JP2022551097A - Targeting B cell activating factor receptor (BAFF-R) using ligand-based chimeric antigen receptor (CAR)-T cells - Google Patents

Abstract

The present disclosure relates generally to ligand-based chimeric antigen receptor (CAR) cells. More specifically, CAR cells express the B-cell activating factor (BAFF) protein for receptor recognition of BAFF on the surface of the cell. CAR cells can include cytotoxic T lymphocytes, natural killer (NK) cells or natural killer T (NKT) cells expressing chimeric receptors that recognize receptors for BAFF. The present disclosure further relates to methods of using the disclosed CAR cells to treat various conditions such as cancer and autoimmune diseases. [Selection drawing] Fig. 1

Description

[0001] Related Application
[0002] This application claims the benefit of U.S. Provisional Application No. 62/980,727 filed February 24, 2020 and U.S. Provisional Application No. 62/908,795 filed October 1, 2019 and all of said patent documents are hereby incorporated by reference in their entirety.

[0003] REFERENCE TO SEQUENCE LISTING The Sequence Listing is provided in this patent document as a text file entitled "60147-SEQ-LIST_ST25.txt" created May 29, 2020 and is 46 kB in size. The contents of this file are incorporated herein by reference in their entirety.

[0004] The present disclosure relates generally to chimeric antigen receptor (CAR) cells that recognize receptors for B-cell activating factor (BAFF) on the surface of cells. More specifically, the present disclosure relates to cytotoxic T lymphocytes, natural killer (NK) cells or natural killer T (NKT) cells that express chimeric receptors that recognize receptors for BAFF. The present disclosure further relates to methods of using the disclosed CAR cells to treat various conditions such as cancer and autoimmune diseases.

[0005] Some cancers are currently incurable. In other cancers, chemotherapy is only partially effective and a significant proportion of patients relapse following treatment. Although some vascular tumors can be treated with hematopoietic stem cell transplantation (HSCT), less than 30% of patients requiring HSCT are of the required age with a matching donor. In another example, about 20% of patients with acute lymphoblastic leukemia (ALL), the most common childhood cancer in the United States and more than 3,000 children diagnosed each year, are resistant to conventional chemotherapy approaches. can. Therefore, there is an urgent need to develop new therapeutic routes to treat patients across a wide range of conditions.

[0006] B-cell activating factor (BAFF) is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. BAFF signaling is essential for the generation of mature B cells and aids in the survival of normal and malignant B cells. BAFF has at least three known receptors, B cell maturation antigen transmembrane activator (BCMA), CAML interactor (TACI) and BAFF receptor (BAFF-R). BAFF-R is specific for BAFF, while BCMA and TACI share another homologous ligand, APRIL. Recent studies have shown that BAFF-R is expressed on the surface of ALL cells, and thus inhibition of BAFF may be a potential candidate for treating ALL and other conditions in which BAFF is implicated. being a potential target.

[0007] According to the present disclosure, in a first aspect, comprising administering a composition comprising cytotoxic T lymphocytes, natural killer (NK) cells, or natural killer T (NKT) cells, There are ways to treat patients in need. Cytotoxic T lymphocytes, NK cells, or NKT cells express chimeric receptors that recognize receptors for B-cell activating factor (BAFF). In addition, chimeric receptors include SEQ ID NO: 1 or partial sequences thereof, or variants having 95% or greater sequence homology to SEQ ID NO: 1 or partial sequences thereof.

[0008] In an example of the first aspect, the partial sequence comprises SEQ ID NO:2 or a variant having 95% or greater sequence homology to SEQ ID NO:2.
[0009] In another aspect of the first aspect, the partial sequence comprises SEQ ID NO:3 or a variant having 95% or greater sequence homology to SEQ ID NO:3.

[0010] In yet another example of the first aspect, the chimeric receptor further comprises one or more hinge domains.
[0011] In another example of the first aspect, the hinge domain is a CD8α hinge domain having SEQ ID NO: 4 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 4 and SEQ ID NO: 5 or is selected from the group consisting of the hinge domain of IgG1 having variants thereof with 95% or greater sequence homology to SEQ ID NO:5.

[0012] In another example of the first aspect, the chimeric receptor further comprises one or more transmembrane domains.
[0013] In another example of the first aspect, the transmembrane domain is the transmembrane domain of CD8α having SEQ ID NO: 6 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 6 and SEQ ID NO: 6 7 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:7.

[0014] In yet another example of the first aspect, the chimeric receptor further comprises one or more intracellular domains.
[0015] In another example of the first aspect, the intracellular domain is the intracellular domain of 41BB having SEQ ID NO: 8 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 8, SEQ ID NO: 9 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 9, SEQ ID NO: 10 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 10 and the intracellular domain of OX40 having SEQ ID NO: 11 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 11.

[0016] In yet another example of the first aspect, the chimeric receptor further comprises a hinge domain, a transmembrane domain and one or more intracellular domains.
[0017] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO: 15 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 15.

[0018] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO:16 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:16.
[0019] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO: 17 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 17.

