide sequence that encodes an extracellular domain and an intracellular domain of said conditional aCAR as separate proteins, wherein each domain is independently fused to a transmembrane motif and comprises a different member of a site binding to a small heterodimerizing molecule.
[00294] [00294] In some modalities, each of the first and the second
[00295] [00295] In yet another aspect, the present invention provides a method for preparing a safe effector immune cell comprising: (i) transfecting an engineer effector immune cell with TCR targeting a tumor associated antigen with a nucleic acid molecule comprising a sequence nucleotide encoding an iCAR or pCAR as defined herein above or transducing cells with a vector or (ii) transfecting a naïve effector immune cell with a nucleic acid molecule comprising a nucleotide sequence encoding an iCAR or pCAR as defined herein above and a nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined herein above; or transducing an effector immune cell with a vector as defined above.
[00296] [00296] In some embodiments, the immune cell for use in engineering includes, but is not limited to, a T cell, a natural killer cell or a cytokine-induced killer cell. In some embodiments, the immune cell for use in engineering includes, but is not limited to, a Jurkat T cell, a Jurkat-NFAT T cell and / or a mononuclear blood cell
[00297] [00297] In yet another aspect, the present invention provides a safe effector immune cell obtained by the method of the present invention, as described above. The safe effector immune cell can be a redirected T cell that expresses an exogenous T cell receptor (TCR) and an iCAR or pCAR, where the exogenous TCR is directed to a non-polymorphic cell surface epitope of an antigen or variant simple allelic of a polymorphic cell surface epitope, wherein said epitope is an antigen associated with a tumor or is shared by at least related tumor cells and normal tissue and iCAR or pCAR is as defined above; or the safe effector immune cell is a redirected effector immune cell, such as a natural killer cell or a T cell that expresses an iCAR or pCAR and an aCAR, as defined above.
[00298] [00298] In some embodiments, the safe effector immune cell, expressed on its surface an aCAR comprising an extracellular domain that specifically binds to a non-polymorphic cell surface epitope of an antigen and an iCAR or pCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of a different antigen to which the extracellular domain of said aCAR binds. In some embodiments, the extracellular domain of iCAR or pCAR that specifically binds to a simple allelic variant of a polymorphic cell surface epitope is the same antigen to which the extracellular domain of said aCAR binds; or the extracellular domain of iCAR or pCAR specifically binds a simple allelic variant other than the same polymorphic cell surface epitope area to which the extracellular domain of said aCAR binds.
[00299] [00299] In some embodiments, the extracellular domain of aCAR expressed on the cell surface specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in
[00300] [00300] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of an HLA-A, HLA-B, HLA-C, HLA-G gene , HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA_DQ or HLA-DR or against a single allelic variant of a gene listed in Table 8.
[00301] [00301] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of an HLA-A gene, HLA-B gene or HLA-C gene or against a simple allelic variant of a gene listed in Table 8; and the extracellular domain of aCAR expressed on the cell surface is directed against or specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in Table 1, such as, for example, but not limited to CD19. In some embodiments, the target for aCAR is any target with an extracellular domain.
[00302] [00302] In some embodiments, aCAR and iCAR are present on the cell surface as separate proteins.
[00303] [00303] In some embodiments, the level of expression on the cell surface of the nucleotide sequence encoding the iCAR is greater than or equal to the level of expression of the nucleotide sequence encoding the aCAR.
[00304] [00304] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of at least one extracellular polymorphic epitope.
[00305] [00305] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a selected gene
[00306] [00306] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP1B, LR2 MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO and TRABD2A.
[00307] [00307] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allele variant of a gene selected from the group consisting of ACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DRY, GP5, GPR128, GPR15, GPR27, GRM2, GRM7, HEG1, HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1, LRRC, NRR1, LRN1 OR5AC1, OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC3A1, SLC5A, TMEM108, TMEM44, TMPRSS7, TNFSF10, UPK1B, VIPR1 and ZPLD1.
[00308] [00308] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR, TL10, TLR, TLR TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1 and UNC5C.
[00309] [00309] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2,
[00310] [00310] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allele variant of a gene selected from the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR126, GPR HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA- F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, ORB OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1 and TREML2.
[00311] [00311] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1,
[00312] [00312] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A10, TNFRSF and TNFRS
[00313] [00313] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2 and VLDLR.
[00314] [00314] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1,
[00315] [00315] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP, LRP4, LRP4, LRP4 MCAM, MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10, OR10 OR10Q1, OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4, OR1, OR4, OR1, OR1, OR4, OR1 OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5B, R5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8, OR8, OR8, OR8, OR8, OR8 OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12, SLCO2B1, SOREM, TM2, TM2, TM2, TM2, TM2, TM2 TRIM5, TRPM5, TSPAN18 and ZP1.
[00316] [00316] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or
[00317] [00317] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A , KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[00318] [00318] In some embodiments, the recognition fraction for use in aCAR, iCAR and / or pCAR provides specificity for at least one extracellular polymorphic epitope in a gene product selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4 and SYNDIG1L.
[00319] [00319] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4,
[00320] [00320] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PDKN, PIE SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219 and TMEM8A.
[00321] [00321] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, LRC, ITGA3, LGA, IT3 LRRC37A, LRRC37B, MRC2, NGFR, OR1A2, OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC26A11, SLCAA, TM2, TR TTYH2 and TUSC5.
[00322] [00322] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12 , DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15 and
[00323] [00323] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3, FCAR, FFAR3 FXYD5, GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LILY, LILY, LILY, LILY LILRB3, LILRB4, LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7, OR7 OR7G3, PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPF2, TSF2, TINT MEM91, TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B and ZNRF4.
[00324] [00324] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a simple allelic variant of a gene selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLCA10, SLCA10
[00325] [00325] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of a selected gene from the
[00326] [00326] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allele variant of a gene selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1 , GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6 and TNFRSF13C.
[00327] [00327] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allele variant of a gene selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB , GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4 and XG.
[00328] [00328] In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allele variant of HLA-A2. In some embodiments, the extracellular domain of iCAR and / or pCAR expressed on the cell surface is directed against or specifically binds to a single allelic variant of CD20. In some modalities, iCAR will target HLA-A2. In some modalities, the iCAR will be directed to CD20. In some modalities, aCAR will be directed to CD19. In some modalities, the iCAR / aCAR set will be HLA-A2 and CD19, respectively. In some modalities, the iCAR / aCAR suite will include CD20 and CD19, respectively. saw. PREPARATION OF TARGET CELLS
[00329] [00329] In some embodiments, the target cells are prepared and tested in an in vitro system. In some modalities, a system
[00330] [00330] In some embodiments, the iCAR / aCAR set will be HLA-A2 and CD19, respectively, recombinant cells that express HLA-A2, CD19 or both will be produced by transfecting cell lines (for example, Hela, Hela-Luciferase or Raji) with expression vector coding for these genes. For detection of recombinant expression of CD19 and HLA-A2, both genes will be fused to a protein marker (for example, HA or Flag or Myc etc.). In some embodiments, the target set of iCAR / aCAR will be CD 20 / CD19 and recombinant cells will express CD19, CD20 or both.
[00331] [00331] In some embodiments, the expression vector comprising the iCAR / aCAR target set is transfected into a cell. In some embodiments, the expression vector is transfected into a cell to produce the target and non-tumor effects.
[00332] [00332] In some embodiments, the expression vector encodes a gene selected from the group consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C, CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B,
[00333] [00333] In some embodiments, the expression vector codes for a gene selected from the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO, MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, SD2, PROM2, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO and TRABD2AD2A.
[00334] [00334] In some embodiments, the expression vector codes for a gene selected from the group consisting of ACKR2, ALCAM, YEAR10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3, GABRR3, GP5, GPR128, GPR15,
[00335] [00335] In some embodiments, the expression vector encodes a gene selected from the group consisting of ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1, TLR6, TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11E, TMPRSS11F, UPRS11F, UPRSS11E
[00336] [00336] In some embodiments, the expression vector codes for a gene selected from the group consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R, IQGAP2, ITGA1, ITGA2, KCNMB1, LIFR, LNPEP, MEGF10, NIPAL4, OR2, NPR3, OR2, PCDH12, PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8, PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB2, PCDHB3, PCD, 6, PCD, 6, PCD, 6, PCD, 6, PCD, 6 SLC1A3, SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2, SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4 and UGT3A1.
[00337] [00337] In some embodiments, the expression vector codes for a gene selected from the group consisting of BAI3, BTN1A1, BTN2A1,
[00338] [00338] In some embodiments, the expression vector codes for a gene selected from the group consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A2, OR2AE1, OR2F2, OR6V1, PILRA, PILR, PILR, PILR, PILR, PILR, PILR, PILD, PILD, PILD RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213, TTYH3, ZAN and ZP3.
[00339] [00339] In some embodiments, the expression vector encodes a gene selected from the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A and TNFRSF10B.
[00340] [00340] In some embodiments, the expression vector codes for a gene selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144,
[00341] [00341] In some embodiments, the expression vector encodes a gene selected from the group consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR, SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TCTN3, TC5,
[00342] [00342] In some embodiments, the expression vector codes for a gene selected from the group consisting of AMICA1, YEAR1, YEAR3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP, MMP26, MPEG1, MRGP4, MPRX, MPRX4, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1, OR10S1, OR1, OR4, OR2, OR2, OR2, OR2, OR2 OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52N1, OR52N1, OR52N1, OR52N1, OR52 OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5, OR5, OR5, OR5, OR5, OR5, OR5, OR5 OR8A1, OR8B12, OR8B2,
[00343] [00343] In some embodiments, the expression vector codes for a gene selected from the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECN1, CLSTN3, CLSTN3 GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1, MANSC4, OLR1, OR10AD1, OR10P1, OR2AP1, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6, OR6 OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8, SLC6A15, SLC8B1, SLCO1A2, SLCO1B2, SLCO1B2, SLCO1B2, SLCO1B2, SLCO1B2, SLCO1 TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7, TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNFRSF1A, TSPAN8 and VSIG10.
[00344] [00344] In some embodiments, the expression vector codes for a gene selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[00345] [00345] In some embodiments, the expression vector encodes a gene selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1 OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4 and SYNDIG1L.
[00346] [00346] In some embodiments, the expression vector codes for a gene selected from the group consisting of ANPEP, CD276, CHRNA7,
[00347] [00347] In some embodiments, the expression vector encodes a gene selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B, SEZ6L2, SLC22A31, SLC5A11, TM2, TM2, TM2, TM2, TM2, TM2, TM2, TM2, TM2, TM2
[00348] [00348] In some embodiments, the expression vector codes for a gene selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2, OR2, OR2, OR2, OR2, OR2, OR2, OR2 OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E, TNFSF12, TRPV3, TTYH2 and TUSC5.
[00349] [00349] In some embodiments, the expression vector codes for a gene selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15 and TNFRSF11A.
[00350] [00350] In some embodiments, the expression vector codes for a gene selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3,
[00351] [00351] In some embodiments, the expression vector codes for a gene selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4 and THBD.
[00352] [00352] In some embodiments, the expression vector codes for a gene selected from the group consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2 and UMODL1.
[00353] [00353] In some embodiments, the expression vector codes for a gene selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCLF, SSTR3, SUSD2, TMPRSS6 and TNFRSF13C.
[00354] [00354] In some modalities, the expression vector codes for
[00355] [00355] In some embodiments, the safe effector immune cells used to treat cancer, as defined above, express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor associated antigen or a cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of an antigen expressed on at least one tissue of tumor origin, such as any of those listed above, which is an antigen other than that to which which extracellular domain of said aCAR binds. In some embodiments, iCAR is expressed in the same tissue as aCAR is expressed. In some modalities, aCAR and iCAR are different alleles of the same gene. In some modalities, aCAR and iCAR are different proteins and hence are different alleles. A. IN VITRO TESTS
[00356] [00356] In some embodiments, iCAR and / or pCAR will be tested for activity on effects, including effectiveness and ability to inhibit, using a variety of assays. In some embodiments, the inhibitory effect of iCAR and / or pCAR will be tested in vitro and / or in vivo. In some embodiments, the inhibitory effect of iCAR and / or pCAR will be tested in vitro. In some embodiments, the inhibitory effect of iCAR and / or pCAR will be tested in vivo. In some embodiments, in vitro assays measure cytokine secretion and / or cytotoxicity effects. In some embodiments, in vivo assays will assess inhibition and protection of iCAR and / or pCAR for xenografts outside of the target tumor. In some embodiments, in vivo assays will assess the inhibition and protection of iCAR and / or pCAR for tissue outside the tumor in the target and / or viral organs.
[00357] [00357] In some embodiments, iCAR and / or pCAR are evaluated using a luciferase cytotoxicity assay. Generally, for a luciferase cytotoxic assay, recombinant target cells (which can be referred to as "T") are engineered to express firefly luciferase. In some embodiments, commercial Hela-Luc cells can be transfected with DNA encoding the target proteins. The in vitro luciferase assay can be performed according to the Bright-Glo Luciferase assay (commercially available from Promega or BPS Biosciences or other commercial suppliers). Transduced effector T (E) cells (which have been transduced with iCAR or pCAR and aCAR or aCAR or simulated CAR) can be incubated for 24-48 h with recombinant target cells that express HLA-A2, CD19 or both CD19 and HLA-A2 or CD20 or both CD20 and CD19 to be tested at different effector to target ratios. In some embodiments, the iCAR / aCAR or pCAR / aCAR pair comprises any of aCAR, pCAR and / or iCAR with the components as described above. In some modalities, the iCAR / aCAR pair comprises an iCAR directed to HLA-A2 and an aCAR directed to CD19. In some modalities, the iCAR / aCAR pair comprises an iCAR directed to CD20 and an aCAR directed to CD19. Cell killing will be quantified indirectly by estimating the number of live cells with the Bright-Glo Luciferase system.
[00358] [00358] In some embodiments, 'out-of-tumor' cytotoxicity can be optimized by classifying populations of transduced T cells according to the level of iCAR / aCAR expression or by selecting subpopulations of recombinant target cells according to their target expression including example, expression of the gene product encoding at least one extracellular polymorphic epitope. In some embodiments, the target of aCAR, iCAR and / or pCAR is any target with a domain
[00359] [00359] In some embodiments, iCAR and / or pCAR is examined to determine whether iCAR-transduced T cells can discriminate between cells 'in the tumor' (eg, tumor cells) and cells 'out of the tumor' (eg non-tumor cells) in vitro. This is usually tested by examining the killing effect of transduced T cells incubated with a mixture of cells 'in the tumor' and 'out of the tumor' in a 1: 1 ratio. In some embodiments, the ratio is 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7 or 1: 8. Recombinant cells in the tumor can be distinguished from 'out-of-tumor' recombinant cells by luciferase expression in modalities where only one population of cells will be engineered to express the luciferase gene at a time). Killing can be quantified after 24-48 h of incubation using the Bright-Glo Luciferase assay (Promega).
[00360] [00360] In some embodiments, T cells transduced from iCAR / aCAR and / or pCAR / aCAR exhibit about 10%, about 20%, about 30%, about 40%, about 50%, about 60 %, about 70%, about 80%, about 90% and / or about 95% less cell killing outside the tumor compared to T cells transduced with aCAR but not transduced with iCAR and / or pCAR. In some embodiments, T cells transduced from iCAR / aCAR and / or pCAR / aCAR exhibit about 1 time, about 2 times, about 3 times, about 4 times, about 5 times or about 10 times less killing of cells outside the tumor compared to T cells transduced with aCAR, but not transduced with iCAR and / or pCAR. ii. Caspase 3
[00361] [00361] In some embodiments, caspase 3 detection assays are employed to examine iCAR and / or pCAR to determine the level of apoptosis of cells 'in the tumor' (e.g., tumor cells) and cells 'out of the tumor' (e.g., non-tumor cells) in vitro. In some
[00362] [00362] Generally, one of the ways in which CTLs kill target cells is by inducing apoptosis via the Fas ligand. The CASP3 protein is a member of the cysteine aspartic acid protease (caspase) family. Typically, sequential activation of caspases plays a significant role in the cell apoptosis execution phase and, as such, the cleavage of pro-caspase 3 to caspase 3 results in conformational change and expression of catalytic activity. The activated cleaved form of caspase 3 can be recognized specifically by a monoclonal antibody.
