Chimeric antigen receptor and CAR-T cells that bind BCMA

Abstract

An isolated chimeric antigen receptor polypeptide (CAR), wherein the CAR comprises an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a B Cell Maturation Antigen (BCMA) polypeptide. The CAR preferably binds an epitope comprising one or more amino acids of residues 13 to 32 of the N-terminus of human BCMA. Also disclosed is a nucleic acid molecule encoding the CAR of the invention, a genetically modified immune cell, preferably a T cell, expressing the CAR of the invention and the use of said cell in the treatment of a medical disorder associated with the presence of pathogenic B cells, such as a disease of plasma cells, memory B cells and/or mature B cells, in particular multiple myeloma, non-Hodgkin's lymphoma or autoantibody-dependent autoimmune diseases.

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) polypeptide, wherein the CAR polypeptide comprises: i. an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a human B Cell Maturation Antigen (BCMA) polypeptide; ii. a transmembrane domain; and iii. an intracellular domain; wherein the extracellular antigen-binding domain comprises: (I) a variable heavy chain (VH) domain comprising: (a) a heavy chain complementary determining region 1 (H-CDR1) comprising SEQ ID NO: 34 (RYWX.sub.1S), wherein X.sub.1 is I, F, L, V, Y, C, G, A, or M; (b) a heavy chain complementary determining region 2 (H-CDR2) comprising amino acids 50-67 of SEQ ID NO: 53 (EINPZ.sub.2SSTINYAPSLKX.sub.11X.sub.12), wherein Z.sub.2 is S, N, T, G, or D; X.sub.11 is D; and X.sub.12 is K or R; and (c) a heavy chain complementary determining region 3 (H-CDR3) comprising SEQ ID NO: 36 (SLYX.sub.4DYGDAX.sub.5DYW), wherein X.sub.4 is Y, and X.sub.5 is Y, L, F, I, V, A, C, or M; and (II) a variable light chain (VL) domain comprising: (a) a light chain complementary determining region 1 (L-CDR1) comprising SEQ ID NO: 37 (KASQSVX.sub.1X.sub.2NVA), wherein X.sub.1X.sub.2 is ES or DS; (b) a light chain complementary determining region 2 (L-CDR2) comprising SEQ ID NO: 29 (SASLRFS), and (c) a light chain complementary determining region 3 (L-CDR3) comprising SEQ ID NO: 30 (QQYNNYPLTFG); wherein expression of the CAR polypeptide in an immune effector cell is effective to increase cytotoxicity of the immune effector cell to both (i) multiple myeloma cells, and (ii) mantle cell lymphoma cells.

2. A genetically modified immune cell comprising the nucleic acid molecule or vector according to claim 1.

3. The genetically modified immune cell according to claim 2, wherein the immune cell is selected from the group consisting of a T lymphocyte or an NK cell.

4. The genetically modified immune cell according to claim 3, wherein the T lymphocyte is a cytotoxic T lymphocyte.

5. The nucleic acid molecule according to claim 1, wherein the CAR polypeptide transduces an intracellular signal in an immune effector cell in response to binding human BCMA.

6. The nucleic acid molecule according to claim 5, wherein: A. the VH domain comprises: (i) H-CDR1 comprising SEQ ID NO: 34 (RYWX.sub.1S), wherein X.sub.1 is I, F, or M; (ii) H-CDR2 comprising amino acids 50-67 of SEQ ID NO: 53 (EINPZ.sub.2SSTINYAPSLKX.sub.11X.sub.12), wherein Z.sub.2 is S, N, or D; X.sub.11 is D; and X.sub.12 is K or R; and (iii) H-CDR3 comprising SEQ ID NO: 36 (SLYX.sub.4DYGDAX.sub.5DYW), wherein X.sub.4 is Y, and X.sub.5 is Y or M; and B. the VL domain comprises: (i) L-CDR1 comprising SEQ ID NO: 37 (KASQSVX.sub.1X.sub.2NVA), wherein X.sub.1X.sub.2 is ES or DS; (ii) L-CDR2 comprising SEQ ID NO: 29 (SASLRFS); and (iii) L-CDR3 comprising SEQ ID NO: 30 (QQYNNYPLTFG).