[0020] In yet another example of the first aspect, the chimeric receptor comprises SEQ ID NO:18 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:18.
[0021] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO:19 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:19.

[0022] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO:20 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:20.
[0023] In another example of the first aspect, the chimeric receptor further comprises a signaling peptide.

[0024] In one example of the first aspect, the signaling peptide comprises SEQ ID NO: 13 or SEQ ID NO: 14, or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 13 or SEQ ID NO: 14.

[0025] In yet another example of the first aspect, the chimeric receptor comprises SEQ ID NO:21 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:21.
[0026] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO:22 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:22.

[0027] In another example of the first aspect, the chimeric receptor comprises SEQ ID NO:23 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:23.
[0028] In yet another example of the first aspect, the T lymphocyte, NK cell or NKT cell is isolated from a human.

[0029] In another example of the first aspect, the T lymphocytes, NK cells or NKTs are autologous.
[0030] In another example of the first aspect, the T lymphocytes, NK cells or NKTs are allogeneic.

[0031] In yet another example of the first aspect, the receptor for BAFF is from B cell maturation antigen transmembrane activator (BCMA), CAML interactor (TACI) and BAFF receptor (BAFF-R) selected from the group consisting of

[0032] In another example of the first aspect, the patient in need thereof has been diagnosed with an autoimmune disorder.
[0033] In another example of the first aspect, the autoimmune disorder is indicative of autoimmune B cells.

[0034] In yet another example of the first aspect, the autoimmune disorder is systemic lupus erythematosus, Sjögren's syndrome, narcolepsy, diabetes, pancreatitis, Crohn's disease, celiac disease, ankylosing spondylitis, psoriasis, Graves' disease, and selected from rheumatoid arthritis;

[0035] In another example of the first aspect, the patient in need thereof has been diagnosed with cancer.
[0036] In another example of the first aspect, the cancer is a hematologic malignancy.
[0037] In yet another example of the first aspect, the hematologic malignancy is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B cell leukemia Selected from the group consisting of 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.

[0038] In another example of the first aspect, the composition is co-administered with one or more chemotherapeutic agents.
[0039] In a second aspect are cytotoxic T lymphocytes, natural killer (NK) cells or natural killer T (NKT) cells expressing chimeric receptors. The chimeric receptor recognizes the receptor for B-cell activating factor (BAFF) and is SEQ ID NO: 1 or a partial sequence thereof, or a variant having 95% or greater sequence homology to SEQ ID NO: 1 or a partial sequence thereof. including.

[0040] In an example of the second aspect, the partial sequence comprises SEQ ID NO:2 or a variant having 95% or greater sequence homology to SEQ ID NO:2.
[0041] In another example of the second aspect, the partial sequence comprises SEQ ID NO:3 or a variant having 95% or greater sequence homology to SEQ ID NO:3.

[0042] In another example of the second aspect, the chimeric receptor further comprises one or more hinge domains.
[0043] In another example of the second aspect, the hinge domain is a CD8α hinge domain having SEQ ID NO:4 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:4 and SEQ ID NO:5 or is selected from the group consisting of the hinge domain of IgG1 having variants thereof with 95% or greater sequence homology to SEQ ID NO:5.

[0044] In yet another example of the second aspect, the chimeric receptor further comprises one or more transmembrane domains.
[0045] In another example of the second aspect, the transmembrane domain is the transmembrane domain of CD8α having SEQ ID NO: 6 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 6 and SEQ ID NO: 6 7 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:7.

[0046] In another example of the second aspect, the chimeric receptor further comprises one or more intracellular domains.
[0047] In another example of the second aspect, the intracellular domain is the intracellular domain of 41BB having SEQ ID NO: 8 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 8, SEQ ID NO: 9 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 9, SEQ ID NO: 10 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 10 and the intracellular domain of OX40 having SEQ ID NO: 11 or a variant thereof with 95% or greater sequence homology to SEQ ID NO: 11.

[0048] In yet another example of the second aspect, the chimeric receptor further comprises a hinge domain, a transmembrane domain and one or more intracellular domains.
[0049] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:15 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:15.

[0050] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:16 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:16.
[0051] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO: 17 or a variant thereof having 95% or greater sequence homology to SEQ ID NO: 17.

[0052] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:18 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:18.
[0053] In yet another example of the second aspect, the chimeric receptor comprises SEQ ID NO:19 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:19.

[0054] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:20 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:20.
[0055] In another example of the second aspect, the chimeric receptor further comprises a signaling peptide. In one example, the signaling peptide comprises SEQ ID NO:13 or SEQ ID NO:14, or a variant thereof having 95% or greater sequence homology to SEQ ID NO:13 or SEQ ID NO:14.

[0056] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:21 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:21.
[0057] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:22 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:22.

[0058] In another example of the second aspect, the chimeric receptor comprises SEQ ID NO:23 or a variant thereof having 95% or greater sequence homology to SEQ ID NO:23.
[0059] In yet another example of the second aspect, the T lymphocytes, NK cells or NKT cells are isolated from humans.

