Regulatory T cells with chimeric antigen receptors that target costimulatory molecules for preventing and / or treating inflammatory conditions - Patent Application 20070123633

Abstract

Described herein are methods and compositions for regulatory T cells (Tregs) that are modified to express a chimeric antigen receptor (CAR) that targets OX40L. Aspects of the invention relate to administering these modified Tregs to subjects with inflammatory or autoimmune conditions. TIFF2025507727000020.tif104128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of U.S. Provisional Patent Application No. 63 / 314,789, filed February 28, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] government support This invention was made with Government support under Grant Nos. HL095791 and AI051731 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Technical Field The technology described herein relates to chimeric antigen receptor (CAR) constructs, for example in regulatory T cells (Tregs), that target costimulatory molecules. [Background technology]

[0004] background The main cause of inflammatory conditions or autoimmunity, as well as transplant rejection after organ transplantation or graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HCT), are autoreactive and alloreactive T cells that attack the patient's or donor's organs or tissues. Current treatments to prevent autoimmunity, transplant rejection, and GVHD involve strict immunosuppressive therapy, which itself can cause severe undesirable side effects. To prevent autoimmunity, transplant rejection, and GVHD without inducing undesirable side effects, it would be advantageous to have engineered T cells, particularly engineered regulatory T cells (Tregs), available to treat patients receiving organ transplants.

[0005] Tregs suppress the immune response of other cells. These cells can inhibit T cell proliferation and cytokine production, halt autoimmunity, or shut down the immune response after successful elimination of an invading microorganism. There are several forms of Tregs, but the best understood are those that express CD4, CD25, Foxp3, and Helios (CD4 + CD25 + By manipulating and directing Tregs to appropriate targets, it is possible to enhance the suppression of aberrant immune responses. Summary of the Invention

[0006] overview As described herein, chimeric antigen receptors (CARs) provide a promising method for targeting APCs and OX40L costimulatory molecules. CARs are engineered receptor proteins in which the natural recognition moiety has been removed and replaced with a specific recognition moiety that redirects the targeting and binding of specific proteins. T cells expressing CARs are called CAR-Ts. CARs and CAR-Ts are most often used to treat tumors and cancers, and to a lesser extent, inflammatory conditions, but more effective CARs are still needed to minimize or eliminate the unwanted immune response to the underlying causes of inflammatory conditions and autoimmune disorders. The creation of OX40L CAR-Tregs aims to more constitutively block cytokine production upon the induction of inflammation or autoimmunity. Compared to some newly developed CAR-Tregs that target specific antigens, such as HLA-A2, CEA, and CD83, CAR-Tregs targeting OX40L cover a wider range of inflammatory conditions and provide a wider range of applications. Furthermore, activated Tregs help restore immune tolerance and maintain the functional balance between conventional T cells (Tcon) and Tregs, making OX40L CAR Treg cells an alternative or superior drug to antibodies for the treatment of inflammatory conditions or autoimmune diseases.

[0007] As described herein, the inventors have created human regulatory T cells (Tregs) that are engineered to express chimeric antigen receptors (CARs) that specifically target OX40L. In some embodiments, described herein is a CAR that comprises an anti-OX40L antibody reagent, e.g., an extracellular recognition portion that comprises scFv, and an intracellular signaling region that allows or induces the activation of CAR-Treg cells when binding with OX40 ligand on APC. Furthermore, to limit the harmful effects of Tcon activation during purification and expansion of Treg cells, a synthetic FoxP3 promoter drives CAR expression. This promoter limits the expression of CAR to Treg cells. Thus, these CAR-Tregs and related compositions can be used in therapeutic methods for treating diseases described herein.

[0008] In one aspect of any of the embodiments, described herein is a chimeric antigen receptor (CAR), comprising, from N-terminus to C-terminus: (a) an extracellular recognition portion that specifically binds to OX40L; (b) a transmembrane portion; and (c) an intracellular signaling portion.

[0009] In some embodiments of any of these aspects, the C-terminus of the intracellular signaling portion of the CAR further comprises a detectable polypeptide.

[0010] In some embodiments of any of these aspects, the detectable polypeptide of the CAR is a fluorescent polypeptide.

[0011] In some embodiments of any of these aspects, the fluorescent polypeptide of the CAR is Neon Green.

