TARGETING B CELL ACTIVATING FACTOR RECEPTOR (BAFF-R) USING LIGAND-BASED CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELLS

Abstract

The disclosure relates generally to ligand-based chimeric antigen receptor (CAR) cells. More specifically, the CAR cells express B-cell activating factor (BAFF) protein for recognition by a receptor of BAFF on the surface of a cell. CAR cells can include cytotoxic T lymphocytes, natural killer (NK) cells or natural killer T (NKT) cells that express a chimeric receptor that recognizes a receptor of BAFF. The disclosure further relates to methods of treating a variety of conditions, such as cancers and autoimmune diseases, using the disclosed CAR cells.

Claims

1-39. (canceled)

40: A method of treating a condition in a patient in need thereof, the method comprising administering to the patient a cell that expresses a chimeric receptor, wherein the chimeric receptor comprises, from N-to-C terminus: (a) an extracellular domain that comprises: (i) a B-cell activating factor (BAFF) partial sequence consisting of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3, and (ii) a hinge domain; (b) a transmembrane domain; (c) a CD28 intracellular domain; and (d) an OX40 intracellular domain.

41: The method of claim 40, wherein the hinge domain is an IgG1 hinge domain.

42: The method of claim 40, wherein the transmembrane domain is a CD28 transmembrane domain.

43: The method of claim 40, wherein the extracellular domain binds to BCMA, TACI, and BAFF-R.

44: The method of claim 40, wherein the BAFF partial sequence consists of the amino acid sequence of SEQ ID NO: 3.

45: The method of claim 40, further comprising a CD3 zeta intracellular domain.

46: The method of claim 40, further comprising a linker peptide between the BAFF partial sequence and the hinge domain.

47: The method of claim 46, wherein the linker peptide comprises the amino acid sequence of SEQ ID NO: 12.

48: The method of claim 40, wherein the cell is a human cell.

49: The method of claim 40, wherein the cell is a T lymphocyte.

50: The method of claim 40, wherein the cell is a cytotoxic T lymphocyte.

51: The method of claim 40, wherein the cell is a natural killer (NK) cell.

52: The method of claim 40, wherein the cell is a natural killer T (NKT) cell.

53: The method of claim 40, wherein the cell is autologous to the subject.

54: The method of claim 40, wherein the cell is allogeneic to the subject.

55: The method of claim 40, wherein the condition is a cancer.

56: The method of claim 40, wherein the condition is a hematologic malignancy.

57: The method of claim 40, wherein the condition is multiple myeloma.

58: The method of claim 40, wherein the condition is non-Hodgkin's lymphoma.

59: The method of claim 40, wherein the condition is a B cell malignancy.

60: The method of claim 40, wherein the condition is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, or Hodgkin's lymphoma.

61: The method of claim 40, wherein the condition is an autoimmune disease.

62: The method of claim 40, wherein the condition is a B cell associated autoimmune disease.

63: The method of claim 40, wherein the condition is systemic lupus erythematosus.

64: The method of claim 40, wherein the condition is systemic lupus erythematosus, Sjogren's syndrome, narcolepsy, diabetes, pancreatitis, Crohn's disease, Celiac disease, ankylosing spondylitis, psoriasis, Grave's Disease, or rheumatoid arthritis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The above and other features, examples and advantages of aspects or examples of the present disclosure are better understood when the following detailed description is read with reference to the accompanying drawings, in which:

[0067] FIG. 1 is an exemplary schematic representation of a CAR construct according to the present disclosure.

[0068] FIG. 2 is a schematic representation of exemplary CAR constructs SEQ ID NOs: 15-17 and 21-23 according to the disclosure.

[0069] FIG. 3 includes graphs demonstrating increased expression of BAFF-R on the surface of ALL cells.

[0070] FIG. 4 shows flow cytometry histograms of BAFF-R expression in newly diagnosed and relapsed ALL patients.

