BAFF-R/CD19 targeted chimeric antigen receptor-modified T cells and use thereof

Abstract

Chimeric antigen receptors targeting both BAFF-R and CD19 are described as are methods for their use.

Claims

1. A chimeric antigen receptor targeted to both B cell activating factor receptor (BAFF-R) and CD19, wherein the chimeric antigen receptor comprises: a targeting domain comprising, from amino to carboxy terminus: a) a scFv targeted to CD19 and a scFv targeted to BAFF-R; b) a VL domain of a CD19 scFv, a scFv targeted to BAFF-R, and a VH of a CD19 scFv; or c) a VH domain of a CD19 scFv, a scFv targeted to BAFF-R, and a VL of a CD19 scFv; followed by: a spacer domain, a transmembrane domain, a costimulatory domain, and a CD35 signaling domain, wherein the scFv targeted to BAFF-R comprises amino acids 135-382 of SEQ ID NO: 62 or amino acids 266-510 of SEQ ID NO: 61 and the scFv targeted to CD19 comprises a VH domain and a VL domain.

2. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 60-63.

3. The chimeric antigen receptor of claim 1, wherein the targeting domain comprising from amino to carboxy terminus a VL domain of a CD19 scFv, a scFv targeted to BAFF-R and a VH of a CD19 scFv further comprises (i) a linker between the VL domain of the CD19 scFv and the scFv targeted to BAFF-R and (ii) a linker between the scFv targeted to BAFF-R and the VH of the CD19 scFv.

4. The chimeric antigen receptor of claim 3, wherein the linker includes only G and S.

5. The chimeric antigen receptor of claim 1, wherein the CD19 scFv comprises amino acids 23-267 of SEQ ID NO: 60.

6. The chimeric antigen receptor of claim 1, wherein the costimulatory domain is: a CD28 costimulatory domain, a 4-1BB costimulatory domain, or an OX40 costimulatory domain.

7. The chimeric antigen receptor of claim 1, wherein the transmembrane domain is a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3 transmembrane domain.

8. The chimeric antigen receptor of claim 1, wherein the spacer domain is IgG4 hinge (S.fwdarw.P), IgG4 hinge, IgG4 hinge (S228P)+linker, CD28 hinge, CD8 hinge-48aa, CD8 hinge-45aa, IgG4(HL-CH3), IgG4(L235E,N297Q), IgG4(S228P, L235E,N297Q), or IgG4(CH3).

9. The chimeric antigen receptor of claim 1, wherein the CD19 scFv VL domain comprises the amino acid sequence TABLE-US-00015 (SEQIDNO:30) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEIT and the CD19 scFV VH domain comprises the amino acid sequence TABLE-US-00016 (SEQIDNO:31) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSS.

10. The chimeric antigen receptor of claim 1, wherein the costimulatory domain comprises SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 or SEQ ID NO: 55 and the CD35 signaling domain comprises SEQ ID NO: 51.

11. The chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises any of SEQ ID NOs: 43-50.

12. The chimeric antigen receptor of claim 1, wherein the spacer domain comprises any of SEQ ID NOs: 32-42.

13. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises a targeting domain comprising, from amino to carboxy terminus: a) a scFv targeted to CD19 and a scFv targeted to BAFF-R; or b) a VL domain of a CD19 scFv, a scFv targeted to BAFF-R, and a VH of a CD19 scFv.

14. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises a targeting domain comprising, from amino to carboxy terminus: a VL domain of a CD19 scFv, a scFv targeted to BAFF-R, and a VH of a CD19 scFv.

15. A chimeric antigen receptor comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 58-63.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 depicts schematic drawings of two example of tandem constructs and one example of a loop construct for BAFF-R/CD19 dual CARs. Also shown is an example of bicistronic construct in which a single cell expresses two CARs, one targeted to BAFF-R and one targeted to CD19.

(2) FIG. 2 depicts the amino acid sequence of an immature 1250 dual CAR, including signal sequence (SEQ ID NO:58). The mature CAR (SEQ ID NO:59) includes, from amino to carboxy terminus: a BAFF-R scFv, a linker, a CD19 scFv (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 4-1BB cytoplasmic domain, a GGG linker, and a CD3 signaling domain.