[0060] In another example of the second aspect, the T lymphocytes, NK cells or NKTs are autologous.
[0061] In another example of the second aspect, the T lymphocytes, NK cells or NKTs are allogeneic.

[0062] In yet another example of the second aspect, the receptor for BAFF is from B cell maturation antigen transmembrane activator (BCMA), CAML interactor (TACI) and BAFF receptor (BAFF-R). selected from the group consisting of

[0063] In yet another example of the second aspect, a method of treating cancer involves a composition comprising cytotoxic T lymphocytes, NK cells or NKT cells according to the second aspect. administering to the patient.

[0064] In another example of the second aspect, a method of treating an autoimmune disease involves a composition comprising cytotoxic T lymphocytes, NK cells or NKT cells according to the second aspect. administering to the patient.

[0065] Additional features and advantages will be set forth in, and in part will be readily apparent to those skilled in the art from, the detailed description that follows, the claims, and the It will be appreciated by practicing the embodiments described herein, including the accompanying drawings.

[0066] It is understood that both the foregoing general description and the detailed description that follows are merely illustrative and are intended to provide a summary or framework for understanding the nature and features of the claims. It should be. The accompanying drawings are included to provide a further understanding, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiments and together with the description serve to explain the principles and operation of the various embodiments. Directional terms used herein, e.g., up, down, right, left, front, back, up (top), bottom (bottom) are used only in connection with the drawings being drawn and are not intended to imply absolute orientation.

[0067] The above and other features, examples and advantages of aspects or examples of the present disclosure will be better understood upon reading the following detailed description with reference to the accompanying drawings.
[0068] FIG. 1 is an exemplary schematic of a CAR construct according to the present disclosure. [0069] Figure 2 is a schematic representation of exemplary CAR constructs SEQ ID NOS: 15-17 and 21-23 according to the present disclosure. [0070] Figure 3 includes graphs showing increased expression of BAFF-R on the surface of ALL cells. [0071] Figure 4 shows flow cytometry histograms of BAFF-R expression in newly diagnosed recurrent ALL patients. [0072] Figure 5 shows killing activity by BAFF-CAR-T expressing SEQ ID NO: 15 and SEQ ID NO: 17 in MCL cells. [0073] Figure 6 is a series of graphs showing cytotoxicity against leukemic cell lines by BAFF-CAR-T cells. [0074] Figure 7 is a scatter plot showing increased degranulation of CAR-T cells following incubation with Jeko-1 cells. [0075] Figure 8 is a graph showing tumor burden following treatment with BAFF-CAR-T cells. [0076] Figure 9 shows photographs of mice engrafted with human leukemic cell lines following treatment with PBS control (left) and BAFF-CAR-T cells (right) according to the present disclosure. [0077] Figure 10 is a line graph showing percent survival following treatment with control and BAFF-CAR-T cells according to the present disclosure.

[0078] Exemplary embodiments will now be described more fully below with reference to accompanying figures in which exemplary embodiments and representative data are shown. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. However, embodiments may take many different forms and should not be construed as limited to the embodiments specifically set forth herein. These exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the claims to those skilled in the art.

[0079] Directional terms used herein (e.g., up, down, right, left, front, back, up, down) refer to the figure being drawn. is used only and is not intended to imply absolute orientation.

[0080] As used herein, the term "about" is not necessarily exact but not exact in terms of amounts, sizes, formulations, parameters, and other contents and characteristics, but includes tolerances, conversion factors, It is meant to be approximate and/or may be larger or smaller, as desired, to reflect rounding off, measurement error and the like, as well as other factors known to those of skill in the art. When the term "about" is used to describe an endpoint of a value or range, it should be understood that the disclosure includes the specific value or endpoint referred to. Whether or not a number or range endpoint states "about" in the specification, any number or range endpoint is qualified one by "about" and one by "about." It is intended to include two embodiments that do not have one. It will be further understood that each endpoint of a range is important with respect to or independent of the other endpoint.

[0081] As used herein, the terms "substantially", "substantially" and variations thereof are intended to refer to the feature being described being equal or approximately equal to the value or description. there is For example, a "substantially planar" surface is intended to indicate a surface that is planar or nearly planar. Additionally, "substantially" is intended to indicate that two values are equal or approximately equal. In some embodiments, "substantially" can refer to values within about 5% of each other, or within about 10% of each other, such as within about 2% of each other.

[0082] The terms "substantially" and "about" are utilized herein to express the inherent degree of uncertainty attributable to any quantitative comparison, value, measurement, or other representation. It is noted that These terms are also utilized herein to denote the degree to which a quantitative expression can deviate from a fixed reference without changing the basic function of the subject matter in question. Thus, for example, cells that are “free” or “substantially free” of T cell contamination are those that have not been actively added or batched into the cell culture, but which have undergone natural cell progression during expansion. cells that may be present in minute amounts as contaminants originating from Similarly, other components can be similarly characterized as "free" or "substantially free."