[0071] FIG. 5 demonstrates killing activity by a BAFF-CAR-T expressing SEQ ID NO: 15 and SEQ ID NO: 17 in MCL cells.

[0072] FIG. 6 is a series of graphs demonstrating cytotoxicity against leukemia cells lines by BAFF-CAR-T cells.

[0073] FIG. 7 is a scatter plot showing increased degranulation of CAR-T cells following incubation with Jeko-1 cells.

[0074] FIG. 8 is a graph showing tumor burden following treatment with BAFF-CAR-T cells.

[0075] FIG. 9 shows photographs of mice implanted with a human leukemia cell line following treatment with PBS control (left) and BAFF-CAR-T cells (right) according to the present disclosure.

[0076] FIG. 10 is a line graph showing survival rates following treatment with control and BAFF-CAR-T cells according to the present disclosure.

DETAILED DESCRIPTION

[0077] Example embodiments will now be described more fully hereinafter with reference to the accompanying figures in which example embodiments and representative data are shown. Whenever possible, the same reference numerals are used throughout the drawings to refer to the same or like parts. However, the embodiments may take on many different forms and should not be construed as limited to those specifically set forth herein. These example embodiments are provided so that this disclosure will be both thorough and complete, and will fully convey the scope of the claims to those skilled in the art.

[0078] Directional terms as used herein (e.g., up, down, right left, front, back, top, bottom) are made only with reference to the figures as drawn and are not intended to imply absolute orientation.

[0079] As used herein, the term about means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. When the term about is used in describing a value or an endpoint of a range, the disclosure should be understood to include the specific value or endpoint referred to. Whether or not a numerical value or endpoint of a range in the specification recites about, the numerical value or endpoint of a range is intended to include two embodiments: one modified by about, and one not modified by about. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0080] The terms substantial, substantially, and variations thereof as used herein are intended to note that a described feature is equal or approximately equal to a value or description. For example, a substantially planar surface is intended to denote a surface that is planar or approximately planar. Moreover, substantially is intended to denote that two values are equal or approximately equal. In some embodiments, substantially may denote values within about 10% of each other, such as within about 5% of each other, or within about 2% of each other.

[0081] It is noted that the terms substantially and about may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. Thus, cells that are free of or substantially free of T cell contamination for example, are cells to which T cells are not actively added or batched into cell culture, but may be present in very small as a contaminant resulting from natural cell progression during expansion. Similarly, other components may be characterized as free of or substantially free of in the same manner.

[0082] Further, as used herein, the term consisting essentially of allows for elements not explicitly recited but excludes element that affect basic or novel characteristics of the inventions. As recited herein, the term consisting of excludes elements not expressly stated.

[0083] B-cell activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. BAFF is abundantly produced by monocytes, macrophages, dendritic cells and stromal cells, which are main cellular components of MCL tumor microenvironment. BAFF signaling is essential for the generation of mature B cells and it helps survival of normal and malignant B cells. BAFF has at least three receptors: transmembrane activator and CAML interactor (TACI), B-cell maturation antigen (BCMA) and BAFF receptor (BAFF-R). Of these, BAFF-R is specific to BAFF while BCMA and TACI share another homologous ligand, APRIL. Signaling through BAFF-R mediates B cell survival. Virtually all mature B cell leukemias and lymphomas express BAFF receptor. Early B cells, which are counterparts of acute lymphocytic leukemia (ALL) do not express BAFF-R, but cells from patients with some cancers express high levels of BAFF-R, including ALL and mantle cell lymphoma (MCL) patients. Further, BAFF-R is expressed only on mature B cells, making it an attractive target for targeting and reducing side effects caused by off targeting. Thus, BAFF-R presents a target opportunity for treating such cancers, as well as autoimmune diseases where increased serum BAFF levels are often present.