(3) FIG. 3 depicts the amino acid sequence of an immature 1296 dual CAR, including signal sequence (SEQ ID NO:60). The mature CAR (SEQ ID NO:61) includes, from amino to carboxy terminus: a CD19 scFv (derived from FMC63), a linker, a BAFF-R scFv, an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 4-1BB cytoplasmic domain, a GGG linker, and a CD3 signaling domain.

(4) FIG. 4 depicts the amino acid sequence of an immature 1316 dual CAR, including signal sequence (SEQ ID NO:62). The mature CAR (SEQ ID NO:63) includes, from amino to carboxy terminus: a CD19 VL (derived from FMC63), a GGGS (SEQ ID NO:70) linker, a BAFF-R scFv, a CD19 VH (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 41BB cytoplasmic domain, a GGG linker, and a CD3 signaling domain.

(5) FIG. 5 depicts the amino acid sequence of: (A) an immature CD19 CAR, including signal sequence (SEQ ID NO:64) and B an immature BAFF-R CAR including a signal sequence (SEQ ID NO:66). The mature CD19 CAR (SEQ ID NO:65) includes, from amino to carboxy terminus: a CD19 scFv (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD28 transmembrane domain, a CD28(GG) cytoplasmic domain, a GGG linker, and a CD3 signaling domain. The mature BAFF-R CAR (SEQ ID NO:67) includes, from amino to carboxy terminus: a BAFF-R scFv, an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 4-1BB cytoplasmic domain, a GGG linker, and a CD3 signaling domain.

(6) FIG. 6 is a schematic drawing of an example of a vector for expressing a BAFF-R/CD19 dual CAR. BAFF-R and CD19 scFv elements can be varied to express the different arrangement of dual CARs. Truncated EGFR (EGFRt) is expressed as a selection marker.

(7) FIG. 7 depicts the results of a study of dual CAR expression. T cells (Jurkat) were transduced with each CAR construct or an empty vector (mock). Cells were stained with Protein L- or EGFR-APC conjugated antibodies. Protein L targets the variable light chain of the scFv, and truncated EGFR is co-expressed by the CAR vector. Constructs that were able to properly express dual CARs were further examined. Dual CAR 1296 and dual CAR 1316 expressed intact CAR and EGFRt selection marker, whereas dual CAR 1250 failed to express intact CAR.

(8) FIG. 8 depicts the result of a FACS assay assessing 1250 dual CAR T-cell degranulation following incubation with target cells. Targets cells are either BAFF-R single positive, CD19 single positive, or BAFF-R and CD19 double negative. Control BAFF-R single CAR and non-transduced T cells (non-CAR) were used as controls. The 1250 dual CAR failed to elicit response against BAFF-R-positive L cells suggesting BAFF-R-targeting scFv is not properly expressed.

(9) FIG. 9 depicts the results of analysis related to development of cells lines used to develop a model for studying BAFF-R/CD19 dual CAR. FACS histograms of Nalm-6 knockout lines used for dual CAR model development. Nalm-6 B-ALL tumor line was gene edited with CRISPR to knockout BAFF-R (left) or CD19 (right). Surface protein expression was confirmed by FACS staining with commercial BAFF-R and CD19 antibodies. Nalm-6 wildtype (WT) was used as controls.

(10) FIG. 10A-10B depicts the results of an analysis of the CTL function of BAFF-R/CD19 dual CAR. Graphs show calculated specific lysis are plotted from a cytotoxic T lymphocyte assay against Nalm-6 ALL tumor lines. Target cell line Nalm-6 (WT, CD19 knockout, or BAFF-R knockout variants) were labeled with chromium-51 and incubated with effector CAR T cells. CARs included BAFF-R/CD19 dual-targeting CARs: A). 1296 and 1250 or B). 1296 and 1316; controls in both panels include single-targeting CARs: BAFF-R CAR and CD19 CAR and non-transduced T cells (non-CAR, allogeneic control). All T cells were derived from a single healthy donor in each panel. Chromium released by target cells due to effector T cell function was measured by a gamma counter and calculated as a percentage of maximum possible release. Experiment was conducted in triplicate and analyzed by a Student's t-test; A. ** P<0.001 compared between dual CARs; and B). ** P<0.001 c/w non-CAR control. 1250 dual CAR CTL data suggests potential BAFF-R targeting deficiency.

