COMPOSITION AND METHOD FOR REDUCING EXPRESSION OF CHECKPOINT INHIBITORS IN T CELLS EXPRESSING A CAR OR CTL

Abstract

Described herein is an approach for targeting multiple critical checkpoint genes involved in T cell exhaustion, for example, PD-1, TIM3, and LAG-3 in a manner that allows knock-down of their expression in T cells that also express a T cell receptor targeted to cancer cells, e.g., a CAR targeted to a cancer antigen or a T cell receptor (TCR).

Claims

1. A nucleic acid vector comprising an shRNA sequence targeted to an mRNA encoding a checkpoint inhibitor selected from the group consisting of PD-1, TIM3 and LAG3 and a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

2. The nucleic acid vector of claim 1, wherein the CAR targets an antigen selected from the group consisting of IL-13Ra, HER2, CD19 and PSCA.

3. The nucleic acid vector of claim 1, wherein each shRNA sequence is operably linked to a promoter.

4. The nucleic acid vector of claim 3, wherein each shRNA sequence is operably linked to the same promoter.

5. The nucleic acid vector of claim 1, wherein the vector comprises two or more different shRNA nucleic acid sequences targeted to the same mRNA encoding a checkpoint inhibitor.

6. The nucleic acid vector of claim 1, wherein each shRNA sequence is targeted to a different mRNA encoding a checkpoint inhibitor.

7. The nucleic acid vector of claim 1, wherein the vector comprises an shRNA sequence targeted to an mRNA encoding PD-1 and an shRNA sequence targeted to an mRNA encoding TIM3.

8. The nucleic acid vector of claim 1, wherein the vector comprises an shRNA sequence targeted to an mRNA encoding PD-1 and an shRNA sequence targeted to an mRNA encoding LAG3.

9. The nucleic acid vector of claim 1, wherein the vector comprises an shRNA sequence targeted to an mRNA encoding TIM3 and an shRNA sequence targeted to an mRNA encoding LAG3.

10. The nucleic acid vector of claim 3, wherein each shRNA sequence is operably linked to a different promoter.

11. The nucleic acid vector of claim 3, comprising two or more different shRNA sequences that are operably linked to the same promoter.

12. The nucleic acid vector of claim 3, wherein the promoter is selected from the group consisting of an H1 DNA POL III promoter, an U6 DNA POL III promoter, and a 75K DNA POL III.

13. The nucleic acid vector of claim 1, wherein the shRNA targeted to an mRNA encoding PD-1 is targeted to SEQ ID NO:25.

14. The nucleic acid vector of claim 1, wherein the shRNA targeted to an mRNA encoding TIM3 is targeted to SEQ ID NO:26.

15. The nucleic acid vector of claim 1, wherein the shRNA targeted to an mRNA encoding LAG3 is targeted to SEQ ID NO:27.

16. The nucleic acid vector of claim 1, wherein the shRNA targeted to PD-1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos 12-14.

17. The nucleic acid vector of claim 1, wherein the shRNA targeted to TIM3 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos: 15-17.

18. The nucleic acid vector of claim 1, wherein the shRNA targeted to LAG3 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos: 18-20.

19. The nucleic acid vector of claim 1, wherein each shRNA sequence comprises: sense sequence and an antisense sequence, wherein the sense and the antisense sequence, wherein the sense sequence comprises a nucleotide sequence identical to a target sequence in an mRNA encoding a checkpoint inhibitor.

20. The nucleic acid vector of claim 1, wherein the vector is a lentiviral vector.

21. A nucleic acid vector comprising an H1 DNA POL III promoter, an U6 DNA POL III promoter, and a 75K DNA POL III promoter, where each promoter is operably linked to an shRNA nucleic acid sequence targeted to an mRNA encoding a checkpoint inhibitor.

22. The nucleic acid vector of claim 21, wherein each shRNA nucleic acid sequence is targeted to the same mRNA encoding a checkpoint inhibitor.

23. The nucleic acid vector of claim 21, wherein each shRNA is targeted to a different mRNA encoding a checkpoint inhibitor.

24. The nucleic acid vector of claim 21, wherein the checkpoint inhibitor is selected from the group consisting of PD-1, TIM3 and LAG3.

25. The nucleic acid vector of claim 21, wherein the shRNA targeted to an mRNA encoding PD-1 is targeted to SEQ ID NO:25.

26. The nucleic acid vector of claim 21, wherein the shRNA targeted to an mRNA encoding TIM3 is targeted to SEQ ID NO:26.

27. The nucleic acid vector of claim 21, wherein the shRNA targeted to an mRNA encoding LAG3 is targeted to SEQ ID NO:27.

28. The nucleic acid vector of claim 25, wherein the shRNA targeted to PD-1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos 12-14.

29. The nucleic acid vector of claim 26, wherein the shRNA targeted to TIM3 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos: 15-17.

30. The nucleic acid vector of claim 27, wherein the shRNA targeted to LAG3 comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos: 18-20.