[0084] In one example according to the present disclosure is a method of treating or preventing a disease or condition by targeting a receptor of BAFF. In another example according to the present disclosure is a method of treating or preventing a disease or condition by targeting a cell expressing or overexpressing a receptor of BAFF, such as BAFF-R. In an exemplary embodiment, the disease or condition is a cancer, such as a hematologic malignancy. In one embodiment, the hematologic malignancy can be any hematologic malignancy wherein the cancer cells express or overexpress a receptor of BAFF, including but not limited to acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B cell malignancies, and multiple myeloma. In another exemplary embodiment, the disease or condition is an autoimmune disease, such as systemic lupus erythematosus, Sjogren's syndrome, narcolepsy, diabetes, pancreatitis, Crohn's disease, Celiac disease, ankylosing spondylitis, psoriasis, Grave's Disease, or rheumatoid arthritis. In another example the disease or condition is an abdominal aortic aneurysm with B cell involvement.

[0085] Conventional chimeric antigen receptor-T (CAR-T) cells use a single chain variable fragment of an antibody (Sc-Fv) to the corresponding tumor target antigen. Indeed, WO 2017/214167 reports a BAFF-R antibody that is capable of binding to human BAFF-R protein and induce antibody-dependent cellular cytotoxicity on BAFF-R expressing cells. These antibodies can form part of a chimeric antigen receptor (CAR) and are said to be used for the treatment of cancer. This antibody treatment approach, however, is limited to only those cells expressing BAFF-R. Because an antibody approach is specific for only one receptor, the prior art disclosure is limited in its approach. Provided herein is a BAFF-ligand-based CAR that can be used to target not only BAFF-R, but any receptor of BAFF, including, e.g., TACI and BCMA.

[0086] Instead of Sc-Fv, the present disclosure uses a ligand approach to hunt for receptors of BAFF, such as BAFF-R, TACI and BCMA, on the surface of, for example, tumor cells for CAR-T cell immunotherapy against a wide variety of cancers and autoimmune diseases. Using BAFF protein, BAFF-ligand-based CARs have been generated and are reported herein.

[0087] In the present disclosure, the BAFF protein or BAFF refers to any of the recombinant or naturally occurring forms of the B-cell activating factor as set forth in SEQ ID NO:1 or variants to homologs thereof that maintain BAFF activity (e.g. within at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparted to BAFF). Optionally, the variants or homologs thereof have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity (i.e. sequence homology) across the whole of SEQ ID NO: 1 or a portion of SEQ ID NO:1.

[0088] As used herein, a partial sequence or BAFF partial sequence refers to a portion of SEQ ID NO: 1 that maintains BAFF activity similar to that of the whole sequence, and in particular, an extracellular portion of BAFF that is responsible for binding with a receptor of BAFF. In one example of a partial sequence is a sequence comprising at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% of the naturally occurring BAFF sequence. Also contemplated are sequences having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the partial sequence. In one example, the partial BAFF sequence comprises amino acids 82-285 (SEQ ID NO:2) or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:2. In another example, the BAFF partial sequence comprises amino acids 134-285 (SEQ ID NO:3) having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:3.

[0089] As used herein, chimeric antigen receptor T cells or CAR-T cells are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. Chimeric antigen receptor or CAR are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. CARs are recombinant receptors that provide both antigen-binding and T-cell activating functions. In an example according to the present disclosure, the CAR-T cells have been engineered to target a receptor of BAFF using BAFF as a ligand. CAR-T cell therapy is based on recognition of specific tumor antigen by genetically modified T cells, followed by intracellular signaling and activation of T cells, which subsequently leads to destruction of the tumor cell. In another example of the present disclosure, natural killer (NK) cells or natural killer T (NKT) cells are modified to express a CAR.

