Patent Application
20240261405
This application contains a Sequence Listing that has been submitted electronically as an XML file named 47902-0007001_ST26.XML/47902-0007001_ST26/47902-0007TW1_ST26. The XML file, created on Jun. 5, 2023, is 269,901 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
This document relates to methods and materials for treating a mammal having an autoimmune disease. For example, this document provides materials and methods for producing an immuno-activatable cell comprising a first chimeric antigen receptor and a second chimeric antigen receptor. This document also provides methods and materials for treating a mammal having an autoimmune disease comprising administering an immuno-activatable cell.
Autoimmunity is a common disease in the United States, with more than 20 million people suffering from any of 81 known autoimmune diseases. B cells are involved in different aspects of autoimmune diseases, including the secretion of autoantibodies, processing and presentation of autoantigen to T cells, and producing inflammatory cytokines. Age-associated B cells (ABCs) are known to have a role in autoimmune disease (Wang et al., DNA Cell Biol., 35:(10):628-35 (2016)). One contribution to autoimmune disease pathology occurs via ABC production of autoantibodies (Rubtsov et al., Immunol. Res., 55:210-16 (2013)). While autoimmune diseases can be treated with immunosuppressive drugs, there remains an unmet need in the management of ABCs in autoimmune disease.
This document provides methods and materials for treating a mammal having an autoimmune disease. For example, this document provides materials and methods for producing an immuno-activatable cell comprising a first chimeric antigen receptor and a second chimeric antigen receptor. This document also provides methods and materials for treating a mammal having an autoimmune disease comprising administering to the mammal an immuno-activatable cell.
In general, one aspect of this document features a method for producing an immuno-activatable cell. The method can comprise (or consist essentially of or consist of) transforming the cell with a first nucleic acid sequence encoding a first chimeric antigen receptor polypeptide. The first chimeric antigen receptor polypeptide can include a first extracellular domain, a first transmembrane domain and a first intracellular domain. The first extracellular domain can include a first antigen binding domain capable of binding to a first antigen on a CD11c+Tbet+ B cell. The first transmembrane domain can include a first CD8α transmembrane domain. The first intracellular domain can include a cytoplasmic signaling domain. The sequence encoding the first chimeric antigen receptor polypeptide can be operably linked to a first promoter. The method can comprise (or consist essentially of or consist of) transforming the cell with a second nucleic acid sequence encoding a second chimeric antigen receptor polypeptide. The second chimeric antigen receptor polypeptide can include a second extracellular domain, a second transmembrane domain, and a second intracellular domain. The second extracellular domain can include a second antigen binding domain capable of binding to a second antigen on the CD11c+Tbet+ B cell. The second transmembrane domain can include a second CD8α transmembrane domain. The intracellular domain can include a co-stimulatory domain. The sequence encoding the second chimeric antigen receptor polypeptide can be operably linked to a second promoter. The first antigen binding domain can be an antibody or an antigen binding fragment. The first antigen binding domain can be an antigen binding fragment selected from the group consisting of a Fab, a F(ab′)2 fragment, a scFV, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. The first antigen can be a B cell receptor. The B cell receptor can be CD19, CD20, or CD45R.
The first antigen can be CD19. In some embodiments, the first antigen binding domain binds CD19. In some embodiments, the first antigen binding domain comprises an scFv comprising a sequence at least 90% identical to one of SEQ ID NOs: 1-10. In some embodiments, the first antigen binding domain comprises one of the following: (a) a heavy chain variable domain comprising SEQ ID NO: 39 and a light chain variable domain comprising SEQ ID NO: 77; (b) a heavy chain variable domain comprising SEQ ID NO: 40 and a light chain variable domain comprising SEQ ID NO: 78; (c) a heavy chain variable domain comprising SEQ ID NO: 41 and a light chain variable domain comprising SEQ ID NO: 79; (d) a heavy chain variable domain comprising SEQ ID NO: 42 and a light chain variable domain comprising SEQ ID NO: 80; (e) a heavy chain variable domain comprising SEQ ID NO: 43 and a light chain variable domain comprising SEQ ID NO: 81; (f) a heavy chain variable domain comprising SEQ ID NO: 44 and a light chain variable domain comprising SEQ ID NO: 82; (g) a heavy chain variable domain comprising SEQ ID NO: 45 and a light chain variable domain comprising SEQ ID NO: 83; (h) a heavy chain variable domain comprising SEQ ID NO: 46 and a light chain variable domain comprising SEQ ID NO: 84; (i) a heavy chain variable domain comprising SEQ ID NO: 47 and a light chain variable domain comprising SEQ ID NO: 85; or (j) a heavy chain variable domain comprising SEQ ID NO: 48 and a light chain variable domain comprising SEQ ID NO: 86.
The second antigen binding domain can be an antibody or an antigen binding fragment. The second antigen binding domain can be an antigen binding fragment selected from the group consisting of Fab, a F(ab′)2 fragment, a scFV, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
The second antigen can be an antigen present on CD11c+Tbet+ B cells. The second antigen can be CD11c. In some embodiments, the first antigen binding domain comprises an scFv comprising a sequence at least 90% identical to SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the first antigen binding domain comprises either (a) a heavy chain variable domain comprising SEQ ID NO: 75 and a light chain variable domain comprising SEQ ID NO: 113; or (b) a heavy chain variable domain comprising SEQ ID NO: 76 and a light chain variable domain comprising SEQ ID NO: 114.