31. A nucleic acid vector of claim 1, wherein the vector comprises the nucleotide sequence of SEQ ID NO:24.

32. The nucleic acid vector of claim 1, wherein each shRNA sequence comprises: sense sequence and an antisense sequence, wherein the sense and the antisense sequence, wherein the sense sequence comprises a nucleotide sequence identical to a target sequence in an mRNA encoding a checkpoint inhibitor.

33. The nucleic acid vector of claim 1, wherein the vector is a lentiviral vector.

34. The nucleic acid vector of claim 21, wherein the vector further comprises a nucleotide sequence encoding a chimeric antigen receptor.

35. The nucleic acid vector of claim 34, wherein the CAR targets an antigen selected from the group consisting of: PSCA, HER2 and CD19.

36. The nucleic acid vector of claim 44, wherein the CAR comprises, from amino to carboxy terminus: a targeting domain for targeting an antigen; a spacer domain; a transmembrane domain; a co-stimulatory domain; and a CD3ζ signaling domain.

37. A T cell harboring the nucleic acid vector of claim 1.

38. A method for reducing the expression of a checkpoint inhibitor in T cell, the method comprising introducing the nucleic acid vector of claim 1 into the T cell.

Description

DESCRIPTION OF DRAWINGS

[0011] FIG. 1 depicts the results of an experiment showing that anti-PD1 antibody, Nivolumab, induces compensatory upregulation of TIM3 in T cells. CD4+ T cells were co-cultured with in vitro differentiated CD14+ dendritic cells, and treated with 0.1, 1, 10 μg/mL of Nivolumab. After 5 days, T cells were analyzed by flow cytometry for cell surface expression of exhaustion markers, PD-1, TIM3, LAG3, and CTLA-4.

[0012] FIG. 2 depicts the results of a study assessing the impact of PSCA-targeted CAR on advanced prostate cancer. (a) Illustration of PSCA-CAR design with PSCA(ΔCH2)BBζ with an anti-PSCA scFv tethered to the membrane by a modified IgG4 Fc linker (deleted CH2 domain), containing a CD4 transmembrane domain, an intracellular 4-1BB co-stimulatory domain and a CD3ζ cytolytic domain. The T2A skip sequence separates the CAR from a truncated CD19 (CD19t) protein for cell tracking. (b-c) NSG male mice bearing PC-3 (c) and DU145 (d) tumors over-expressing human PSCA were treated with a single intravenous dose of 1, 2.5 or 5×106 PSCA(ΔCH2)BBζ CAR or 5×10.sup.6 Mock (untransduced) T cells, and tumor volume was monitored by caliper measurement. (d-e) PC-3-PSCA (d) and DU145-PSCA (e) tumors were stained for CD3 (human T cells), PD-1, and PD-L1 expression by immunohistochemistry, and representative images (20× magnification) are shown from Mock (untransduced) and PSCA (ΔCH2)BBζ CAR T cell-treated mice.

[0013] FIG. 3 depicts the amino acid sequence of the PSCA CAR used in the studies described herein.

[0014] FIG. 4 depicts the results of a study assessing the impact of anti-PD-1 antibody blockade in combination with PSCA CAR T cells in vitro. (a) IFNγ production quantified by ELISA in supernatants from Mock (untransduced) or PSCA-CAR T cells cultured overnight with DU145 or DU145-PSCA tumor cells in the presence or absence of indicated concentrations of anti-human PD-1 antibody Nivolumab. (b) DU145-PSCA tumor killing (left), and PD-1 (middle) and 4-1BB expression in PSCA-CAR T cells (right), following a 4 and 8 day co-culture in the presence or absence of Nivolumab. (c) NSG male mice bearing DU145-PSCA tumors were treated with a single intravenous dose of 1×10.sup.6 PSCA(ΔCH2)BBζ CAR or Mock (untransduced) T cells, and treated with 4 doses with 100 ug Nivolumab, and tumor volume was monitored by caliper measurement. (d) Percentage of human CD3+ cells (top) and PD-1 expression in tumor-infiltrating T cells from mice treated with PSCA-CAR T cells alone or with Nivolumab.

[0015] FIG. 5 depicts the results of a study examining the impact of M1 and M2 macrophages on PD-L1 expression and PSCA-CAR T cell mediated anti-tumor responses in vitro (a) Schematic of assay used. Macrophages, CAR T cells, and tumors were co-cultured for 6 or 10 days, and evaluated for functional assays using flow cytometry. (b) Representative bright field images of M1 and M2 macrophages. (c) Phenotypes of M1 and M2 macrophages using indicated cell surface markers, evaluated by flow cytometry. (d) CAR T cell killing of DU145-PSCA tumor cells in the presence or absence of M1 or M2 macrophages. (e) Mock (untransduced) and PSCA-CAR T cell proliferation and activation (indicated by expression of 4-1BB) in the presence or absence of M1 and M2 macrophages. (f) PD-L1 expression in DU145-PSCA tumor cells and M1 or M2 macrophages following co-culture with Mock or PSCA-CAR T cells.