[0090] The present disclosure relates to CARs that have been engineered to express a BAFF ligand. FIG. 1 shows a schematic example of how a CAR according to the present disclosure can be designed. In one example, the CAR expresses the native human BAFF full sequence, i.e. SEQ ID NO:1. In other examples according to the present disclosure, the CAR expresses a partial sequence of BAFF. In one embodiment the partial sequence comprises an extracellular domain of BAFF. In another embodiment, the partial sequence comprises a domain involved in receptor binding, such as a BAFF-R, TACI or BCMA binding domain. In one embodiment, the partial sequence comprises amino acids 82-285 (SEQ ID NO:2) of BAFF. In another embodiment, the partial sequence comprises amino acids 134-285 (SEQ ID NO:3) of BAFF. In other examples, the BAFF sequence (full or partial) can be incorporated into the construct such that the BAFF domain is transposed so that the amino acid sequence is reversed. In other words, the amino acids that are at the C-terminal end in the native sequence become the amine terminus of the sequence, and the amino acids that are normally at the N-terminus become the C-terminal end of the sequence.

[0091] The CAR can further comprise one or more hinge domains. In one example, the hinge domain comprises a hinge domain of CD8a (SEQ ID NO:4), or a partial sequence thereof. In another example, the hinge domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:4. In another example, the hinge domain comprises a hinge domain of IgG1 (SEQ ID NO:5), or a partial sequence thereof. In another example, the hinge domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5. In yet another example, the CAR cells of the present disclosure comprise a hinge domain of CD28. In yet another example, the CAR cells of the present disclosure comprise a hinge domain of FCyRIII.

[0092] The CAR further comprises one or more transmembrane domains. In one example, the CAR comprises a transmembrane domain of CD8?, such as that of SEQ ID NO:6. In another example, the transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:6. In another example, the CAR comprises a transmembrane domain of CD28, such as that of SEQ ID NO: 7. In another example, the transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:7. In one embodiment, the CAR comprises a hinge domain and a transmembrane domain.

[0093] The CAR can further comprise one or more intracellular domains. In one example, the CAR comprises an intracellular domain of 41BB, such as SEQ ID NO:8. In another example, the intracellular domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:8. In another example, the CAR comprises an intracellular domain of CD28, such as that disclosed in SEQ ID NO:9. In another example, the intracellular domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:9. In yet another embodiment, the CAR comprises an intracellular domain of CD3-zeta, such as that disclosed in SEQ ID NO:10. In another example, the intracellular domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:10. In yet another example, the CAR comprises an intracellular domain of OX40 (SEQ ID NO:11). In another example, the intracellular domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:11. According to the present disclosure, the CAR can comprise more than one intracellular domain. In one embodiment, the CAR comprises a hinge domain, a transmembrane domain, and one or more intracellular domain(s).

[0094] The CAR can further comprise a signaling peptide. In one example, the signaling peptide is a peptide according to SEQ ID NO:21 or a peptide having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:21. In another example, the signaling peptide is a peptide according to SEQ ID NO:22 or a peptide having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:22. In one embodiment, the CAR comprises a hinge domain, signaling domain, a transmembrane domain, and one or more intracellular domain(s).

[0095] Because distance between T cell and target cell is an important factor affecting tumor recognition and cytotoxicity, a linker domain can be incorporated between BAFF and the hinge domain or between the hinge domain and the transmembrane domain. In some embodiments, the linker comprises a simple alkyl chain, such as (CH.sub.2).sub.nCH.sub.3 unit, wherein n is the number of CH.sub.2 groups and can vary from 1-100, preferably 1-50, 1-20, or 1-10. In another example, the linker can be a peptide of 1-50 amino acids, such as 1-20 amino acids, or 1-10 amino acids. In a preferred embodiment, the linker comprises SEQ ID NO: 12, or a variant thereof having at least 85% sequence homology to SEQ ID NO: 12.