7. The nucleic acid molecule according to claim 6, wherein the CAR polypeptide comprises the following sequences: TABLE-US-00022 SEQIDNO.25 i. H-CDR1:(RYWFS), SEQIDNO.26 ii. H-CDR2:(EINPSSSTINYAPSLKDK), SEQIDNO.27 iii. H-CDR3:(SLYYDYGDAYDYW), SEQIDNO.28 iv. L-CDR1:(KASQSVESNVA), SEQIDNO.29 v. L-CDR2:(SASLRFS), and SEQIDNO.30 vi. L-CDR3:(QQYNNYPLTFG).

8. The nucleic acid molecule according to claim 1, wherein the CAR polypeptide comprises a VH domain with at least 80% sequence identity to SEQ ID NO: 11; and a VL domain with at least 80% sequence identity to SEQ ID NO: 12.

9. The nucleic acid according to claim 8, wherein the VH domain comprises at least W36, E50, L99, Y100, Y101 and A106 of SEQ ID NO: 11, and the VL domain comprises at least S31, A34, S50, L53, Q89, Y91, Y94 and L96 of SEQ ID NO: 12.

10. The nucleic acid molecule according to claim 8, wherein the VH domain comprises at least the CDR sequences of SEQ ID NOs: 25 to 27, and the VL domain comprises at least the CDR sequences of SEQ ID NOs: 28 to 30.

11. The nucleic acid molecule according to claim 8, wherein the CAR polypeptide comprises the VH and VL domains according to SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

12. The nucleic acid molecule according to claim 1, wherein when the CAR polypeptide is expressed in a genetically modified immune cell, said immune cell binds human BCMA on the surface of a B-cell non-Hodgkin's lymphoma (B-NHL) via said CAR and is activated, thereby inducing cytotoxic activity against said B-NHL.

13. The nucleic acid molecule according to claim 12, wherein the B-NHL is JeKo-1, DOHH-2, SU-DHL4, JVM-3 and/or MEC-1 cell lines.

14. The nucleic acid molecule according to claim 1, wherein the extracellular antigen-binding domain further comprises a linker polypeptide positioned between the VH and VL domains.

15. The nucleic acid molecule according to claim 14, wherein said linker is selected from a Whitlow (SEQ ID NO: 13; GSTSGSGKPGSGEGSTKG) or Gly-Ser (SEQ ID NO: 14; SSGGGGSGGGGSGGGGS) linker, or linkers with at least 80% sequence identity to SEQ ID NO: 13 or 14.

16. The nucleic acid molecule according to claim 1, wherein the CAR polypeptide further comprises a spacer polypeptide positioned between the extracellular antigen-binding domain and the transmembrane domain, wherein said spacer is selected from the group consisting of: TABLE-US-00023 IgG1-CD28spacer(SEQIDNO15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK), IgG1-4-1BBspacer(SEQIDNO16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK), IgG4(Hi-CH2CH3)spacer(SEQIDNO17; ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK), IgG4(Hi-CH3)spacer(SEQIDNO18; ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK), IgG4(Hi)spacer(SEQIDNO19; ESKYGPPCPPCP), and a spacer with at least 80% sequence identity to any one of SEQ ID NOs: 15 to 19.

17. The nucleic acid molecule according to claim 1, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, and a transmembrane domain with at least 80% sequence identity to SEQ ID NO: 20 or 21.

18. The nucleic acid molecule according to claim 1, wherein the intracellular domain comprises a co-stimulatory domain selected from the group consisting of a 4-1BB co-stimulatory domain, a CD28 co-stimulatory domain, and a co-stimulatory domain with at least 80% sequence identity to SEQ ID NO: 22 or 23.

19. The nucleic acid molecule according to claim 1, wherein the CAR polypeptide further comprises a signaling domain, wherein said signaling domain comprises a CD3 zeta signaling domain, or a signaling domain with at least 80% sequence identity to SEQ ID NO: 24.