[0016] FIG. 6 depicts the results of a study examining the impact of the HUSKY vector. (a) Diagram of pHIV7-backbone with HUSKY cassette and GFP reporter. (b) HEK293T cells were co-transfected with a dual luciferase plasmid (Firefly and Renilla) and one of three HUSKY cassette vectors containing shRenilla driven by H1, U6, or 7SK promoters. Empty HUSKY was used as a negative control. Cells were analyzed for luminescence signal 48 hours post-transfection and the Renilla/Firefly ratio determined. Results with p<0.05 were considered significant by one-way ANOVA analysis N=3.

[0017] FIG. 7 depicts the results of an analysis of HUSKY-mediated knockdown of PD1, TIM3, and LAG3. HEK293T cells were co-transfected with a dual luciferase plasmid containing either the 100 bp sense or antisense targets for each shRNA in the Renilla 3′UTR and a HUSKY vector containing one of three candidate 7SK-driven shPD1, H1-driven shTIM3, or U6-driven shLAG3. HUSKY-shRenilla vectors for each promoter were used as positive controls and empty HUSKY as a negative control. Sense vs. antisense target strand knockdown was assessed. Cells were analyzed for luminescence signal 48 hours post-transfection and the Renilla/Firefly ratio determined. Results with p<0.05 were considered significant by one-way ANOVA analysis. N=6.

[0018] FIG. 8 depicts the results of an examination of knock-down of PD-1 by HUSKY shRNA constructs in HEK 293T-PD-1 cells. PD-1 and GFP were overexpressed in 293T cells by lentivirus transduction, and double positive cells were obtained by FACS. HEK 293T-PD-1 cells were used to screen shRNA candidates against PD-1 by Lipofectamine transfection of plasmids with shRNA candidates on day 0, and on day 5, PD-1 expression was evaluated by flow cytometry.

[0019] FIG. 9 depicts the nucleotide sequence of the HUSKY cassette without any snRNA inserts SEQ ID NO: 24). The H1 (SEQ ID NO: 21), U6 (SEQ ID NO: 22) and 7SK (SEQ ID NO:23) promoters are indicated with italics and underlining.

DETAILED DESCRIPTION

[0020] An example of a HUSKY cassette with three promoters for expression of shRNA targeted to checkpoint inhibitors is depicted in FIG. 9.

[0021] A HUSKY cassette can include and shRNA sequence targeted to Human PD-1 (Genbank NM_005018) mRNA (SEQ ID NO:25).

[0022] The HUSKY cassette can include and shRNA sequence targeted to Human TIM3 (Genbank NM_032782) mRNA (SEQ ID NO: 26).

[0023] The HUSKY cassette can include and shRNA sequence targeted to Human LAG3 (Genbank NM_002286) mRNA (SEQ ID NO:27).

[0024] HUSKY cassette can be used to reduce expression of one or more checkpoint inhibitors, e.g., one or more (e.g., all) of PD-1, TIM3 and LAG3 in T cells, e.g., T cells expressing a CAR. Reduced expression of one or of PD-1, TIM3 and LAG3 can be useful in conjunction with expression of a CAR or a TCR targeted to a cancer antigen, for example, PSCA, CD19 or HER2. Suitable CAR include, but are not limited to, those described in: WO 2017/079694 (HER2); WO 2017/062628 (PSCA); and US 2016/0340649 (IL-13Ralpha2). Each CAR includes a targeting sequence, which can e an svFv or a receptor ligand; a spacer sequence, a transmembrane domain, a co-stimulatory domain and a CD3 zeta domain. Examples of each are provided below.

scFv Sequences

[0025] A variety of scFv targeting sequences can be used CAR. Suitable sequences for targeting PSCA

TABLE-US-00001 include: (SEQ ID NO: 28) DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAP KRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQWGSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEVQLV EYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWV AWIDPENGDTEFVPKFQGRATMSADTSKNTAYLQMNSLRAEDT AVYYCKTGGFWGQGTLVTVSS.

[0026] Suitable sequences for targeting CD19 include:

TABLE-US-00002 FMC63 scFv: (SEQ ID NO: 29) IPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLI YHTSRLHSGV PSRFSGSGSGTDYSLTIS NLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKP GSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTII KDN SKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSS. FMC63 VL: (SEQ ID NO: 30) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ EDIATYFCQQGNTLPYTFGGGTKLEIT. FMC63 VH: (SEQ ID NO: 31) EVK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN SKSQV FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTV SS.

[0027] Additional scFv that bind CD19 are described in US 2016/0152723 and in WO 2016/033570.

Spacer Region

[0028] The CAR can include a spacer located between the targeting domain (e.g., the scFv) and the transmembrane domain. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can be used.

TABLE-US-00003 TABLE 1 Examples of Spacers Name Length Sequence a3   3 aa AAA linker  10 aa GGGSSGGGSG (SEO ID NO: 32) IgG4 hinge (S.fwdarw.P)  12 aa ESKYGPPCPPCP (SEQ ID NO: 33) (S228P) IgG4 hinge  12 aa ESKYGPPCPSCP (SEQ ID NO: 34) IgG4 hinge  22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 35) (S228P) + linker CD28 hinge  39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 36) CD8 hinge-48aa  48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACD (SEQ ID NO: 37) CD8 hinge-45aa  45 aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 38) IgG4(HL-CH3) 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQV (includes S228P SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS in hinge) RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 39) IgG4 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVV (L235E, N2970) DVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFQSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 40) IgG4(S228P, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVV L235E, N2970) DVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFQSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41) IgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 42)

[0029] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0030] The hinge/linker region can also comprise a IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:34) or ESKYGPPCPPCP (SEQ ID NO:33).