[00363] [00363] In some embodiments, transduced T cells can be incubated with any of the cells 'in the tumor' (eg, mimicking tumor) and 'out of the tumor' (eg mimicking non-tumor) recombinant cells. In some embodiments, cells 'in the tumor' (eg, tumor) and 'out of the tumor' (eg, non-tumor) recombinant cells were previously labeled with CFSE ((5 (6) -Carboxyfluorescein N-hydroxysuccinimidyl ester)) or another cell marker dye (for example, CellTrace Violet). In some embodiments, the matching of target cells to effector cells occurs for about 1 hour to about 6 hours, about 2 hours to about 5 hours, or about 2 to about 4 hours. In some embodiments, apoptosis of the target cell is quantified by flow cytometry. The cells can be permeabilized and fixed by an internal staining kit (Miltenyi or BD bioscience) and stained with an antibody to activated caspase 3 (BD bioscience).
[00364] [00364] In some embodiments, T cells transduced from iCAR / aCAR and / or pCAR / aCAR induce about 10%, about 20%, about 30%, about 40%, about 50%, about 60 %, about 70%, about 80%, about 90% and / or about 95% less apoptosis of cells outside the
[00365] [00365] Time-passing microscopy of T cells transduced from iCAR and / or pCAR can be employed in order to discern target binding. In some embodiments, the target cells will be labeled with a reporter gene (for example, but not limited to a fluorescent protein, such as mCherry). In some embodiments, the transduced T cells are incubated with either 'on tumor' or 'off tumor' cells for up to 5 days. In some embodiments, time-lapse microscopy can be used to visualize the killing. In some modalities, flow cytometry analysis will be performed using viable cell number staining and CountBright (Invitrogen) beads to determine the number of target cells at the end point time.
[00366] [00366] In some embodiments, in order to determine whether T cells transduced from aCAR / iCAR or aCAR / pCAR can discern targets in vitro, each recombinant target cell ('in the tumor' or 'out of the tumor') is marked with a different reporter protein (for example, GFP and mCherry). In some embodiments, any reporter protein pair would work, as long as the reporter pair contains two reporters that are easily distinguishable. In some embodiments, the transduced T cells (effector cells) will be matched with the recombinant cells (target cells) in a 1: 1 E / T ratio. In some embodiments, the ratio of effector to target (E / T) includes, but is not limited to 16: 1, 12: 1, 10: 1, 8: 1, 6: 1, 4: 1, 2: 1 or 1: 1. In
[00367] [00367] Cytokine release can be examined to determine T cell activation. In some embodiments, T cells transduced from iCAR / aCAR and / or pCAR / aCAR are incubated with recombinant target cells and cytokine production for one or more cytokines are quantified, for example, by measuring cytokine secretion in cell culture supernatant according to the BioLegend MAXTM Deluxe Set kit or by flow cytometric analysis of the percentage of T cells producing cytokines. For flow cytometric analysis, a Golgi arrest is usually employed to prevent cytokine secretion. In some embodiments, after an incubation of 6 hours and 18 hours to 24 hours of the T cells transduced with target cells, the T cells will be permeabilized and fixed by an internal staining kit (Miltenyi) and stained with antibodies to the T cell markers. (CD3 and CD8) and for one or more cytokines. In some embodiments, cytokines include, but are not limited to, IL-2, INFγ and / or TNFα. v. CD107a staining
[00368] [00368] Staining for CD107a can also be examined to determine the cytolytic activity of the transduced T cells. Generally, degranulation of T cells can be identified by the surface expression of CD107a, a lysosome-associated membrane protein (LAMP-1) and surface expression of LAMP-1 that has been shown to correlate with CD8 T cell cytotoxicity. In addition, this molecule is located on the luminal side of the lysosomes. Typically, upon activation, CD107a is transferred to the cell membrane surface of activated lymphocytes. Furthermore, CD107a is expressed on the cell surface transiently and is rapidly re-internalized via the endocytic pathway. Therefore, although
[00369] [00369] In some embodiments, the transduced T cells transduced from aCAR / iCAR and / or aCAR / pCAR are incubated with the target cells for about 6 hours at about 24 h and the expression of CD107a in the CD8 T cells is examined. In some embodiments, the target cells express only a target protein recognized by aCAR (as in tumor cells) or target cells expressing both target proteins recognized by aCAR and iCAR (as in normal cells). In some embodiments, the transduced T cells transduced from iCAR and / or pCAR are incubated with the target cells for about 6 minutes in about 24 hours in the presence of monensin and the expression of CD107a in CD8 T cells is followed by flow cytometry. using conjugated antibodies against T cell surface markers (for example, CD3 and CD8) and a conjugated antibody to CD107a. saw. Quantification of Secreted Cytokines by ELISA
[00370] [00370] In some embodiments, after the co-cultivation of transduced T cells (Jurkat, or primary T cells) that express iCAR or aCAR or both aCAR and iCAR with modified target cells expressing iCAR or aCAR antigens or both aCAR and iCAR on their surface cell, the conditioned medium will be collected and the cytokine concentration will be measured by cytokine ELISA. In some modalities, the cytokine is selected from the group consisting of IL-2, INFγ and / or TNFα. In some embodiments, the cytokine is selected from the group consisting of IL-2. In some modalities, the cytokine is selected from the group consisting of INFγ. In some embodiments, the cytokine is selected from the group consisting of TNFα. In some modalities, a decrease of about 20%, about
[00371] [00371] The Cytometric Account Matrix (CBA) is used to measure a variety of soluble and intracellular proteins, including cytokines, chemokines and growth factors. In some embodiments, T cells (primary T cells or Jurkat cells) transduced with aCAR or both aCAR and iCAR constructs (effector cells) are stimulated with modified target cells expressing both iCAR and aCAR or aCAR or iCAR target antigens in your cell surface. In some modalities, the effective to target ratio ranges from 20: 1 to 1: 1. In some embodiments, the effective to target ratio ranges from 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11: 1 , 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1,2: 1 or 1: 1. In some modalities, after several hours of coincubation, the effector cells produce and secrete cytokines that indicate their effector status. In some embodiments, the reaction supernatant is collected and the secreted IL-2 was measured and quantified by a multiplex CBA assay.
[00372] [00372] In some modalities, a decrease of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% is demonstrated with cells transduced from Double CAR (aCAR / iCAR) that were matched with target cells expressing both high antigens compared to IL-2 secretion resulted from the matching of the same effector cells with target cells expressing only one target. In some modalities, a decrease of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
[00373] [00373] In some embodiments, degranulation of T cells can be identified by the surface expression of CD107a, a membrane protein associated with lysosomes (LAMP-1). In some embodiments, the surface expression of LAMP-1 correlates with the cytotoxicity of CD8 T cells. In some modalities, granulation (CD107a) is a marker for killing potential. B. IN VIVO TESTS
[00374] [00374] In some modalities, the iCAR / aCAR and / or iCAR / pCAR pairs are tested for effectiveness in vivo. In some embodiments, NOD / SCID / γc- or similar mice are inoculated intravenously with tumor cells. In some embodiments, the tumor cells are CD19 positive NALM 6 cells (ATCC, human B-ALL cell line) that are engineered to express firefly luciferase. In some modalities, for establishment and / or differentiation
[00375] [00375] For the trial, mice will be divided into study groups; one group will be injected with NALM 6 cells, while the other will be injected with NALM-6 which expresses the iCAR epitope. Several days later, the mice will be infused intravenously with T cells transduced with aCAR, aCAR / iCAR and a control group of untranslated T cells or no T cells. Mice will be sacrificed and the tumor burden will be quantified according to the total flow. .
[00376] [00376] According to an assay modality, in order to test whether T cells expressing the iCAR and / or pCAR construct could discriminate between target cells and off-target cells in vivo within the same organism, mice are injected with a 1: 1 mixture of NALM-6 cells' in the tumor '/' outside the tumor, followed by injection of transduced T cells that express either aCAR alone or both aCAR and iCAR. With this modality, by sacrificing the mice, the presence of cells' in the tumor 'and' outside the tumor in the spleen and bone marrow will be analyzed by flow cytometry for the two markers, CD19 and the iCAR epitope. i. CTL In Vivo Assay in Human Xenograft Mouse Models
[00377] [00377] In some modalities, to test whether the T cells that express both the aCAR and iCAR constructs discriminate between the
[00378] [00378] In some embodiments, T cells transduced with iCAR or aCAR or both iCAR and aCAR will be injected iv into NOD / SCID / γc-naïve or similar mice and until several hours later, the target cells that express iCAR, aCAR or both will be injected. In some modalities, these targets will be marked with any CFSE / CPDE or similar cell trace dye in different concentrations (high, medium and low), which will allow for additional discrimination between them. In some embodiments, the specific kill percentage will be calculated, as described in Example 5. ii. Kinetics of tumor growth in human xenograft mouse models
[00379] [00379] In some embodiments, tumor cells express either an iCAR target, aCAR target, or both. In some embodiments, an aCAR tumor cell line could be NALM 6 CD19 positive (ATCC, human BALL cell line). In some embodiments, tumor cells that express both aCAR and iCAR (i.e., 'non-tumor' cells) are NALM 6 engineered to express the iCAR epitope (e.g., HLA-A2), thereby representing healthy cells. In some embodiments, NALM 6 and NALM 6-HLA-A2 can also be engineered to express a reporter gene (eg, firefly luciferase), for easy detection.
[00380] [00380] In some modalities, monitoring will be conducted by measuring the tumor volume by mechanical means (caliper) and also using in vivo imaging systems (IVIS). In some embodiments, the tumor burden can be quantified and infiltrating T cell populations can be analyzed by FACS.
[00381] [00381] In some embodiments, transgenic mice that express the targets of human aCAR and iCAR will also be used to determine the effectiveness of the transduced T cells. In some modalities, the system will allow us to monitor problems of effectiveness and toxicity. C. IN VIVO USES: TREATMENT, BIOMARKERS
[00382] [00382] In yet another aspect, the present invention provides a method for selecting a personalized biomarker for a subject having a tumor characterized by LOH, the method comprising (i) obtaining a tumor biopsy of the subject; (ii) obtaining a sample of normal tissue from the subject, for example, PBMCs; and (iii) identifying a simple allelic variant of a polymorphic cell surface epitope that is not expressed by the tumor cells due to LOH, but that is expressed by the cells of normal tissue, thereby identifying a personalized biomarker for the subject.
[00383] [00383] In some modalities, the biomarker is used to personalize a subject's treatment, so that the method further comprises the steps of treating cancer in a patient having a tumor characterized by LOH, comprising administering to the patient an effector immune cell, as defined above, in which the iCAR is directed to the simple allelic variant identified in (iii). In some embodiments, the present invention provides a method for selecting a personalized biomarker for a subject having a tumor characterized by LOH, the method comprising (i) obtaining a tumor biopsy from the subject; (ii) obtaining a sample of normal tissue from the subject, for example, PBMCs; (iii) identify a simple allelic variant of a polymorphic cell surface epitope that is not expressed by tumor cells due to LOH, but is expressed by cells in normal tissue, based on the LOH candidate score, in
[00384] [00384] In a further aspect, the present invention provides a method for treating cancer in a patient having a tumor characterized by LOH, comprising administering to the patient an effector immune cell as defined above, wherein the iCAR is directed to an allelic variant simple encoding a polymorphic cell surface epitope absent from tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of the patient's normal related mammalian tissue.
[00385] [00385] In a similar aspect, the present invention provides a method for reducing the tumor burden in a subject having a tumor characterized by LOH, comprising administering to the patient an effector immune cell as defined above, wherein the iCAR is directed to a simple allelic variant encoding a polymorphic cell surface epitope absent from tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of the patient's normal related mammalian tissue, or at least in vital tissues in that aCAR is expressed.
[00386] [00386] In another similar aspect, the present invention provides a method for increasing survival of a subject having a tumor characterized by LOH, comprising administering to the patient an effector immune cell as defined above, wherein the iCAR is directed to an allelic variant simple encoding a polymorphic cell surface epitope absent from tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of the patient's normal related mammalian tissue.
[00387] [00387] In yet an additional aspect, the present invention is directed to a safe effector immune cell, as defined above, for use in
[00388] [00388] In still a further aspect, the present invention is directed to a method for treating cancer in a patient having a tumor characterized by LOH comprising: (i) identifying or receiving information identifying a simple allelic variant of a polymorphic cell surface epitope which is not expressed by tumor cells due to LOH, but which is expressed by cells of normal tissue, (ii) identifying or receiving information identifying a non-polymorphic cell surface epitope of an antigen or a simple allelic variant of an epitope of polymorphic cell surface, wherein said epitope is a tumor-associated antigen or is shared by cells of at least relative tumor and normal tissue in said cancer patient; (iii) selecting or receiving at least one nucleic acid molecule defining an iCAR as defined above and at least one nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined above, or at least one vector as defined above, where iCAR comprises an extracellular domain that specifically binds to a (i) cell surface epitope and aCAR comprises an extracellular domain that specifically binds to a (ii) cell surface epitope; (iv) prepare or receive at least one population of safe redirected effector immune cells by transfecting immune effector cells with the nucleic acid molecules of (iii) or transducing effector immune cells with the vectors of (iii); and (v) administering to said cancer patient at least one population of immune redirected immune effector cells safe from (iv).
[00389] [00389] In a similar aspect, the present invention provides at least one population of safe redirected immune effector cells to treat cancer in a patient having a tumor characterized by LOH, in which the safe redirected immune cells are obtained by (i) identifying or receiving information identifying a simple allelic variant of a polymorphic cell surface epitope that is not expressed by tumor cells due to LOH, but is expressed by cells in normal tissue, (ii) identifying or receiving information identifying a cell surface epitope non-polymorphic antigen or a simple allelic variant of a polymorphic cell surface epitope, wherein said epitope is a tumor-associated antigen or is shared by cells of at least a relative tumor and normal tissue in said cancer patient; (iii) selecting or receiving at least one nucleic acid molecule defining an iCAR as defined above and at least one nucleic acid molecule comprising a nucleotide sequence encoding an aCAR as defined above, or at least one vector as defined above, where iCAR comprises an extracellular domain that specifically binds to a (i) cell surface epitope and aCAR comprises an extracellular domain that specifically binds to a (ii) cell surface epitope; (iv) prepare or receive at least one population of safe redirected effector immune cells by transfecting immune effector cells with the nucleic acid molecules of (iii) or transducing effector immune cells with the vectors of (iii).
[00390] [00390] In some modalities referring to any of the above modalities aimed at treating cancer or safe immune effector cells for use in cancer treatment (i) the extracellular domain of iCAR specifically binds to a simple allelic variant of a surface epitope polymorphic cell of an antigen, which is an antigen different from that to which the extracellular domain of aCAR binds; (ii) the domain
[00391] [00391] In some embodiments, the treatment results in reduced reactivity on the target outside the tumor, compared to a treatment that comprises administering to the cancer patient at least one population of immune effector cells that express an (iii) aCAR, but lacking of iCAR from (iii).
[00392] [00392] In some embodiments, the safe effector immune cells used to treat cancer, as defined above, express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-pylimorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of tumor origin or a housekeeping protein, which is an antigen other than the one to which the extracellular domain of said aCAR alloys.
[00393] [00393] In some embodiments, the safe effector immune cells used to treat cancer, as defined above, express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-pylimorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of tumor origin or a housekeeping protein, such
[00394] [00394] In some embodiments, the safe effector immune cells used to treat cancer, as defined above, express on their surface an aCAR comprising an extracellular domain that specifically binds to a tumor-associated antigen or a non-pylimorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of tumor origin, such as an HLA-A, which is a different antigen the one to which the extracellular domain of said aCAR binds.
[00395] [00395] In some embodiments, more than one population of immune effector cells are administered and the different populations express different pairs of aCARs and iCARs having specific binding to cell surface epitopes of different gene products.