[0083] Furthermore, as used herein, the term "consisting essentially of" permits the explicitly recited elements, but excludes elements that affect the basic or novel characteristics of the invention. do. As recited herein, the term “consisting of” excludes elements not explicitly stated.

[0084] B-cell activating factor (BAFF) is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. BAFF is abundantly produced by monocytes, macrophages, dendritic cells and stromal cells, which are the major cellular components of the MCL tumor microenvironment. BAFF signaling is essential for the generation of mature B cells and aids in the 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 for 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 receptors. Early B cells are the counterpart of acute lymphocytic leukemia (ALL) and do not express BAFF-R, but cells from patients with several cancers, including ALL and mantle cell lymphoma (MCL) patients Expresses high levels of BAFF-R. Furthermore, BAFF-R is only expressed on mature B cells, making it an attractive target for targeting and reducing side effects caused by off-targets. BAFF-R thus provides a targeted opportunity for treating such cancers, as well as autoimmune diseases in which increased serum BAFF levels are often present.

[0085] One example according to this disclosure is a method of treating or preventing a disease or condition by targeting the receptor for BAFF. Another example according to the present disclosure is a method of treating or preventing a disease or condition by targeting cells expressing or overexpressing a receptor for BAFF, such as BAFF-R. . In an exemplary embodiment, the disease or condition is cancer, such as a hematologic malignancy. In one embodiment, the hematologic malignancy is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma the cancer cell expresses a receptor for BAFF, including but not limited to, marginal zone lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell malignancies, and multiple myeloma; or Any hematological malignancy that is overexpressed is possible. In another exemplary embodiment, the disease or condition is autologous, such as systemic lupus erythematosus, Sjögren's syndrome, narcolepsy, diabetes, pancreatitis, Crohn's disease, celiac disease, ankylosing spondylitis, psoriasis, Graves' disease, or rheumatoid arthritis. It is an immune disease. In another example, the disease or condition is B-cell mediated abdominal aortic aneurysm.

[0086] Conventional chimeric antigen receptor-T (CAR-T) cells use single-chain variable fragments (Sc-Fv) of antibodies against the corresponding tumor-targeting antigens. Indeed, WO2017/214167 reports BAFF-R antibodies capable of binding human BAFF-R protein and inducing 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 in the treatment of cancer. However, this antibody treatment approach is restricted to only cells expressing BAFF-R. Since the antibody approach is specific for only one receptor, the prior art disclosure is limited to that approach. Provided herein are BAFF-ligand-based CARs that can be used to target not only BAFF-R, but also any receptor for BAFF, including, for example, TACI and BCMA.

[0087] Instead of Sc-Fv, the present disclosure provides CAR-T cell immunotherapy against a wide variety of cancers and autoimmune diseases, such as BAFF-R, TACI and BCMA, on the surface of tumor cells. using a ligand approach that seeks the receptor for BAFF in . A BAFF-ligand-based CAR has been produced using the BAFF protein and is reported herein.

[0088] In the present disclosure, "BAFF protein" or "BAFF" refers to a recombinant or naturally occurring form of the B cell activator shown in SEQ ID NO: 1 or that maintains BAFF activity (e.g. within at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity) of any variant or analog thereof. Optionally, the variant or analogue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over all of SEQ ID NO:1 or a portion of SEQ ID NO:1. (ie, sequence homology).

[0089] As used herein, "partial sequence" or "BAFF partial sequence" refers to a portion of SEQ ID NO: 1 that retains BAFF activity similar to that of the entire sequence, particularly The extracellular portion of BAFF responsible for binding to the BAFF receptor. Examples of partial sequences include at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40% of the naturally occurring BAFF sequence. %, 35%, 30%, 25%, 20%, 15% or 10%. Sequences having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the partial sequences are also envisioned. In one example, the partial BAFF sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to amino acids 82-285 (SEQ ID NO:2) or SEQ ID NO:2 Contains arrays. In another example, the BAFF partial sequence has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to amino acids 134-285 (SEQ ID NO:3) or SEQ ID NO:3. contains an array with

[0090] As used herein, "chimeric antigen receptor T cells" or "CAR-T" cells are genetically engineered to produce artificial T cell receptors for use in immunotherapy. These are T cells. A "chimeric antigen receptor" or "CAR" is a receptor protein that has been engineered to give T cells a new ability to target specific proteins. CAR is a recombinant receptor that provides both antigen binding and T cell activation functions. In examples according to the present disclosure, CAR-T cells are engineered to target BAFF's receptor using BAFF as a ligand. CAR-T cell therapy is based on the recognition of specific tumor antigens by genetically modified T cells, followed by intracellular signaling and activation of T cells, which subsequently destroy tumor cells. In another example of the present disclosure, natural killer (NK) cells or natural killer T (NKT) cells are engineered to express a CAR.