[0031] The hinge/linger region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO:33) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:32) followed by IgG4 CH3 sequence GQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:41). Thus, the entire linker/spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:______) In some cases, the spacer has 1,2,3,4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO:______. In some cases, the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).

Transmembrane Domain

[0032] A variety of transmembrane domains can be used in CAR. Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain is located carboxy terminal to the spacer region.

TABLE-US-00004 TABLE 2 Examples of Transmembrane Domains Name Accession Length Sequence CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 43) CD28 NM_006139 27 aa FWVLVVVGGVLACYSLLVTVAFII FWV (SEQ ID NO: 44) CD28(M) NM_006139 28 aa MFWVLVVVGGVLACYSLLVTVAFI IFWV (SEQ ID NO: 45) CD4 M35160 22 aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 46) CD8tm NM_001768 21 aa IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 47) CD8tm2 NM_001768 23 aa IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 48) CD8tm3 NM_001768 24 aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 49) 41BB NM_001561 27 aa IISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO: 50)

Costimulatory and CD3zeta Domain

[0033] The costimulatory domain can be any domain that is suitable for use with a CD3ζ signaling domain. In some cases, the costimulatory domain is a CD28 costimulatory domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:52; LL to GG amino acid change double underlined). In some cases, the CD28 co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative and preferably not in the underlined GG sequence) compared to SEQ ID NO:23. In some cases the co-signaling domain is a 4-1BB co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:54). In some cases, the 4-1BB co-signaling domain has 1, 2, 3, 4 or 5 amino acid changes (preferably conservative) compared to SEQ ID NO:54.

[0034] The costimulatory domain(s) are located between the transmembrane domain and the CD3ζ signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3ζ signaling domain.

TABLE-US-00005 TABLE 3 CD3ζ Domain and Examples of Costimulatory Domains Name Accession Length Sequence CD3ζ J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 51) CD28 NM_006139  42 aa RSKRSRLLHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRS (SEQ ID NO: 52) CD28gg* NM_006139  42 aa RSKRSRGGHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRS (SEQ ID NO: 53) 4-1BB NM_001561  42 aa KRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCEL (SEQ ID NO: 54) OX40  42 aa ALYLLRRDQRLPPDAHKPPGGGSFRT PIQEEQADAHSTLAKI (SEQ ID NO: 55)

[0035] In various embodiments: the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present. In some embodiments there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. The costimulatory domain is amino terminal to the CD3ζ signaling domain and in some cases a short linker consisting of 2-10, e.g., 3 amino acids (e.g., GGG) is positioned between the costimulatory domain and the CD3ζ signaling domain.

CD3ζ Signaling Domain

[0036] The CD3ζ Signaling domain can be any domain that is suitable for use with a CD3 ζ signaling domain. In some cases, the CD3ζ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGRDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:51). In some cases, the CD3ζ signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:51.

Truncated EGFR

[0037] The CD3ζ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:56) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNKHFKNCTSISGDLHILPVAFRGD SFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRG PDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO:57). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:57.

[0038] An amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence. An “amino acid substitution” or “substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

[0039] The CAR can include a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to the amino acid sequence depicted in FIG. 3 (SEQ ID Nos:______), either including or excluding the GMCSFRa signal sequence and either including or excluding the T2A ribosomal skip sequence and the truncated EGFRt).

[0040] In some cases, the CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail. In this arrangement, co-expression of EGFRt provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt incorporated in the CAR lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity.

[0041] The vectors can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line.

[0042] Various T cell subsets isolated from the patient can be transduced with a vector for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+/CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3/CD28, transduced with, for example, a lentiviral vector that directs the expression of the CAR as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated/genetically modified central memory T cells can be expanded in vitro with IL-2/IL-15 and then cryopreserved.

EXAMPLES

Example 1: Impact of Targeting PD1 on Expression of Checkpoint Inhibitors

[0043] Efforts to target a checkpoint inhibitor can lead to upregulation of one or more other checkpoint inhibitors. Nivolumab, an anti-PD1 antibody can cause upregulation of TIM3. In this study, the results of which are depicted in FIG. 1, CD4+ T cells were co-cultured with in vitro differentiated CD14+ dendritic cells, and treated with 0.1, 1, 10 μg/mL of Nivolumab. After 5 days, T cells were analyzed by flow cytometry for cell surface expression of exhaustion markers, PD-1, TIM3, LAG3, and CTLA-4.