[00396] [00396] In some embodiments, the safe effector immune cells used in the method to treat cancer are selected from T cells, natural killer cells or killer cells induced by cytokines. In some embodiments, the safe effector immune cell is an autologous or universal (allogeneic) effector cell. In some embodiments, the iCAR used in any of the cancer treatment methods defined above is targeted at all patient tissues in which the aCAR target antigen is present, in which the aCAR target antigen is a surface epitope of non-polymorphic cell of an antigen or a simple allelic variant of a polymorphic cell surface epitope is present and said epitope is a tumor-associated antigen or is shared at least by cells of
[00397] [00397] In some modalities, the cancer is selected from Acute Myeloid Leukemia [LAML], Adrenocortical Carcinoma [ACC], Urothelial Carcinoma of the Bladder [BLCA], Lower Grade Brain Glioma [LGG], Invasive Breast Carcinoma [BRCA] , Cervical squamous cell carcinoma and endocervical adenocarcinoma [CESC], Cholangiocarcinoma [CHOL], Colon adenocarcinoma [COAD], Esophageal carcinoma [ESCA], Glioblastoma multiforme [GBM], Head and neck squamous cell carcinoma [HNSC], Chromophobia Kidney [KICH], Renal clear cell carcinoma of the kidneys [KIRC], Renal papillary cell carcinoma of the kidneys [KIRP], Hepatocellular carcinoma of the liver [LIHC], Lung adenocarcinoma [LUAD], Squamous cell carcinoma of the lung [ LUSC], B-Cell Lymphoma, Large Diffuse Neoplastic Lymphoid [DLBC], Mesothelioma [MESO], Serous Ovarian Cystadenocarcinoma [OV], Pancreatic Adenocarcinoma [PAAD], Pheochromocytoma and Paraganglioma [PCPG], Adenocarcinoma of the prostate, PRAD] [READ], S arcoma [SARC], Cutaneous Skin Melanoma [SKCM], Stomach adenocarcinoma [STAD], Testicular Germ Cell Tumors [TGCT], Thymoma [THYM], Thyroid Carcinoma [THCA], Uterine Carcinoma [UCS], Endometrial Carcinoma Uterine Body [UCEC], Uveal Melanoma [UVM].
[00398] [00398] In some embodiments, the iCAR and / or pCAR for use in the treatment of cancer is any iCAR and / or pCAR described here. In some embodiments, the iCAR and / or pCAR used to treat cancer, like any of the types of cancer mentioned above, is directed against or specifically binds to a simple allele variant of the HLA genes (including, for example, the HLA- A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR , HLA-B gene or HLA-C gene or against a simple allelic variant of a listed gene Table 8 In some embodiments, the iCAR used to treat the
[00399] [00399] For oral administration, the pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions, or it can be presented as a drug product for reconstitution with water or another suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binding agents (for example, pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets can be coated by methods well known in the art.
[00400] [00400] Preparations for oral administration can be
[00401] [00401] For oral administration, the compositions can take the form of tablets or lozenges formulated in a conventional manner.
[00402] [00402] The compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injection formulations can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions can take forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulation agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
[00403] [00403] The compositions can also be formulated in rectal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases, such as cocoa butter or other glycerides.
[00404] [00404] For administration by inhalation, the compositions for use according to the present invention are conveniently distributed in the form of an aerosol spray presentation from pressurized packages or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator can be formulated containing a mixture of powder of the compound and a suitable powder base, such as lactose or starch.
[00405] [00405] For the sake of clarity, and in no way limiting the scope
[00406] [00406] The term “about”, as used here, means that values of 10% or less above or below the indicated values are also included. EXEMPLARY MODALITIES
[00407] [00407] In some embodiments, the methods of the present invention provide the following exemplary embodiments.
[00408] [00408] 1. A nucleic acid molecule comprising a nucleotide sequence encoding an inhibitory chimeric antigen receptor (iCAR) or protective chimeric antigen receptor (pCAR) capable of preventing or attenuating unwanted activation of an effector immune cell, wherein iCAR or pCAR comprises an extracellular domain that specifically binds to a simple allelic variant of a polymorphic cell surface epitope absent in mammalian tumor cells due to loss of heterozygosity (LOH), but present at least in all cells of normal tissue related mammal; and an intracellular domain comprising at least one signal transducing element that inhibits an effector immune cell.
[00409] [00409] 2. The nucleic acid molecule of claim 1, wherein the polymorphic epitope of the cell surface is a genetic cleaning product, such as an HLA gene, a G protein-coupled receptor (GPCR), an ion channel or a tyrosine kinase receptor, preferably an HLA-A,
[00410] [00410] 3. The nucleic acid molecule of claim 1, wherein said extracellular domain comprises (i) an antibody, derivative or fragment thereof, such as a humanized antibody; a human antibody; a functional fragment of an antibody; a single domain antibody, such as a Nanobody; a recombinant antibody; and a single chain variable fragment (ScFv); (ii) an antibody mimetic, such as an affibody molecule; an affilin; an affimer; an affitin; an alphabody; an anticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide; and a monobody; or (iii) an aptamer.
[00411] [00411] 4. The nucleic acid molecule of claim 1, wherein the mammalian tissue is human tissue and said related normal mammalian tissue is the normal tissue from which the tumor has developed.
[00412] [00412] 5. The nucleic acid molecule of claim 1, wherein said effector immune cell is a T cell, a natural killer cell or a cytokine-induced killer cell.
[00413] [00413] 6. The nucleic acid molecule of claim 1, wherein said at least one signal transducing element capable of inhibiting an effector immune cell is homologous to a signal transducing element of an immune checkpoint protein .
[00414] [00414] 7. The nucleic acid molecule of claim 6, wherein the checkpoint protein is selected from the group consisting of PD1; CTLA4; BTLA; 2B4; CD160; CEACAM, such as CEACAM1; KIRs, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LIR1, LIR2, LIR3, LIR5, LIR8 and CD94– NK2; LAG3; TIM3; T-cell activation domain V suppressor (VISTA); STimulator of INterferon Genes (STING); immunoreceptor tyrosine-based inhibitory motif proteins (ITIM),
[00415] [00415] 8. The nucleic acid molecule of claim 1, wherein said extracellular domain is fused through a transmembrane motif and flexible hinge to said intracellular domain.
[00416] [00416] 9. A vector comprising a nucleic acid molecule of any one of claims 1 to 8 and at least one control element, such as a promoter, operably linked to the nucleic acid molecule.
[00417] [00417] 10. The vector of claim 9, further comprising a nucleic acid molecule comprising a nucleotide sequence encoding an aCAR comprising an extracellular domain that specifically binds a non-polymorphic cell surface epitope to an antigen or a simple allelic variant of a polymorphic cell surface epitope, wherein said epitope is a tumor-associated antigen or is shared by at least related tumor cells and normal tissue and an intracellular domain comprising at least one signal transducing element that activates and / or co-stimulates an effector immune cell.
[00418] [00418] 11. The vector of claim 10, wherein the extracellular domain of aCAR specifically binds to a non-polymorphic cell surface epitope of an antigen and the extracellular domain of iCAR specifically binds to a simple allelic variant of a surface epitope of a polymorphic cell of an antigen other than that to which the extracellular domain of said aCAR binds.
[00419] [00419] 12. The vector of claim 10 or 11, wherein the extracellular domain of aCAR specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in Table 1, such as CD19.
[00420] [00420] 13. The vector of claim 10, wherein said at least one signal transducing element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM), for example, CD3ζ or FcRγ chains; a killer cell immunoglobulin-like receptor (KIR), such as KIR2DS and KIR3DS, or an adapter molecule, such as DAP12; or a co-stimulating signal transducing element of, for example, CD27, CD28, ICOS, CD137 (4-1BB) or CD134 (OX40).
[00421] [00421] 14. The vector of claim 10, wherein the nucleotide sequence comprises an internal ribosome entry site (IRES) between the nucleotide sequence encoding aCAR and the nucleotide sequence encoding iCAR.
[00422] [00422] 15. The vector of claim 14, wherein the nucleotide sequence encoding aCAR is downstream of the nucleotide sequence encoding iCAR.
[00423] [00423] 16. The vector of claim 10, wherein the nucleotide sequence comprises a viral autoclivable 2A peptide between the nucleotide sequence encoding the aCAR and the nucleotide sequence encoding the iCAR.
[00424] [00424] 17. The vector of claim 16, wherein the viral autoclivable peptide 2A is selected from the group consisting of Thosea asigna virus (TaV) T2A, Foot and mouth disease virus (FMDV) F2A, Equine virus E2A rhinitis A (ERAV) and Porcine teschovirus-1 (PTV1) P2A.
[00425] [00425] 18. The vector of claim 10, comprising a nucleotide sequence encoding said constitutive aCAR linked via a flexible linker to said iCAR.
[00426] [00426] 19. A method for preparing an inhibitory chimeric antigen (iCAR) receptor capable of preventing or attenuating unwanted activation of an effective immune cell, as defined in claims 1 to 8, the
[00427] [00427] 20. The method of claim 19, wherein the minor allele frequency for each variant is equal to or exceeds 1, 2, 3, 4 or 5%.
[00428] [00428] 21. A method for preparing a safe effector immune cell comprising: (i) transfecting a TCR engineered effector immune cell targeting a tumor associated antigen with a nucleic acid molecule comprising a nucleotide sequence encoding an iCAR of any one of claims 1 to 8 or transducing the cells with a vector of claim 9; or (ii) transfecting a naïve effector immune cell with a nucleic acid molecule comprising a nucleotide sequence encoding an iCAR of any one of claims 1 to 8 and a nucleic acid molecule comprising a nucleotide sequence encoding a defined aCAR in any one claims 10 to 13; or transducing an effector immune cell with a vector of any one of claims 10 to 18.
[00429] [00429] 22. A safe effector immune cell obtained by the method of claim 21.
[00430] [00430] 23. The safe effector immune cell of claim 22, expressing on its surface an aCAR comprising an extracellular domain that specifically binds to a non-polymorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds an simple allelic variant of a polymorphic cell surface epitope of a different antigen to which the extracellular domain of the aCAR binds.
[00431] [00431] 24. The safe effector immune cell of claim 22 or 23, wherein the extracellular domain of aCAR specifically binds to a non-polymorphic cell surface epitope selected from the antigens listed in
[00432] [00432] 25. The safe effector immune cell of claim 22, wherein aCAR and iCAR are present on the cell surface as separate proteins.
[00433] [00433] 26. The safe effector immune cell of claim 22, wherein the level of expression of said nucleotide sequence encoding the iCAR is greater than or equal to the level of expression of the nucleotide sequence encoding the aCAR.
[00434] [00434] 27. Method for selecting a personalized biomarker for a subject having a tumor characterized by LOH, the method comprising (i) obtaining a tumor biopsy of the subject; (ii) obtaining a sample of normal tissue from the subject, for example, PBMCs; (iii) identifying a simple allelic variant of a polymorphic cell surface epitope that is not expressed by the tumor cells due to LOH, but which is expressed by the cells of normal tissue, thereby identifying a personalized biomarker for the subject.
[00435] [00435] 28. A method of treating cancer in a patient having a tumor characterized by LOH, comprising administering to the patient an effector immune cell of claim 22, wherein the iCAR is directed to a simple allelic variant encoding a surface epitope of polymorphic cell absent from tumor cells due to loss of heterozygosity (LOH), but present in at least all cells of the patient's normal related mammalian tissue.
[00436] [00436] 29. A safe effector immune cell of claim 22 for use in treating a patient having a tumor characterized by LOH, wherein the iCAR is directed to a simple allelic variant encoding a polymorphic cell surface epitope absent from the tumor cells due to loss
[00437] [00437] 30. The safe effector immune cell for use in claim 29, wherein the treatment results in reduced reactivity in the target outside the tumor, compared to a treatment comprising administering to the cancer patient at least one population of immune effector cells that express an aCAR of (iii), but lacking an iCAR of (iii).
[00438] [00438] 31. The safe effector immune cell for use in claim 29, expressing on its surface an aCAR comprising an extracellular domain that specifically binds to a tumor associated antigen or a non-pylimorphic cell surface epitope of an antigen and an iCAR comprising an extracellular domain that specifically binds a simple allelic variant of a polymorphic cell surface epitope of an antigen expressed at least in a tissue of tumor origin or a hosekeeping protein, such as an HLA-A, which is a different antigen the one to which the extracellular domain of said aCAR binds.
[00439] [00439] 32. The safe effector immune cell for use in claim 28, which is an autologous effector cell or a universal (allogeneic) cell.
[00440] [00440] 33. The safe effector immune cell for the use of any of claims 28 to 32, selected from a T cell, natural killer cell or cytokine-induced killer cell.
[00441] [00441] 34. A combination of two or more nucleic acid molecules, each comprising a nucleotide sequence encoding a different member of a controlled effector immune cell activation system, said nucleic acid molecules forming a single acid molecule continuous nucleic, or comprising two or more separate nucleic acid molecules, in which the controlled effector immune activation system directs effector immune cells to kill tumor cells that have lost one or more chromosomes or fractions thereof due to
[00442] [00442] 35. The combination of claim 34, wherein the first member is selected from: (a) a constitutive aCAR further comprising an intracellular domain comprising at least one signal transducing element that activates and / or co-stimulates an effector immune cell ; and (b) a conditional aCAR further comprising an intracellular domain comprising a first member of a binding site for a small heterodimerizing molecule and optionally at least one co-stimulatory signal transduction element, but lacking an activation signal transduction element; and the second member is: (c) an inhibitory chimeric antigen receptor (iCAR) further comprising an intracellular domain comprising at least one signal transducing element that inhibits an effector immune cell; or
[00443] [00443] 36. The combination of claim 34 or 35, wherein: (i) the extracellular domain of iCAR or pCAR specifically binds to a simple allelic variant of a polymorphic cell surface epitope of an antigen that is an antigen other than that to which the extracellular domain of aCAR binds (ii) the extracellular domain of said pCAR or iCAR specifically binds to a simple allelic variant of a polymorphic cell surface epitope other than the same antigen to which the extracellular domain of said aCAR binds; or (iii) the extracellular domain of said pCAR or iCAR specifically binds to a simple allelic variant other than the same polymorphic cell surface epitope to which the extracellular domain of said aCAR binds.
[00444] [00444] 37. The combination of claim 34, wherein said substrate for a sheddase is a substrate for a disintegrin and metalloproteinase (ADAM) or a beta-secretase 1 (BACE1).
[00445] [00445] 38. The combination of claim 37, wherein said substrate forms part of the extracellular domain and comprises Lin 12 / Notch repeats and an ADAM protease cleavage site.
[00446] [00446] 39. The combination of claim 34, wherein said substrate for an intramembrane cleavage protease is a substrate for
[00447] [00447] 40. The combination of claim 39, wherein said substrate forms part of the transmembrane motif canon and is homologous / derived from a Notch transmembrane domain, ErbB4, E-cadherin, N-cadherin, ephrin-B2, precursor protein amyloid or CD44.
[00448] [00448] 41. The combination of claim 34, comprising a nucleotide sequence encoding an extracellular domain and an intracellular domain of said conditional aCAR as separate proteins, wherein each domain is independently fused to a transmembrane motif and comprises a different member of a binding site for a small heterodimerizing molecule.
[00449] [00449] 42. The combination of claim 34, wherein each of said first and second members of said binding site for a small heterodimerizing molecule is derived from a protein selected from: (i) tacrolimus binding protein (FK506) and FKBP; (ii) catalytic A subunit of FKBP and calcineurin (CnA); (iii) FKBP and cyclophilin; (iv) protein associated with FKBP and FKBP-rapamycin (FRB); (v) gyrase B (GyrB) and GyrB; (vi) dihydrofolate reductase (DHFR) and DHFR; (vii) DmrB (DmrB) and DmrB homodimerization domain; (viii) a PYL protein (also known as an abscisic acid receptor and as RCAR) and ABI; (ix) GAI Arabidopsis thaliana protein (also known as Gibberellic Acid Insensitive protein and DELLA GAI; GAI) and GID1 protein Arabidopsis thaliana (also known as Giberelin GID1 receptor; GID1).
[00450] [00450] The patent application contains a section of long tables. Copies of the tables are presented simultaneously with this one on CD-ROM. EXAMPLES
[00451] [00451] With respect to the examples, the following terminology is used.
[00452] [00452] When the term chromosome is used, it usually refers to the chromosome on which the SNP is found. For SNP analysis, position refers to the SNP genomic position (set GRCh37.p13). Snp_id when used refers to the dbSNP rs ID, where one exists.
[00453] [00453] The term "ref" refers to the reference nucleotide allele. The term "alt" refers to the alternative nucleotide allele.
[00454] [00454] The term “quality” refers to the quality score of the Exome Aggregation Consortium (ExAC). The term “filter_status” refers to ExAC filter information.
[00455] [00455] The term “allele_frequency” refers to the global allele frequency of ExAC. The term "max_allele_frequency" refers to the overall allele frequency of the most common alternative allele (generally, this is only relevant when the SNP has more than two alternative alleles in the same site and this can often mean sequencing errors anyway).