(11) FIG. 11A-11B depicts the activity of BAFF-R/CD19 Dual CAR in BAFF-R-plus CD19-deficiant mixed B-ALL tumor. A) Bioluminescence images of NSG mice following IV tumor challenge on day 0 with a mixture of 110.sup.5 RFP-negative, luciferase-expressing Nalm-6-CD19KO plus 2.510.sup.5 RFP-positive, luciferase-expressing Nalm-6-BAFF-RKO tumor cells. Groups of 5 tumor-bearing mice each were then randomly assigned to treatment with either 2.510.sup.6 CD4 T.sub.N CAR-T+10.sup.6 CD8 T.sub.N 1296 or 1316 dual CART cells/mouse IV on day 10, as a single infusion. Non-transduced CD4/CD8 T cells from the same donor were used as allogeneic controls (non-CAR). B) Kaplan-Meier plots of overall survival are shown. Log-rank test compare experimental groups as shown. 1316 treatment conferred significant prolonged survival compared to 1296 treatment.

(12) FIG. 12 depicts the activity of 1316 BAFF-R/CD19 Dual CAR against knock-out tumors. FACS plots of BAFF-R CAR T cell functional potency as measured by a CD107a degranulation assay. CD4 or CD8 BAFF-R CAR T cells were coincubated with either CD19.sup.BAFF-R.sup.+ Nalm-6 or CD19.sup.+BAFF-R.sup. Nalm-6 lines. Single targeting CD19 or BAFF-R CAR T cells were used as controls.

(13) FIG. 13A-13B depicts the activity of T.sub.N/MEM 1316 BAFF-R/CD19 Dual CAR in mixed B-ALL tumor. A) Bioluminescence images of NSG mice following IV tumor challenge on day 0 with a mixture of 110.sup.5 RFP-negative, luciferase-expressing Nalm-6-CD19K0 plus 110.sup.5 RFP-positive, luciferase-expressing Nalm-6-BAFF-RKO tumor cells. Groups of 5 tumor-bearing mice each were then randomly assigned to treatment with 1316 dual CAR T cells/mouse IV on day 9, as a single infusion of either low dose (2.810.sup.6 T.sub.N/MEM), high dose (5.610.sup.6 T.sub.N/MEM), which yielded 110.sup.6 and 210.sup.6 BAFF-R CAR T cells, respectively. 2.5 or 510.sup.6 non-transduced T.sub.N/MEM cells from the same donor were used as allogeneic controls (non-CAR). B) Kaplan-Meier plots of overall survival are shown. Log-rank test compare experimental groups as shown. No significant difference in survival between the two dosing were observed.

DETAILED DESCRIPTION

(14) The BAFF-R/CD19 dual CAR and BAFF-R/CD19 bicistronic CAR can employ any of a variety of BAFF-R scFv and CD19 scFv and, in the case of BAFF-R/CD19 dual CAR in which a first scFv is located between the variable domains of a second scFv, any of a variety of VL and VH can be used.

(15) FIG. 1 depicts schematic drawings of two example of tandem construct, one in which the BAFF-R scFv is amino terminal to the CD19 scFv and one in which the CD19 scFv is amino terminal to the BAFF-R scFv. Also depicted is one example of a loop construct for BAFF-R/CD29 dual CARs. In this example a BAFF-R is located between the CD19 VL (amino terminal to the scFv) and the CD19 VH domain (carboxy terminal to the scFv). Also shown is an example of bicistronic construct in which a single cell expresses two CARs, one targeted to BAFF-R and one targeted to CD19.

BAFF-R scFv Sequences

(16) The BAFF-R scFv sequences used in BAFF-R/CD19 dual CAR can be derived from two monoclonal antibodies, Clone 90 and Clone 55 described in greater detail in US PCT/US2017/036181. For example, the VL can include the C90 CDR sequences or the C55 CDR sequences described below.

(17) TABLE-US-00003 C90CDRL1: (SEQIDNO:1) ESVDNYGISF C90CDRL2: AAS C90CDRL3: (SEQIDNO:3) QQSKEVPWT C90CDRH1: (SEQIDNO:4) GDSITSGY C90CDRH2: (SEQIDNO:5) ISYSGST C90CDRH3: (SEQIDNO:6) ASPNYPFYAMDY C55CDRL1: (SEQIDNO:7) QDISNY C55CDRL2: YTS C55CDRL3: (SEQIDNO:9) FSELPWT C55CDRH1: (SEQIDNO:10) GFSLSTSGMG C55CDRH2: (SEQIDNO:11) IWWDDDK C55CDRH3: (SEQIDNO:12) ARSFGYGLDY

(18) Among the suitable heavy chain variable domains (VH) for use in the BAFF-R scFv of a dual CAR are the following heavy chain variable domains derived from monoclonal antibody Clone 90 (Described in greater detail in PCT/US2017/036181. Of these, Hu90 HC-1, HC-2 and HC-3 are humanized.