[0096] FIG. 2 shows a schematic of six exemplary CAR constructs according to the disclosure. The top schematic includes a BAFF full sequence, of which SEQ ID NO: 15 is an example. The top middle schematic utilizes a BAFF partial sequence but is otherwise identical to the top schematic. SEQ ID NOs: 16 & 17 are examples of such a construct. The middle bottom and bottom schematics employ the BAFF and partial BAFF sequences, respectively, but incorporate different hinge, transmembrane and intracellular regions. SEQ ID NO: 21 and SEQ ID NOs:22 & 23, respectively, are represented by these schematics.

[0097] The following examples are illustrative and are not intended to limit the scope of the invention as claimed.

EXAMPLES

Example 1ALL Cells Express BAFF-R

[0098] To confirm BAFF-R expression in ALL cells, 359 pediatric and 147 adult ALL patient samples were compared with PBMCs from 74 healthy donors using the Haferlach Leukemia Oncomine database. In the present disclosure, the peripheral blood mononuclear cells, PBMCs or mononuclear cells refer to mononuclear cells separated from peripheral blood typically used for anti-cancer immunotherapy. PBMCs may be obtained from a healthy person, a patient at risk of cancer, or a cancer patient. Collected blood samples (30 mL) from healthy donors were processed to isolate peripheral blood mononuclear cells (PBMCs) by density gradient separation using Ficoll-Paque. Samples were diluted with 1? volume of PBS (pH 7.2) and diluted blood (30 mL) was layered over 15 mL of Ficoll-Paque in a 50 ml conical tube. Samples were centrifuged (400?g, 30 min) at 20? C. in a swing-bucket rotor without brake. The resulting upper layer was aspirated, leaving the PBMC layer undisturbed. The PBMC layer was transferred to a new 50 mL conical tube, which was then filled with PBS and centrifuged (300?g, 10 min) at 20? C. The cell pellet was resuspended with PBS and centrifuged (300?g, 10 min) at 20? C. The cells were resuspended in 1? MojoSort buffer at density of 1?10.sup.8/mL. The CD3+ T cells were then purified by MojoSort human CD3 T cell isolation kit (Biolegend).

[0099] As seen in FIG. 3, mRNA expression was significantly increased in both ALL patient samples. FIG. 3 show a significant increase in BAFF-R expression in pediatric (FIG. 3, 2) and adult (FIG. 3) as compared to healthy donors (1), showing that BAFF-R levels are increased in ALL cells as compared to healthy cells.

[0100] BAFF-R expression was also measured in newly diagnosed ALL cells at the time of diagnosis. Using flow cytometry (see Vicioso, et al. Combination Therapy for Treating Advanced Drug-Resistant Acute Lymphoblastic Leukemia Cancer Immun. Res 7, 1106-1119 (2019), hereby incorporated by reference, for methods), levels of BAFF-R expression in newly diagnosed (FIG. 4 bottom left) and relapsed (FIG. 4, bottom right) ALL patients were measured and compared to Jurkat (negative control, top left) and Jeko-1 (positive control, top right) cells. In all graphs, the left histogram represents an unstained BAFF-R positive control; the right histogram represents cells stained with an anti-BAFF-R antibody.

Example 2Development of BAFF-CAR-T Cells

[0101] The design of various BAFF-CAR-T cells was discussed above. Each construct was designed to include the BAFF protein (whole or partial sequence), a hinge domain, a transmembrane domain and one or more intracellular domains. The construct may also optionally include an extracellular signaling domain and a linker domain to optimize spacing. Each construct was cloned into a 3.sup.rd generation lentivirus vector and packaged in HEK293T cells.

[0102] Human PBMC cells were used to collect and purify CD3+ T cells as described above. The T cells were activated using Dynabeads human T-Activator CD3/CD28 (Gibco/Invitrogen). For activation of 1?10.sup.6 of CD3+ T cells, mixing 25 ?L Dynabeads with the T cells in 1.5 mL of complete culture medium (advanced RPMI medium 1640 with 2 mM L-glutamine, 10% FBS and 100 U/mL penicillin/streptomycin) in the presence of 50 U/mL of interleukin-2 (IL-2) (PeproTech). Cells were resuspended with beads in medium and distributes to one well of a 24-well plate. Cells were cultured in 5% CO.sub.2 incubator at 37? C. for 24-48 hours to prepare for virus infection with concentrated lentivirus containing BAFF CAR.