[00456] [00456] The term "het_allele_count" refers to the number of participants in ExAC who were heterozygous. The term "AFR_AF" refers to the lowest allele frequency of African genomes. The term “AMR_AF” refers to the lowest frequency of alleles in Latin genomes. The term "EAS_AF" refers to the lowest frequency of alleles in East Asian genomes. The term “FIN_AF” refers to the lowest frequency of alleles in Finnish genomes. The term "NFE_AF" refers to the lowest frequency of alleles in non-Finnish European genomes. The term “OTH_AF” refers to the lowest frequency of allele
[00457] [00457] The term "max_AF" refers to the maximum minor allele frequency among populations categorized in ExAC (0.5 is the maximum permissible allele frequency).
[00458] [00458] The term "gene" refers to the HUGO symbol of the gene on which the SNP falls.
[00459] [00459] The term “hgnc_ID” refers to the numerical ID of the HUGO Genetic Nomenclature Committee of the gene on which the SNP falls.
[00460] [00460] The term "consequence" refers to the impact of the SNP on the translated protein product. It can be one of several, including: missense_variant, frameshift_variant, inframe_deletion, stop_gained.
[00461] [00461] The term "protein_consequence" reports the amino acid substitution and its location in the reference protein transcript (for example, pArg482Gln).
[00462] [00462] The term "aa_affected" refers to the numerical location of the affected amino acid in the consensus protein transcript.
[00463] [00463] The term "allele_1" refers to the amino acid encoded by the reference allele.
[00464] [00464] The term "allele_2" refers to the amino acid encoded by the alternative allele.
[00465] [00465] The term “sift_score” refers to the score and interpretation of the predicted functional effect of amino acid substitution by the SIFT algorithm. Uses sift5.2.2 version. Scores range from 0-1. A low score means that an amino acid substitution is more likely to be tolerated.
[00466] [00466] The term “polyphen_score” refers to the score and interpretation of the predicted functional effect of amino acid substitution by the polyphen algorithm. Uses PolyPhen (v2.2.2). Scores range from 0-1. A low score means that an amino acid substitution is more likely to be
[00467] [00467] The term “polyphen_numeric” refers to the numerical score extracted from the polyphen algorithm.
[00468] [00468] The term "protein_domains_affected" refers to the protein domains predicted based on the following algorithms: Gene3D, hmmpanther, Prosite.
[00469] [00469] The term “BLOSUM_score” refers to the score for amino acid substitution based on the BLOSUM62 matrix from https://www.ncbi.nlm.nih.gov/IEB/ToolBox/C_DOC/lxr/source/data/BLOSU M62. A negative score indicates an amino acid substitution that occurred less frequently over time in evolution (more likely to affect protein function).
[00470] [00470] The term "allele_1_one_letter" refers to the amino acid code of a letter of the reference amino acid allele.
[00471] [00471] The term "allele_2_one_letter" refers to the amino acid code of a letter of the alternative amino acid allele.
[00472] [00472] The term "mono_allelic_expression" refers to whether or not the gene on which the SNP falls suffers monoallelic expression in humans. The database established by Savova et al. was used for this annotation7. A 1 in this column indicates that the gene exhibits monoallelic expression. A 0 in this column indicates that the gene did not exhibit monoallelic expression in the database of Savova et al. An NA in this column means that the gene was not noted in the article by Savova et al.
[00473] [00473] The term "extracellular" refers to whether or not the SNP falls into an extracellular domain of the affected protein. A 1 in this column indicates that the SNP is in an extracellular domain and a 0 indicates that it is not. Uniprot was used to annotate protein domains.
[00474] [00474] The term "Pdb_id" refers to the protein database ID of the affected protein, if it exists. In the event that there are many
[00475] [00475] The term "aa_context_21aa_allele_1" refers to window A 21 of the amino acid surrounding the amino acid SNP in the sequence of the consensus protein. The sequence consists of the 10 amino acids from the preceding part of the consensus protein sequence. A check was made to ensure that the reference amino acid corresponded to the consensus protein sequence at the affected position. If these two amino acids were not the same, then the entry reads "discrepancy with uniprot fasta based on consensus isoform".
[00476] [00476] The term “aa_context_21aa_allele_2: the same amino acid window above, but inserting amino acid 2 allele in the middle.
[00477] [00477] The term “gtex_mean: expression of average gene through tissues (in RPKM). This consists of the average value of the median RPKM values across the GTEX tissues. For example, if the values for a given gene were Lung (median) = 3, Breast (median) = 2, Pancreas (median) = 5, then the value reported in this entry would be 3.33.
[00478] [00478] The term “gtex_min: the lowest gene expression for a tissue across all tissues. This value is derived from the list of median values of gene expression across all tissues. For example, if the values for a given gene were Lung (median) = 3, Breast (median) = 2, Pancreas (median) = 5, then the value reported in this entry would be 2.
[00479] [00479] The term “gtex_max: the highest gene expression for a tissue across all tissues. This value is derived from the list of median values of gene expression across all tissues. For example, if the values for a given gene were Lung (median) = 3, Breast (median) = 2, Pancreas (median) = 5, then the value reported in this entry would be 5.
[00480] [00480] The term “gtex_std_dev: the standard deviation of gene expression values across tissues for a given gene. For example, if the values for a given gene were Lung (median) = 3, Breast (median) = 2, Pancreas (median) = 5, then the value reported in this entry would be 1.5.
[00481] [00481] The term “cell_surface_protein_atlas: a binary marker for whether or not the protein was noted as a membrane protein in the cell surface protein atlas (wlab.ethz.ch/cspa/). A1 indicates that the gene was noted as a membrane protein in this database.
[00482] [00482] The term “human_protein_atlas_membrane_proteins: a binary marker for whether or not the protein was noted as a membrane protein in the human protein atlas (https://www.proteinatlas.org/). A1 indicates that the gene was noted as a membrane protein in this database.
[00483] [00483] The term “subcellular_map_proteome_membrane_proteins: a binary marker for whether or not the protein was noted as a membrane protein in the subcellular map of the proteome (http://science.sciencemag.org/ content / early / 2017/05/05 / 10 / science) .aal3321 /). A1 indicates that the gene was noted as a membrane protein in this database.
[00484] [00484] The term “n_membrane_databases_w_gene: The total number of databases with the gene noted as a gene that is expressed on the cell membrane. Maximum = 3, minimum = 0.
[00485] [00485] The term “membrane_protein_call: A textual interpretation of the number of membrane databases that included the gene. If the gene was included in a database, then the link is a "low confidence" membrane protein. If the gene was included in two databases, then the link is a "medium-trust" membrane protein. If the gene was included in three databases, then the link is a "high-confidence" membrane protein.
[00486] [00486] The term “ratio_gtex_std_dev_to_mean: the reason for the deviation
[00487] [00487] The term “universally_expressed: a binary marker of whether a gene appears to be universally expressed. A gene is said to be universally expressed if gtex_mean is> 10, gtex_min. The term “> 1 and ratio_gtex_std_dev_to_mean <1. A 1 in this column indicates that the gene in question met these criteria.
[00488] [00488] The term “disease: the TCGA barcode for the disease analyzed for LOH data in this row of the spreadsheet.
[00489] [00489] The term “mean_expression_in_tissue: The expression of the average gene in the analyzed tissue. Several tissue categorizations can map to a single type of TCGA tumor. The mapping of tissues in GTEX to TCGA tumor types is given in the file “tcga_disease_tissue_lookup.txt”. A representative sample is given below: tcga_disease gtex_tissues acc Gland.Adrenal blca Bladder brca Breast ... Tissue.Maria cesc Cérvix ... Endocérvix, Cérvix ... Ectocérvix
[00490] [00490] The term “mean_expression_in_other_tissues: The average gene expression in all other tissues, except in the analyzed tissue. For example, if the gene being analyzed was PSMA (a prostate specific gene), then this value would be very low when the type of tumor analyzed was PRAD (prostate adenocarcinoma).
[00491] [00491] The term "cohens_d: Cohen's measure d of expression separation in the analyzed tissue versus all other tissues. This means
[00492] [00492] The term “proportion_w_LOH_relative: The proportion of tumors in the type of tumor analyzed that exhibit evidence of LOH. The threshold for calling a genomic segment that suggests that LOH was -0.1 (in units of relative copy number). The number of relative copies of a segment was the log of the sign of the number of copies in the tumor divided by the sign of the number of copies in the corresponding normal. These data were obtained from the cbio portal and the technique was validated in part 1.
[00493] [00493] The term “CI_95_low_relative: The lower limit of the 95% confidence interval in the proportion of tumors suffering LOH in this locus. The prop.test function in R was used for this calculation. This function calculates a binomial confidence interval with Yates' continuity correction.
[00494] [00494] The term “CI_95_high_relative: the upper limit of the 95% confidence interval in the proportion of tumors suffering LOH in this locus. The prop.test function in R was used for this calculation. This function calculates a binomial confidence interval with Yates' continuity correction.
[00495] [00495] The term “mutsig_hits_on_chr: the genes on the same chromosome as the SNP that pass statistical significance (q value <0.25) for being cancer-inducing. The Mutsig 2.0 algorithm was used. The format is “Gene symbol, q = q-value; Gene symbol 2,… ”
[00496] [00496] The term “tsg_on_chr_mutated_in_disease: a binary indicator variable for whether or not one of the genes that passes statistical significance on mutsig is a tumor suppressor gene. The list of tumor suppressor genes used for this annotation was the list in the table published by Vogelstein et al9. A 1 in this column indicates that the gene is noted as a tumor suppressor gene.
[00497] [00497] The term “hallmark_tsg_on_chr_mutated_in_disease: a binary indicator variable for whether any of the genes identified as significantly mutated in the type of tumor analyzed and on the same chromosome as the SNP are“ striking ”tumor suppressor genes. The "striking" tumor suppressor genes are a short list of very well-validated tumor suppressor genes that are more likely to be mutated early in tumor development. These genes were: TP53, PTEN, APC, MLL3, MLL2, VHL, CDKN2A and RB1. A 1 in this column indicates that one of these striking TSGs exists on the same chromosome as the SNP in question and is significantly mutated in the type of tumor analyzed.
[00498] [00498] The term “gistic_deletion_n_peaks: the number of GISTIC peaks on the chromosome on which the SNP falls. A higher number suggests (vaguely) that there are more selective forces leading to the loss of genetic material from this chromosome.
[00499] [00499] The term “gistic_deletion_best_q_value: the lowest q GISTIC value for genomic loss on the chromosome on which the SNP falls. A very low q value suggests that there is significant selective pressure to lose genomic material somewhere on the chromosome.
[00500] [00500] The term “proportion_of_patients_eligible: the estimated proportion of patients who would have i) SNP germline heterozygosity and ii) SNP LOH in the tumor. The estimate of the proportion of patients with heterozygosity of the SNP germline assumes the Hardy-Weinberg balance, using the equation of proportion of heterozygote = 2pq. Where p is the global allele fraction of the SNP and q = 1-p.
[00501] [00501] The term “proportion_of_patients_eligible_max_ethnicity_targeted: the estimated proportion of patients who would have i) SNP germline heterozygosity and ii) SNP LOH in the tumor. The estimate of the proportion of patients with heterozygosity of the SNP germline assumes the Hardy-Weinberg balance, using the
[00502] [00502] The term “cumulative_score: a score that quantifies the degree to which an SNP is a good candidate for an iCAR target. The scores range from 0 to theoretical 1. For more information on the calculation of this score, please see the section “Cumulative score to classify candidate SNPs”. EXAMPLE 1. LOH RATE ASSESSMENT OF HLA GENES
[00503] [00503] A therapeutic strategy is proposed to address vulnerabilities incurred by genomic loss in cancer cells. The proposed strategy uses a combination of activation T CAR cells (aCAR) and inhibitory T CAR cells (iCAR) to more safely target tumors that have lost genomic segments encoding heterozygous cell membrane proteins for maternal and paternal alleles (ie , with changes in the polymorphic protein coding).
[00504] [00504] iCARs can decrease non-tumor toxicity of CAR-T therapy without decreasing anti-tumor efficacy if the iCAR target is expressed only by non-tumor tissues. One such scenario in which iCAR targets are expressed only by non-tumor cells occurs when the iCAR antigen is encoded by a portion of the genome that has been deleted in tumor cells. A highly polymorphic family of genes known to be expressed in all cells is HLA.
[00505] [00505] HLA proteins are almost universally expressed by mammalian cells to allow the presentation of non-autoantigens to cells of the immune system. HLA genes also tend to be quantitatively highly expressed, making them more amenable to therapeutic targeting. The RNA expression of HLA genes is higher
[00506] [00506] The purpose of this section is to identify types of cancer in which the HLA gene is frequently deleted. Secondary analyzes include attempts to identify drivers of genomic loss at the HLA locus.
[00507] [00507] We executed a detailed plan to identify cancers with selective pressures that led to frequent copy loss of HLA genes (Figure 5). Frequency of loss of HLA through tumor types using ABSOLUTE data:
[00508] [00508] We used TCGA copy number profiles that were processed by the ABSOLUTE algorithm to evaluate true estimates of the HLA-A allelic loss rate. Publicly available ABSOLUTE segmented copy number data was downloaded from (https://www.synapse.org/#!Synapse:syn1710464.2)1. The ABSOLUTE algorithm generates the entire copy level of each allelic segment within a single cancer genome. In the case of the loss of a single copy of chromosome 6 (harboring the HLA locus), then the numbers of allelic copies would be: 1 for the retained segment and 0 for the segment that was lost. In the case of neutral copy loss of heterozygosity, then, the retained segment would have copy number 2 and the lost segment would have number 0. Publicly available copy number data, processed by ABSOLUTE, were available for 12 tumor types (Table 4 ). Lung squamous cell carcinoma (LUSC) had the highest frequency of HLA-A LOH compared to the other types of tumor (Figure 6). Uterine / endometrial cancers (UCEC) had the lowest HLA-A LOH frequency of all evaluable tumors (AML samples were not included
[00509] [00509] We try to obtain the LOH frequency of as many types of tumors as publicly available. However, these data were not processed by ABSOLUTE and the raw data to be processed by ABSOLUTE are not publicly available. Instead, we used relative copy number data on 32 types of TCGA tumors (Figure 13). These data were downloaded from cbioportal (cbioportal.org/data_sets.jsp). Relative copy number data was obtained from Affymetrix SNP 6.0 matrices of tumor samples.
[00510] [00510] In order to determine whether accurate LOH estimates could be obtained from relative copy number data, we calculated the LOH rate with relative data for tumors that had previously had ABSOLUTE LOH data. This data consisted of a segmented copy number file. Each segment receives a relative copy ratio. The copy ratio is defined as the log of the signal density ratio in the tumor compared to the corresponding normal (in Affymetrix matrices). Standardization for combined control (usually of peripheral blood) helps to remove any germline copy number variants from being misinterpreted as somatic. A segment is said to have suffered genomic loss if the relative copy number of that genomic segment is below a given threshold. For example, if
[00511] [00511] First, we try to determine the ideal cut in the number of copies to label segments of relative copy numbers as having suffered LOH. The agreement of the ABSOLUTE estimates and the relative number of copies of LOH was higher with a cut of -0.1 for the relative number of copies (Table 5 and Figure 9). This threshold is also the threshold used by the TCGA copy number group to define copy loss on the TCGA Tumorscape portal (http://portals.broadinstitute.org/tcga/home). The correlation between the fraction of individuals with HLA-A LOH in the relative data versus ABOSLUTE data was 0.55. This reasonably high correlation allowed us to proceed with the analysis of all types of tumor with relative data on the number of copies available. Fraction of HLA-LOH patients across 32 tumor types using relative copy number data
[00512] [00512] The portion of patients who had HLA-A LOH was computed for all 32 available tumors of the TCGA (Figure 10A; COAD and READ were analyzed together). The tumor with the highest HLA-A LOH rate was chromophobic kidney cancer. The tumor with the lowest HLA-A LOH rate was uveal melanoma (Table 6). To ensure that the LOH rate we derived in these analyzes was robust for minor genomic position disruption, we analyzed the LOH rate of the upstream and downstream HLA-A genes to see if their HLA-LOH rate was similar to HLA -THE. As expected, the LOH rate of the upstream and downstream genes, HLA-G and ZNRD1, respectively, was exactly the same as for HLA-A. (Figure 3 A-C). These data demonstrate that HLA-A LOH calls are robust to small deviations in the genomic position. In
[00513] [00513] Intratumoral genomic heterogeneity is a feature recently appreciated in almost all human cancers analyzed to date2, 3. Therapies aimed at genetic alterations present only in a fraction of tumor cells can affect only the tumor cells that harbor the referred alterations. An iCAR strategy that targets antigens not present in tumor cells can protect some tumor cells from attack by aCAR if the antigen is not deleted clonally. Therefore, we seek to identify tumors in which the HLA genes are likely to suffer clonal LOH. LOH that occurs at the beginning of the evolution is likely to be driven by selective forces at the beginning and / or maintenance of the tumor. Therefore, we look for tumor suppressors on chromosome 6 (harboring the HLA locus) in three ways. First, we looked for genes that were significantly mutated on chromosome 6 in each of the tumor types evaluated4. The spreadsheet reports the genes with significant mutation on chromosome 6 in the “chr6_mutsig_sig_genes” column.