(19) TABLE-US-00004 Chi90HC: (SEQIDNO:13) MYRMQLLSCIALSLALVTNSEVQLQESGPSLVKPSQTLSLTCSVTGDSI TSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQY YLQLNSVTPEDTATYYCASPNYPFYAMDYWGQGTSVTVSSDI Hu90HC-1: (SEQIDNO:14) MDPKGSLSWRILLFLSLAFELSYGQVQLQESGPGLVKPSQTLSLTCTVS GDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTS KNQFSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS Hu90HC-2: (SEQIDNO:15) MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSQTLSLTCTVS GDSITSGYWNWIRQHPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTS KNQYSLKLSSVTAADTAVYYCASPNYPFYAMDYWGQGTLVTVSS Hu90HC-3: (SEQIDNO:16) MDPKGSLSWRILLFLSLAFELSYGEVQLQESGPGLVKPSETLSLTCSVS GDSITSGYWNWIRQPPGKGLEYIGYISYSGSTYYNPSLKSRVTISRDTS KNQYSLRLSSVTAADTALYYCASPNYPFYAMDYWGQGTRVTVSS

(20) Among the suitable light chain variable domains (VL) for use in the BAFF-R scFv of a dual CAR are the following heavy chain variable domains derived from monoclonal antibody Clone 90 (Described in greater detail in PCT/US2017/036181. Of these, Hu90 LC-1, LC-2 and LC-3 are humanized.

(21) TABLE-US-00005 Chi90LC: (SEQIDNO:17) MYRMQLLSCIALSLALVTNSDIVLTQSPASLAVSLGQRATISCRASESV DNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLN IHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIKTMEIKR HuC90LC-1: (SEQIDNO:18) METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASESV DNYGISFLNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTV Hu90LC-2: (SEQIDNO:19) METDTLLLWVLLLWVPGSTGDIVLTQSPATLSLSPGERATLSCRASESV DNYGISFMNWFQQKPGQAPRLLIYAASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQSKEVPWTFGGGTKVEIKRTV HuC90LC-3: (SEQIDNO:20) METDTLLLWVLLLWVPGSTGDIVNITQSPSSLSASVGDRVTITCRASES VDNYGISFMNWFQQKPGKAPKLLIYAASNLGSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQSKEVPWTFGQGTKVEIKRTV

(22) Also among the suitable heavy chain variable domains (VH) for use in the BAFF-R scFv of a dual CAR are the following heavy chain variable domains derived from monoclonal antibody Clone 55 (described in greater detail in PCT/US2017/036181). Of these, Hu55 HC-1, HC-2 and HC-3 are humanized.

(23) TABLE-US-00006 Chi55HC: (SEQIDNO:21) MYRMQLLSCIALSLALVTNSQVTLKESGPGILKPSQTLSLTCSFSGFSL STSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNSSLKSHLTISKDTSRN QVFLKITSVDTADTATYYCARSFGYGLDYWGQGTTLTVSSAS Hu55HC-1: (SEQIDNO:22) MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFS GFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTITKD TSKNQVVLTMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS Hu55HC-2: (SEQIDNO:23) MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPTLVKPTQTLTLTCTFS GFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNSSLKSRLTITKD TSKNQVVLTMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS Hu55HC-3: (SEQIDNO:24) MDPKGSLSWRILLFLSLAFELSYGQVTLKESGPALVKPTQTLTLTCTFS GFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNTSLKSRLTITKD TSKNQVVLKMTNMDPVDTATYYCARSFGYGLDYWGQGTLVTVSS

(24) Also among the suitable light chain variable domains (VL) for use in the BAFF-R scFv of a dual CAR are the following heavy chain variable domains derived from monoclonal antibody Clone 90 (described in greater detail in PCT/US2017/036181). Of these, Hu55 LC-1, LC-2 and HC-3 are humanized.