[0103] Production of the lentivirus supernatants containing the BAFF-CAR was accomplished as follows. Lentiviral supernatant was produced using the packaging cell line, 293 FT. Healthy 293 FT cells were cultured in DMEM medium with 10% FBS with 100 U/mL penicillin/streptomycin. The day before vector transfection, 293 FT cells were split to 5?10.sup.6/10 cm tissue culture plate. Four plates of 293 FT cells were needed to make enough supernatants for concentration of lentivirus. For one 10 cm plate, 200 ?L of Opti-MEM medium was added to an Eppendorf tube, followed by 3750 ng packaging plasmid psPax2, 1250 ng envelop plasmid pMD2G and 5000 ng of BAFF CAR vector. The tubes were mixed gently and then 10 ?L of X-tremeGENE HP transfection reagent (Sigma catalog #06 366 236 001) was added, mixed gently and incubated for 20 minutes at RT. The DNA mixture was added dropwise into a 10 cm 293 FT plate and incubated at 5% CO.sub.2 incubator at 37? C. After an overnight culture, the medium was removed and replaced with 6 mL of fresh complete DMEM medium. Supernatants were collected at 48 h and 72 h after vector transfection from the four plates (23 mL in total) into 50 mL conical tube and centrifuge at 500?g for 10 min to remove cells debris. 23 mL of the clear supernatants was transferred to a new 50 ml conical tube, to which 7.6 mL of Lenti-X Concentrator (Clontech) was added, mixed and incubated at 4? C. overnight. The supernatants were centrifuged (1500?g, 45 min, 4? C.) and the supernatants removed to afford an off-white pellet. Pellets were resuspended in 1 mL complete advanced RMPI medium with IL-2 and prepared for virus transduction into CD3+ T cells.

[0104] Around 1?10.sup.6 activated T cells together with Dynbeads were resuspended in 1 mL of complete advanced RPMI medium containing concentrated BAFF CAR and distributed in an amount of 100 ?L into each well into total of 10-12 wells in round-bottom 96 well plate. T cells were spinoculated (3480 rpm, 22? C., 90 min). After centrifugation, cells were resuspended and collected with Dynabeads, then reseparated into 24-well plates again. 1 mL more complete advanced RPMI medium was added together with IL-2 50 U/mL, IL-7 10 ng/ml and IL-15 5 ng/mL. T cells were cultured in an incubator at 5% CO.sub.2, 37? C. Fresh media with cytokines was added when the media in the culture became yellow. The Dynabeads was removed 4-5 days after initial T cells activation. The transduced T cells expanded exponentially 5-8 days after activation. T cell culture was maintained at density below 3?10.sup.6/mL and more complete media with cytokines was added as needed. BAFF CAR transduction efficiency in T cells was monitored 72 hours after virus transduction. Transduction was confirmed by either presence of GFP positive cells or BAFF staining using an anti-human BAFF antibody-APC conjugate (Biolegend). BAFF CAR T cells were used at day 7-10 for in vitro and in vivo experiments or frozen in 95% FBS+5% DMSO in liquid nitrogen for later use.

[0105] In one example, T cells were isolated from human blood, activated and transduced with SEQ ID NO:15 lentiviral particles. Transduction efficiency was estimated by GFP expression (data not shown). About 27% of CAR-T cells expressed the protein corresponding to SEQ ID NO:15.