20. The nucleic acid molecule according to claim 1, wherein the intracellular domain comprises a CD28 co-stimulatory domain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is demonstrated by way of the example by the examples and figures disclosed herein. The figures provided herein represent particular embodiments of the invention and are not intended to limit the scope of the invention. The figures are to be considered as providing a further description of possible and potentially preferred embodiments that enhance the technical support of one or more non-limiting embodiments.

(2) FIG. 1: Schematic representation of preferred CAR structures.

(3) FIG. 2: Schematic representation of preferred CAR constructs IX, X; XI, XV, XVI, XVII.

(4) FIG. 3: List of preferred constructs and potential combinations of the various structural elements of the CARs as described herein.

(5) FIG. 4: Sequence comparisons between the mAb binding regions and the preferred humanized sequences employed in the present CAR.

(6) FIG. 5: GeneArt Plasmid with the BCMA-CAR Sequence.

(7) FIG. 6: Gel electrophoresis of the construct and vectors after restriction.

(8) FIG. 7: Confirmation of BCMA CAR-expression on human T cells following retroviral transduction: CAR Expression, constructs IX-XII, CD19, SP6.

(9) FIG. 8: Co-cultures of CAR-transduced human T cells with different target cell lines show specific T cell activation by distinct BCMA.sup.+ multiple myeloma (MM) and B-NHL cell lines. Functional in vitro co-cultivation and IFN-gamma ELISA.

(10) FIG. 9: CD107a (LAMP1) staining of co-cultured CAR-T cells with multiple myeloma cells: detection of activated degranulating CD8.sup.+ T cells upon antigen-specific (BCMA) stimulation by flow cytometry. Functional in vitro co-cultivation and LAMP1 detection, as determined by FACS.

(11) FIG. 10: Cytotoxicity assays reveal selective killing of BCMA-postive cell lines; essentially no killing was seen in BCMA-negative cell lines. Functional in vitro co-cultivation and 51Cr release assay.

(12) FIG. 11: BCMA and CD19 expression on the cell types assessed in the functional assays. Also shown are the results of MACS-based B-Cell isolation from PBMCs, together with anti-BCMA and anti-CD19 staining.

(13) FIG. 12: Schematic representation of the binding interaction between the scFV of the CAR and the BCMA epitope.

(14) FIG. 13: Sequence alignment of preferred humanized sequences of the HC compared to J22.9-xi.

(15) FIG. 14: Sequence alignment of preferred humanized sequences of the LC compared to J22.9-xi.

(16) FIG. 15: BCMA redirected CAR-T cells are effective against MM tumors in a xenografted NSG mouse model. (A) Engraftment of MM tumors in a xenografted NSG mouse model. Mice were challenged by i.v. transplantation of MM. 1S cells. At day 8 after tumor inoculation, tumor cell growth was visualized by IVIS imaging. To measure tumor burden, imaging was extended to 300 sec (day 1). (B) To follow treatment efficacy and to scale down bioluminescence intensity for better presentation, mice as in (A) were again imaged for 30 sec at day 1. Subsequent IVIS-exposures after CAR-T cell transfer, control SP6 CAR-T cells (n=4) and BCMA CAR-T cells (n=6), were done at 30 sec to allow better comparisons between day 1 and day 17 which has the highest intensity. White cross, animal was sacrificed because of advanced disease and animal protection laws. (C) Mean values of bioluminescence signal intensities obtained from regions of interests covering the entire body of each mouse are plotted for each group at each time point.

(17) FIG. 16: BCMA redirected CAR-T cells are effective against B-NHL tumors in a xenografted NSG mouse model. (A) Engraftment of mantle cell lymphomas in a xenografted NSG mouse model. Mice were challenged by i.v. transplantation of 610.sup.5 JeKo-1 cells. At day 7 after tumor inoculation, tumor cell growth was visualized by IVIS imaging. IVIS exposure, 120 sec. (B) To follow treatment efficacy and to scale down bioluminescence intensity for better presentation, mice as in (A) were again imaged for 30 sec at day 0. Subsequent IVIS-exposures after CAR-T cell transfer, control SP6 CAR-T cells (n=7) and BCMA CAR-T cells (n=7), were done for 30 sec to allow better comparisons between day 0 and day 16 which has the highest intensity. (C) Mean values of bioluminescence signal intensities obtained from regions of interests covering the entire body of each mouse are plotted for each group at each time point.