[00514] [00514] Second, we look for regions of significantly deleted genes, meaning tumor suppressors probably deleted. We use the results of running GISTIC2.0 on this data. The spreadsheet reports the number of GISTIC deletion peaks on chromosome 6 (q <0.25) and the lowest q value of these exclusion peaks. Generally, the more GISTIC deletion peaks and the lower the q value, the stronger the selection pressure. However, it is also possible to have the scenario in which a
[00515] [00515] Third, we overlapped the set of genes that were significantly mutated in each tumor with a list of known tumor suppressor genes to determine whether any of the mutated genes would likely lead to chromosome 65 loss. We were able to identify two types of tumor with possible mutational drivers . In adrenocortical carcinoma, the DAXX gene was significantly mutated (q = 0.0571) and in Large Diffuse B-Cell Lymphoma, the TNFAIP3 gene was significantly mutated (q = 0.00278). DAXX encodes a chaperone histone, whose mutations are associated with longer telomeres in adrenocortical carcinoma6. TNFAIP3 encodes a negative signaling regulator NF-capaB. Mutations of this gene that occur in DLBCL have so far been shown to increase NF-capaB7 signaling. Table 3. Genomic loci analyzed for LOH. The genomic coordinates are in the hg19 human genome set. Gene Protein Chromosome Start Position End Position RNA Expression (RPKM) HLA-A HLA-A 6 29941260 29945884 226.6 HLA-B HLA-B 6 31353872 31357188 422.4 HLA-C HLA-C 6 31268749 31272130 193.4 Table 4. Tumor types with ABSOLUTE data Disease name Abbreviation Number of Number of TCGA samples Finished samples ABSOLUTE Bladder urothelial carcinoma BLCA 138 90 Invasive breast carcinoma BRCA 880 750 Colon adenocarcinoma COAD 422 349 Glioblastoma multiforme GBM 580 485 Cell carcinoma scaly head eHNSC 310 270 neck Renal clear cell carcinoma of the kidneys KIRC 497 373 Acute Myeloid Leukemia LAML 200 0 Lung adenocarcinoma LUAD 357 292 Lung squamous cell carcinoma LUSC 344 261 Serous ovarian cystadenocarcinoma OV 567 457 READ Adenocarcinoma 147 Endometrial carcinoma of the uterine body UCEC 498 378
[00516] [00516] Based on the above, we conclude that LOH in the HLA region is a common event in many tumors, however, and the percentage of LOH varies between tumor types. Therefore, HLA genes are good candidates for
[00517] [00517] 1. Zack TI, Schumacher SE, Carter SL, Cherniack AD, Saksena G, Tabak B, Lawrence MS, Zhsng CZ, Wala J, Mermel CH, Sougnez C, Gabriel SB, Hernandez B, Shen H, Laird PW, Getz G, Meyerson M, Beroukhim R. Pan-cancer patterns of somatic copy number alteration. Nature genetics. 2013; 45: 1134-1140
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[00522] [00522] 6. Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L,
[00523] [00523] 7. Compagno M, Lim WK, Grunn A, Nandula SV, Brahmachary M, Shen Q, Bertoni F, Ponzoni M, Scandurra M, Califano A, Bhagat G, Chadburn A, Dalla-Favera R, Pasqualucci L. Mutations of multiple genes cause deregulation of nf-kappab in diffuse large b-cell lymphoma. Nature. 2009; 459: 717-721 EXAMPLE 2. GENERAL IDENTIFICATION OF ALLEL GENOME LINEAR LINKS THAT CODE PROTEINS EXPRESSES OF CELL SURFACE THAT SUFFER LOSS
[00524] [00524] Inhibitory CAR-T cells can decrease toxicity outside the tumor of DE CAR-T therapy without decreasing anti-tumor efficacy if the iCAR target is expressed only by non-tumor tissues. One such scenario in which iCAR targets will be expressed only by non-tumor cells is where the iCAR antigen is encoded by a portion of the genome that has been deleted in tumor cells. The purpose of this section of the workflow is to identify these alleles. Allele Identification:
[00525] [00525] We used the Exome Aggregation Consortium (ExAC) database as an entry for the analysis (exac.broadinstitute.org). The ExAC database is a compilation of exomes from various studies of
[00526] [00526] The following filters were applied to the variants of the ExAC database: i) the variant must affect the amino acid composition of the encoded protein ii) the variant must have an allelic frequency less than 0.05 (5%) in at least one of the populations in Table 6. The analysis was corrected for scenarios where the minor allele had an allele fraction greater than 0.5 (50%). If more than three alleles at one site were observed, then the most prevalent substitution was used (these sites are generally sequencing error sites and should be interpreted with caution).
[00527] [00527] An SNP was considered to have an impact on protein composition if any SNP produced any of the following classes of variants: 'missense_variant', 'inframe_deletion', 'start_lost', 'stop_gained', 'inframe_insertion', 'stop_retained_variant', 'frameshift_variant', 'stop_lost', 'coding_sequence_variant', 'protein_altering_variant'. The analysis started with 9,362,319 variants and 29,904 variants passed
[00528] [00528] We used the Genotype-Tissue Expression (GTEX) database v6p (dbGaP Accession phs000424.v6.p1) to identify genes that are expressed in various types of tissues (https://gtexportal.org/home/) two. The GTEX database consists of RNA sequencing of 8,555 human samples from different types of healthy tissues. Several notes were obtained from this database. First, we determine the average expression of each gene across all tissues. The average expression for each gene was calculated by taking the median expression data per tissue and computing the average of these values across the tissues. These data were obtained from the file GTEx_Analysis_v6p_RNA-seq_RNA-SeQCv1.1.8_gene_median_rpkm.gct from available at https://gtexportal.org/home/datasets.
[00529] [00529] The average expression of each gene corresponding to each type of tumor was also included. To obtain this data, we created a mapping of tumor types to corresponding normal tissues. For example, pancreatic cancer TCGA data would be annotated with pancreatic tissue from GTEX. In some cases, the mapping was more approximate. For example, glioblastoma expression data was mapped from all tissues noted as brain in GTEX. A table with these mappings (titled tcga_disease_tissue_lookup.txt) is attached. Several measures were computed to assess the homogeneity or overexpression of each gene in each type of tissue / tumor. For each type of tumor, a D cohen score was computed to establish a possible overexpression of the gene. Genes overexpressed in particular tissues are likely to be good targets for aCAR. On the other hand, we measure the standard deviation of gene expression across tissues and compare this with the
[00530] [00530] A gene was called "universally expressed" if it met the following criteria: (i) the mean expressed through tissues was greater than 10 RPKM. (ii) The tissues with the least expression had an RPKM greater than 1. (iii) The ratio of the standard deviation in median RPKM across tissues compared to the mean RPKM was less than 1. Only 1,092 genes were noted as universally expressed.
[00531] [00531] Candidates were selected only on the basis of the UniProt annotation. For transmembrane proteins, there is usually a clear prediction for segments of the protein that are extracellular.
[00532] [00532] Table 8 presents a list of 1,167 good candidate genes identified by the above method having extracellular polymorphic epitopes classified according to the location of the chromosome. Allele annotation Impact of allele on protein function:
[00533] [00533] For an iCAR to effectively recognize only cancer cells that have lost an allele of a membrane protein, the structure of the protein is sufficiently different based on which allele is encoded. Several measures were taken to quantify the effect of each SNP on the resulting protein. First, the SNP variant class reported (for example, missense, nonsense) was reported in the 'consequence' column. The effect on consensus protein translation was included in the 'protein_consequence' column (e.g., p.Arg482Gln). The SIFT algorithm attempts to predict whether a protein variant will have an effect on protein structure and, therefore, function6. The score can vary from 0 (harmful) to 1 (benign). SIFT scores (version sift5.2.2) were included for each SNP for which a score
[00534] [00534] A classic measure of an amino acid substitution probability of inducing structural change is to use the BLOSUM62 substitution matrix. We downloaded the BLOSUM62 matrix at https://www.ncbi.nlm.nih.gov/IEB/ToolBox/C_DOC/lxr/source/data/BLOSU M62. Each SNP was noted with the BLOSUM62 score corresponding to its replacement. Allele classification as falling in the extracellular portion of the protein:
[00535] [00535] For an iCAR to recognize an allele, the allele must fall into the extracellular portion of the protein. For each SNP, we extract the position of the affected amino acid in the consensus translation and compare this to domains noted as extracellular from the Uniprot database. The Uniprot database was downloaded from www.uniprot.org/downloads. Many false negatives are possible due to a lack of characterization of all protein domains. A total of 3,288 SNPs in 1,167 genes were noted as extracellular (Table 8). SNP Peptide Context Annotations:
[00536] [00536] The peptide context of the analyzed alleles is likely to matter when trying to generate antibodies that recognize these sequences. We included as a reference the 10 amino acids that precede and flank the amino acid encoded by the SNP (total of the sequence of 21 amino acids). The uniprot database was used for the consensus amino acid sequence. We noted any conflicts where the uniprot database sequence did not match the amino acid encoded by any SNP at the position
[00537] [00537] Finding patients whose tumors could benefit from the proposed therapy would require an iCAR target if it were an SNP that suffers loss of heterozygosity (LOH) in a large fraction of tumors. Copy number file segments were downloaded from the cbio cancer genomics portal http://www.cbioportal.org/ 8. As an example, the proportion of uveal melanoma tumors suffering LOH for all SNPs is shown in Figure 12. Potential conductor changes in chromosomes that harbor candidate SNPs
[00538] [00538] A possible mechanism of therapy genomically directed to resistance is if one of the intended genomic changes were present only in a fraction of the cancer cells. One mechanism for trying to identify targets that may be present in the early stages of tumor development is to identify conductive events for each tumor. The most frequent mechanism of tumor suppression gene inactivation is mutation and subsequent LOH of the non-mutated chromosome. We try to find conductive genes, particularly tumor suppressor genes (TSGs) likely to undergo this process in each type of tumor. We used the results of running MUTSIG 2.0 on all tumors in this analysis to identify significantly mutated genes in each type of tumor. We noted whether or not one of the genes that was significantly mutated was included in a list of “striking” tumor suppressor genes, including TP53, PTEN, APC, MLL3, MLL2, VHL, CDKN2A, RB1. Finally, the list of conducting genes, TSG and TSGs
[00539] [00539] Although mutations in conductive genes that subsequently undergo LOH are a mechanism that can mark events that are likely to occur early in the evolution of the tumor, focal deletion of genomic segments containing a tumor suppressor gene is another. We use the GISTIC algorithm to identify regions of DNA that undergo genomic deletion at a higher than average rate. The GISTIC algorithm identifies “peaks” of statistical significance along the chromosomal arms that suggest a negative selective pressure in these regions. For each SNP, we record the number of deletion peaks on the chromosome on which the SNP fell. We also recorded the lowest q value of any of these peaks. A lower q value suggests stronger selective pressure. Cumulative score to classify candidate SNPs:
[00540] [00540] In an effort to provide a continuous “score” for candidate SNPs, we have combined several different metrics that should be associated with better SNP candidates. The score consists of the product of the percentile classification of each of the following: 1. proportion of tumors with LOH in that SNP (higher is better) 2. prevalence of allele (higher is better)
[00541] [00541] To illustrate, we will calculate the score for a theoretical SNP. If only 32% of SNPs have a tumor suppressor gene on the chromosome, then the percentile rating for having one would be 0.68. If the allele had a lower allele fraction of 0.49 (where 0.5 is the highest possible), then the percentile rating would be 0.99. If the LOH rate was 0.10 and 75% of the SNPs had more LOH than that, then the percentile rating would be 0.25. If the standard deviation ratio of expression values using
[00542] [00542] Any SNP with a score greater than 0.4 was considered "top-hit". Table 8. Exemplary iCAR targets Gen. No. 1 ABCA4 1 ADAM30 1 ASTN1 1 C1orf101 1 CACNA1S 1 CATSPER4 1 CD101 1 CD164L2 1 CD1A 1 CD1C 1 CD244 1 CD34 1 CELSR2 1 CHRNB2 1 CLCA2 1 CLSTN1 CR1B2 CSF3R 1 CSMD2 1 ECE1 1 ELTD1 1 EMC1 1 EPHA10 1 EPHA2 1 ERMAP 1 FCAMR 1 FCER1A 1 FCGR1B 1 FCGR2A 1 FCGR2B 1 FCGR3A 1 FCRL1 1 FCRL3 1 FCRL4 1 FCRL5 1 FCRL6 1 GJB4 1 GPA33 1 GPR1 IGSF3 1 IGSF9 1 IL22RA1
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[00549] [00549] For that matter, the HLA allotype was determined for DNA derived from 6 frozen KICH combination samples (Normal and Cancer) RC-001-RC003, TNEABA1l, TNEABNWE, 2rDFRAUB, 2RDFRNQG, IOWT5AVJ, IOWT5N74. In addition, two combined samples of DNA OG-001-OG-002 (Normal and Cancer) were also analyzed. A DNA library was prepared, sequence analysis was performed in order to identify the HLA typing of the sample. DNA was extracted from 6 frozen combined KICH samples (Normal and tumor) and a library was prepared as described below.
[00550] [00550] HLA TruSight Sequencing Libraries were prepared using the TruSight® HLA v2 Sequencing Panel (Illumina, San Diego, California, U.S.A.) at Genotypic Technology Pvt. Ltd., Bangalore, India.
[00551] [00551] Briefly, HLA Amplicons were generated using the primers provided in the HLA TruSight Sequencing Kit. The amplicons were confirmed on Agarose Gel followed by cleaning the amplicons using Sample Purification Beads provided in the kit. The amplicons were normalized and fragmented by Tagmentation reaction. Post Tagmentation different amplicons from each individual sample were collected and continued until PCR enrichment. The bar code of
[00552] [00552] The table below represents the HLA genotype of the samples above.
[00553] [00553] As seen below, we can infer that the lost allele of the analysis, for example, patient # RC001 exhibits loss of HLA-A30 allele in the tumor samples and becomes hemizygous for HLA-32; patient # RC003 lost HLA-1 in the tumor sample and becomes hemizygous for HLA-30. The lost allele identified will determine the relevant iCAR for each patient. Cases in which tumor samples were contaminated with normal cells, could exhibit a clear loss of HLA allele in this method. Table 9: HLA genotype of the corresponding KICH samples Sample_ID HLA-A HLA-B HLA-C OG_001_NAT_NORMAL 02:06:01: - 7:02:01 03:04:01: - 24:02:01: - 15:01:01: - 07:02:01: - OG_001_TUM_TUMOR 02:06:01: - 7:02:01 03:04:01: - 24:02:01: - 15:01: 01: - 07:02:01: - OG_002_NAT_NORMAL 02:01:01: - 15:01:01: - 3:03:01 24:02:01: - 55:01:01 X OG_002_TUM_TUMOR 02 : 01: 01: - 15:01:01: - 3:03:01 24:02:01: - 55:01:01 X RC_002_NAT_A_NORMAL 03:01:01: - 7:02:01 06: 02:01: - 68:02:01: - 58:02:01 7:18:00
[00554] [00554] Final raw paired Illumina readings (150X2, HiSeq) were checked for quality using FastQC. Illumina raw readings were processed by Trim Galore software to secure adapter
[00555] [00555] Variants have been identified using SAMtools and BCFtools. In this case, joint genotyping is done to identify variants in each pair of samples (each normal and tumor pair). Therefore, for each pair, a merged .vcf is generated. Potential variants are identified from each of these merged .vcf files using reading depth threshold> 20 and mapping quality> 30. For each pair of the filtered merged .vcf, .vcf files were generated in the sense of the sample. The filtered variants were also noted for genes, protein changes and the impact of variations using Variant Studio.