Example 3CAR-T Cells Kill Leukemia Cells In Vitro

[0106] The MCL cell line Jeko-1 and the ALL cell line RS4; 11 were used as tumor targets to test the efficacy of the BAFF CAR-T cells. The cells were grown and transduced as described above using SEQ ID NO: 15 and SEQ ID NO: 17. Tumor cell killing by BAFF CAR-T cells was analyzed using a calcein-AM assay purchased from Life Technologies. Target tumor cells (10?10.sup.6) were labeled with (0.5 ?mol/L) calcein-AM for 30 min at 37? C. Following staining, cells were washed with PBS, counted using Trypan blue (Sigma), and incubated with BAFF CAR-T cells for 4-6 hours at 5:1 or 10:1 ratio as indicated. The percentage of live tumor cells was analyzed by Annexin/PI negative and CD19 positive staining and the percentage lysis determined according to the following equation:

[00001]$\%\mathrm{specific}\mathrm{lysis}=100x\frac{\mathrm{AFU}\mathrm{mean}\mathrm{experimental}\mathrm{release}-\mathrm{AFU}\mathrm{mean}\mathrm{spontaneous}\mathrm{release}}{\mathrm{AFU}\mathrm{mean}\mathrm{maximal}\mathrm{release}-\mathrm{AFU}\mathrm{mean}\mathrm{spontaneous}\mathrm{release}}$

wherein AFU mean spontaneous release is calcein-AM release by target cells alone (in the absence of T cells); [0107] AFU mean maximal release is calcein-AM release by target cells upon lysis by detergent; and [0108] AFU mean experimental release is calcein-AM release by target cells mixed with BAFF CAR-T cells.

[0109] FIG. 5 shows the % cell killing (y-axis) for control T cells (left histogram), CAR-T cells expressing SEQ ID NO: 15 (left middle), CAR-T cells expressing SEQ ID NO:17 (right middle), and anti CD19 CAR-T cells (right). For the MCL Jeko-1 cell line, cell killing following treatment with the control T cells resulted in about 45% MCL cell death and treatment with the CD19 CAR-T cells resulted in over 90% cell death. Treatment with the CAR-T cells expressing SEQ ID NO:15 and SEQ ID NO:17 resulted in approximately 60% cell death for both constructs. For the RS4; 11 ALL cells, the CAR-T cells resulted in approximately 80% and 85% cell death. Treatment with the negative control demonstrated cell death of about 60% and treatment with the positive control resulted in nearly 100% cell death. These results demonstrate the viability of treatment of cancer cells with the CAR-T cells of the disclosure.

[0110] The cytotoxicity of RS4; 11 cells was further studied following exposure to CAR-T cells expressing the construct of SEQ ID NO:15 and SEQ ID NO:22. FIG. 6 (top) shows the effect of treatment of RS4 cells with T cell control (left), CAR-T cells expressing SEQ ID NO: 15 (middle) and CAR-T cells expressing SEQ ID NO:22 (right). Treatment with the negative control resulting in cell death of less than 10% of cells. Treatment with CAR-T cells expressing SEQ ID NO: 15 and SEQ ID NO:22 resulted in cell killing of about 40% and 25%, respectively.

[0111] The cytotoxicity of Jeko-1 and RS4; 11 cells were also studied following exposure to BAFF CAR-T cells expressing the construct expressing the partial BAFF sequence of SEQ ID NO:17. Following overnight incubation with control T cells (left), CAR-T cells (5:1 BAFF CAR-T:Jeko-1) (middle) and 10:1 (right), it was found that in all cases, the CAR-T cells more efficiently killed MCL and ALL cells compared to control. More specifically, at a 5:1 ratio of BAFF-CAR-T cells to tumor cells, Jeko-1 and RS4; 11 cells had a cell death of about 40% and about 30%, respectively. Cell death was expectedly higher for 10:1 exposure, about 60% and about 40% for Jeko-1 and RS4; 11 cells, respectively. Comparatively, the cell death for control T cells was less than 20% in both cases.

Example 5Degranulation of CAR-T Cells

[0112] The degranulation of CAR-T cells following incubation with Jeko-1 cells was studied. Lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been described as a marker of CD8+ T cell degranulation following stimulation. Cells were incubated with anti CD107 antibody and analyzed for CD107 positive staining using flow cytometry.