Example 2: Evaluation of Anti-PD-1 Antibody Blockade in Combination with PSCA CAR T Cells In Vitro and In Vivo

[0044] A CAR (PSCA(ΔCH2)BBζ) CAR targeting PSCA was developed. This CAR is depicted schematically in FIG. 2A. The PSCA(ΔCH2)BBζ includes: the MB1 scFv targeting PSCA, IgG4(HL-CH3) spacer, lacks the CH2 domain and has a short linker sequence located between the hinge region and the CH3 region, a CD4 transmembrane domain, the 4-1BB co-stimulatory domain and the CD3ζ cytoplasmic signaling domain. The CAR coding sequence is followed by the T2A ribosomal skip sequence and a truncated CD19 sequence to permit co-expression of surface, signaling incompetent, truncated CD19 as a marker. The complete amino acid sequence of this CAR is depicted in FIG. 3, with the various domains indicated.

[0045] To assess impact on tumor growth, NSG male mice bearing PC-3 (FIG. 2B) and DU145 (FIG. 2C) tumors over-expressing human PSCA were treated with a single intravenous dose of 1, 2.5 or 5×10.sup.6 PSCA(ΔCH2)BBζ CAR or 5×10.sup.6 Mock (untransduced) T cells, and tumor volume was monitored by caliper measurement. To asses CD3, PD-1 and PD-L1 expression, PC-3-PSCA (FIG. 2D) and DU145-PSCA (FIG. 2E) tumors were stained for CD3 (human T cells), PD-1, and PD-L1 expression by immunohistochemistry, and representative images (20× magnification) are shown from Mock (untransduced) and PSCA (ΔCH2)BBζ CAR T cell-treated mice.

[0046] The impact of the anti-human PD-1 antibody Nivolumab and PSCA (ΔCH2)BBζ CAR T was examined by measuring IFNγ production in Mock (untransduced) or PSCA-CAR T cells cultured overnight with DU145 or DU145-PSCA tumor cells in the presence or absence of indicated concentrations of Nivolumab. As can be seen in FIG. 4A, IFNγ production was increased at 1 μg/ml Nivolumab, but decreased at higher concentrations. DU145-PSCA tumor killing, PD-1 expression and 4-1BB expression in PSCA-CAR T cells following a 4 and 8 day co-culture in the presence or absence of Nivolumab was also measured. The results of this analysis are shown in FIG. 4B. NSG male mice bearing DU145-PSCA tumors were treated with a single intravenous dose of 1×10.sup.6 PSCA(ΔCH2)BBζ CAR or Mock (untransduced) T cells, and treated with 4 doses with 100 ug Nivolumab, and tumor volume was monitored by caliper measurement. The results of this study are shown in FIG. 2C. In addition, both the percentage of human CD3+ cells and PD-1 expression in tumor-infiltrating T cells from mice treated with PSCA-CAR T cells alone or with Nivolumab was assessed (FIG. 2D).

[0047] The impact of M1 and M2 macrophages on PD-1 expression and PSCA-CAR T cell mediated anti-tumor responses in vitro was examined. Exposure of human monocytes to Th1 response-promoting IFN-γ or tumor necrosis factor alpha as well as the endotoxin lipopolysaccharide (sometimes referred to as “classical activation”) leads to M1 macrophages that function to produce pro-inflammatory mediators that provide host protection against bacteria and viruses. Human monocytes can also be differentiated to M2 macrophages by encountering Th2 response-promoting cytokines such as interleukin-10 and transforming growth factor-β (sometimes referred to as “alternative activation”). M2 macrophages express high levels of CD206 (mannose receptor) and CD163, produce low levels of pro-inflammatory cytokines, and promote wound healing and matrix remodeling.

[0048] FIG. 5A is a schematic depiction of the assay used. The M1 and M2 macrophages were differentiated as described in Zarif et al. (Biotechniques 61:33, 2016). Macrophages, CAR T cells, and tumors were co-cultured for 6 or 10 days, and evaluated for functional assays using flow cytometry. Representative bright field images of M1 and M2 macrophages are shown in FIG. 5B and the phenotypes of M1 and M2 macrophages as assessed by certain cell surface markers are shown in FIG. 5C. PSCA(ΔCH2)BBζ CAR T cell killing of DU145-PSCA tumor cells in the presence or absence of M1 or M2 macrophages was assessed and the results are shown in FIG. 5D. PSCA-CAR T cell proliferation and activation (indicated by expression of 4-1BB) in the presence or absence of M1 and M2 macrophages is shown in FIG. 5E. Finally, PD-L1 expression in DU145-PSCA tumor cells and M1 or M2 macrophages following co-culture with Mock or PSCA-CAR T cells was assessed and the results of the study are shown in FIG. 5F. These studies show that M2 macrophages inhibit PSCA(ΔCH2)BBζ CAR T cell killing of DU145-PSCA and induce PDL-1 expression.

Example 3: Vector for Knock-Down of PD-1, TIM3 and LAG3 Expression

[0049] To create a vector useful for reducing expression of certain checkpoint inhibitors in cells expressing a CAR. shRNA sequences targeting PD-1, TIM-3, and LAG-3 were designed and used to create a cassette that can be installed in a lentiviral vector. Several potential shRNAs for each of PD-1, TIM-3, and LAG-3 were created and assessed. The shRNA are expressed under the control of three independent DNA Polymerase III promoters: the H1 promoter, the U6 promoter, and the 7SK promoter. The lentiviral vector has a pHIV7-backbone, a GFP reporter, and the H1, U6, and 7SK promoter cassette (referred to as “HUSKY”) with digestion sites for shRNAs following each promoter. The vector is schematically depicted in FIG. 6A and the sequence of the expression cassette without shRNA inserts in FIG. 9.