The cytoplasmic signaling domain can be a CD3 zeta, CD3 epsilon, CD3 delta, TCR zeta, FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b, or CD66d domain. The co-stimulatory domain can be a CD28, 4-1BB, CD97, CD11a-CD18, CD2, CD27, ICOS, CD154, CD5, or OX40 signaling domain. The first and second CD8α transmembrane domains can include a CD8α hinge domain and a CD8α stalk domain. The cell can be further transformed by transforming the cell with a third nucleic acid encoding a first chemokine receptor polypeptide. The cell can be further transformed by transforming the cell with a fourth nucleic acid encoding a second chemokine receptor polypeptide. The cell can be further transformed by transforming the cell with a third nucleic acid encoding a first chemokine receptor polypeptide and a fourth nucleic acid encoding a second chemokine receptor polypeptide. The first or second chemokine receptor polypeptides can be a receptor present on a lymphoid cell. The chemokine receptor can be a CXCR5 or CCR7. The third nucleic acid can encode a CXCR5 polypeptide and the fourth nucleic acid can encode a CCR7 polypeptide. The immuno-activatable cell can be an immune cell selected from the group consisting of a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, and a cytotoxic T cell.
In another aspect, this document features an immuno-activatable cell produced by the methods described herein.
In another aspect, this document features a method for treating a mammal having an autoimmune disease. The method can comprise (or consist essentially of or consist of) administering to the mammal an effective amount of the immuno-activatable cell as produced by the methods and materials as described herein. For example, the immuno-activatable cell can express a first chimeric antigen receptor polypeptide having a first antigen binding domain that binds a first antigen on a CD11c+Tbet+ B cell with low affinity, where the binding activates the immuno-activatable cell. The immuno-activatable cell also can express a second chimeric antigen receptor polypeptide having a second antigen binding domain that binds a second antigen on a CD11c+Tbet+ B cell and stimulates the immuno-activatable cell, thereby treating or preventing an autoimmune disorder in the mammal. The mammal can be human. The autoimmune disease can result from production of autoantibodies by Age-associated B cells. The autoimmune disease can be lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa. The method can reduce the number of age-associated B-cells.
In another aspect, this document features a method for producing a T cell targeting Age-associated B cells. The method can comprise (or consist essentially of or consist of) transforming the T cell with a first nucleic acid sequence encoding a first chimeric antigen receptor polypeptide. The first chimeric antigen receptor can include an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain can include a first antigen binding domain capable of binding to a CD19 antigen on the surface of a CD11c+Tbet+ B cell. The first transmembrane domain can include a first CD8α transmembrane domain. The first intracellular domain comprises CD3zeta domain. The sequence encoding the first chimeric antigen receptor polypeptide can be operably linked to a first promoter. The method can comprise (or consist essentially of or consist of) transforming the cell with a second nucleic acid sequence encoding a second chimeric antigen receptor polypeptide. The second chimeric antigen receptor can include a second extracellular domain, a second transmembrane domain and a second intracellular domain. The second extracellular domain can include a second antigen binding domain capable of binding to a CD11c antigen on the surface of a CD11c+Tbet+ B cell. The second transmembrane domain can include a second CD8α transmembrane domain. The second intracellular domain comprises a CD28 signaling domain. The sequence encoding the second chimeric antigen receptor polypeptide can be operably linked to a second promoter. The cell can be further transformed by transforming the cell with a third nucleic acid encoding a first chemokine receptor polypeptide. The cell can be further transformed by transforming the cell with a fourth nucleic acid encoding a second chemokine receptor polypeptide. The cell can be further transformed by transforming the cell with a third nucleic acid encoding a first chemokine receptor polypeptide and a fourth nucleic acid encoding a second chemokine receptor polypeptide. The first or second chemokine polypeptide can be a receptor present on a lymphoid cell. The chemokine receptor polypeptide can be a CXCR5 or CCR7. The third nucleic acid can encode a CXCR5 polypeptide and the fourth nucleic acid can encode a CCR7 polypeptide.
In another aspect, this document features a method for treating a mammal having an autoimmune disease. The method can comprise (or consist essentially of or consist of) administering to the mammal an effective amount of the immuno-activatable cell as produced by methods and materials described herein, where the T cell expresses a first chimeric antigen receptor polypeptide having a first antigen binding domain that binds a CD19 antigen on a CD11c+Tbet+ B cell with low affinity, where the binding activates the T cell, and where the T cell expresses a second chimeric antigen receptor polypeptide having a second antigen binding domain that binds a CD11c antigen on a CD11c+Tbet+ B cell and stimulates the T cell, thereby treating or preventing an autoimmune disease in the mammal. The mammal can be a human. The autoimmune disease can result from production of autoantibodies by age-associated B cells. The autoimmune disease can be lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, or polyarteritis nodosa. The method can reduce the number of age-associated B cells in the mammal.
In another aspect, this document features a composition containing a chimeric antigen receptor polypeptide that includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain can include an antigen binding domain capable of binding to an antigen on a CD11c+Tbet+ B cell. The transmembrane domain can include a CD8α transmembrane domain. The intracellular domain can include a cytoplasmic signaling domain. The antigen binding domain can be an antibody or antigen binding fragment. The antigen binding domain can be an antigen binding fragment selected from the group consisting of a Fab, a F(ab′)2 fragment, a scFV, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. The antigen can be a B cell receptor. The B cell receptor can be CD19, CD20, or CD45R. The antigen can be CD19. The antigen can be an antigen present on CD11c+Tbet+ B cells. The antigen can be CD11c. The cytoplasmic signaling domain can be a CD3zeta, CD3epsilon, CD3delta, TCRzeta, FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b or CD66d domain.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD3 zeta cytoplasmic signaling domain, the CD3 zeta cytoplasmic signaling domain has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 274 (NCBI Reference No: NP_932170) (SEQ ID NO: 274) or a fragment thereof that has activating or stimulatory activity.