(25) TABLE-US-00007 Chi55LC: (SEQIDNO:25) MYRMQLLSCIALSLALVTNSDIQMTQTTSSLSASLGDRVTISCSASQDI SNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISSL EPEDIATYYCHQFSELPWTFGGGTKLEIKRT Hu55LC-1: (SEQIDNO:26) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQASQDI SNYLNWYQQKPGKAPKLLIYYTSSLHTGVPSRFSGSGSGTDYTFTISSL QPEDIATYYCHQFSELPWTFGGGTKVEIKRTV Hu55LC-2: (SEQIDNO:27) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCSASQDI SNYLNWYQQKPGKAPKLLIYYTSSLHTGVPSRFSGSGSGTDYTLTISSL QPEDIATYYCHQFSELPWTFGGGTKVEIKRTV Hu55LC-3: (SEQIDNO:28) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCQASQDI SNYLNWYQQKPGKTPKLLIYYTSSLHTGVPSRFSGSGSGTDYTLTISSL QPEDIATYYCHQFSELPWTFGGGTKVEIKRTV

(26) The VH and VL domains of the BAFF-R scFv can be modified. Thus, each of Hu90 LC-1, Hu90 LC-2, Hu90 LC-3, Hu90 HC-1, Hu90 HC-2 and Hu90 HC-3, Hu55 LC-1, Hu55 LC-2, Hu55 LC-3, Hu55 HC-1, Hu55 HC-2 and Hu90 HC-3 in a scFv can include 1, 2, 3, 4 or 5 single amino acid substitutions. In some cases, the substitutions are confined to the framework regions (FRs) rather than the CDRs. In some cases, the substitutions are conservative substitutions.

(27) The position of CDRs and FRs may be defined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)). Likewise, the positions occupied by individual residues within the light or the heavy chain of an antibody may be defined by the Kabat numbering system. Therefore, the location of residues required for binding within a humanized light chain and a humanized heavy chain of a humanized antibody may be defined by the position of the residue according to the Kabat numbering system as is well known in the art. As described above, a humanized antibody may be an antibody having CDRs from a donor antibody (e.g. mouse) and variable region framework (FR) from a human antibody. The framework regions (FRs) are said to hold the CDRs in place in a humanized antibody. Proceeding from the amino-terminus, these regions are designated FR L1, FR L2, FR L3, and FR L4 for the light chain and FR H1, FR H2, FR H3, and FR H4, for the heavy chain, respectively.

CD19 scFv Sequences

(28) A variety of scFv targeting CD19 can be used in BAFF-R/CD19 dual CAR.

(29) TABLE-US-00008 FMC63scFv: (SEQIDNO:29) IPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVA PSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS. FMC63VL: (SEQIDNO:30) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEIT. FMC63VH: (SEQIDNO:31) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

(30) Additional scFv that bind CD19 are described in US 2016/0152723 and in WO 2016/033570

Spacer Region

(31) The dual CAR and bicistronic CAR described herein 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 in the CARs described herein.

(32) TABLE-US-00009 TABLE1 ExamplesofSpacers a3 3aa AAA linker 10aa GGGSSGGGSG(SEQIDNO:32) IgG4hinge(S.fwdarw.P) 12aa ESKYGPPCPPCP(SEQIDNO:33) (S228P) IgG4hinge 12aa ESKYGPPCPSCP(SEQIDNO:34) IgG4hinge(S228P)+linker 22aa ESKYGPPCPPCPGGGSSGGGSG(SEQIDNO:35) CD28hinge 39aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(SEQID NO:36) CD8hinge-48aa 48aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC D(SEQIDNO:37) CD8hinge-45aa 45aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQIDNO:38) IgG4(HL-CH3) 129aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQV (includesS228Pinhinge) SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:39) IgG4(L235E,N297Q) 229aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHQAKTKPREEQFQSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQIDNO:40) IgG4(S228P,L235E,N297Q) 229aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHQAKTKPREEQFQSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLIVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQIDNO:41) IgG4(CH3) 107aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK(SEQIDNO:42)

(33) 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.

(34) 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).

(35) 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 GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:41). Thus, the entire linker/spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 39). In some cases, the spacer has 1,2,3,4, or 5 single amino acid changes (e.g., conservative changes). 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

(36) A variety of transmembrane domains can be used in the dual CAR and biscistonic CAR described herein. 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.