[0091] The present disclosure relates to CARs that have been engineered to express BAFF ligands. FIG. 1 shows a schematic example of how a CAR according to this disclosure can be designed. In one example, the CAR expresses the full native human BAFF sequence, ie SEQ ID NO:1. In other examples according to this disclosure, the CAR expresses a partial sequence of BAFF. In one embodiment, the partial sequence comprises the extracellular domain of BAFF. In another embodiment, the partial sequence comprises a domain involved in receptor binding, such as the BAFF-R, TACI or BCMA binding domains. In one embodiment, the partial sequence comprises amino acids 82-285 of BAFF (SEQ ID NO:2). In another embodiment, the partial sequence comprises amino acids 134-285 of BAFF (SEQ ID NO:3). In another example, a BAFF sequence (complete or partial) can be incorporated into the construct such that the BAFF domain is replaced to reverse the amino acid sequence. In other words, the amino acid at the C-terminus in the native sequence becomes the amine-terminus of the sequence and the amino acid normally at the N-terminus becomes the C-terminus of the sequence.

[0092] A CAR can further comprise one or more hinge domains. In one example, the hinge domain comprises the hinge domain of CD8α (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 the 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 this disclosure comprise the hinge domain of CD28. In yet another example, the CAR cells of this disclosure comprise the hinge domain of FCγRIII.

[0093] CARs further comprise one or more transmembrane domains. In one example, the CAR comprises the transmembrane domain of CD8α, such as the transmembrane domain 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 the transmembrane domain of CD28, such as the transmembrane domain 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.

[0094] A CAR can further comprise one or more intracellular domains. In one example, the CAR includes the 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 the intracellular domain of CD28, such as the intracellular domain 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 the intracellular domain of CD3-zeta, such as the intracellular domain 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 the 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, a CAR can contain more than one intracellular domain. In one embodiment, a CAR comprises a hinge domain, a transmembrane domain, and one or more intracellular domains.

[0095] 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 is. In one embodiment, a CAR comprises a hinge domain, a signaling domain, a transmembrane domain, and one or more intracellular domains.

[0096] Since the distance between T cells and target cells is an important factor influencing tumor recognition and cytotoxicity, the linker domain is either between BAFF and the hinge domain or between the hinge domain and the transmembrane domain. can be incorporated into In some embodiments, the linker comprises a simple alkyl chain such as —(CH ₂ ) _n CH ₃ units, where n is the number of CH ₂ groups, 1-100, preferably 1-50, 1 ~20, or 1-10. In another example, a 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 with at least 85% sequence homology to SEQ ID NO:12.

[0097] Figure 2 shows a schematic representation of six exemplary CAR constructs according to the present disclosure. The schematic above contains the complete BAFF sequence, of which SEQ ID NO: 15 is an example. The second schematic from the top utilizes a BAFF partial sequence, but is otherwise identical to the schematic above. SEQ ID NOs: 16 and 17 are examples of such constructs. The third from top and bottom schematics use BAFF and partial BAFF sequences, respectively, but incorporate different hinge, transmembrane and intracellular regions. SEQ ID NO: 21 and SEQ ID NOS: 22 and 23, respectively, are represented by these schematics.

[0098] The following examples are illustrative and are not intended to limit the scope of the claimed invention.
[0099]

[00100] Example 1 ALL cells express BAFF-R
[00101] To confirm BAFF-R expression in ALL cells, 359 pediatric and 147 adult ALL patient samples were compared to PBMCs from 74 healthy donors using the Haferlach Leukemia Oncomine database. In the present disclosure, "peripheral blood mononuclear cells", "PBMCs" or "mononuclear cells" refer to mononuclear cells isolated from peripheral blood typically used for anti-cancer immunotherapy. be. PBMCs may be obtained from healthy individuals, patients at risk for cancer, or cancer patients. 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 onto 15 mL of Ficoll-Paque in a 50 mL conical tube. Samples were centrifuged at 20° C. (400×g, 30 min) in a swing-type rotor without brakes. The resulting top layer was aspirated and the PBMC layer was left undisturbed. The PBMC layer was transferred to a new 50 mL conical tube, which was then filled with PBS and centrifuged at 20° C. (300×g, 10 minutes). Cell pellets were resuspended in PBS and centrifuged at 20°C (300 xg, 10 minutes). Cells were resuspended in 1×MojoSort buffer at a density of 1×10 ⁸ /mL. CD3+ T cells were then purified by MojoSort Human CD3 T Cell Isolation Kit (Biolegend).

[00102] As seen in Figure 3, mRNA expression was significantly increased in both ALL patient samples. Figure 3 shows a significant increase in BAFF-R expression in children (Figures 3, 2) and adults (Figure 3) when compared to healthy donors (1), demonstrating that BAFF-R levels It shows an increase in ALL cells when compared to normal cells.

[00103] BAFF-R expression was also measured in newly diagnosed ALL cells at diagnosis. using flow cytometry (see Vicioso et al., "Combination Therapy for Treating Advanced Drug-Resistant Acute Lymphoblastic Leukemia" Cancer Immun. Res 7, pp. 1106-1119 (2019), for methods hereby incorporated by reference). , levels of BAFF-R expression in newly diagnosed (Fig. 4, bottom left) and relapsed (Fig. 4, bottom right) ALL patients were measured and Jurkat (negative control, top left) and Jeko-1 (positive control) , upper right) compared with cells. In all graphs, the left histogram represents unstained BAFF-R positive control and the right histogram represents cells stained with anti-BAFF-R antibody.