In another aspect, this document features a composition containing a second chimeric antigen receptor polypeptide containing an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain can include an antigen binding domain capable of binding to an antigen on a CD11c+Tbet+ B cell. The transmembrane domain can include a CD8α transmembrane domain. The intracellular domain can include a co-stimulatory domain. The antigen binding domain can be an antibody or antigen binding fragment. The antigen binding domain can be an antigen binding fragment selected from the group consisting of a Fab, a F(ab′)2 fragment, a scFV, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. The antigen can be a B cell receptor. The B cell receptor can be CD19, CD20, or CD45R. The antigen can be CD19. In some embodiments, the antigen binding domain comprises an scFv comprising a sequence at least 90% identical to one of SEQ ID NOs: 1-10. In some embodiments, the antigen binding domain comprises one of the following: (a) a heavy chain variable domain comprising SEQ ID NO: 39 and a light chain variable domain comprising SEQ ID NO: 77; (b) a heavy chain variable domain comprising SEQ ID NO: 40 and a light chain variable domain comprising SEQ ID NO: 78; (c) a heavy chain variable domain comprising SEQ ID NO: 41 and a light chain variable domain comprising SEQ ID NO: 79; (d) a heavy chain variable domain comprising SEQ ID NO: 42 and a light chain variable domain comprising SEQ ID NO: 80; (e) a heavy chain variable domain comprising SEQ ID NO: 43 and a light chain variable domain comprising SEQ ID NO: 81; (f) a heavy chain variable domain comprising SEQ ID NO: 44 and a light chain variable domain comprising SEQ ID NO: 82; (g) a heavy chain variable domain comprising SEQ ID NO: 45 and a light chain variable domain comprising SEQ ID NO: 83; (h) a heavy chain variable domain comprising SEQ ID NO: 46 and a light chain variable domain comprising SEQ ID NO: 84; (i) a heavy chain variable domain comprising SEQ ID NO: 47 and a light chain variable domain comprising SEQ ID NO: 85; or (j) a heavy chain variable domain comprising SEQ ID NO: 48 and a light chain variable domain comprising SEQ ID NO: 86.
The antigen can be an antigen present on CD11c+Tbet+ B cells. The antigen can be CD11c. In some embodiments, the scFv fragment comprises a sequence at least 90% identical to SEQ ID NO: 37 or SEQ ID NO: 38. In some embodiments, the scFv fragment comprises either: (a) a heavy chain variable domain comprising SEQ ID NO: 75 and a light chain variable domain comprising SEQ ID NO: 113; or (b) a heavy chain variable domain comprising SEQ ID NO: 76 and a light chain variable domain comprising SEQ ID NO: 114.
The co-stimulatory domain can be a CD28, 4-1BB, CD97, CD11a-CD18, CD2, CD27, ICOS, CD154, CD5, or OX40.
Also provided herein are nucleic acids encoding any of the chimeric antigen receptor polypeptides described herein. Some embodiments include a vector containing a nucleic acid as described herein. In some embodiments, the vector can be an expression vector. Also provided herein are cells transformed with the nucleic acids and/or vectors described herein. The cells can be selected from the group consisting of a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, and a cytotoxic T cell. Also provided herein is a pharmaceutical composition including one or more chimeric antigen receptor polypeptides as described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document relates to methods and materials for treating a mammal having an autoimmune disease. For example, this document provides materials and methods for producing an immuno-activatable cell that expresses or contains a first chimeric antigen receptor and a second chimeric antigen receptor. This document also provides methods and materials for treating a mammal identified as having an autoimmune disease, where the methods include administering an immuno-activatable cell as described herein. In some cases, the methods and compositions provided herein include treating a disease in a patient, including an autoimmune disease or cancer, by administering to the patient a cell transformed with any of the nucleic acids, vectors, or polypeptides described herein.
In some embodiments, this document provides methods and materials for treating a mammal having an autoimmune disease resulting from the production of autoantibodies by age-associated B cells (ABCs). In some cases, a method of treating an autoimmune disease, as described herein, can include by administering to the mammal an immune cell (e.g., a T cell) genetically altered to encode and express two or more chimeric antigen receptors (e.g., an immuno-activatable cell). In some cases, an immune cell (e.g., a T cell) with two or more CARs expressed on its surface is capable of inducing and sustaining an immune response. In some cases, an immune cell can be genetically altered to contain two CARs designed to bind to antigens on the surface of ABCs. In some cases, binding of CARs to antigens on the surface of ABCs can result in an immune response against the ABCs, and possibly elimination of the ABCs. In some cases, only when both antigens are present on the surface of an ABC will CAR signaling occur (e.g., activated and sustained immune response against the ABC). In some cases, only when both antigens are present on the surface of an ABC and the cell contains the appropriate combination of two or more CARs will CAR signaling occur (e.g., activated and sustained immune response against the ABC). For example, an activated and sustained immune response can be achieved when a first CAR containing a cytoplasmic signaling domain activates CAR signaling upon binding of the CAR to an antigen, and a second CAR containing a co-stimulatory domain that stimulates CAR signaling upon binding of the CAR to a second antigen.
To ensure that CAR activation does not occur due to only one of the antibodies or antigen binding fragments binding its target antigen, the affinity of the antibodies can be adjusted so that CAR activation, and thus ABC killing, occurs only when there is a combined effect of both antibodies. In general, CAR activation can depend on the function of an antigen binding fragment, antibody affinity, antigen density of either the antigens for either CARs, and/or the antigen selectively of the CARs. In some cases, altering the binding affinity of a CAR can be achieved by empirically determining the antigen density and antigen selectivity. In some cases, one CAR can be designed to have lower affinity. In some cases, one CAR can be designed to have higher affinity.
In some cases, empirical testing can be done to determine which CAR (e.g., which antigen binding fragment) contains should contain which intracellular domain. In some cases, the scFv CD19 can have a binding affinity in the micromolar range, while the scFv CD11c will preferably be in the nanomolar range. In some cases, the actual affinities of both scFvs can be determined by determining their corresponding antigen densities present on an ABC cell. For example, empirical determination of antigen density on an ABC can allow determination of actual binding affinities favorable for CAR activation, and thus the elimination or reduction of ABC cells (see, e.g., Kloss et al., Nat. Biotechnol., 31:71-75 (2013)).