(37) TABLE-US-00010 TABLE2 ExamplesofTransmembraneDomains Name Accession Length Sequence CD3z J04132.1 21aa LCYLLDGILFIYGVILTALFL(SEQIDNO:43) CD28 NM_006139 27aa FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQID NO:44) CD28(M) NM_006139 28aa MFWVLVVVGGVLACYSLLVIVAFIIFWV(SEQID NO:45) CD4 M35160 22aa MALIVLGGVAGLLLFIGLGIFF(SEQIDNO:46) CD8tm NM_001768 21aa IYIWAPLAGTCGVLLLSLVIT(SEQIDNO:47) CD8tm2 NM_001768 23aa IYIWAPLAGTCGVLLLSLVITLY(SEQIDNO:48) CD8tm3 NM_001768 24aa IYIWAPLAGTCGVLLLSLVITLYC(SEQIDNO:49) 41BB NM_001561 27aa IISFFLALTSTALLFLLFFLTLRFSVV(SEQIDNO:50)
Costimulatory and CD3zeta Domain

(38) The costimulatory domain can be any domain that is suitable for use with a CD3 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:24.

(39) The costimulatory domain(s) are located between the transmembrane domain and the CD3 signaling domain. Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3 signaling domain.

(40) TABLE-US-00011 TABLE3 CD4DomainandExamplesofCostimulatoryDomains Name Accession Length Sequence CD3 J04132.1 113aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR(SEQIDNO:51) CD28 NM_006139 42aa RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRS(SEQIDNO:52) CD28gg* NM_006139 42aa RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRS(SEQIDNO:53) 4-1BB NM_001561 42aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCEL(SEQIDNO:54) OX40 42aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHST LAKI(SEQIDNO:55)

(41) 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 CD3 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 CD3 signaling domain.

CD3 Signaling Domain

(42) The CD3 Signaling domain can be any domain that is suitable for use with a CD3 signaling domain. In some cases, the CD3 signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

(43) TABLE-US-00012 (SEQIDNO:51) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGEIDGLYQGLSTAT KDTYDALHMQALPPR.
In some cases, the CD3 signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:51.

Truncated EGFR

(44) The CD3 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:

(45) TABLE-US-00013 (SEQIDNO:57) LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCT SISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPE NRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDV IISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALC SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCH PECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVW KYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LVVALGIGLFM.
In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:57.

(46) 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.

(47) The CAR can include a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to the mature amino acid sequence depicted in FIG. 9 (SEQ ID Nos: 29-40), either including or excluding the GMCSFRa signal sequence and either including or excluding the T2A ribosomal skip sequence and the truncated EGFRt).

(48) 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.

(49) The CAR described herein 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.

(50) 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.

Example 1: Preparation of T Cell Populations for Expression of Dual CAR

(51) The following T cell populations can be prepared for expression of BAFF-CD19 dual CAR: CD4+ nave T cells (CD4+ T.sub.N), CD8+ nave T cells (CD8+ T.sub.N), CD8+ central memory T cells (CD8+ T.sub.CM), CD8+ memory stem cells (CD8+ MSC) and Pan T cells (Pan T). Briefly, 5 mL of a blood sample are added to 5 mL histopaque-1077 (Sigma Aldrich). The mixture is centrifuged for 20 min at 2500 RPM (room temperature (RT), no brake). The middle peripheral blood mononuclear cell (PBMC) layer is collected, washed with 50 mL PBS (Corning), centrifuged for 5 min at 1500 RPM (RT). The collected cells are combined with 10 mL RBC lysis buffer (Qiagen) and incubated for 7 min. The cells are then washed with PBS and centrifuged for 5 min (1500 RPM, RT).

(52) Various T cell populations can be prepared using the following kits available from StemCell Technologies, Inc. using the manufacturer's instructions: EasySep Human Nave CD4+ T Cell Enrichment Kit (CD4+T.sub.N), EasySep Human Nave CD8+ T Cell Enrichment Kit (CD8+T.sub.N), and EasySep Human T Cell Enrichment Kit (Pan T). CD8+ T.sub.CM can be prepared by isolating CD8+ T cells using the EasySep Human CD8+ T Cell Enrichment Kit from StemCell Technologies, Inc. using the manufacturer's instructions and then stained with: CD8-PerCP-Cy5.5, CD45 RO-APC, and CD62L-PE. The stained cells are then sorted to isolate CD8+/CD45+/CD62L+ triple positive cells. CD8+ memory stem cells (CD8+ MSC) can be generated from CD8+ T.sub.N using the culture conditions shown in Table 4. The other T cell populations can be cultured as indicated in Table 4.