[00556] [00556] The table below describes the extent of chromosomal loss for the samples above. RC001, RC002 and RC003 exhibit extensive chromosome loss including chromosome 6 that codes for HLA genes, hence, for these samples, HLA can be used as an iCAR target, in addition to many other targets encoded on chromosomes 1, 2, 3, 4 (for RC002), 5, 6, 8 (for RC003), 9 (RC001, RC002), 10 (RC001, RC003), 11 (RC003), 13 (RC001, RC003), 14 (RC002), 17 (RC001, RC003), 19 (RC001), 21 (RC001, RC003), 22 (RC001, RC002). Table 10 Chrosomal loss Chr RC001 RC002 RC003 OG001 OG002 2RD IOW TNE 1 ++ ++ ++ 2 ++ + ++ + 3 ++ ++ + 4 ++ 5 ++ ++ 6 ++ + ++ + 7 8 ++ 9 ++ ++ ++ 10 ++ ++
[00557] [00557] For RC001, Fig. 14 represents the loss of a chromosomal region adjacent to the tumor suppressor protein TP53, encoded on chromosome 17. The genes encoded on chromosome 17, which have been identified as targets for iCAR can be used to treat RC001 patient. Abbreviations: ADP, adenosine diphosphate; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; APRIL, a proliferation-inducing ligand; BAFF, B cell activation factor of the TNF family; BCMA, B cell maturation antigen; BCR, B cell receptor; BM, bone marrow; CAIX, carbonic anhydrase IX; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; CLL, chronic lymphocytic leukemia; CNS, central nervous system; CSPG4, chondroitin sulfate proteoglycan 4; DC, dendritic cell; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EGFRvIII, variant III of EGFR; EphA2, erythropoietin-producing hepatocellular A2 carcinoma; FAP, fibroblast-activating protein; FR-α, alpha folate receptor; GBM, glioblastoma multiforme; GPI, glycophosphatidylinositol; H&N, head and neck; HL, Hodgkin's lymphoma; Ig, immunoglobulin; L1-CAM, L1 cell adhesion molecule; MM, multiple myeloma; NB, neuroblastoma; NF-KB, nuclear factor-KB; NHL, non-Hodgkin's lymphoma; NK, natural killer; ligating NKG2D-L, NKG2D; PBMC, peripheral blood mononuclear cell; PC, plasma cell; PLL, prolymphocytic leukemia; PSCA, prostate stem cell antigen; PSMA,
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[00649] [00649] LOH can be detected at the protein level by differential staining of normal versus tumor cell samples using allele-specific antibodies. For example, checking for HLA-LOH in samples
[00650] [00650] The samples will be subjected to immunohistochemical staining (HCI), as described in the HCI protocol below. Table 11: Allele-specific anti-HLA antibodies Manufacturer Antibody HLA-A2 anti-human APC (BB7.2) eBiosciences HLA-A2 PE-cy7 anti-human (BB7.2) eBiosciences HLA-A3 anti-human FITC (GAP A3 ) Anti-human HLA-A3 PE eBiosciences (GAP A3) Anti-HLA Mouse Class A25 Antigen, Antibody A32 US Biological Antigen Class 1 HLA A30, A31 MyBioSource HLA-B7-PE anti-human mouse (BB7.1 ) Millipore Antibody HLA-A2 (BB7.2) Novus Antibody HLA B7 (BB7.1) Novus HLA-B27-FITC mouse anti-human (clone HLA.ABC.m3) Millipore Protocol IHC Frozen tissue samples -
[00651] [00651] Frozen tissues are often fixed in a formalin-based solution and incorporated in OCT (optimum cutting temperature compound), which allows the sample to freeze. OCT tissues are kept frozen at -80oC. The frozen blocks are removed from -80oC before sectioning, balanced in the cryostat chamber and cut into thin sections (usually 5-15μm thick). The sections are mounted on a histological slide. The slides can be stored at -20oC to -80oC. Before IHC staining, the slides are thawed at room temperature (RT) for 10-20 min. Paraffin-embedded fabrics -
[00652] [00652] Fabrics are incorporated in a Formaldehyde Fixative Solution. Before adding paraffin wax, the tissues are dehydrated by gradual immersion in increasing concentrations of ethanol (70%, 90%, 100%) and xylene for specific times and durations at RT. Then, the fabrics are soaked in paraffin wax.
[00653] [00653] The tissues embedded in paraffin are cut in a microtome to sections from 5 to 15μm thick, floated in a water bath at 56oC and mounted on a histological slide. The blades can be kept at RT.
[00654] [00654] Before IHC staining, paraffin-soaked sections require a rehydration step. REHYDRATION - the sections are rehydrated by immersion in xylene (2 X 10min.), Followed by decreasing concentrations of ethanol - 100% X2, each for 10min. 95% ethanol - 5min. 70% ethanol - 5min. 50% ethanol - 5min. rinse in dH2O. Detection of immunofluorescence:
[00655] [00655] Protocol:
[00656] [00656] 1. Rehydrate slides in wash buffer (PBSX1) for 10 min. Drain the wash buffer.
[00657] [00657] 2. Perform antigen recovery - if necessary (heat-induced antigen recovery or enzymatic recovery).
[00658] [00658] 3. For intracellular antigens, perform permeabilization - incubate the slides in 0.1% X-100 triton in PBSX1 for 10 min. to RT.
[00659] [00659] 4. BLOCKING - Block the tissue in blocking buffer for 30 min. to RT. The blocking buffer depends on the detection method, usually 5% animal serum in PBSX1 or 1% BSA in PBSX1
[00660] [00660] 5. PRIMARY ANTIBODY - primary antibody diluted in incubation buffer (eg 1% BSA, 1% donkey serum in PBS, other incubation buffers can also be used), according to the manufacturer's instructions of antibody. Incubate the tissue in the diluted primary antibody at 4oC overnight. The primary antibody can be an anti-HLA-A, anti-HLA-B or allele-specific anti-HLA-C monoclonal antibody, as detailed above. • If a conjugated primary antibody is used, protect from light and proceed to step 8.
[00661] [00661] 6. 6. WASH - wash the slides in the wash buffer - 3 X 5-15 min.
[00662] [00662] 7. 7. SECONDARY ANTIBODY - Dilute the secondary antibody in incubation buffer according to the instructions of the antibody manufacturer. Incubate the tissue in the diluted secondary antibody for 30-60 min at RT. Protect from light.
[00663] [00663] 8. 8. WASH - wash slides in wash buffer - 3 X 5-15 min.
[00664] [00664] 9. 9. DAPI staining - Dilute the DAPI incubation buffer (~ 300nM - 3μM). Add 300μl of DAPI solution to each section. Incubate at RT for 5-10 min.
[00665] [00665] 10. 10. WASH - wash the slide once with X1 PBS.
[00666] [00666] 11. 11. Mount with antifade mounting media.
[00667] [00667] 12. 12. Keep the blades protected from light.
[00668] [00668] 13. 13. View the slides using a fluorescence microscope. Chromogenic detection: Protocol:
[00669] [00669] 1. 1. Rehydrate slides in wash buffer (PBSX1) for 10 min. Drain the wash buffer.
[00670] [00670] 2. 2. Perform antigen recovery - if necessary - see above.
[00671] [00671] 3. 3. For HRP reagents, block the endogenous activity of peroxidase with 3.0% hydrogen peroxide in methanol for at least 15 min.
[00672] [00672] 4. 4. Wash the sections by immersing them in dH2O for 5 minutes.
[00673] [00673] 5. 5. For intracellular antigens, perform permeabilization - incubate the slides in 0.1% X-100 triton in PBSX1 for 10 min. to RT.
[00674] [00674] 6. 6. BLOCKING - Block the tissue in blocking buffer for 30 min. to RT. The blocking buffer depends on the detection method, usually 5% animal serum in PBSX1 or 1% BSA in PBSX1.
[00675] [00675] 7. 7. PRIMARY ANTIBODY - primary antibody diluted in incubation buffer (for example, 1% BSA, 1% donkey serum in PBS, other incubation buffers can also be used), according to the instructions from the antibody manufacturer. Incubate the tissue in the diluted primary antibody at 4 ° C overnight.
[00676] [00676] 8. 8. WASH - wash slides in wash buffer - 3 X 5-15 min.
[00677] [00677] 9. 9. SECONDARY ANTIBODY - Incubate the tissue in secondary antibody conjugated with HRP for 30-60 min. to RT.
[00678] [00678] 10. 10. WASH - wash slides in wash buffer - 3 X 5-15min min.
[00679] [00679] 11. 11. Add the ABC-HRP reagent according to the manufacturer's guidelines. Incubate at RT for 60 min.
[00680] [00680] 12. 12. Prepare the DAB solution (or another chromogen) according to the manufacturer's guidelines and apply to the tissue sections. The chromogenic reaction turns the epitope sites brown (usually a few seconds - 10 minutes). Proceed to the next step when the signal strength is appropriate for imaging
[00681] [00681] 13. 13. WASH - wash slides in wash buffer - 3 X 5-15 min.
[00682] [00682] 14. 14. Wash the slides in dH2O - 2 X 5-15min.
[00683] [00683] 15. 15. Nucleus staining - add hematoxylin solution. Incubate at RT for 5 min.
[00684] [00684] 16. 16. Dehydrate tissue sections - 95% ethanol - 2 x 2 min. 100% Ethanol - 2 x 2 min. Xylene - 2 X 2 min.
[00685] [00685] 17. 17. Mount with antifade mounting media
[00686] [00686] 18. 18. View the slides using bright field lighting EXAMPLE 5. CAR-T DESIGN AND CONSTRUCTION
[00687] [00687] The purpose of the study is to create a synthetic receptor that inhibits the 'out-of-tumor' effect on the target of CAR-T therapy. To that extent, a library of CAR constructs composed of activating and inhibitory CARs was established.
[00688] [00688] The first set of constructs included an inhibitory CAR targeting the HLA type I sequence (HLA-A2) and an activating CAR targeting the tumor antigen (CD19). The next set of constructs to be used for proof of concept purposes includes CD19 activation sequences targeting CD19 and an inhibitory CAR sequence targeting CD20. Additional constructs targeting target antigens identified by future bioinformatics analyzes will be built. Target candidates will be prioritized according to established criteria (exemplary criteria include, but are not limited to, target expression pattern, target expression level, antigenicity and more). A CD19 aCAR, CD20 iCAR and HLA-A2 iCAR were constructed, as described in Figures 15 and 21.
[00689] [00689] The iCAR constructs were designed and synthesized using commercial DNA synthesis. The transmembrane and intracellular domains up to the first annotated PD-1 extracellular domain (amino acid 145-288) were fused downstream with HLA-A2 scFv (DNA sequence encoding HLA-A2, was recovered from the hybridoma BB7.2, ( ATCC cat #: HB-82), producing anti HLA-A2).
[00690] [00690] Similar constructs with CTLA4 (amino acids 161-223) or with other sequences derived from negative immune regulators
[00691] [00691] For iCAR detection and classification, a reporter gene (eg, eGFP) was integrated downstream with the iCAR sequence via IRES sequences and followed by an antibiotic resistance gene (ie hygromycin) separated by sequence of P2A, as illustrated in Figure 15.
[00692] [00692] For the aCAR construct, scFv of CD19 was merged with the 2nd generation CAR construct composed of hinge sequence CD8 followed by transmembrane CD28 and co-stimulation 1 of 41BB and CD3ζ. Additional aCAR constructs composed of other signaling or structural elements will also be designed and constructed (for example, CD28 hinge, CD28 signaling domain or both CD28 and 41BB signaling domains). For detection and classification of aCAR, the RFP reporter gene was integrated downstream of the aCAR sequence by means of IRES sequences followed by the antibiotic resistance gene (Puromicyn resistance) separated by the P2A sequence (Figure 15).
[00693] [00693] Both the aCAR and iCAR sequences were cloned into a lentivirus transfer vector and then used to produce viral particles using HEK-293T packaging cells. EXAMPLE 6. EFFECTIVE CELL PRODUCTION
[00694] [00694] To study the effect of iCAR constructs on modulation of CD19 CAR activation, recombinant Jurkat effector cells were constructed as detailed in Table 12 below. Jurkat (ATCC TIB152), a CD4 + T cell line and lJurkat-NFAT (a Jurkat cell line purchased from BPS Biosciences, engineered to express a firefly luciferase protein, under the control of NFAT response elements) were transduced using plates linked to a lentiviral vector coated with retronectin (Takara) or in the presence of polybrene. The transduced cells were also subjected to antibiotic selection to produce the
[00695] [00695] In addition, activated T cells, derived from peripheral blood obtained from healthy donors, will be transduced with viral particles that code for aCAR, iCAR or both, in different multiplicities of infection (MOI). The selection of FACS based on the expression of the reporter gene will be used for classification and selection of the population of cells that express different levels of aCAR, iCAR or both. EXAMPLE 7. PREPARATION OF TARGET CELLS
[00696] [00696] An in vitro recombinant system has been established to test the functionality of iCAR constructs in inhibiting aCAR activity towards cells outside the target. For this purpose, target cells were produced that express the aCAR epitope, iCAR epitope or both. Recombinant cells that express the aCAR epitope represent cells 'on target' 'in the tumor', while cells expressing both aCAR and iCAR epitopes represent healthy cells 'on target' 'out of tumor'.
[00697] [00697] As our first set of iCAR / aCAR is based on HLA-A2 and CD19, respectively, recombinant cells expressing HLA-A2 or CD19 or both were produced by transfecting the strain of
[00698] [00698] To detect the expression of recombinant HLA A-2, the Myc marker was inserted. For the second iCAR / aCAR set composed of CD20 iCAR / CD19 aCAR, recombinant cells that express CD20 or CD19 or both were constructed (the target cells are detailed in Table 15). Table 13 - Target cell lineage Set # Parental cell Protein Protein Objective Modeling target 1 target 2 1 Raji CD19 None A model for cancer cells In the tumor expressing endogenous CD19 Raji CD19 HLA-A2 A model for normal cells Outside the tumor expressing endogenous CD19 ; Recombinant HLA-A2 Thp1 None HLA_A2 A model for normal cells Negative control expressing endogenous and negative HLA-A2 for CD19 2 Hela HLA-A2 None A model for normal cells Negative control expressing endogenous and negative HLA-A2 for CD19 Hela HLA-A2 CD19 A model for normal cells outside the tumor expressing recombinant CD19; HLA-A2 4 Hela CD19 None A model for cancer cells In the tumor expressing recombinant CD19 Hela CD19 CD20 A model for normal cells Outside the tumor expressing recombinant CD19; CD20 Hela CD20 None A model for normal cells Negative control expressing endogenous CD20 negative for CD19 3 Hela-Luciferase HLA-A2 None Negative control to be used in the Negative control kill test Hela-Luciferase HLA-A2 CD19 A model for normal cells Outside tumor expressing recombinant CD19; HLA-A2 (kill assay) 5 Hela-Luciferase CD19 None A model for cancer cells In the tumor expressing recombinant CD19 (kill assay) Hela-Luciferase CD19 CD20 A model for normal cells Outside the tumor expressing recombinant CD19; CD20 (kill test) Hela-Luciferase CD20 No Negative control (Negative kill control test) Tests
[00699] [00699] The inhibitory effect of iCAR will be tested both in vitro and in vivo.
[00700] [00700] In in vitro assays, we will focus on measuring cytokine secretion and the effects of cytotoxicity, while in vivo, we will assess the inhibition and protection of iCAR for xenografts 'on the target outside the tumor'. We will limit T cells that lack iCAR to contaminate the results by classifying T cells to be iCAR / aCAR double positive using reporter genes. As a negative control for iCAR blocking activity, we can use T cells transduced with CAR lacking the scFv domain (ie, simulated transduction). EXAMPLE 8 IN VITRO TESTS Luciferase cytotoxic T lymphocyte (CTL) assay
[00701] [00701] The assay will be performed using recombinant Hela-Luc target cells described above, engineered to express firefly luciferase and one or two CAR target antigens. The in vitro luciferase assay will be performed according to the manufacturer's protocol for the Bright-Glo Luciferase (Promega) assay and bioluminescence as a reading.
[00702] [00702] T cells (transduced with both iCAR and pCAR or iCAR and aCAR or aCAR or simulated CAR) will be incubated for 24-48 h with the recombinant target cells expressing HLA-A2 or CD19 or both HLA-A2 and CD19 or CD20 or both CD20 and CD19 in different ratios from target to target. Cell killing will be quantified with the Bright-Glo Luciferase system.