[0012] In some embodiments of any of these aspects, the CAR further comprises a cleavage site between the intracellular signaling moiety and the detectable polypeptide.

[0013] In some embodiments of any of these aspects, the cleavage site of the CAR is a cleavable T2A site or a tandem P2A-T2A site.

[0014] In some embodiments of any of these aspects, the recognition moiety of the CAR is an antibody reagent or a ligand functional domain.

[0015] In some embodiments of any of these aspects, the antibody reagent of the CAR is an scFV.

[0016] In some embodiments of any of these aspects, the antibody reagent of the CAR is an anti-OX40L antibody reagent.

[0017] In some embodiments of any of these aspects, the CAR antibody reagent comprises CDR sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the six CDRs of SEQ ID NOs: 1-6.

[0018] In some embodiments of any of these aspects, the CAR antibody reagent comprises a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the amino acid sequence of SEQ ID NOs: 7-12.

[0019] In some embodiments of any of these aspects, the intracellular signaling portion of the CAR comprises one or more of a CD28 co-signaling domain, a 41BB co-signaling domain, an IL2Rα JAK3 and IL2Rβ STAT5 complex docking site, a TGFβ-R SMAD2 / 3 docking site, and a CD3ζ signaling domain.

[0020] In some embodiments of any of these aspects, the intracellular signaling portion of the CAR comprises a CD28 co-signaling domain and a CD3 zeta domain.

[0021] In some embodiments of any of these aspects, the intracellular signaling portion of the CAR comprises a 41BB co-signaling domain and a CD3 zeta domain.

[0022] In some embodiments of any of these aspects, the nucleic acid molecule encodes the CAR.

[0023] In some embodiments of any of these aspects, expression of the nucleic acid molecule is controlled by a Treg-specific promoter or an MND promoter.

[0024] In some embodiments of any of these aspects, the Treg-specific promoter of the nucleic acid molecule encoding a CAR comprises a FoxP3 promoter or a FoxP3 and IKZF2 / Helios hybrid promoter.

[0025] In some embodiments of any of these aspects, the vector comprises a nucleic acid molecule encoding the CAR.

[0026] In some embodiments of any of these aspects, the cell comprises the CAR, or a nucleic acid molecule encoding a CAR, or a vector comprising a nucleic acid molecule encoding a CAR.

[0027] In some embodiments of any of these aspects, the cells are Tregs.

[0028] In some embodiments of any of these aspects, the Tregs express Foxp3.

[0029] In some embodiments of any of these aspects, the Tregs express CD4 and CD25.

[0030] In some embodiments of any of these aspects, the population of cells, at least 80% of which are cells of any of these aspects.

[0031] In some embodiments of any of these aspects is a method of treating an autoimmune or inflammatory condition in a subject in need thereof comprising administering to the subject a cell or cell population of any of these aspects.

[0032] In some embodiments of any of these aspects, the autoimmune or inflammatory condition comprises allograft or xenograft rejection, or graft-versus-host disease (GVHD).

[0033] In some embodiments of any of these aspects, the autoimmune or inflammatory condition is selected from the group consisting of inflammatory bowel disease; rheumatoid arthritis; type I diabetes or autoimmune insulitis; multiple sclerosis; autoimmune thyroiditis; autoimmune gastritis; autoimmune uveitis or uveoretinitis; autoimmune orchitis; autoimmune oophoritis; psoriasis; vitiligo; autoimmune prostatitis; any unwanted immune response; tissue rejection; and an inflammatory condition.

[0034] In some embodiments of any of these aspects, the population of Tregs is autologous to the subject.

[0035] In some embodiments of any of these aspects, the population of Tregs is allogeneic to the subject.

[0036] In some embodiments of any of these aspects, the cell or cell population is for use in a method of treating an autoimmune or inflammatory condition in a subject in need thereof comprising administering the cell or cell population to the subject.

[0037] In some embodiments of any of these aspects, the autoimmune or inflammatory condition comprises allograft or xenograft rejection, or graft-versus-host disease (GVHD).

[0038] In some embodiments of any of these aspects, the autoimmune or inflammatory condition is selected from the group consisting of inflammatory bowel disease; rheumatoid arthritis; type I diabetes or autoimmune insulitis; multiple sclerosis; autoimmune thyroiditis; autoimmune gastritis; autoimmune uveitis or uveoretinitis; autoimmune orchitis; autoimmune oophoritis; psoriasis; vitiligo; autoimmune prostatitis; any unwanted immune response; tissue rejection; and an inflammatory condition.

