T cell modifying compounds and uses thereof

An isolated genetically modified human T-cell that comprises:

• (I) an exogenous sequence encoding a chimeric antigen receptor (CAR) integrated into an endogenous programmed death receptor PDCD1 (PD1) or CTLA-4 gene;
• and
• (II)
• (a) a pair of zinc finger nucleases (ZFNs) that cleave the PD1 gene, the pair comprising:
• (i) ZFN 12942 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• QSGHLSR (SEQ ID NO:34);
• F2:
• RSDSLSV (SEQ ID NO:35);
• F3:
• HNDSRKN (SEQ ID NO:36);
• F4:
• RSDDLTR (SEQ ID NO:37);
• and
• F5:
• RSDHLTQ (SEQ ID NO:38);
• and
• ZFN 12946 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• RSAALSR (SEQ ID NO:39);
• F2:
• RSDDLTR (SEQ ID NO:37);
• F3:
• RSDHLTT (SEQ ID NO:40);
• F4:
• DRSALSR (SEQ ID NO:6);
• and
• F5:
• DRSALAR (SEQ ID NO:41);
• or
• (ii) ZFN 12942 and
• ZFN 12947 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• RSAALAR (SEQ ID NO:42);
• F2:
• RSDDLSK (SEQ ID NO:3);
• F3:
• RNDHRKN (SEQ ID NO:43);
• F4:
• DRSALSR (SEQ ID NO:6);
• and
• F5:
• DRSALAR (SEQ ID NO:41);
• or
• (iii) ZFN 12934 comprising four zinc finger domains designated F1 to F4, each domain comprising a recognition helix region ordered as follows:
• F1:
• RSDHLSE (SEQ ID NO:44);
• F2:
• TSSDRTK (SEQ ID NO:45);
• F3:
• RSDHLSE (SEQ ID NO:44);
• and
• F4:
• QSASRKN (SEQ ID NO:46);
• and
• ZFN 12971 comprising six finger domains designated F1 to F6, each domain comprising a recognition helix region ordered as follows:
• F1:
• RSDVLSE(SEQ ID NO:47);
• F2:
• RSANLTR (SEQ ID NO:48);
• F3:
• RSDHLSQ (SEQ ID NO:49);
• F4:
• TSSNRKT (SEQ ID NO:50);
• F5:
• DRSNLSR (SEQ ID NO:9);
• and
• F6:
• RSDALAR (SEQ ID NO:7);
• or
• (iv) ZFN 12934 and
• ZFN 12972 comprising six zinc finger domains designated F1 to F6, each domain comprising a recognition helix region as follows:
• F1:
• DDWNLSQ (SEQ ID NO:51);
• F2:
• RSANLTR (SEQ ID NO:48);
• F3:
• RSDHLSQ (SEQ ID NO:49);
• F4:
• TSSNRKT (SEQ ID NO:50);
• F5:
• DRSNLSR (SEQ ID NO:9);
• and
• F6:
• RSDALAR(SEQ ID NO:7);
• or
• (v) ZFN 12942 and
• ZFN 25029 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• RNAALTR (SEQ ID NO:73);
• F2:
• RSDELTR (SEQ ID NO:57);
• F3:
• RHHHLAA (SEQ ID NO:77);
• F4:
• TRPVLKR (SEQ ID NO:54);
• and
• F5:
• DRSALAR (SEQ ID NO:41);
• or
• (b) a pair of transcription activator-like effector nucleases (TALENs) that cleave the PD1 gene, wherein the pair of TALENs comprises:
• (i) a TALEN comprising 17 TALE repeat units comprising the following repeat variable diresidues (RVDs):
• NI-NN-HD-NI-HD-HD-NN-HD-HD-HD-NI-NN-NI-HD-NN-NI-HD and
• a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NN-HD-NG-HD-HD-NI-NN-NN-HD-NI-NG-NN-HD-NI-NN-NI-NG;
• or
• (ii) a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NG-NN-NG-NI-NN-HD-NI-HD-HD-NN-HD-HD-HD-NI-NN-NI-HD;
• and
• a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NN-HD-NG-HD-HD-NI-NN-NN-HD-NI-NG-NN-HD-NI-NN-NI-NG or a TALEN comprising 15 TALE repeat units comprising the following RVDs:
• NN-HD-NI-NN-NI-NG-HD-HD-HD-NI-HD-NI-NN-NN-HD;
• or
• (iii) a TALEN comprising 16 TALE repeat units comprising the following RVDs:
• NG-NN-NG-NI-NN-HD-NI-HD-HD-NN-HD-HD-HD-NI-NN-NI;
• and
• a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NN-HD-NG-HD-HD-NI-NN-NN-HD-NI-NG-NN-HD-NI-NN-NI-NG or a TALEN comprising 15 TALE repeat units comprising the following RVDs:
• NN-HD-NI-NN-NI-NG-HD-HD-HD-NI-HD-NI-NN-NN-HD;
• or
• (iv) a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NN-NINI-NN-NN-NG-NN-NN-HD-NN-NG-NG-NN-NG-HD-HD-HD;
• and
• a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NG-HD-NG-HD-HD-HD-HD-NI-NN-HD-HD-HD-NG-NN-HD-NG-HD;
• or
• (v) a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• HD-NG-HD-NG-NG-NG-NN-NI-NG-HD-NG-NN-HD-NN-HD-HD-NG;
• and
• a TALEN comprising 17 TALE repeat units comprising the following RVDs:
• NI-HD-HD-NG-HD-NG-NN-NG-NN-NN-NN-NN-HD-HD-NI-NG-HD;
• or
• (c) a pair of ZFNs that cleave the CTLA-4 gene, the pair comprising:
• (i) ZFN 20186 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• QSSDLSR (SEQ ID NO:1);
• F2:
• RSDNLRE (SEQ ID NO:2);
• F3:
• RSDDLSK (SEQ ID NO:3);
• F4:
• QSSDLRR (SEQ ID NO:4);
• and
• F5:
• LKQHLNE (SEQ ID NO:5);
• and
• ZFN 20185 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• DRSALSR (SEQ ID NO:6);
• F2:
• RSDALAR (SEQ ID NO:7);
• F3:
• QSGDRNK (SEQ ID NO:8);
• F4:
• DRSNLSR (SEQ ID NO:9);
• and
• F5:
• RSDDRKT (SEQ ID NO:10);
• or
• (ii) ZFN 20190 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• QSGSLTR (SEQ ID NO:11);
• F2:
• RSDNLTT (SEQ ID NO:12);
• F3:
• QNATRIK (SEQ ID NO:13);
• F4:
• RSDVLSA (SEQ ID NO:14);
• and
• F5:
• DRSNRIK (SEQ ID NO:15);
• and
• ZFN 20189 comprising five zinc finger domains designated F1 to F5, each domain comprising a recognition helix region ordered as follows:
• F1:
• RSANLAR (SEQ ID NO:16);
• F2:
• TNQNRIT (SEQ ID NO:17);
• F3:
• TSGHLSR (SEQ ID NO:18);
• F4:
• RSDSLLR (SEQ ID NO:19);
• and
• F5:
• RNDDRKK (SEQ ID NO:20).