[00703] [00703] Cytotoxicity 'outside the tumor' is optimized by classifying the population of transduced T cells according to the level of expression iCAR / aCAR or selecting the subpopulation of recombinant target cells according to their level of expression of CD19, HLA-A2 or CD20. To test whether iCAR-transduced T cells can discriminate between ‘in tumor’ and ‘out of tumor’ cells in vitro, we’ll test the cell killing effect
[00704] [00704] One of the ways in which cytotoxic T cells kill target cells is by inducing apoptosis through the Fas ligand. Sequential activation of caspases plays a significant role in the stage of cell apoptosis. The cleavage of pro-caspase 3 to caspase 3 results in conformational change and expression of catalytic activity. The activated cleaved form of caspase 3 can be specifically recognized by a monoclonal antibody.
[00705] [00705] Transduced T cells will be co-cultured for 2-4 h with any of the recombinant cells 'in the tumor' or 'outside the tumor', previously labeled with CFSE or another cell marker dye (for example, CellTrace Violet). Then the cell permeabilization and fixation by an internal staining kit (for example, Miltenyi or BD bioscience) the activated CASP3 will be detected by specific antibody staining (BD bioscience), and the apoptotic target cells will be detected and quantified by cytometry of flow. CTL Time-Delay Microscopy
[00706] [00706] Transduced T cells will be incubated with any of the cells 'in the tumor' or 'out of the tumor' for up to 5 days. Time-lapse microscopy will be used to visualize the kill. Alternatively, flow cytometry analysis will be performed using viable cell number staining and CountBright (Invitrogen) beads to determine the number
[00707] [00707] In order to demonstrate the efficacy of T cells transduced by aCAR / iCAR on discerning targets in vitro, each recombinant target cell ('in the tumor' or 'out of the tumor') is labeled with a different reporter protein (for example, GFP and mCherry). The transduced T cells (effector cells) will be matched with a mixture of recombinant cells expressing one or two target antigens (Target cells) in different E / T ratios. The fate of each cell line will be followed by microscopic imaging. Cytokine Release
[00708] [00708] Through T cell activation, cells secrete cytokines that can be quantified and used to evaluate T cell activation and inhibition. Cytokines can be detected intracellularly by flow cytometry or by measuring the proteins secreted in the medium by ELISA or Cytometric. Bead Array (CBA). Quantification of Secreted Cytokines by ELISA
[00709] [00709] Following co-cultivation of transduced T cells (Jurkat, or primary T cells) expressing iCAR or aCAR or both aCA and iCAR with modified target cells expressing iCAR or aCAR or both aCAR and iCAR antigens on their cell surface, the conditioned medium will be collected and the cytokine concentration will be measured by cytokine ELISA (IL-2, INFγ and or TNFα) according to the manufacturing instructions (for example, BioLegened or similar) and by Cytometric Bead Array (Miltenyi or similar) . Specific iCAR inhibition as measured by IL-2 ELISA
[00710] [00710] The effector cells Jurkat CD19 aCAR and Jurkat CD19 aCAR / HLA-A2 iCAR were co-cultured with Raji, Raji-HLA-A2 and Thp1 target cells and the corresponding supernatants were collected for IL-2 measurement by ELISA, as illustrated in Figure 16A. The incubation of Jurkat CD19-aCAR / HLA-A2-iCAR with Raji target cells ('tumor') expressing CD19 showed IL-2 secretion, however, the incubation of these cells
[00711] [00711] Transduced T cells (Jurkat, or primary T cells) expressing iCAR or aCAR or both aCAR and iCAR cultured for 6-24 h with recombinant target cells, which express iCAR or aCAR antigens or both aCAR and iCAR on their surface cell, will be subjected to the Golgi transport blocker (for example, Brefeldina A, monensin) to allow the intracellular accumulation of cytokine. T cells will then be permeated and fixed by an internal staining kit (for example, Miltenyi) and stained with anti CD3 and CD8 and for IL-2 and or INFγ and or TNFα. Cytokine Secretion Measured by Cytometric Account Matrix (CBA)
[00712] [00712] The Cytometric Account Matrix (CBA) is used to measure a variety of soluble and intracellular proteins, including cytokines, chemokines and growth factors.
[00713] [00713] T cells (primary T cells or Jurkat cells) transduced with aCAR or both the aCAR and iCAR constructs (effector cells) were stimulated with modified target cells that express both iCAR and aCAR or aCAR or iCAR target antigens on their surface cell (Figure 17A). Following several hours of coincubation, the effector cells produce and secrete cytokines that indicate their effector status. The reaction supernatant was collected and the secreted IL-2 was measured and quantified by a multiplex CBA assay.
[00714] [00714] As shown in Figure 17B, a specific inhibition of IL-2 secretion has been demonstrated for Jurkat T cells transduced by aCAR / iCAR co-cultured with target cells that express both target antigens. An 86% decrease in IL-2 secretion was demonstrated when double cells transduced from double CAR (aCAR / iCAR) were matched with target cells that express both target antigens compared to the IL-2 secretion resulting from the matching effector cells with target cells expressing only one target. NFAT activation test
[00715] [00715] To determine T cell activation, as measured by NFAT activation, Jurkat-NFAT cells were transduced with different combinations of aCAR and iCAR, as detailed in Table 12. Effector Jurkat-NFAT cell lines expressing CD19 aCAR, HLA -A2 iCAR or both, were cultured with target cells expressing either CD19 (Raji cells' in the target ') both CD19 and HLA-A2 (Raji-HLA-A2' outside the tumor ') or HLA-A2 (Thp1' outside the tumor ') as described in Table 13. As a positive control, effector cells were stimulated in the presence of PMA and Ionomycin, which trigger the release of calcium necessary for NFAT signaling. After 16 h of incubation at 37oC, luciferase was quantified using the BPS Biosciences kit “One step luciferase assay system” according to the manufacturer's instructions. As expected, the Jurkat NFAT cell line that expresses the CD19-CAR construct was specifically activated in the presence of the Raji cell line that expresses CD19, while no activation was shown when these cells were co-cultured with the Thp1 cell line that does not express CD19 ( Figure 18).
[00716] [00716] The inhibitory effect of HLA-A2 iCAR on CD19 aCAR-induced NFAT activation can be seen in Figure 21. The Jurkat-NFAT cell line that expresses both CDC aCAR and HLA-A2 iCAR was
[00717] [00717] The effect of different E / T ratios has been tested. The test was repeated several times with E / T ratios of 10: 1, 5: 1, 1: 1. The results given in Figure 20 indicate that an increased inhibitory effect can be obtained with a higher E / T ratio. The results are presented as a ratio of the average luminescence value of the coculture of each effector cell line with target cells 'out of the tumor' to the average value of the coculture with presentation cells 'on the target'. As shown, the Jurkat-NFAT cell line that expresses both CDC aCAR and HLA-A2 iCAR was specifically inhibited when matched with Raji-HLA-A2 expressing CD19 and HLA-A2 proteins, however, no inhibition was detected when this cell was co-cultured with the Raji cell line expressing only CD19. In contrast, the Jurkat-NFAT cell line expressing CD19 aCAR was also activated regardless of the target cell line expressing CD19 with which it was co-cultured (Raji or Raji-HLA-A2). T cell degranulation assay, measured by CD107a staining
[00718] [00718] T-cell degranulation can be identified by the surface expression of CD107a, a membrane protein associated with lysosomes (LAMP-1). The surface expression of LAMP-1 has been shown to correlate with CD8 T cell cytotoxicity. This molecule is located on the luminal side of the lysosomes. Upon activation, CD107a is transferred to the cell membrane surface of activated lymphocytes. CD107a is expressed on the cell surface transiently and is rapidly re-internalized via the endocytic pathway. Therefore, the detection of
[00719] [00719] Transduced T cells will be incubated with target cells for 6-24 h in the presence of monensin and will follow CD107a expression in CD8 T cells by flow cytometry using conjugated antibodies against T cell surface markers (CD3, CD8) and a conjugated antibody to CD107a.
[00720] [00720] Granulation (CD107a) as a marker for the killing potential. The most critical function of cytolytic T cells is the ability to kill target cells. Cytotoxic CD8 + T lymphocytes mediate target cell killing via two main pathways: perforin-granzyme-mediated activation of apoptosis and ligand-mediated induction of apoptosis. The induction of these pathways depends on the release of cytolytic granules from the responding CD8 + T cells. Degranulation is a prerequisite for perforin-granzyme-mediated killing and is necessary for immediate lytic function mediated by responding antigen-specific CD8 + T cells. Cytotoxicity does not require de novo protein synthesis by the effector CD8 + T cell; instead, preformed lytic granules located within the cytoplasm are released in a polarized manner towards the target cell. Lytic granules are secretory lysosomes attached to the membrane that contain a dense nucleus composed of several proteins, including perforin and granzymes. The granule core is surrounded by a lipid bilayer containing numerous lysosome-associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1), CD107b (LAMP-2) and CD63 (LAMP-3). During the degranulation process, the lytic granule membrane fuses with the activated CD8 + T cell plasma membrane and the granule content is then released at the immune synapse between the CD8 + T cell and the target cell. As a result of this process, the membrane
[00721] [00721] PBMC’s transduced with iCAR + aCAR / aCAR constructs (effector cells) are stimulated with either PMA + Ionomycin (Positive Control) or modified target cells that express iCAR + aCAR / aCAR / iCAR antigens on their cell surface. During several hours of coincubation, effector cells degranulate, CD107a can be detected on the cell surface. This expression is transient and CD107a is rapidly reinternalized via the endocytic pathway. Therefore, detection of CD107a is maximized by antibody staining during cell stimulation and by the addition of monensin (to prevent acidification and subsequent degradation of endocyte CD107a antibody complexes). BFA is necessary for optimal cytokine expression. EXAMPLE 9. IN VIVO MODELS In vivo CTL assay in mouse models of human xenografts
[00722] [00722] To test whether T cells expressing both the aCAR and iCAR constructs could discriminate between target cells and 'off-target' cells within the same organism and effectively kill the target cells, while saving the 'off-target' cells will be assessed by a CTL in assay
[00723] [00723] T cells transduced with iCAR or aCAR or both iCAR and aCAR, will be injected i.v. into naïve NOD / SCID / γc mice or similar. Several hours later, target cells that express iCAR, aCAR or both will be injected. These targets will be marked with any CFSE / CPDE or similar cell trace dye in different concentrations (high, medium and low), which will allow for additional discrimination between them. 18 h after the injection of the targets, the mice will be sacrificed, the spleens will be harvested and the elimination of the specific target will be evaluated by FACS. The specific kill percentage will be calculated according to the formula below:   %% pop pop (day 1) (day1)  % pop (day 1)   1 -   ÷ % popmedium (day1)   × 100 high medium high   %% pop    %% pop (day 0) (day0) pop high high (day 0) (day0)     pop medium average de Tumor growth kinetics in mouse models of human xenografts
[00724] [00724] NOD / SCID / γc or similar mice will be inoculated with tumor cells. Inoculation can be i.p. / i.v. or s.c. Tumor cells express either the iCAR target, the aCAR target, or both. An example for a possible aCAR tumor cell line could be NALM 6 CD19 positive (ATCC, human BALL cell line). Example of tumor cells that express both aCAR and iCAR (i.e., 'non-tumor' cells) is NALM 6 engineered to express the iCAR epitope (e.g., HLA-A2), thereby representing healthy cells . NALM 6 and NALM 6-HLA-A2 can also be engineered to express a reporter gene (eg, firefly luciferase), for easy detection. Mice will be divided into several study groups inoculated with all possible combinations of target cells. As an example, one group will be injected with NALM 6 cells, while the other will be injected with NALM-6 which expresses the iCAR epitope. Several days later, while the tumor was already established, the mice will be infused intravenously
[00725] [00725] Transgenic mice that express the targets of human aCAR and iCAR will also be used to determine the effectiveness of the transduced T cells. Under these adjustments, the mice have a fully functional immune system and the potential toxicity of T cells transduced by iCAR / aCAR can be evaluated. The CAR construct will contain scFv that corresponds to human antigens, while signaling domains will be modified to activate or inhibit murine T cells. An example for this model is the HHD-HLA-A2 mice that express only the human HLA-A2 molecule, while all other proteins are murine only. The CD19 aCAR scFv will be targeted in this case
[00726] [00726] Several pairs of preserved and lost allelic variants identified in different tumors are selected and their polypeptide products will serve for the generation of variant-specific mAbs using mAb production techniques. The discriminatory power of candidate mAbs will be assessed by double staining and flow cytometry or immunohistochemistry experiments, as determined by binding to recombinant cell lines that express the selected alleles.
[00727] [00727] All section titles and designations are used for clarity and reference purposes only and will not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects of different titles and sections, as appropriate, in accordance with the spirit and scope of the invention described in this document.
[00728] [00728] All references cited in this document are incorporated by reference in this document in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
[00729] [00729] Many modifications and variations of this order can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific modalities and examples described in this document are offered by way of example only, and the application
242/242 will be limited only by the terms of the appended claims, together with the full scope of equivalents to which the claims are entitled.

权利要求:
Claims (66)
[1]
1. Method to identify a target to prepare an inhibitory chimeric antigen receptor (iCAR) or a protective chimeric antigen receptor (pCAR) capable of preventing or attenuating the unwanted activation of an effector immune cell, in which the target is identified by a method, characterized by the fact that it comprises: (i) identifying a gene with at least two expressed alleles that encode a protein comprising an extracellular polymorphic epitope; (ii) determining that at least one of the expressed alleles exhibits a change in the amino acid sequence in the extracellular polymorphic epitope sequence with respect to an extracellular polymorphic epitope reference sequence; (iii) determining that the gene is located in a chromosomal region that suffers loss of heterozygosity (LOH) in a type of tumor; and (iv) determine that the gene is expressed in the tissue of origin of the tumor type in which the chromosomal region was considered to suffer LOH.
[2]
2. Method according to claim 1, characterized by the fact that the LOH position is selected from the group consisting of substitution, deletion and insertion.
[3]
3. Method according to claim 1, characterized by the fact that the LOH position is a SNP.
[4]
Method according to claim 1, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA gene.
[5]
5. Method according to claim 4, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA gene -K, HLA-L,
2/14 HLA-DM, HLA-DO, HLA-DP, HLA_DQ or HLA-DR.
[6]
6. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-A gene.
[7]
Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-B gene.
[8]
8. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-C gene.
[9]
Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-G gene.
[10]
10. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-E gene.
[11]
11. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-F gene.
[12]
12. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-K gene.
[13]
13. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-L gene.
[14]
14. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-DM gene.
[15]
15. Method according to claim 5, characterized
3/14 due to the fact that the gene comprising the extracellular polymorphic epitope is an HLA-DO gene.
[16]
16. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-DP gene.
[17]
17. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-DQ gene.
[18]
18. Method according to claim 5, characterized in that the gene comprising the extracellular polymorphic epitope is an HLA-DR gene.
[19]
19. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C, CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP, FCAMR, FCER1A, FCGRB2 FCGR3A, FCRL1, FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1, GPR88, HCRTR1, IGSF3, IGSF9, IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013, LDLRR2, LRRR2, LEPR LRRC8B, LRRN2, LY9, MIA3, MR1, MUC1, MXRA8, NCSTN, NFASC, NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4, OR10K1, OR10R2, OR10T2, OR10X1, OR11L1, OR14A2, OR1, OR14A2 OR2G2, OR2G3, OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29, OR2T33, OR2T34, OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2, OR6, OR6, OR6, OR6, OR6, OR6, OR6 OR6Y1, PDPN, PEAR1, PIGR, PLXNA2, PTCH2, PTCHD2, PTGFRN, PTPRC, PTPRF, PVRL4, RHBG, RXFP4,
4/14 S1PR1, SCNN1D, SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7, SLC5A9, TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1, USF, 2
[20]
20. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO, MERTK, NRP2, OR6B2, PLA2R1, PLA2R1, PLA2R1, PLA2R1 PROM2, SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO and TRABD2A.
[21]
21. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3, GABRR3, GP5, GRPR, G27, GRPR HEG1, HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1, LRRC15, LRRN1, MST1R, NAALADL2, NRROS, OR5, OR5AC1, OR5, OR5AC1, OR5AC OR5K3, OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC4A7, SLITRK3, STAB1, SUSD5, TFRC, TLR9, TMR10, TMEM108, TMEM108 ZPLD1.