EXAMPLES

(18) The invention is demonstrated by way of the examples disclosed herein. The examples provide technical support for and a more detailed description of potentially preferred, non-limiting embodiments of the invention. In order to demonstrate the functionality of the CAR described herein, the inventors have performed the following experiments: co-cultures of CAR-transduced human T cells with different target cell lines show specific T cell activation by distinct BCMA+ MM and NHL cell lines; readout was release of IFN-gamma as effector cytokine from T cells; cytotoxicity assays reveal selective killing of BCMA+ cell lines; essentially no killing was seen in BCMA-negative cell lines or primary cells, e.g. HUVECs (endothelial origin), HEK293 (kidney), peripheral blood B cells, peripheral blood total leukocytes, T- and B-ALL, colon carcinoma. CD107a staining of co-cultured CAR-T cells with multiple myeloma cells, detection of degranulating CD8+ T cells upon antigen-specific (BCMA) stimulation by flow cytometry. In vivo experiments relate to using a xenotransplantation NSG mouse model to generate data on i) functionality, ii) off-target reactivity, iii) T cell memory, and iv) biosafety of adoptively transferred CAR-T cells against B-NHL and myeloma cell lines. For B-NHL the cytolytic capacity of anti-BCMA CAR-T cells is compared with an established anti-CD19 CAR-T cell product.

Example 1: Cloning and Plasmid Preparation

(19) CAR sequences were synthesized using GeneArt (Gene Synthesis Service). Restriction digestion of the CAR construct was carried out using Notl and EcoRI (FIG. 5). The retroviral vector MP71 was also digested with Notl and EcoRI, and subsequently dephosphorylated. The CAR and vector were separated using gel electrophoresis (FIG. 6.) and the fragments were purified. The CAR construct was subsequently ligated into the vector (50 ng) at a ratio of 3:1. Transformation of the ligation mixture into MACH-1 was carried out (FIG. 3.). A control digest was conducted and the Mini-Preparation was sequenced. The constructs were subsequently re-transformed into MACH-1. A maxi-Preparation of the MP71-BCMA-CAR plasmid was produced.

(20) MP71 is a single (+)-strand-RNA-Virus. Reverse-Transcriptase converts the retroviral RNA-Genome into a DNA copy. The DNA integrates as a provirus at a random position into the target genome. Through cell division the virus reproduces stably as a provirus.

Example 2: Transfection and Transduction

(21) Day 0: Seeding HekT (293T)- or GalV-cells for virus production in 6 well plates

(22) Day 1: Transient 3-plasmid transfection for retrovirus production (calcium phosphate transfection). Per well, 18 g of DNA was used, in 250 mM Cacl2, 150 l H2O, according to standard protocols. Cells are incubated for 6 h at 37 C., medium is exchanged, further incubation carried out for 48 h at 37 C.

(23) Coating of 24-well non-tissue culture plates with anti-huCD3 und anti-huCD28 antibodies: Prepare anti-CD3/anti-CD28-antibody solution in PBS (5 g/ml anti-CD3, 1 g/ml anti-CD28), 0.5 ml per well. Incubate each well with 0.5 ml antibody solution for 2 h at 37 C., replace with sterile 2% BSA-solution (in water), incubation: 30 min (37 C.). Remove BSA-solution and wash wells with 2 ml PBS.

(24) Purification of PBMCs from 40 ml Blood (2.510.sup.7 PBMCs):

(25) Prepare 12.5 ml Ficoll-Gradient medium in 250 ml Falcon-Tube, dilute blood with RPMI (+100 IU/ml Penicillin, Streptomycin) to 45 ml, mix and coat with 22.5 ml Blood-Medium-mixture, centrifuge (20 min, 20 C., 1800 rpm, RZB *648, G 17.9). Discard 15 ml upper phase. Transfer remainder of the upper phase with white-milky PBMC-containing intermediate phase to a new 50 ml Falcon-Tube, fill to 45 ml with RPMI (+100 IU/ml Penicillin, Streptomycin) and centrifuge. Re-suspend pellets in 45 ml RPMI (+100 IU/ml Penicillin, Streptomycin), centrifuge, combine pellets in 10-20 ml T cell medium, stain one sample with trypan blue, count cells and add cells at a concentration of 1-1.510.sup.6 cells/ml (T-cell medium (+100 IU/ml IL-2) corresponds to 400U/ml clinic-IL2) to the anti-CD3, anti-CD28 coated wells. Centrifuge remainder of PBMCs, suspend in freezing medium and store in Cryo tubes at 80 C.