(53) TABLE-US-00014 TABLE 4 Additional Population Media Serum Cytokines Supplement CD4+ T.sub.N, X- 10% human 100 U/mL 100 U/mL CD8+ T.sub.N, VIVO 15 Ab serum hIL-2 penicillin CD8+ T.sub.CM (Lonza) (Valley 100 g/mL Pan T Biomedical) streptomycin CD8+ MSC AIM-V 5% human 5 ng/mL 2 mM glutamax (Thermo Ab serum IL-7 (Thermo Fisher Fisher) (Valley 30 ng/mL Scientific) Biomedical) IL-21 5 mM TWS119 (Cellgenix) (Cayman Chemical)

Example 2: Sequence of BAFF-R/CD19 Dual CAR

(54) A variety of BAFF-R/CD19 dual CAR were prepared. The 1250 dual CAR (SEQ ID NO:59) includes, from amino to carboxy terminus: a BAFF-R scFv, a linker, a CD19 scFv (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 41BB cytoplasmic domain, a GGG linker, and a CD3 zeta signaling domain. (FIG. 2). The 1296 dual CAR (SEQ ID NO:61) includes, from amino to carboxy terminus: a CD19 scFv (derived from FMC63), a linker, a BAFF-R scFv, an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 41BB cytoplasmic domain, a GGG linker, and a CD3 zeta signaling domain (FIG. 3). The 1316 dual CAR (SEQ ID NO:63) includes, from amino to carboxy terminus: a CD19 VL (derived from FMC63), a GGGS (SEQ ID NO:70) linker, a BAFF-R scFv, a CD19 VH (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 41BB cytoplasmic domain, a GGG linker, and a CD3 zeta signaling domain.

(55) A biscitronic CAR expresses a BAFF-R CAR and a CD19 CAR using the lentiviral vector. The two CAR are expressed in the same T cell. The mature CD19 CAR (SEQ ID NO:65) can include, from amino to carboxy terminus: a CD19 scFv (derived from FMC63), an IgG4 (SmP/L235E,N297Q) spacer domain, a CD28 transmembrane domain, a CD28(GG) cytoplasmic domain, a GGG linker, and a CD3 signaling domain. The mature BAFF-R CAR (SEQ ID NO:67) can include, from amino to carboxy terminus: a BAFF-R scFv, an IgG4 (SmP/L235E,N297Q) spacer domain, a CD4 transmembrane domain, a 4-1BB cytoplasmic domain, a GGG linker, and a CD3 signaling domain.

Example 2: Preparation of Lentiviral Vectors Expressing BAFF-R/CD19 Dual CAR

(56) FIG. 5 is a schematic diagram of the lentiviral vector used to express 1250 dual CAR. The 1296 dual CAR, and the 1316 dual CAR were produced using similar lentiviral vectors (with replacement of the svFv portion).

Example 3: Preparation of CAR Expressing Cells

(57) Cells were activated in preparation for transduction with lenitviral vectors expressing a dual CAR by combining the cells with CD3/CD28 human T-cell activation beads (Thermo Fisher) at a 1:1 bead to cell ratio and incubating overnight (humidified, 5% CO.sub.2, 37 C.). After incubation, the cells were counted and distributed 110.sup.6 cell/well in a 48-well plate. Cells were infected at an MOI of 1. In each case the total culture media was supplemented to 250 L, centrifuged for 30 min (800 g, RT). Cells are incubated overnight (humidified, 5% CO.sub.2, 37 C.) and then cultured for 10 days in the media indicated in Table 1. Cultures of CD4+ T.sub.N, CD8+ T.sub.N, CD8+T.sub.CM, and Pan T cells included CD3/CD28 beads at a 1:1 cell to bead ratio. The culture of CD8+ T MSC did not include CD3/CD28 beads. Expression of the CAR was assessed by the percentage of GFP positive cells using flow cytometry.

Example 4: Expression of CAR

(58) T cells (Jurkat) were transduced with each CAR construct or an empty vector. To assess expression of the CAR, cells were stained with Protein L- or EGFR-APC conjugated antibodies. Protein L targets the variable light chain of the scFv, and truncated EGFR is co-expressed by the CAR vector. Constructs that were able to properly express dual CARs were further examined. As can be seen in FIG. 7, Dual CAR 1296 and dual CAR 1316 expressed intact CAR and EGFRt selection marker, whereas dual CAR 1250 failed to express intact CAR.