As used herein, the term “ABC cells” also termed double negative B cells, atypical memory B-cells, or tissue-like memory B-Cells that exhibit the cell surface receptor CD11c and the T-Box transcription factor (T-bet). Unlike other B cells, ABCs express CD11c, a receptor also expressed in myeloid cells. T-bet is a transcription factor known for its role as a master regulator of commitment of T cells to the T helper 1 cell lineage. ABC cells are also referred to as CD11c+T-bet+ B cells (see, Karnell et al., Cellular Immunol., 321: 40-50 (2017)).
As used herein, the term “chimeric antigen receptor” or “CAR” as used herein refers to comprising chimeric receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain. In some cases, the extracellular domain can comprise an antigen binding domain as described herein. In some cases, the transmembrane domain can comprise a transmembrane domain derived from a natural polypeptide obtained from a membrane-binding or transmembrane protein. For example, a transmembrane domain can include, without limitation, a transmembrane domain from a T cell receptor alpha or beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide. In some cases, the intracellular domain can comprise a cytoplasmic signaling domain as described herein. In some cases, the intracellular domain can comprise a co-stimulatory domain as described herein.
In some embodiments, the scFv comprises a light chain variable domain comprising a sequence that is at least 90% identical (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to one of SEQ ID NOs: 77-114. In some embodiments, the scFv comprises a heavy chain variable domain comprising a sequence that is at least 90% identical (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to one of SEQ ID NOs: 39-76.
As used herein, the term “activation” refers to induction of a signal on an immune cell (e.g., a B cell or T cell) that can result in initiation of the immune response (e.g., T cell activation). In some cases, upon binding of an antigen (e.g., CD19 or CD11c) to a T cell receptor (TCR) or an exogenous chimeric antigen receptor (CAR), the immune cell can undergo changes in protein expression that result in the activation of the immune response. In some cases, a TCR or CAR includes a cytoplasmic signaling sequence that can drive T cell activation. For example, upon binding of the antigen, a chimeric antigen receptor comprising an intracellular domain that includes a cytoplasmic signaling sequence (e.g., an immunoreceptor tyrosine-based inhibition motifs (ITAM)) that can be phosphorylated. A phosphorylated ITAM results in the induction of a T cell activation pathway that ultimately results in a T cell immune response. Examples of ITAMs include, without limitation, CD3 gamma, CD3 delta, CD3 epsilon, TCR zeta, FcR gamma, FcR beta, CD5, CD22, CD79a, and CD66d.
The term “affinity” as used herein, refers to the binding of mutant porcine IL-2 to the human IL-2 receptor, trimeric or dimeric forms. Affinity can be measured using any suitable method. See, e.g., Shanafelt et al., 2000 Nature Biotechnol 18: 1197-1202.
As used herein, the term “stimulation” refers to stage of TCR or CAR signaling where a co-stimulatory signal can be used to achieve a robust and sustained TCR or CAR signaling response. As described herein, a co-stimulatory domain can be referred to as a signaling domain. In some cases, a signaling domain (e.g., co-stimulatory domain) can be a CD27, CD28, OX40, CD30, CD40, B7-H3, NKG2C, LIGHT, CD7, CD2, 4-1BB, PD-1, or LFA-1.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain, the CD28 co-stimulatory domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 273.
In some embodiments where the chimeric antigen receptor polypeptide includes a OX40 co-stimulatory domain, the OX40 co-stimulatory domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 278.
In some embodiments where the chimeric antigen receptor polypeptide includes a 4-1BB co-stimulatory domain, the 4-1BB co-stimulatory domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 279.
In some cases, the transformed cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), and wherein the intracellular domain of the mutated chimeric NOTCH polypeptide is a transcriptional activator or repressor. In some cases, the nucleic acid sequence encoding the CAR is operably linked to a transcriptional control element that is activated by the intracellular domain of the mutated chimeric NOTCH polypeptide. In some embodiments, the cell to be transformed already stably expresses a CAR. Any appropriate mammal having an autoimmune disease can be treated by administering to the mammal an immuno-activatable cell comprising two or more chimeric antigen receptors (CARs). For example, humans or other primates such as monkeys having an autoimmune disease can be treated by administering to the mammal an immuno-activatable cell comprising two or more CARs as described herein. In some cases, dogs cats, horses, cows, pigs, sheep, mice, or rats having an autoimmune disease can be treated by administering an immuno-activatable cell as described herein.
In some embodiments, the intracellular domain comprises a transcriptional activation domain. In some embodiments, the transcriptional activation domains is selected from the group comprising a VP 16 activation domain, a VP64 activation domain, a p65 activation domain, a MyoDl activation domain, a Tbx21 activation domain a HSF1 activation domain, a RTA activation domain, a SET7/9 activation domain, a Gal4 DNA binding domain (DBD)-VP64 domain, a tTA-VP64: tetR-VP64 domain, a VP64-p65-Rta (VPR) activation domain, a mini VPR activation domain, a yeast GAL4 activation domain, a yeast HAP1 activation domain, a histone acetyltransferase, or any combination thereof.
In some embodiments where the chimeric antigen receptor polypeptide includes a Tbx21 transcriptional activation domain, the Tbx21 transcriptional activation domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 284.
In some embodiments where the chimeric antigen receptor polypeptide includes a E2S-VP64 transcriptional activation domain, the E2S-VP64 transcriptional activation domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 285.
In some embodiments where the chimeric antigen receptor polypeptide includes a GAL4-VP64 transcriptional activation domain, the GAL4-VP64 transcriptional activation domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 286.