(26) Day 3: Transduction of PBLs

(27) Remove and filter (0.45 m filter) viral supernatant from Hekt- or GalV-cells. Treat stimulated PBMCs with 1.5 ml viral supernatant.

(28) Day 4: Transduction of PBLs

(29) Filter remaining viral supernatant (4 C.) and second supernatant from Hekt- or GalV-cells (0.45 m). Collect 1 ml to 1.5 ml supernatant from the PBLs. Treat stimulated PBMCs with 1 ml to 1.5 ml viral supernatant and centrifuge in the CD3-/CD28-coated wells (90 min, 32 C., 2000 rpm). Final concentration of 100 IU/ml IL2 (1 ul von 400U/l) or 10 ng/l IL7 und 10 ng/l IL15, and additionally 4 g/ml (8 l) Protamine sulfate. Centrifuge at 90 min 2000 rpm 32 C.

(30) Day 7 to Day 13: Culture PBLs, treat T cell medium with fresh IL2 or IL7/IL15.

(31) Day 13: End T-cell stimulation.

(32) Rinse PBL-cultures from the cell culture flasks, centrifugation, re-suspend pellet in T-cell medium (+10 IU/ml IL2).

(33) As of Day 15: Functional assays

Example 3: Functional In Vitro Testing of Anti-BCMA CAR T Cells

(34) I. Confirmation of BCMA CAR-Expression on Human T Cells Following Retroviral Transduction

(35) Evidence was obtained of folding and transport of the CAR receptor in context of human T cells; the functionality of retrovirus transduction protocol was assessed.

(36) Human peripheral blood leukocytes were purified via a Ficoll gradient. Cells were cultured, stimulated and retrovirally transduced as described above. Following transduction, cells were further cultured in either IL-2 or IL-7/IL-15 containing medium prior to the analysis of BCMA-CAR expression.

(37) Transduction rate and viability were assessed by flow cytometry (FACS) analysis. To detect BCMA-CAR expression, cells were stained with anti-human Ig-antibody that recognizes selectively the human IgG1 or IgG4 section in the spacer region of the CAR construct. A co-staining for CD3/CD8/CD4 T cells was performed. For the results refer to FIG. 7.

(38) II. Co-Cultures of CAR-Transduced Human T Cells with Different Target Cell Lines Show Specific T Cell Activation by Distinct BCMA.sup.+ Multiple Myeloma (MM) and B-NHL Cell Lines

(39) The readout was release of IFN-gamma as effector cytokine from T cells.

(40) Generate retrovirus-transduced human T cells, as detailed before; employ all BCMA CAR-receptor variants (IX-XVII), SP6-negative control CAR, CD19 CAR, UT=untransduced T cells. Use the following human cell lines as target cells in co-culture:

(41) TABLE-US-00021 Cell line Origin BCMA-positivity NCI-H929 multiple myeloma (MM) yes MM.1S MM yes OPM-2 MM yes RPMI 8226 MM yes REH B acute lymphoblastic leukemia no (B-ALL) REH-BCMA REH stably transduced with yes BCMA DOHH-2 immunoblastic B cell lymphoma yes, weakly progressed from follicular centroblastic/centrocytic lymphoma (FL) JVM-3 B cell chronic lymphocytic yes, weakly leukemia (B-CLL) SU-DHL4 diffuse large B cell lymphoma yes, weakly (DLBCL), germinal center type NALM-6 B acute lymphoblastic leukemia no (B-ALL) RS4 B-ALL no Jurkat T cell acute lymphoblastic no leukemia (T-ALL) normal peripheral healthy donor no B cells MEC-1 B-CLL yes, weakly JEKO-1 mantle cell lymphoma (MCL), B- yes, weakly NHL HUVEC human umbilical vein endothelial no cells, healthy donor SW620 colon carcinoma no HT116 colon carcinoma no HEK293 human embryonic kidney epithelial no cells PBMC human peripheral blood no mononuclear cells, healthy donor