Example 5: In Vitro Cell Killing by BAFF-R Targeted CAR T Cells

(59) An in vitro assay using various T cell populations expressing 1296 Dual CAR, 1316 Dual CAR, CD19 CAR or BAFF-R CAR cells. Target cell line Nalm-6 (WT, CD19 knockout, or BAFF-R knockout variants) were labeled with chromium-31 and incubated with effector CAR T cells. CARs included BAFF-R/CD19 dual-targeting CARs: 1296 and 1316; single-targeting CARs: BAFF-R CAR and CD19 CAR. Non-transduced T cells (non-CAR) were used as an allogeneic control. All T cells were derived from a single healthy donor. Chromium released by target cells due to effector T cell function was measured by a gamma counter and calculated as a percentage of maximum possible release. As can be seen in FIG. 8, 1250 dual CAR failed to elicit response against BAFF-R-positive L cells suggesting BAFF-R-targeting scFv is not properly expressed.

Example 6: BAFF-R- and CD19-Deficient Murine Model

(60) Nalm-6 B-ALL tumor line was gene edited with CRISPR to knockout BAFF-R or CD19. Surface protein expression was confirmed by FACS staining with commercial BAFF-R and CD19 antibodies. Nalm-6 wild-type (WT) was used as controls. As shown in FIG. 9, FACS analysis confirms the knockout.

Example 7: CTL Function of Dual CAR T Cells

(61) In this study, the results of which are shown in FIG. 10, target cell lines Nalm-6, WT, CD19 knockout, or BAFF-R knockout variants, were labeled with chromium-51 and incubated with effector CART cells. CARs included BAFF-R/CD19 dual-targeting CARs. The 1250 dual CAR CTL data suggests potential BAFF-R targeting deficiency.

Example 8: Activity of BAFF-R/CD19 Dual CAR in BAFF-R-Positive CD19-Negative Mixed B-ALL Tumor

(62) FIG. 11A-11B shows the results of a study examining the impact of 1316 Dual CAR and 1296NSG mice following IV tumor challenge on day 0 with a mixture of 110.sup.5 RFP-negative, luciferase-expressing Nalm-6-CD19KO plus 2.510.sup.5 RFP-positive, luciferase-expressing Nalm-6-BAFF-RKO tumor cells. Groups of 5 tumor-bearing mice each were then randomly assigned to treatment with either 2.510.sup.6 CD4 T.sub.N CAR-T+10.sup.6 CD8 T.sub.N 1296 or 1316 dual CART cells/mouse IV on day 10, as a single infusion. Non-transduced CD4/CD8 T cells from the same donor were used as allogeneic controls (non-CAR). As can be seen, 1316 treatment conferred significant prolonged survival compared to 1296 treatment.

Example 9: Degranulation of 1316 BAFF-R/CD19 Dual CAR Against Knock-Out Tumors

(63) A CD107a degranulation was used to assess Dual CAR 1316 potency against knockout tumors. CD4 or CD8 BAFF-R CAR T cells were incubated with either CD19.sup.BAFF-R.sup.+ Nalm-6 or CD19.sup.+BAFF-R.sup. Nalm-6 lines. Single targeting CD19 or BAFF-R CAR T cells were used as controls.

Example 10: Activity of TN/MEM 1316 BAFF-R/CD19 Dual CAR in Mixed B-ALL Tumor

(64) NSG mice were challenged on day 0 with a mixture of 1105 RFP-negative, luciferase-expressing Nalm-6-CD19KO plus 1105 RFP-positive, luciferase-expressing Nalm-6-BAFF-RKO tumor cells. Groups of 5 tumor-bearing mice each were then randomly assigned to treatment with 1316 dual CAR T cells/mouse IV on day 9, as a single infusion of either low dose (2.8106 TN/MEM), high dose (5.6106 TN/MEM), which yielded 1106 and 2106 BAFF-R CART cells, respectively.2.5 or 5106 non-transduced TN/MEM cells from the same donor were used as allogeneic controls (non-CAR). 1316 Dual CAR conferred significant prolonger survival. No significant difference in survival between the two dosing were observed.