[00104] Example 2 Development of BAFF-CAR-T cells
[00105] The design of various BAFF-CAR-T cells was discussed above. Each construct was designed to contain the BAFF protein (full or partial sequence), hinge domain, transmembrane domain and one or more intracellular domains. The constructs may optionally also include extracellular signaling domains and linker domains to optimize spacing. Each construct was cloned into a third generation lentiviral vector and packaged into HEK293T cells.

[00106] Human PBMC cells were used to collect and purify the CD3+ T cells described above. T cells were activated using Dynabeads human T-activator CD3/CD28 (Gibco/Invitrogen). For activation of 1×10 ⁶ CD3+ T cells, 25 μL Dynabeads were added to T cells and 1.5 mL complete culture medium (2 mM Mix in advanced RPMI medium 1640) with L-glutamine, 10% FBS and 100 U/mL penicillin/streptomycin. Cells were resuspended in media with beads and dispensed into one well of a 24-well plate. Cells were cultured for 24-48 hours at 37° C. in a 5% CO ₂ incubator and prepared for viral infection with concentrated lentivirus containing BAFF CAR.

[00107] Generation of lentiviral supernatant containing BAFF CAR was accomplished as follows. Lentiviral supernatants were generated using the packaging cell line, 293FT. Healthy 293FT cells were cultured in DMEM with 10% FBS and 100 U/mL penicillin/streptomycin. The day before vector transfection, 293FT cells were split into 5×10 ⁶ /10 cm tissue culture plates. Four plates of 293FT cells were required to generate sufficient supernatant for lentiviral enrichment. In one 10 cm plate, 200 μL of Opti-MEM medium was added to an eppendorf tube followed by 3750 ng of packaging plasmid psPax2, 1250 ng of envelope plasmid pMD2G and 5000 ng of BAFF CAR vector. The tubes were mixed gently, then 10 μL of X-tremeGENE HP Transfection Reagent (Sigma, Catalog No. 06 366 236 001) was added, mixed gently and incubated at room temperature for 20 minutes. The DNA mixture was added dropwise into a 10 cm 293FT plate and incubated at 37°C in a 5% _CO2 incubator. After overnight culture, the medium was removed and replaced with 6 mL of fresh complete DMEM medium. Supernatants from 4 plates (23 mL total) were collected in 50 mL conical tubes at 48 and 72 hours after vector transfection and centrifuged at 500 xg for 10 minutes to remove cell debris. 23 mL of clear supernatant was transferred to a new 50 mL conical tube to which 7.6 mL of Lenti-X concentrator (Clontech) was added, mixed and incubated overnight at 4°C. The supernatant was centrifuged (1500×g, 45 min, 4° C.) and decanted to give an off-white pellet. The pellet was resuspended in 1 mL complete advanced RMPI medium with IL-2 and prepared for viral transduction into CD3+ T cells.

[00108] Approximately 1 x 10 ⁶ activated T cells were resuspended with Dynbeads in 1 mL of complete advanced RPMI medium containing concentrated BAFF CAR and plated in a volume of 100 μL for a total of 10-10 to 96-well round bottom plates. Dispensed into each well of 12 wells. T cells were spinoculated (3480 rpm, 22° C., 90 minutes). After centrifugation, cells were resuspended, harvested with Dynabeads and then re-separated into 24-well plates. An additional 1 mL of complete advanced RPMI medium was added along with IL-2 50 U/mL, IL-7 10 ng/mL and IL-15 5 ng/mL. T cells were cultured at 37° C. with 5% CO ₂ in an incubator. Fresh medium with cytokines was added when the medium in culture turned yellow. Dynabeads were removed 4-5 days after initial T cell activation. Transduced T cells expanded exponentially 5-8 days after activation. T cell cultures were maintained at densities below 3×10 ⁶ /mL and supplemented with additional complete medium with cytokines as needed. BAFF CAR transduction efficiency in T cells was monitored for 72 hours after viral transduction. Transduction was confirmed by the presence of GFP-positive cells or by BAFF staining using an anti-human BAFF antibody APC conjugate (Biolegend). BAFF CAR T cells were used on days 7-10 for in vitro and in vivo experiments and were frozen in 95% FBS + 5% DMSO in liquid nitrogen for later use.

[00109] In one example, T cells were isolated from human blood, activated, and transduced with lentiviral particles of SEQ ID NO:15. Transduction efficiency was assessed by GFP expression (data not shown). Approximately 27% of CAR-T cells expressed a protein corresponding to SEQ ID NO:15.