(42) Co-culture retrovirally transduced T cells for 18-20 hrs in the presence of the listed cell lines or primary cells at a ratio 1:1. After that time, take cell-free culture supernatant; max. release is induced by PMA/ionomycin stimulation of effector T cells; minimum release is T cells only. Determine IFN-gamma release in the supernatant by ELISA. Refer to FIG. 8 for the results.

(43) III. CD107a (LAMP1) Staining of Co-Cultured CAR-T Cells with Multiple Myeloma Cells: Detection of Activated Degranulating CD8.sup.+ T Cells Upon Antigen-Specific (BCMA) Stimulation by Flow Cytometry

(44) Generate retrovirus-transduced human T cells, as detailed above; employ BCMA CAR-receptor variants (IX-XI), SP6-negative control CAR.

(45) Co-culture retrovirally transduced T cells for 18 hrs in the presence of the listed cell lines at a ratio of 1:1.

(46) Add for overnight culture anti CD107a (LAMP1) antibody into cell medium; antibody binds continuously on T cells when secretory lysosomes are fusing with the plasma membrane and release the enzymatic content of their vesicles. These vesicles contain cytolytic mediators such as granzymes and perforin. On the next day, T cells are co-stained with anti CD8 and/or CD3.

(47) Analysis by flow cytometry: higher CD107a reactivity, expressed as mean fluorescence intensity (MFI), indicates stronger activation of T cells. The antigen-dependent activation of T cells can be confirmed. For results refer to FIG. 9.

(48) IV. Cytotoxicity Assays Reveal Selective Killing of BCMA-Postive Cell Lines; Essentially No Killing was Seen in BCMA-Negative Cell Lines

(49) Use of .sup.51Cr-release assay for quantitation of cytotoxic T lymphocyte activity. Measure target cell cytolysis.

(50) Generate retrovirus-transduced human T cells, as detailed before; employ BCMA CAR-receptor variants (IX-XI), SP6-negative control CAR; CD19 CAR as control Label target cells with .sup.51Cr. Co-culture then CAR-T cells and labeled target cells for 4 hrs. Titrate the effector to target ratio. E:T 80:1 40:1 20:1 10:1 5:1 2.5:1

(51) Harvest cell-free cell culture supernatant. Transfer supernatant to LUMA-scintillation plates, measure released .sup.51Cr in a gamma-scintillation counter. Max. release: target cells lysed by Triton X-100 permeabilisation. Min. release: target cells alone. For the results, refer to FIG. 10.

(52) Furthermore, FIG. 11 provides results showing the amount of BCMA and CD19 expressed on the surface of each of the cell types assessed for cytotoxicity. FIG. 12 provides a schematic representation of the interaction between the scFV binding region of the CAR and the BCMA epitope.

Example 4: In Vivo Experiments Using a Xenotransplantation NSG Mouse Model to Assess Adoptively Transferred CAR-T Cells Against B-NHL and Myeloma Cell Lines In Vivo Experiments Using Xenotransplantation into NSG Mice

(53) 1) To demonstrate that CAR T cells equipped with the diverse anti BCMA-variants have effector activity also under in situ conditions, multiple myeloma cells with different BCMA antigen densities are transplanted via an i.v. route into NSG-mice (NOD.Cg-Prkdc.sup.scid II2rg.sup.tm1 Wjl/SzJ). The multiple myeloma cell lines that may be employed are: RPMI-8226, low BCMA; MM1S, intermediate BCMA density; NCI-H229, high BCMA density.

(54) 2) To confirm anti BCMA CAR T cell reactivity against B-NHL cell lines in situ, NSG mice are injected i.v. with luciferase-transduced cell lines, such as SU-DHL4 (DLBCL), JEKO-1 (mantle cell lymphoma), JVM3 (CLL), MEC1 (CLL), DOHH-2 (FL).