As used herein, the term “antibody,” “antigen binding domain,” or antigen binding fragment” refers to an intact immunoglobulin or to an antigen binding portion thereof. Antigen binding portions are well known in the art and may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Examples of antigen binding portions include Fab, Fab′, F(ab′)2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. As used herein, the term “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Included in the definition are single domain antibody, including camelids. In some cases, the antibody is human or humanized.
In some embodiments, any of the “antigen binding domain,” “antibody,” or “ligand binding domain” described herein can bind specifically to a target selected from the group of: CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3ε, and CD3γ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGFDD, a ligand of TGF-β receptor II (TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAMI, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NK30, a ligand for a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for a ULP16-binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.
In some embodiments of these chimeric antigen receptor polypeptides described herein, the first antigen binding domain and the second antigen binding domain bind specifically to the same antigen. In some embodiments of these chimeric antigen receptor polypeptides, the first antigen binding domain and the second antigen binding domain bind specifically to the same epitope. In some embodiments of these chimeric antigen receptor polypeptides, the first antigen binding domain and the second antigen binding domain include the same amino acid sequence. In some embodiments of any of these chimeric antigen receptor polypeptides described herein, the first antigen binding domain and the second antigen binding domain bind specifically to different antigens.
The antigen-binding domains present in any of the chimeric antigen receptor polypeptides described herein are each independently selected from the group consisting of: a VHH domain, a VNAR domain, and a scFv.
As described herein, any appropriate method of producing immune cells (e.g., T cells) comprising chimeric antigen receptors (CAR) and chemokine receptor polypeptides can be used to generate the immuno-activatable cells as described herein. In some cases, a combination of nucleic acid sequences encoding the domains listed in
Methods of introducing nucleic acids and expression vectors into a cell (e.g., a eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.
In some embodiments, the transformed cell can be an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some embodiments, the transformed cell is an immune cell selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell and a cytotoxic T cell. In some examples, the immune cell is a NK cell, and the detection of a memory NK cell can include, e.g., the detection of the level of one or more of IL-12, IL-18, IL-33, STAT4, Zbtb32, DNAM-1, BIM, Noxa, SOCS1, BNIP3, BNIP3L, interferon-γ, CXCL16, CXCR6, NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. A description of NK memory cells and methods of detecting the same is described in O'Sullivan et al., Immunity 43:634-645, 2015. In some examples, the immune cell is a T cell, and the detection of memory T cells can include, e.g., the detection of the level of expression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin αeβ7, CD43, CD27, CD28, IL-7Rα, CD95, IL-2Rβ, CXCR3, and LFA-1. In some examples, the immune cell is a B cell and the detection of memory B cells can include, e.g., the detection of the level of expression of CD27. Other types and markers of memory or memory-like immune cells are known in the art.
As described herein, this document features a method for generating an immuno-activatable cell comprising two or more CARs and one or more chemokine receptor polypeptides. In some cases, an immuno-activatable cell can be generated by transforming an immune cell (e.g., a T cell) with nucleic acid sequences encoding two or more CARs as described herein and nucleic acid sequences encoding one or more chemokine receptor polypeptides. In some cases, chemokine receptor polypeptides can be specifically designed so that when expressed on the surface the polypeptides can target the transformed immune cell to lymphoid tissue. For example, an immuno-activatable cell can be generated by transforming an immune cell (e.g., a T cell) with nucleic acid sequences encoding two or more CARs and a nucleic acid sequence encoding a CXCR5 chemokine receptor polypeptide. A CXCR5 chemokine receptor polypeptide can direct the transformed immune cell to lymphoid tissue follicles (Hughes et al., FEBS J., 285(16):2944-71 (2018)). In another example, an immuno-activatable cell can be generated by transforming an immune cell (e.g., a T cell) with nucleic acid sequences encoding two or more CARs and a nucleic acid sequence encoding a CCR7 chemokine receptor polypeptide. A CCR7 chemokine receptor polypeptide can direct the transformed immune cell (e.g., T cell) to lymphatic tissue and/or the thymus (Hughes et al., FEBS J., 285(16):2944-71 (2018)). Other examples of chemokine receptor polypeptides that can be used to direct a transformed immune cell to a specific location in the body (e.g., lymphoid tissue follicles or thymus tissue) include, without limitation, CCR2, CCR3, CCR4, CCR5, CCR8, CCR9, CXCR1, CXCR2, CXCR, 3 CXCR4, CXCR6, and CRTH2.
Also provided herein are nucleic acids sequences that encode any of the chimeric antigen receptor polypeptides described herein. Also provided herein are vectors that include any of the nucleic acids encoding any of the chimeric antigen receptor polypeptides described herein.
Any of the vectors described herein can be an expression vector. For example, an expression vector can include a promoter sequence operably linked to the sequence encoding the chimeric antigen receptor polypeptides. Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, e.g., include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any of the immuno-activatable cells as described herein. Any appropriate promoter (e.g., EF1 alpha) can be operably linked to any of the nucleic acid sequences described herein. As used herein, the term “operably linked” is well known in the art and refers to genetic components that are combined such that they carry out their normal functions. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter. In another example, a nucleic acid sequence can be operable linked to another nucleic acid sequence by a self-cleaving 2A polypeptide. In such cases, the self-cleaving 2A polypeptide allows the second nucleic acid to be under the control of the promoter operably linked to the first nucleic acid sequence and allows the second nucleic acid to be in frame with the first nucleic acid.
In some embodiments the T2A cleavage sequence (GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 280)), a P2A cleavage sequence (GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 281)), a E2A cleavage sequence (GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 282)) or a F2A cleavage sequence GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 283)).