[0050] In order to assess each promoter in the cassette, HEK293T cells were co-transfected with a dual luciferase plasmid (Firefly and Renilla) and one of three HUSKY cassette vectors, each containing shRenilla driven by the H1 promoter, the U6 promoter, or the 7SK promoter. Empty HUSKY was used as a negative control. The results of this study are shown in FIG. 6B.

[0051] For each of the three targets, three different shRNA sequences were designed: PD1-C, -D and -E; TIM3-1, -2 and -3; and LAG3-A, -B and -C. HEK293T cells were co-transfected with a dual luciferase plasmid containing either the 100 bp sense or antisense targets for each shRNA in the Renilla 3′UTR and a HUSKY vector containing one of three candidate 7SK-driven shPD1, H1-driven shTIM3, or U6-driven shLAG3. HUSKY-shRenilla vectors for each promoter were used as positive controls and empty HUSKY as a negative control. Sense and antisense target strand knockdown was assessed. The results are presented in FIG. 7A-C. The shRNA sequences are in Table 4.

TABLE-US-00006 TABLE 4 snRNA Sequences Target Name Sequence PD-1 PD1-C CCAACACATCGGAGAGCTTCGTTGTGTACGAAG CTCTCCGATGTGTTGG (SEQ ID NO: 12) PD1-D GAGTATGCCACCATTGTCTTTTTGTGTAAAAGA CAATGGTGGCATACTC (SEQ ID NO: 13) PDI-E CGTCCAGCTCCCTGAATCTCTTTGTGTAAGAGA TTCAGGGAGCTGGACG (SEQ ID NO: 14) TIM3 TIM3-1 CGTGGACCAAACTGAAGCTATTTGTGTAATAGC TTCAGTTTGGTCCACG (SEQ ID NO: 15) TIM3-2 GCACTGAACTTAAACAGGCATTTGTGTAATGCC TGTTTAAGTTCAGTGC (SEQ ID NO: 16) TIM3-3 CAAATGCAGTAGCAGAGGGAATTGTGTATTCCC TCTGCTACTGCATTTG (SEQ ID NO: 17) LAG3 LAG3-A GTGACTGGAGCCTTTGGCTTTTTGTGTAAAAGC CAAAGGCTCCAGTCAC (SEQ ID NO: 18) LAG3-B GGATCTCAGCCTTCTGCGAAGTTGTGTACTTCG CAGAAGGCTGAGATCC (SEQ ID NO: 19) LAG3-C GCAAGATAGAGGAGCTGGAGCTTGTGTAGCTCC AGCTCCTCTATCTTGC (SEQ ID NO: 20)

[0052] To assess knock-down of PD-1 by HUSKY shRNA constructs, PD-1 and GFP were overexpressed in 293T cells by lentivirus transduction, and double positive cells were obtained by FACS. HEK 293T-PD-1 cells were used to screen shRNA candidates against PD-1 by Lipofectamine transfection of plasmids with shRNA candidates on day 0. On day 5, PD-1 expression was evaluated by flow cytometry. The results of this assessment are presented in FIG. 8.