(55) BCMA CAR-T Cells Mediate In Vivo Antitumor Activity in Mouse Models of Multiple Myeloma (MM) and B-Cell Non Hodgkin's Lymphoma (B-NHL):

(56) To provide proof-of-concept that the strong in vitro activity of T cells modified with the BCMA CAR translates into efficient antitumor activity in vivo, we inoculated cohorts of NOD.Cg-Prkdc.sup.scid II2rg.sup.tm1 Wjl/SzJ (NSG) mice i.v. with the human MM.1S cell line (FIG. 15) or the B-NHL cell line JeKo-1 (mantle cell lymphoma) (FIG. 16), transduced with the luciferase gene in tandem with GFP. NSG mice do not develop T, B, and NK cells and are therefore suitable for tolerance and growth of xenotransplantated human cells. Within the experimental time frame presented here, graft-versus-host (GvHD) reactions (xenoreactivity) was not observed (data not shown). Tumor growth was monitored by IVIS imaging and luciferin injection 7-8 days thereafter. Following tumor growth confirmation, CAR-T cells were i.v. injected one day later (=day 0). For functional in vivo experiments CAR construct IX (B IX) was used. Total numbers never exceeded 6-710.sup.6/animal CAR-T cells, and the average transduction rate for T cells in this population was 40-60%. Per donor, SP6 and BCMA transduction rates were matched within a range of +/10%. For the two experiments shown, an effective rate of 310.sup.6 transduced CAR-T cells was used. Control mice received SP6 CAR-T cells.

(57) In the MM1.S experiment (FIG. 15), 310.sup.6 transduced CAR-T cells (as above, total: 6-710.sup.6) were transplanted and the observation interval was extended to 17 days. While essentially all SP6 CAR treated animals had progressive MM disease, characterized by strong luminescence signals over the spine, pelvis, and hind legs, or were sacrificed because of disease progression in accordance with (Berlin State) animal protection laws, this was clearly not the case for the BCMA CAR treatment group. We conclude that at this comparably low CAR-T cell number the BCMA CAR-T cells already have anti-myeloma activity (FIGS. 15A-C).

(58) Due to the high affinity and avidity of the anti-BCMA CAR-T cell, even low BCMA-expressing mature B cell NHL can be recognized, allowing for T cell activation and tumor cell killing. Such mature B-NHL entities include certain stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia (see FIGS. 11, 10, 9, 8). To prove the suitability of BCMA as a target structure in B-NHL entities, transduced CAR-T cells (total: 6-710.sup.6) were transplanted in NSG mice which had been challenged with the mantle cell lymphoma cell line JeKo-1. While essentially all SP6 CAR treated animals had progressive lymphoma disease, characterized by strong luminescence signals over the liver, thoracical organs, bone marrow in hind limbs, and spleen, this was clearly not the case for the BCMA CAR treatment group. With this we provide the first pre-clinical in vivo proof that BCMA CAR-T cells have anti-tumor activity beyond multiple myeloma and extending to B-NHL lymphoma entities (FIGS. 16A-C).

Example 5: Determination of Surface Density of BCMA Molecules

(59) The high affinity and avidity of the anti-BCMA CAR-T cells allow for the recognition of even low BCMA-expressing mature B cell NHL entities, resulting in T cell activation and tumor cell killing. Such mature B-NHL entities include certain stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

(60) To quantify the surface density of the BCMA molecules, we have applied the PE Phycoerythrin Fluorescence Detection Kit, also referred to as BD Quantibrite assay (BD Bioscience). The number of PE molecules per cell can be converted to antibodies per cell, which is a quantitative estimate of the number of antigens per cell. A flow cytometry detection method was applied.

(61) Using this method, we find that the multiple myeloma cell line NCI-H929 has a relative surface BCMA antigen density of 12555, the multiple myeloma cell line OPM-2 has 3443 BCMA molecules, and the multiple myeloma cell line MM.1S has a relative value of 3181.

(62) The BCMA antigen densities for the mentioned B-NHL cell lines, relative to NCI-H929, are: DOHH-2: 1/20, JeKo-1: 1/250, MEC-1: 1/34