In some cases, an exemplary nucleic acid sequence used to make an immuno-activatable cell as described herein can include a promoter operably linked to nucleic acid sequences encoding a CAR comprising an antigen binding domain capable of binding to antigen on a CD11c+Tbet+ B cell, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a cytoplasmic signaling domain. In some cases, an exemplary nucleic acid sequence used make an immuno-activatable cell as described herein can include a promoter operably linked to nucleic acid sequences encoding a CAR comprising an antigen binding domain capable of binding to antigen on a CD11c+Tbet+ B cell, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a co-stimulatory domain. In some cases, an exemplary nucleic acid sequence used to make an immuno-activatable cell as described herein can include a promoter operably linked to nucleic acid sequences encoding a CAR comprising an antigen binding domain capable of binding to antigen on a CD11c+Tbet+ B cell, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, a cytoplasmic signaling domain, and a chemokine receptor polypeptide operably linked to the CAR (e.g., in frame) with a self-cleaving 2A sequence (e.g., a P2A, a T2A, a E2A or a F2A) (
In some cases, the nucleic acid sequences encoding a CAR as used herein can include a sequence from 5′ to 3′ a promoter operably linked to nucleic acid sequences encoding a scFv antigen binding domain capable of binding to CD19, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a CD3zeta cytoplasmic signaling domain (
In some embodiments, a CAR can include a transmembrane domain. The transmembrane domain may be derived from a natural polypeptide, or may be artificially designed. If the transmembrane domain is derived from a natural polypeptide it can be obtained from a membrane-binding or transmembrane protein. For example, useable transmembrane domains can be from a T cell receptor alpha or beta chain, a CD3 zeta chain, CD28, CD3-epsilon, or numerous others known in the art. See, U.S. Pat. Nos. 9,670,281 and 9,834,608, both of which are incorporated by reference in their entireties. In some embodiments, the transmembrane domain is derived from CD28 or CD8. In some embodiments where the chimeric antigen receptor polypeptide includes a CD8 alpha transmembrane domain, the CD8 alpha transmembrane domain has an amino acid sequence is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to NCBI Reference No: NP_001759 or a fragment thereof. In some embodiments where the chimeric antigen receptor polypeptide includes a CD28 transmembrane domain, the CD28 transmembrane domain has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 277.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD8 alpha transmembrane domain, the CD8 alpha transmembrane domain has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 275 or 276.
Other transmembrane domains are known in the art and include CD16, NKG2D, NKp44, NKp46, CD27, DAP10, and DAP12 transmembrane domains.
In some cases, a nucleic acid sequence encoding a CAR as used herein can include a sequence from 5′ to 3′ a promoter operably linked to nucleic acid sequence encoding a scFv antigen binding domain capable of binding to CD11c, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a CD28 co-stimulatory domain (
In some cases, an immuno-activatable cell can be generated by transforming the cell with nucleic acid sequences encoding two or more CARs and one or more chemokine receptor polypeptides wherein the nucleic acid sequences are on one continuous nucleic acid sequence (e.g., a polycistronic vector). For example, a nucleic acid sequence can include: (a) a nucleic acid sequences encoding a CAR comprising an antigen binding domain capable of binding to antigen on a CD11c+Tbet+ B cell, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a cytoplasmic signaling domain, (b) a self-cleaving polypeptide (e.g., a P2A, a T2A, a E2A or a F2A), (c) a nucleic acid sequences encoding a CAR comprising an antigen binding domain capable of binding to antigen on a CD11c+Tbet+ B cell, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, and a co-stimulatory domain, (d) a second self-cleaving polypeptide (e.g., a P2A, a T2A, a E2A or a F2A), and (e) a chemokine receptor polypeptide.
In some cases, a CAR comprising an antigen binding domain capable of binding to CD19 can include a co-stimulatory domain, and a CAR comprising an antigen binding domain capable of binding to CD11c can include a cytoplasmic signaling domain. For example, a nucleic acid sequence encoding a CAR can include a promoter operably linked to nucleic acid sequences encoding a scFv antigen binding domain capable of binding to CD19, a CD8α transmembrane domain comprising a CD8α stalk and a CD8 hinge region, a CD28 co-stimulatory domain. In such cases, the nucleic acid sequence can include a chemokine receptor polypeptide (e.g., CXCR5 or CCR7) operably linked by a 2A self-cleaving polypeptide. In another example, For example, a nucleic acid sequence encoding a CAR can include a promoter operably linked to nucleic acid sequences encoding a scFv antigen binding domain capable of binding to CD11c, a CD8α transmembrane domain comprising a CD8α stalk and a CD8α hinge region, a CD3zeta cytoplasmic signaling domain. In such cases, the nucleic acid sequence can include a chemokine receptor polypeptide (e.g., CXCR5 or CCR7) operably linked by a 2A self-cleaving polypeptide.
Exemplary CD19 antibodies or antigen binding fragments thereof are described in U.S. Pat. Nos. 11,623,956, 11,618,788, U.S. Patent Publication Number 2023/0099646, U.S. Patent Publication Number 2023/0087263, and U.S. Patent Publication Number 2023/0086030, each of which is incorporated herein by reference in its entirety. Exemplary CD20 antibodies or antigen binding fragments thereof are described in U.S. Pat. Nos. 11,623,005, 11,608,383, 11,603,411, and U.S. Patent Publication No. 2023/0056900, each of which is incorporated herein by reference in its entirety. Exemplary CD45R antibodies or antigen binding fragments thereof are described in U.S. Pat. Nos. 10,093,743, 7,160,987, and 6,010,902, each of which is incorporated herein by reference in its entirety.
In various other embodiments, the autoimmune disease can be SLE, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitus, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, or polyarteritis nodosa.
As used herein, the terms “percent identity” and “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same. Percent identity can be determined using sequence comparison software or algorithms or by visual inspection.
In general, percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid or polypeptide sequences, dividing the number of matched positions by the total number of aligned nucleotides or amino acids, respectively, and multiplying by 100. A matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned sequences. The total number of aligned nucleotides or amino acids refers to the minimum number of NOTCH nucleotides or amino acids that are necessary to align the second sequence, and does not include alignment (e.g., forced alignment) with non-NOTCH sequences, such as those fused to NOTCH. The total number of aligned nucleotides or amino acids may correspond to the entire NOTC sequence or may correspond to fragments of the full-length NOTCH sequence.
Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res, 25:3389-3402, 1997) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches or alignments can be performed to determine percent sequence identity between a NOTCH nucleic acid or polypeptide and any other sequence or portion thereof using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST programs to calculate the percent identity between a NOTCH sequence and another sequence, the default parameters of the respective programs are used.
In some cases, the nucleic acid sequences encoding the CARs and the chemokine receptor polypeptides can include a nucleic acid sequence encoding a linker. This linker can provide conformational flexibility.
Also provided herein are compositions (e.g., pharmaceutical compositions) that include at least one of any of the chimeric antigen receptor polypeptides, any of the cells, or any of the nucleic acids described herein. In some embodiments, the compositions include at least one of the any of chimeric antigen receptor polypeptide described herein. In some embodiments, the compositions include any of the cells (e.g., any of the immune cells described herein including any of the immune cells produced using any of the methods described herein).
In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline).
Also provided herein are cells (e.g., any of the exemplary cells described herein) containing any of the nucleic acids described herein that encode any chimeric antigen receptor polypeptides described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein) that include any of the vectors described herein that encode any chimeric antigen receptor polypeptides described herein.
In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).
Also provided herein are methods of treating a mammal (e.g., a human) having an autoimmune disease that includes administering to the mammal a therapeutically effective amount of a cell (e.g., an immuno-activatable cell) transformed with the chimeric antigen receptor polypeptides or nucleic acids described herein, or that includes administering any of the compositions (e.g., pharmaceutical compositions) described herein.
In some embodiments of these methods, a mammal (e.g., a human) can be identified as having an autoimmune disease. In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of an autoimmune disease in the mammal (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the autoimmune disease in the subject prior to treatment). For example, a mammal having an autoimmune disease having been administered an immuno-activatable cell as described here can experience a reduction in inflammation or B cell autoantibody production (e.g., B cell antibody production inhibition or reduction in the number of B cells).
Any appropriate method of administration can be used to administer the immuno-activatable cells to a mammal (e.g. a human) having an autoimmune disease. Examples of methods of administration include, without limitation, parenteral administration and intravenous injection.
A pharmaceutical composition containing the immuno-activatable cells and a pharmaceutically acceptable carrier can be administered to a mammal (e.g., a human) having an autoimmune disease. For example, a pharmaceutical composition (e.g., immuno-activatable cell along with a pharmaceutically acceptable carrier) to be administered to a mammal having an autoimmune disease can be formulated in an injectable form (e.g., emulsion, solution and/or suspension).
Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in a pharmaceutical composition described herein can include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
Effective dosage can vary depending on the severity of the autoimmune disease, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating physician. An effective amount of an immuno-activatable cell can be any amount that reduces inflammation and B cell autoantibody production (e.g., B cell antibody production inhibition or reduction in the number of B cells) within a mammal having an autoimmune disease without producing significant toxicity to the mammal. For example, an effective amount of immuno-activatable cells administered to a mammal having an autoimmune disease can be from about 1×106 cells to about 1×1010 (e.g., from about 1×106 to about 1×109, from about 1×106 to about 1×108, from about 1×106 to about 1×107, from about 1×107 to about 1×1010, from about 1×107 to about 1×109, from about 1×107 to about 1×108, from about 1×108 to about 1×1010, from about 1×108 to about 1×109, or form about 1×109 to about 1×1010). In some cases, the immuno-activatable cells can be a purified population of immune cells generated as described herein. In some cases, the purity of the population of immuno-activatable cells can be assessed using any appropriate method, including, without limitation, flow cytometry. In some cases, the population of immuno-activatable cells to be administered can include a range of purities from about 70% to about 100%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100%, from about 80% to about 100%, from about 80% to about 90%, or from about 90% to 100%. In some cases, the dosage (e.g., number of immuno-activatable cells to be administered) can adjusted based on the level of purity of the immuno-activatable cells in the overall population of cells.
In some cases, two or more (e.g., two, three, four, five, six, or more) different immuno-activatable cells can be administered to a mammal having an autoimmune disease. For example, a percentage (e.g., from about 1.0% to about 99.0%) of the cells to be administered can include a first CAR and a second CAR and a second percentage of cells (e.g., the remaining percentage of the administered cells) can include a third CAR and a fourth CAR. In such cases, each CAR may be designed with a different antigen binding domain that binds to different antigens (e.g., different antigens on the same cell or different antigens on different cells).
The frequency of administration of an immuno-activatable cell can be any amount that reduces inflammation or B cell autoantibody production (e.g., B cell antibody production inhibition or reduction in the number of B cells) within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the actual frequency of administration can vary depending on various factors including, without limitation, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in frequency of administration.
An effective duration for administering a composition containing an immuno-activatable cell can be any duration that reduces inflammation or B cell autoantibody production (e.g., B cell antibody production inhibition or reduction in the number of B cells) within a mammal having an autoimmune disease without producing toxicity to the mammal. In some cases, the effective duration can vary from several days to several months. In general, the effective treatment duration for administering a composition containing an immuno-activatable cell to treat an autoimmune disease can range in duration from about one month to about five years (e.g., from about two months to about five years, from about three months to about five years, from about six months to about five years, from about eight months to about five years, from about one year to about five years, from about one month to about four years, from about one month to about three years, from about one month to about two years, from about six months to about four years, from about six months to about three years, or from about six months to about two years).
In some cases, a course of treatment and/or the severity of one or more symptoms related to autoimmune disease can be monitored. Any appropriate method can be used to determine whether the autoimmune disease is being treated. For example, immunological techniques (e.g., ELISA) can be performed to determine if the level of autoantibodies produced by the age-associated B cells present within a mammal being treated as described herein is reduced following the administration of the immuno-activatable cells. Remission and relapse of the disease can be monitored by testing for one or more markers of autoimmune disease.