[0113] FIG. 7 shows the results of incubation of CAR-T cells alone (left) and CAR T and Jeko-1 cells (right). As can be seen, the percentage of CAR-T cells showing degranulation was about 6-fold higher when incubated with the cancer cells as compared to incubation alone. This suggests that CAR-T cells according to the present disclosure recognize and are activated in the presence of the MCL cells tested.

Example 6Granzyme B Release Studies

[0114] Granzyme B is an important molecule that is responsible for T cell mediated cell death. It is a serine protease commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells along with the pore forming protein perforin to mediate apoptosis in target cells. Thus, release of granzyme B was measured following the incubation of cancer cells in the presence of BAFF-CAR-T cells. Supernatant was collected from CAR-T cell or CAR-T+ target cell co-culture plates. Human GranzymeB Elisa kit (Biolegend) was used and experiments performed as specified by the manufacturer's instructions. Binding of GranzymeB was detected using a secondary antibody, streptavidin-HRP, and TMB substrate solution (provided with specified ELISA kit). Substrate conversion was stopped after 20 minutes with 100 ?L stop solution (2 N H.sub.2SO.sub.4) (Biolegend). Plates were washed with PBS plus 0.05% Tween20 in between incubations. Assay diluent provided by the manufacturer or RPMI medium (Sigma) was used as negative controls, and specific standard proteins were used as positive controls. Standard reconstitutions and curves were generated as per manufacturer's instructions for each assay. Optical density values were obtained using a microplate reader set to 450 nm (Bio-Rad iMark Microplate reader).

DETAILS

[0115] FIG. 8 shows that granzyme B is released when the CAR-T cells according to the present disclosure are incubated with both RS4; 11 or Jeko-1 cells. The cell ratio (5:1 or 10:1) did not appear to make a different in released granzyme levels, nor did cell type. In all cases, granzyme B was released at about the same amount. When CAR-T cells were incubated alone, no granzyme B excretion was detected (left). This suggests that the CAR-T cells according to the present disclosure recognize and are activated in the presence of the leukemic cells tested.

Example 7BAFF-CAR T Cells Inhibit Tumor Growth In Vivo

[0116] BAFF-CAR T cells expressing SEQ ID NO: 17 were tested in an in vivo study to determine whether the in vitro studies translate in vivo. Jeko-1 cells (1?10.sup.6 cells) were injected subcutaneously into immunocompromised mice. At day 14 following tumor cell implantation, the mice had palpable tumor. Mice were given an intratumor injection of either PBS solution (control), control T cells or BAFF-CAR-T cells. Tumor size was monitored on days 15, 18, 20, 22 and 25 post tumor cell injection.

[0117] FIG. 9 shows a plot of tumor volume (mm.sup.3, y-axis) vs. day (x-axis). As can be seen, the tumors treated with PBS control and T cell control showed continued, uncontrolled tumor growth following treatment. The tumors treated with BAFF-CAR-T cells continued to grow for one day following treatment, at which time, tumor growth not only was stopped, but the tumors began to shrink. At day 25, 11 days following treatment, the tumors treated with BAFF-CAR-T cells were nearly entirely gone. FIG. 10 shows images of mice having Jeko-1 tumors treated with PBS (left) and BAFF-CAR-T cells (right).

[0118] Finally, survival of mice treated with BAFF-CAR-T cells was prolonged as compared to PBS and control T cells. The PBS treatment group exhibited 100% survival until about 35 days post tumor inoculation, and by 38 days post inoculation, all of the mice had died or been humanely euthanized due to tumor burden. Treatment with control T cells prolonged survival by about 2 days. The mice treated with BAFF-CAR-T cells experienced 100% survival until about 45 days, with all mice having died or been euthanized at day 60 post inoculation.

[0119] Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and various principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.