TABLE-US-00007 Human PD-1 (Genbank NM_005018) mRNA (SEQ ID NO: 25):    1 gctcacctcc  gcctgagcag  tggagaaggc ggcactctgg tggggctgct ccaggcatgc   61 agatcccaca ggcgccctgg ccagtcgtct gggcggtgct acaactgggc tggcggccag  121 gatggttctt agactcccca gacaggccct ggaacccccc caccttctcc ccagccctgc  181 tcgtggtgac cgaaggggac aacgccacct tcacctgcag cttctccaac acatcggaga  241 gcttcgtgct aaactggtac cgcatgagcc ccagcaacca gacggacaag ctggccgcct  301 tccccgagga ccgcagccag cccggccagg actgccgctt ccgtgtcaca caactgccca  361 acgggcgtga cttccacatg agcgtggtca gggcccggcg caatgacagc ggcacctacc  421 tctgtggggc catctccctg gcccccaagg cgcagatcaa agagagcctg cgggcagagc  481 tcagggtgac agagagaagg gcagaagtgc ccacagccca ccccagcccc tcacccaggc  541 cagccggcca gttccaaacc ctggtggttg gtgtcgtggg cggcctgctg ggcagcctgg  601 tgctgctagt ctgggtcctg gccgtcatct gctcccgggc cgcacgaggg acaataggag  661 ccaggcgcac cggccagccc ctgaaggagg acccctcagc cgtgcctgtg ttctctgtgg  721 actatgggga gctggatttc cagtggcgag agaagacccc ggagcccccc gtgccctgtg  781 tccctgagca gacggagtat gccaccattg tctttcctag cggaatgggc acctcatccc  841 ccgcccgcag gggctcagct gacggccctc ggagtgccca gccactgagg cctgaggatg  901 gacactgctc ttggcccctc tgaccggctt ccttggccac cagtgttctg cagaccctcc  961 accatgagcc cgggtcagcg catttcctca ggagaagcag gcagggtgca ggccattgca 1021 ggccgtccag gggctgagct gcctgggggc gaccggggct ccagcctgca cctgcaccag 1081 gcacagcccc accacaggac tcatgtctca atgcccacag tgagcccagg cagcaggtgt 1141 caccgtcccc tacagggagg gccagatgca gtcactgctt caggtcctgc cagcacagag 1201 ctgcctgcgt ccagctccct gaatctctgc tgctgctgct gctgctgctg ctgctgcctg 1261 cggcccgggg ctgaaggcgc cgtggccctg cctgacgccc cggagcctcc tgcctgaact 1321 tgggggctgg ttggagatgg ccttggagca gccaaggtgc ccctggcagt ggcatcccga 1381 aacgccctgg acgcagggcc caagactggg cacaggagtg ggaggtacat ggggctgggg 1441 actccccagg agttatctgc tccctgcagg cctagagaag tttcagggaa ggtcagaaga 1501 gctcctggct gtggtgggca gggcaggaaa cccctccacc tttacacatg cccaggcagc 1561 acctcaggcc ctttgtgggg cagggaagct gaggcagtaa gcgggcaggc agagctggag 1621 gcctttcagg cccagccagc actctggcct cctgccgccg cattccaccc cagcccctca 1681 caccactcgg gagagggaca tcctacggtc ccaaggtcag gagggcaggg ctggggttga 1741 ctcaggcccc tcccagctgt ggccacctgg gtgttgggag ggcagaagtg caggcaccta 1801 gggcccccca tgtgcccacc ctgggagctc tccttggaac ccattcctga aattatttaa 1861 aggggttggc cgggctccca ccagggcctg ggtgggaagg tacaggcgtt cccccggggc 1921 ctagtacccc cgccgtggcc tatccactcc tcacatccac acactgcacc cccactcctg 1981 gggcagggcc accagcatcc aggcggccag caggcacctg agtggctggg acaagggatc 2041 ccccttccct gtggttctat tatattataa ttataattaa atatgagagc atgctaa Human TIM3 (Genbank NM_032782) mRNA (SEQ ID NO: 26)    1 atttggagag ttaaaactgt gcctaacaga ggtgtcctct gacttttctt ctgcaagctc   61 catgttttca catcttccct ttgactgtgt cctgctgctg ctgctgctac tacttacaag  121 gtcctcagaa gtggaataca gagcggaggt cggtcagaat gcctatctgc cctgcttcta  181 caccccagcc gccccaggga acctcgtgcc cgtctgctgg ggcaaaggag cctgtcctgt  241 gtttgaatgt ggcaacgtgg tgctcaggac tgatgaaagg gatgtgaatt attggacatc  301 cagatactgg ctaaatgggg atttccgcaa aggagatgtg tccctgacca tagagaatgt  361 gactctagca gacagtggga tctactgctg ccggatccaa atcccaggca taatgaatga  421 tgaaaaattt aacctgaagt tggtcatcaa accagccaag gtcacccctg caccgactcg  481 gcagagagac ttcactgcag cctttccaag gatgcttacc accaggggac atggcccagc  541 agagacacag acactgggga gcctccctga tataaatcta acacaaatat ccacattggc  601 caatgagtta cgggactcta gattggccaa tgacttacgg gactctggag caaccatcag  661 aataggcatc tacatcggag cagggatctg tgctgggctg gctctggctc ttatcttcgg  721 cgctttaatt ttcaaatggt attctcatag caaagagaag atacagaatt taagcctcat  781 ctctttggcc aacctccctc cctcaggatt ggcaaatgca gtagcagagg gaattcgctc  841 agaagaaaac atctatacca ttgaagagaa cgtatatgaa gtggaggagc ccaatgagta  901 ttattgctat gtcagcagca ggcagcaacc ctcacaacct ttgggttgtc gctttgcaat  961 gccatagatc caaccacctt atttttgagc ttggtgtttt gtctttttca gaaactatga 1021 gctgtgtcac