[00110] Example 3 CAR-T cells kill leukemic cells in vitro
[00111] The MCL cell line Jeko-1 and the ALL cell line RS4;11 were used as tumor targets to test the efficacy of BAFF CAR-T cells. 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 the Calcein-AM assay purchased from Life Technologies. Target tumor cells (10×10 ⁶ ) were labeled with calcein-AM (0.5 μmol/L) for 30 minutes at 37°C. Following staining, cells were washed with PBS, counted using trypan blue (Sigma), and incubated with BAFF CAR-T cells at 5:1 or 10:1 as indicated for 4-6 hours. The percentage of viable tumor cells was analyzed by Annexin/PI-negative and CD19-positive staining methods and the percentage lysis was determined according to the formula below.

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

[00112] Figure 5. Control T cells (left histogram), CAR-T cells expressing SEQ ID NO: 15 (middle left), CAR-T cells expressing SEQ ID NO: 17 (middle right), and % cell killing (y-axis) for anti-CD19 CAR-T cells (right). In the MCL Jeko-1 cell line, cell killing following treatment with control T cells resulted in approximately 45% MCL cell death and treatment with CD19 CAR-T cells resulted in greater than 90% cell death. Treatment with CAR-T cells expressing SEQ ID NO: 15 and SEQ ID NO: 17 resulted in approximately 60% cell death for both constructs. In RS4;11 ALL cells, CAR-T cells caused approximately 80% and 85% cell death. Treatment with the negative control showed approximately 60% cell death and treatment with the positive control resulted in nearly 100% cell death. These results demonstrate the feasibility of treating cancer cells with CAR-T cells of the present disclosure.

[00113] The cytotoxicity of RS4;11 cells was further studied following exposure to CAR-T cells expressing the constructs of SEQ ID NO:15 and SEQ ID NO:22. Figure 6 (top) 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). shows the effect of treatment of Treatment with the negative control resulted in cell death in less than 10% of cells. Treatment with CAR-T cells expressing SEQ ID NO: 15 and SEQ ID NO: 22 resulted in approximately 40% and 25% cell killing, respectively.

[00114] The cytotoxicity of Jeko-1 and RS4;11 cells was also studied following exposure to BAFF CAR-T cells expressing a construct expressing a partial BAFF sequence of SEQ ID NO:17. In all cases, following overnight incubation with control T cells (left), CAR-T cells (5 vs. 1 BAFF CAR-T vs. Jeko-1) (middle) and 10 vs. 1 (right), CAR - T cells were found to kill MCL and ALL cells more efficiently compared to controls. More specifically, at a 5:1 ratio of BAFF CAR-T cells to tumor cells, cell death of Jeko-1 and RS4;11 cells was about 40% and about 30%, respectively. Cell death was higher at the 10 to 1 exposure as expected, approximately 60% and 40% in Jeko-1 and RS4;11 cells, respectively. By comparison, cell death in control T cells was less than 20% in both cases.

[00115] Example 5 Degranulation of CAR-T cells
[00116] Degranulation of CAR-T cells following incubation with Jeko-1 was examined. Lysosomal 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.

[00117] Figure 7 shows the results of incubation of CAR-T cells alone (left) and CAR T with Jeko-1 cells (right). As can be seen, the percentage of CAR-T cells exhibiting degranulation was approximately 6-fold when incubated with cancer cells when compared to incubation alone. This suggests that CAR-T cells according to the present disclosure recognize the tested MCL cells and become activated in the presence of these cells.

[00118] Example 6 Granzyme B release studies
[00119] Granzyme B is a key molecule responsible for T cell-mediated cell death. This molecule is a serine protease commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. This molecule is co-secreted by these cells with the pore-forming protein perforin to mediate apoptosis in target cells. Granzyme B release was therefore measured following incubation of cancer cells in the presence of BAFF-CAR-T cells. Supernatants were collected from CAR-T cell or CAR-T+ target cell co-culture plates. A human GranzymeB Elisa kit (Biolegend) was used and experiments were performed as specified in the manufacturer's instructions. GranzymeB binding was detected using a secondary antibody, streptavidin-HRP, and TMB substrate solution (provided in the specified ELISA kit). Substrate conversion was stopped after 20 minutes with 100 μL of stop solution (2N H ₂ SO ₄ ) (Biolegend). Plates were washed with PBS plus 0.05% Tween 20 between incubations. Assay diluent or RPMI media (Sigma) provided by the manufacturer were used as negative controls and specific standard proteins were used as positive controls. Standard reconstructions and curves were generated according to the manufacturer's instructions for each assay. Optical density values were obtained using a microplate reader (Bio-Rad iMark Microplate reader) set at 450 nm.

detail
[00120] Figure 8 shows that granzyme B is released when CAR-T cells according to the present disclosure are incubated with both RS4;11 or Jeko-1 cells. Cell ratios (5 to 1 or 10 to 1) did not appear to make a difference in released granzyme levels, nor did cell type. Granzyme B was released in approximately the same amount in all cases. Granzyme B shedding was not detected when CAR-T cells were incubated alone (left). This suggests that CAR-T cells according to the present disclosure recognize the tested leukemic cells and become activated in the presence of these cells.