Also provided herein are methods of killing, removing, and/or eliminating age-associated B cells (e.g., CD11c+ T-bet+ B cells). The methods can include administering to a subject (e.g., a mammal) a therapeutically effective amount of any of the immuno-activatable cells or compositions (e.g., pharmaceutical compositions) described herein.
Any appropriate autoimmune disease can be treated with an immuno-activatable cell as described herein. In some cases, an autoimmune disease caused by the accumulation of autoantibodies produced by age-associated B cells can be treated with an immuno-activatable cell as described herein. Examples of autoimmune diseases caused, at least in part, by age-associated B cells include, without limitation, lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, Crohn's disease, Celiac disease, and polyarteritis nodosa.
In some embodiments where the chimeric antigen receptor polypeptide includes an anti-CD19 scFv antigen binding domain, the anti-CD19 scFv binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the anti-CD19 scFv VH domain can have an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 287: LKPREWKLVESGGGLVOPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINLD SSTINYTPSLKDKFIISRDNAKNTLYLOMSKVRSEDTALYYCARRYDAMDYWGQGTSV TVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKASO. In some embodiments, the anti-CD19 scFv VL domain can have an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 288: ASDIWLTOSPASLAVSLGORATISCRASESVDDYGISFMNWFOOKPGQ PPKLLIYAAPNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKDVRWRHQA GDQTG. In some embodiments, any appropriate linker can be used to couple the VH and VL domains of the anti-CD19 scFv (e.g., GGGGSGGGGSGGGGS; SEQ ID NO: 272).
In some embodiments, where the chimeric antigen receptor polypeptide includes a CD11c scFv antigen binding domain, the anti-CD11c scFv can have an amino acid sequence that is least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to the variable heavy chain and variable light chain of hamster anti-mouse CD11c mAb (cloneN418) joined by a linker (-GGGGSGGGGSGGGGS; SEQ ID NO: 272).
In some embodiments where the chimeric antigen receptor polypeptide includes a CD8 alpha transmembrane domain, the CD8 alpha transmembrane domain has an amino acid sequence is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to NCBI Reference No: NP_001759 or a fragment thereof.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD3 zeta cytoplasmic signaling domain, the CD3 zeta cytoplasmic signaling domain has an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to NCBI Reference No: NP_932170 or a fragment thereof that has activating or stimulatory activity.
In some embodiments where the chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain, the CD28 co-stimulatory domain is at least 80% (e.g., at least 85%, 90%, 95%, 99% and 100%) identical to SEQ ID NO: 273:
As used herein, “CXCR5” refers to a C-X-C motif chemokine receptor 5 polypeptide. When preparing the immuno-activatable cell or treating a mammal with the immuno-activatable cell, “CXCR5” refers to human CXCR5. An example of a human CXCR5 polypeptide includes, without limitation, the sequence set forth in NCBI reference sequence NP_001707 (e.g., version NP_001707.1) or a fragment thereof. The CXCR5 sequence set forth in NCBI reference sequence NP_001707.1 is MNYPLTLEMDLENLEDLFWELDRLDNYNDTSL VENHLCPATE GPLMASFKAVFVPVAYSLIFLLGVIGNVLVL VILERHRQTRSSTETFLFHLAVADLLLVFI LPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRR LLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYH VAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFL DTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKL GCTGPASLCQLFPSWRRSSLSESENATSLTTF (SEQ ID NO: 289)
As used herein, “CCR7” refers to C-C motif chemokine receptor 7 polypeptide. When preparing the immuno-activatable cell or treating a mammal with the immuno-activatable cell, “CCR7” refers to human CCR7. An example of a human CCR7 polypeptide includes, without limitation, NCBI reference sequence NP_001288643 (e.g., version NP_001288643.1) or a fragment thereof. The CCR7 sequence set forth in NCBI reference NP_001288643.1 is MYSIIC
Here, a single lentiviral vector is used to transduce two CARs and a chemokine receptor polypeptide into a T cell.
Any appropriate method for isolating and culturing primary human T cell can be used. T cell placed in culture are transduced with the lentivirus. After lentiviral transduction, the expression of both the CD19 CAR and the CD11c Car can be assessed by staining with an anti-CD19 fluorescently-labeled antibody and a CD11c fluorescently-labeled antibody and analyzed using flow cytometry.
A mammal suffering from SLE will be assayed to determine the presence of SLE autoantibodies prior to and after treatment with an immuno-activatable T cell generated in Example 1. Human ABCs from peripheral blood samples of SLE patients can be isolated by magnetic cell sorting system (Miltenyi et al., Cytometry 11: p231-238 (1990)). The isolated cells are then stimulated for 5-7 days with CD40L (R &D system, Cat #6420-CL) and CpG ODN 2006 (InvivoGen, Cat #tlrl-2006) in the presence or absence immuno-activatable T cell comprising a CD19 CAR, a CD11c CAR and a CXCR5 receptor. Culture supernatants will be tested for secreted Immunoglobulins (e.g., IgM, IgA and IgG) by ELISA assay and ANA autoantibodies by immunofluorescence analysis as described previously (Capolunghi et al., Rheumatology 49: p2281-89 (2010)). A patient is administered a dose of the immuno-activatable T cell comprising the CD19 CAR, the CD11c CAR and a CXCR5 receptor, in the range of 10-100 million T cells. The dose will be empirically determined depending on a number of factors, including side effects, and indications of efficacy. The modified T-cells can be administered by any method known in the art including, without limitation, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal and directly to the thymus. A single dose or multiple doses may be administered.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Provisional Patent Application No. 63/350,141, filed on Jun. 8, 2022, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63350141 | Jun 2022 | US |