[22]
22. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ANTXR2, BTC, CNGA1, CORIN,
5/14 EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF1, TEN10 , TLR1, TLR6, TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1 and UNC5C.
[23]
23. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R, IQGAP2, ITGA1, ITGA2, KCNMB1, NPR, LIFR, NIF, NRG2, OR2V1, OR2Y1, OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8, PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCH, PCH, PCH, PCH, PCH, PCH PRLR, SEMA5A, SEMA6A, SGCD, SLC1A3, SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2, SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4 and UGT3A1.
[24]
24. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B, HLA-B, HLA-B, HLA-B, HLA-B, HLA-B HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2,
6/14 NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2, OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROSLCA, SIMA, SDIM1, , TREM1, TREML1 and TREML2.
[25]
25. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A, P2, OR2, OR2, P2, OR2 PLXNA4, PODXL, PTPRN2, PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213, TTYH3, ZAN and ZP3.
[26]
26. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A and TNFRSF10B.
[27]
27. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2, OR1K1, OR1L1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2 and VLDLR.
7/14
[28]
28. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR, SLCRAA, SLC16A9, TSPAN15, UNC5B and VSTM4.
[29]
29. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRPP, MMP26 MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR1, OR1, OR1, OR1, OR1, OR1, OR1, OR1 OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1, OR52E1, OR52E1, OR52E1, OR52E1, OR52 OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5, OR5, OR5, OR5, OR5, OR5, OR5, OR5, OR5 3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1, OR8D2, OR8H1, OR8H2, OR8H3, OR8I2, OR8J1, OR8J2, OR8, OR8, OR8, OR8, OR8, OR8, OR8, OR8 OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12,
8/14 SLCO2B1, SORL1, ST14, SYT8, TENM4, TMEM123, TMEM225, TMPRSS4, TMPRSS5, TRIM5, TRPM5, TSPAN18 and ZP1.
[30]
30. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1, MANSC4, OL1, OR1, OR6, OR1, OR1, OR1 OR6C4, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLCOA, SLCO1, SLCO1, SLCO1, SLCO1 STAB2, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7, TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNPRS8
[31]
31. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6 and TNFRSF19.
[32]
32. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4 and SYNDIG1L.
[33]
33. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is
9/14 selected from the group consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN, IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4, OR4M4, OR4M4 PRTG, RHCG, SCAMP5, SEMA4B, SEMA6D, SLC24A1, SLC24A5, SLC28A1, SPG11, STRA6, TRPM1 and TYRO3.
[34]
34. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B, SLCA, TMZ, SLCA TMC7, TMEM204, TMEM219 and TMEM8A.
[35]
35. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A2, LRRC37A2, LRRC37A2, LRRC37A2, LRRC37A2 OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E, TNFSF12, TRPV3, TTY2.
[36]
36. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15 and TNFRSF11A.
10/14
[37]
37. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5, GFY, GP6, GPR42, GRIN3, GIN42, GRIN3 IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1, LDLR, LILRA1, LILRA2, LILRA, LILA, LILA, LILA, LILA, LILA, LILA MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, PTV, PTY, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, TGFBR3L, TMC4, TMEM91, TMFR, TNFM, TMR9 G10L, VSTM2B and ZNRF4.
[38]
38. Method according to Claim 1, characterized in that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4 and THBD.
[39]
39. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS15,
11/14 TMPRSS2, TMPRSS3, TRPM2 and UMODL1.
[40]
40. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6 and TNFRSF13C.
[41]
41. Method according to claim 1, characterized by the fact that the gene comprising the extracellular polymorphic epitope is selected from the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4 and XG.
[42]
42. Method according to any of claims 1 to 41, characterized in that the tumor is selected from the group consisting of a breast tumor, a prostate tumor, an ovarian tumor, a cervical tumor, a skin tumor, a pancreatic tumor, a colorectal tumor, a kidney tumor, a liver tumor, a brain tumor, lymphoma, leukemia, a lung tumor and a glioma.
[43]
43. Method according to any of claims 1 to 41, characterized in that the tumor is selected from the group consisting of a tumor of the adrenal gland, a kidney tumor, a melanoma, DLBC, a breast tumor, a sarcoma , an ovarian tumor, a lung tumor, a bladder tumor and a liver tumor.
[44]
44. Method according to claim 43, characterized by the fact that the tumor of the adrenal gland is an adrenocortical carcinoma.
[45]
45. The method of claim 43, characterized in that the kidney tumor is a chromophobic renal cell carcinoma.
[46]
46. Method according to claim 43, characterized by the fact that melanoma is uveal melanoma.
[47]
47. Safe effector immune cell, characterized by the fact that
12/14 expresses (i) an iCAR or pCAR of any of claims 1 to 46 and (ii) an activation chimeric antigen receptor (aCAR).
[48]
48. Safe effector immune cell according to claim 47, characterized in that the aCAR is directed against or specifically binds to a tumor-associated antigen or a non-polymorphic cell surface epitope.
[49]
49. Safe effector immune cell according to claim 47, characterized by the fact that aCAR is directed against or specifically binds to a tumor-associated protein, a CAR target as listed in table 1, any cell surface protein that is expressed in a tumor tissue in which iCAR is also expressed.
[50]
50. Safe effector immune cell according to claim 49, characterized by the fact that the non-polymorphic cell surface epitope is selected from the group consisting of CD19, CD20, CD22, CD10, CD7, CD49f, CD56, CD74, CAIX Igκ , ROR1, ROR2, CD30, LewisY, CD33, CD34, CD38, CD123, CD28, CD44v6, CD44, CD41, CD133, CD138, NKG2D-L, CD139, BCMA, GD2, GD3, hTERT, FBP, EGP-2, EGP -40, FR- α, L1-CAM, ErbB2,3,4, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, kRas, MAGE-A1, MUC1 MUC16, PDL1, PSCA, EpCAM, FSHR, AFP, AXL, CD80, CD89, CDH17, CLD18, GPC3, TEM8, TGFB1, NY-ESO-1, WT-1 and EGFR.
[51]
51. Safe effector immune cell according to any of claims 47 to 50, characterized in that the safe effector immune cell is an autologous effector cell or a universal (allogeneic) cell.
[52]
52. Safe effector immune cell according to any of claims 47 to 51, characterized in that the safe effector immune cell is selected from the group consisting of a T cell, a natural killer cell and a cytokine-induced killer cell.
[53]
53. Safe effector immune cell according to any of the
13/14 claims 47 to 52, characterized by the fact that the expression level of iCAR or pCAR is greater than or equal to the expression level of aCAR.
[54]
54. Safe effector immune cell according to any of claims 47 to 53, characterized by the fact that iCAR or pCAR is expressed by a first vector and aCAR is expressed by a second vector.
[55]
55. Safe effector immune cell according to any of claims 47 to 53, characterized by the fact that iCAR or pCAR and aCAR are both expressed by the same vector.
[56]
56. A safe effector immune cell according to claim 55, characterized in that the nucleotide sequence encoding for aCAR is downstream of the nucleotide sequence encoding for iCAR or pCAR.
[57]
57. A safe effector immune cell according to claim 55, characterized in that the nucleotide sequence comprises a viral autoclivable 2A peptide between the nucleotide sequence encoding for aCAR and the nucleotide sequence encoding for iCAR or pCAR.
[58]
58. Safe effector immune cell according to claim 57, characterized by the fact that the viral autoclivable peptide 2A is selected from the group consisting of T2A of Thosea asigna virus (TaV), F2A of the foot and mouth disease virus (FMDV) , E2A from Equine rhinitis A virus (ERAV) and P2A from Porcine teschovirus-1 (PTV1).
[59]
59. A safe effector immune cell according to claim 55, characterized in that the nucleotide sequence encoding aCAR is linked via a flexible linker to iCAR or pCAR.
[60]
60. Safe effector immune cell according to any of claims 47 to 59, characterized by the fact that aCAR comprises at least one signal transducing element that activates or co-stimulates an effector immune cell
14/14
[61]
61. Safe effector immune cell according to claim 60, characterized by the fact that at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to an immunoreceptor tyrosine-based activation motif (ITAM) of, for example, CD3ζ or FcRγ chains.
[62]
62. Safe effector immune cell according to claim 60, characterized in that the at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to an activation killer cell immunoglobulin-like receptor (KIR) , such as KIR2DS and KIR3DS.
[63]
63. Safe effector immune cell according to any one of claims 60, characterized by the fact that the at least one signal transduction element that activates or co-stimulates an effector immune cell is homologous to or an adapter molecule, such as DAP12.
[64]
64. Safe effector immune cell according to claim 60, characterized in that the at least one signal transducing element that activates or co-stimulates an effector immune cell is homologous to or a CD27, CD28 co-stimulating signal transduction element , ICOS, CD137 (4-1BB), CD134 (OX40) or GITR.
[65]
65. Method for treating cancer in a patient having a tumor, with the characteristic of LOH, characterized by the fact that it comprises administering to the patient a safe effector immune cell expressing the iCAR of any of claims 1 to 64.
[66]
66. Method for treating cancer in a patient having a tumor with the characteristic of LOH, characterized by the fact that it comprises administering to the patient a safe effector immune cell of any of claims 47 to 64.
Petition 870200061625, of 18/05/2020, p. 265/345 Figure
Target cells Cells off target
Cytokines T Cell Proliferation 1/79
Temporarily restricted functionality
Cytotoxicity
Petition 870200061625, of 18/05/2020, p. 266/345 Figure
Tumor cell Tumor cell
Co-stimulation 2/79
Heterodimerizer
T cell T cell T cell Without Activation Activation Without Activation
3/79
Figure
Tumor Type
(upstream gene)
4/79
Figure
Tumor Type
5/79
Figure
Tumor Type
(downstream gene)
6/79
Figure
Frequency
Log gene expression
7/79
Figure Loss of HLA protein heterozygosity through cancers
Loss of HLA genes- Loss of HLA-A, B, C genes by ABSOLUTE A, B, C by relative copy through 12 cancers through 12 cancers TCGA (pancan 12) TCGA (pancan 12)
LOH comparison frequency of both methods
If consistent
LOH frequency calculation for 33 tumor types based on relative copy number
Mutated tumor suppressors
Search for suppressors Tumor suppressors Deleted suppressors List of tumor in tumor deleted 6p chromosomes tumor suppressors in 6p
Tumor suppressants cured in the literature (Cancer gene census)
List of tumors with 6p who suffer LOH due to tumor suppressor
8/79
Figure
Proportion of Tumors with HLA-A LOH
Tumor Type
9/79
Figure
LOH spanning the gene
All LOH identified are of this type (n = 588)
Deletion break point in the middle of the gene
No such deletions identified
10/79
Figure
HLA-A LOH Deletions
Length Chr6p Frequency
Number of bases in a deleted segment overlapping HLA-A
11/79
Figure
Combined non-individual
Regression Proportion of individuals with LOH (absolute)
Proportion of individuals with LOH (relative)
12/79
Figure
Tumor Type
13/79
Figure
Tumor Type
14/79
Figure
Tumor Type
15/79
Figure
No deletion of chromosome 6p (selected samples with deletions at the top)
Samples without any change in the number of copies
16/79
Figure
Uveal Melanoma Frequency
LOH proportion
17/79
Figure
TCGA Study Abbreviations
Study Abbreviation Study Name
Acute Myeloid Leukemia Adrenocortical Carcinoma Urothelial Carcinoma of the Bladder Glioma of the Lower Brain Invasic Breast Carcinoma Cervical squamous cell carcinoma and endocervical adenocarcinoma Cholangiocarcinoma Chronic myelogenous cell Adenocarcinoma Chromatographic carcinomama Esophageal cancer Kidney Chromophobe Kidney clear cell carcinoma of the kidneys Renal papillary cell carcinoma of the kidneys Hepatocellular carcinoma of the lung Lung adenocarcinoma Lung squamous cell carcinoma B cell lymphoma Large Diffuse Neoplastic Lymphoid Mesothelioma Miscellaneous Serous Ovarian Cytokine Cell Carcinoma Adeno Carcinoma prostate adenocarcinoma of the rectum sarcoma cutaneous melanoma of the stomach stomach adenocarcinoma of the testicular germ cell tumors thymoma thyroid carcinoma uterine carcino-sarcoma endometrial carcinoma of the body U Uveal Melanoma
18/79
Figure
19/79
Figure
ICAR constructs
ACAR construct
Petition 870200061625, of 18/05/2020, p. 284/345 Figure
Schematic illustration of the IL-2 Secretion assay as measured by ELISA
IL-2 Secretion ELISA Analysis
Effector Cells
Target Cells T Cell T Cell
Non-target cell 20/79
"Normal"
Target Cell Activation “Normal” Inhibition
Target Cell Activation Activation “Cancer”
Petition 870200061625, of 18/05/2020, p. 285/345 Figure
IL-2 secretion as measured by CBA Schematic illustration of the assay
Analysis of CBA of IL-2 Secretion
Effector Cells
Target Cells T Cell T Cell
Non-target cell 21/79
"Normal"
Target Cell Activation “Normal” Inhibition
Conc. of IL-2 in Mean [pg / ml]
Target Cell Activation Activation “Cancer”
22/79
Figure
Without stim 731 Luminescence
23/79
Figure
Without stim 1121 983 Luminescence
24/79
Figure
Response rate to trge T cells
25/79 FIGURE 21
DNA SEQUENCE
26/79
PROTEIN SEQUENCE
PROTEIN SEQUENCE
STRENGTH PROTEIN SEQUENCE
DNA SEQUENCE
27/79
PROTEIN SEQUENCE
PROTEIN SEQUENCE
28/79
SEQUENCE OF HYDROMYCIN RESISTANCE PROTEIN
DNA SEQUENCE
29/79
PROTEIN SEQUENCE
30/79
Figure 22
31/79
Figure 22 (Continued)
32/79
Figure 22 (Continued)
33/79
Figure 22 (Continued)
34/79
Figure 22 (Continued)
35/79
Figure 22 (Continued)
36/79
Figure 22 (Continued)
37/79
Figure 22 (Continued)
38/79
Figure 22 (Continued)
39/79
Figure 23
Position Position
40/79
Figure 23 (Continued)
Position Position
41/79
Figure 23 (Continued)
Position Position
42/79
Figure 23 (Continued)
Position Position
43/79
Figure 23 (Continued)
Position Position
44/79
Figure 23 (Continued)
Position Position
45/79
Figure 23 (Continued) Position Position
46/79
Figure 23 (Continued)
Position Position
47/79
Figure 23 (Continued)
Position Position
48/79
Figure 23 (Continued) Position Position
49/79
Figure 23 (Continued)
Position Position
50/79
Figure 23 (Continued)
Position Position
51/79
Figure 23 (Continued) Position Position
52/79
Figure 23 (Continued)
Position Position
53/79
Figure 23 (Continued)
Position Position
54/79
Figure 23 (Continued)
Position Position
55/79
Figure 23 (Continued)
Position Position
56/79
Figure 23 (Continued) Position Position
57/79
Figure 23 (Continued)
Position Position
58/79
Figure 23 (Continued)
Position Position
59/79
Figure 23 (Continued) Position Position
60/79
Figure 23 (Continued) Position Position
61/79
Figure 23 (Continued)
Position Position
62/79
Figure 23 (Continued) Position Position
63/79
Figure 23 (Continued) Position Position
64/79
Figure 23 (Continued) Position Position
65/79
Figure 23 (Continued)
Position Position
66/79
Figure 23 (Continued)
Position Position
67/79
Figure 23 (Continued) Position Position
68/79
Figure 23 (Continued) Position Position
69/79
Figure 23 (Continued)
Position Position
70/79
Figure 23 (Continued)
Position Position
71/79
Figure 23 (Continued)
Position Position
72/79
Figure 23 (Continued)
Position Position
73/79
Figure 23 (Continued)
Position Position
74/79
Figure 23 (Continued)
Position Position
75/79
Figure 23 (Continued)
Position Position
76/79
Figure 23 (Continued)
Position Position
77/79
Figure 23 (Continued)
Position Position
78/79
Figure 23 (Continued)
Position Position
79/79
Figure 23 (Continued)
Position Position

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法律状态:
2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762564454P| true| 2017-09-28|2017-09-28|

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US62/564,454|2017-09-28|

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US201862649429P| true| 2018-03-28|2018-03-28|

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US62/649,429|2018-03-28|

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PCT/US2018/053583|WO2019068007A1|2017-09-28|2018-09-28|A universal platform for preparing an inhibitory chimeric antigen receptor |

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