ctgactggtt ttggaggttc tgtccactgc tatggagcag agttttccca 1081 ttttcagaag ataatgactc acatgggaat tgaactggga cctgcactga acttaaacag 1141 gcatgtcatt gcctctgtat ttaagccaac agagttaccc aacccagaga ctgttaatca 1201 tggatgttag agctcaaacg ggcttttata tacactagga attcttgacg tggggtctct 1261 ggagctccag gaaattcggg cacatcatat gtccatgaaa cttcagataa actagggaaa 1321 actgggtgct gaggtgaaag cataactttt ttggcacaga aagtctaaag gggccactga 1381 ttttcaaaga gatctgtgat ccctttttgt tttttgtttt tgagatggag tcttgctctg 1441 ttgcccaggc tggagtgcaa tggcacaatc tcggctcact gcaagctccg cctcctgggt 1501 tcaagcgatt ctcctgcctc agcctcctga gtggctggga ttacaggcat gcaccaccat 1561 gcccagctaa tttgttgtat ttttagtaga gacagggttt caccatgttg gccagtgtgg 1621 tctcaaactc ctgacctcat gatttgcctg cctcggcctc ccaaagcact gggattacag 1681 gcgtgagcca ccacatccag ccagtgatcc ttaaaagatt aagagatgac tggaccaggt 1741 ctaccttgat cttgaagatt cccttggaat gttgagattt aggcttattt gagcactgcc 1801 tgcccaactg tcagtgccag tgcatagccc ttcttttgtc tcccttatga agactgccct 1861 gcagggctga gatgtggcag gagctcccag ggaaaaacga agtgcatttg attggtgtgt 1921 attggccaag ttttgcttgt tgtgtgcttg aaagaaaata tctctgacca acttctgtat 1981 tcgtggacca aactgaagct atatttttca cagaagaaga agcagtgacg gggacacaaa 2041 ttctgttgcc tggtggaaag aaggcaaagg ccttcagcaa tctatattac cagcgctgga 2101 tcctttgaca gagagtggtc cctaaactta aatttcaaga cggtataggc ttgatctgtc 2161 ttgcttattg ttgccccctg cgcctagcac aattctgaca cacaattgga acttactaaa 2221 aatttttttt tactgtt Human LAG3 (Genbank NM_002286) mRNA (SEQ ID NO: 27)    1 agagaccagc agaacggcat cccagccacg acggccactt tgctctgtct gctctccgcc   61 acggccctgc tctgttccct gggacacccc cgcccccacc tcctcaggct gcctgatctg  121 cccagctttc cagctttcct ctggattccg gcctctggtc atccctcccc accctctctc  181 caaggccctc tcctggtctc ccttcttcta gaaccccttc ctccacctcc ctctctgcag  241 aacttctcct ttacccccca ccccccacca ctgccccctt tccttttctg acctcctttt  301 ggagggctca gcgctgccca gaccatagga gagatgtggg aggctcagtt cctgggcttg  361 ctgtttctgc agccgctttg ggtggctcca gtgaagcctc tccagccagg ggctgaggtc  421 ccggtggtgt gggcccagga gggggctcct gcccagctcc cctgcagccc cacaatcccc  481 ctccaggatc tcagccttct gcgaagagca ggggtcactt ggcagcatca gccagacagt  541 ggcccgcccg ctgccgcccc cggccatccc ctggcccccg gccctcaccc ggcggcgccc  601 tcctcctggg ggcccaggcc ccgccgctac acggtgctga gcgtgggtcc cggaggcctg  661 cgcagcggga ggctgcccct gcagccccgc gtccagctgg atgagcgcgg ccggcagcgc  721 ggggacttct cgctatggct gcgcccagcc cggcgcgcgg acgccggcga gtaccgcgcc  781 gcggtgcacc tcagggaccg cgccctctcc tgccgcctcc gtctgcgcct gggccaggcc  841 tcgatgactg ccagcccccc aggatctctc agagcctccg actgggtcat tttgaactgc  901 tccttcagcc gccctgaccg cccagcctct gtgcattggt tccggaaccg gggccagggc  961 cgagtccctg tccgggagtc cccccatcac cacttagcgg aaagcttcct cttcctgccc 1021 caagtcagcc ccatggactc tgggccctgg ggctgcatcc tcacctacag agatggcttc 1081 aacgtctcca tcatgtataa cctcactgtt ctgggtctgg agcccccaac tcccttgaca 1141 gtgtacgctg gagcaggttc cagggtgggg ctgccctgcc gcctgcctgc tggtgtgggg 1201 acccggtctt tcctcactgc caagtggact cctcctgggg gaggccctga cctcctggtg 1261 actggagaca atggcgactt tacccttcga ctagaggatg tgagccaggc ccaggctggg 1321 acctacacct gccatatcca tctgcaggaa cagcagctca atgccactgt cacattggca 1381 atcatcacag tgactcccaa atcctttggg tcacctggat ccctggggaa gctgctttgt 1441 gaggtgactc cagtatctgg acaagaacgc tttgtgtgga gctctctgga caccccatcc 1501 cagaggagtt tctcaggacc ttggctggag gcacaggagg cccagctcct ttcccagcct 1561 tggcaatgcc agctgtacca gggggagagg cttcttggag cagcagtgta cttcacagag 1621 ctgtctagcc caggtgccca acgctctggg agagccccag gtgccctccc agcaggccac 1681 ctcctgctgt ttctcatcct tggtgtcctt tctctgctcc ttttggtgac tggagccttt 1741 ggctttcacc tttggagaag acagtggcga ccaagacgat tttctgcctt agagcaaggg 1801 attcaccctc cgcaggctca gagcaagata gaggagctgg agcaagaacc ggagccggag 1861 ccggagccgg aaccggagcc cgagcccgag cccgagccgg agcagctctg acctggagct 1921 gaggcagcca gcagatctca gcagcccagt ccaaataaac tccctgtcag cagcaa