[00121] Example 7 BAFF-CAR T cells inhibit tumor growth in vivo
[00122] BAFF-CAR T cells expressing SEQ ID NO: 17 were tested in in vivo studies to determine if in vitro studies translate into in vivo. Jeko-1 cells (1×10 ⁶ cells) were injected subcutaneously into immunocompromised mice. On day 14 following tumor cell implantation, mice had palpable tumors. Mice received intratumoral injections of PBS solution (control), control T cells or BAFF-CAR-T cells. Tumor size was monitored on days 15, 18, 20, 22 and 25 after tumor cell injection.

[00123] Figure 9 shows a plot of tumor volume ( ^mm3 , y-axis) versus days (x-axis). As can be seen, the PBS control and T cell control treated tumors exhibited continued, uncontrolled tumor growth following treatment. Tumors treated with BAFF-CAR-T cells continued to grow for 1 day following treatment, at which point not only did tumor growth stop, but tumors began to shrink. On day 25, 11 days after treatment, tumors treated with BAFF-CAR-T cells were almost completely cleared. FIG. 10 shows images of Jeko-1 tumor-bearing mice treated with PBS (left) and BAFF-CAR-T cells (right).

[00124] Finally, survival of mice treated with BAFF-CAR-T cells was prolonged when compared to PBS and control T cells. The PBS-treated group showed 100% survival until approximately 35 days post-tumor inoculation, and by 38 days post-inoculation all mice were dead or humanely euthanized due to tumor burden. Treatment with control T cells prolonged survival by approximately 2 days. Mice treated with BAFF-CAR-T cells experienced 100% survival until about day 45, and all mice died or were euthanized 60 days after inoculation.

[00125] 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.

Claims (20)

An immune cell expressing a chimeric antigen receptor that recognizes the receptor for B cell activating factor (BAFF), said immune cell selected from cytotoxic T lymphocytes, natural killer cells and natural killer T cells wherein said chimeric antigen receptor is SEQ ID NO: 18, a variant having 95% or greater sequence homology to SEQ ID NO: 18, SEQ ID NO: 19, 95% or greater sequence homology to SEQ ID NO: 19 SEQ ID NO:20, and variants having 95% or greater sequence homology to SEQ ID NO:20. 2. The immune cell of claim 1, wherein said chimeric antigen receptor further comprises a signaling peptide. Said chimeric antigen receptor is SEQ ID NO: 21, variants having 95% or greater sequence homology to SEQ ID NO: 21, SEQ ID NO: 22, variants having 95% or greater sequence homology to SEQ ID NO: 22 , SEQ ID NO:23, and variants having 95% or greater sequence homology to SEQ ID NO:23. 2. The immune cell of claim 1, wherein said immune cell is isolated from a human. 2. The immune cell of claim 1, wherein said immune cell is a cytotoxic T lymphocyte. 2. The immune cell of claim 1, wherein said immune cell is a natural killer cell or a natural killer T cell. 2. The immune cell of claim 1, wherein said receptor for BAFF is selected from the group consisting of B cell maturation antigens, transmembrane activators and CAML interactors, and BAFF receptors. A method comprising administering to a patient a therapeutically effective amount of a composition comprising immune cells expressing chimeric antigen receptors that recognize receptors for B-cell activating factor (BAFF), wherein the immune cells are cytotoxic SEQ ID NO: 18, a variant wherein said chimeric antigen receptor has 95% or greater sequence homology to SEQ ID NO: 18, SEQ ID NO: 19 , a variant having 95% or greater sequence homology to SEQ ID NO: 19, SEQ ID NO: 20, and a variant having 95% or greater sequence homology to SEQ ID NO: 20, The above method of treating a patient in need of. 9. The method of claim 8, wherein said patient has been diagnosed with cancer. 10. The method of claim 9, wherein said cancer is a hematologic malignancy. 11. The method of claim 10, wherein said hematologic malignancy is leukemia. 9. The method of claim 8, wherein said patient has been diagnosed with an autoimmune disease. 13. The autoimmune disease of claim 12, wherein the autoimmune disease is selected from systemic lupus erythematosus, Sjögren's syndrome, narcolepsy, diabetes, pancreatitis, Crohn's disease, celiac disease, ankylosing spondylitis, psoriasis, Graves' disease, and rheumatoid arthritis. Method. 9. The method of claim 8, wherein said composition is co-administered with one or more chemotherapeutic agents. 9. The method of claim 8, wherein said chimeric antigen receptor further comprises a signaling peptide. Said chimeric antigen receptor is SEQ ID NO: 21, variants having 95% or greater sequence homology to SEQ ID NO: 21, SEQ ID NO: 22, variants having 95% or greater sequence homology to SEQ ID NO: 22 , SEQ ID NO:23, and variants having 95% or greater sequence homology to SEQ ID NO:23. 9. The method of claim 8, wherein said immune cells are isolated from humans. 9. The method of claim 8, wherein said immune cells are cytotoxic T lymphocytes. 9. The method of claim 8, wherein said immune cells are natural killer cells or natural killer T cells. 9. The method of claim 8, wherein said receptor for BAFF is selected from the group consisting of B cell maturation antigens, transmembrane activators and CAML interactors, and BAFF receptors.

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