[0039] In some embodiments of any of these aspects, the population of Tregs is autologous to the subject.

[0040] In some embodiments of any of these aspects, the population of Tregs is allogeneic to the subject. [Brief description of the drawings]

[0041] [Figure 1A]Construction and expansion of OX40L CAR-Treg cells. (Figure 1A) Overview of constructs encoding Treg-specific chimeric antigen receptors (CARs): Single-chain fragment variants derived from the antigen-binding fragment of the anti-OX40L antibody were linked to intracellular CD3ζ and 4-1BB or CD28 signaling domains, and to the fluorescent protein reporter neon green via a cleavable T2A site. Expression of the CAR and fluorescent reporter protein was controlled by either a Treg-specific promoter or a synthetic non-specific MND promoter. Constructs encoding only neon green under a Treg-specific promoter or MND promoter were used as controls. (Figure 1B) Demonstration of cell surface expression of OX40L-specific CAR constructs: OX40L soluble protein binding to the cell surface of Jurkat lymphoid cells in vitro was observed in more than 85% of neon green positive cells (successfully transduced with CAR / reporter encoding constructs), whereas cells transduced with neon green encoding constructs demonstrated negligible OX40L protein binding. (Figure 1C) Human FACS-purified CD25+CD127- Treg cells were transduced with OX40L-specific CAR constructs carrying signaling CD3ζ / 4-1BB (BBz) or CD3ζ / CD28 (28z) domains under the control of a FoxP3-specific promoter and then expanded in vitro with CD3 / CD28 microbeads and IL-2. Bar graph depicts successful expansion of CAR-transduced Treg cells. (Figure 1D) Expanded human Treg cells stably express FoxP3 promoter-controlled CAR constructs. Human FACS-purified CD25+CD127- Treg cells and CD25-CD127+ conventional CD4 (Tconv) cells were transduced with OX40L-specific CAR constructs carrying signaling CD3ζ / 4-1BB (BBz) or CD3ζ / CD28 (28z) domains under the control of a FoxP3-specific promoter and subsequently expanded in vitro with CD3 / CD28 microbeads and IL-2.The percentage of neon green+ cells in Treg and Tconv cell cultures was assessed by flow cytometry at different time points. The graph shows stable expression of FoxP3 promoter-controlled CAR construct in Treg cells, whereas in resting Tconv cells, expression of the CAR construct decreased upon removal of CD3 / CD28 beads (D18). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2A] Large-scale ex vivo expansion of OX40L CAR-Tregs. (Figure 2A) Experimental overview of large-scale CAR-Treg cell expansion experiment. (Figure 2B) Efficacy of lentiviral transduction procedure: Human FACS-purified CD25+CD127- Treg cells and CD25-CD127+ Tconv cells were transduced with lentiviruses containing a gene construct encoding a CAR fused to a neon green reporter fluorescent protein or a neon green reporter (see Figure 1A). The percentage of neon green+ cells was measured by flow cytometry on day 13 in Treg and Tconv cultures. (Figure 2C) Large-scale ex vivo expansion of CAR-Treg cells: Untransduced human Treg cells or Treg cells transduced with a neon green reporter or a CAR construct were expanded ex vivo for 34 days. The plot demonstrates a similar expansion capacity of CAR-expressing Treg cells. (Figure 2D) Ex vivo expanded human CAR-Treg cells demonstrate stable FoxP3 expression. Human FACS purified CD25+CD127- Treg cells were transduced with BBz or 28z CAR constructs or neon green reporter and expanded ex vivo with CD3 / CD28 + IL-2. FoxP3 protein expression was measured by flow cytometry, confirming that FoxP3 expression in CAR-Treg cells was at the same level as endogenous Treg control cells. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3A] Functional characteristics of CAR-Treg cells are shown. (Figure 3A) CAR stimulation induces expression of Treg activation / suppression markers: Treg cells transduced with BBz or 28z CAR constructs were stimulated in vitro via the CAR by co-culturing with K562 cells expressing OX40L (K562-L) or via the endogenous TCR by co-culturing with CD3 / CD28 activation beads. Anti-OX40 antibodies were added to Treg / K562-L cultures (K562-L + Ab) to block potential signaling via the endogenous OX40L / OX40 pathway, thus confirming that CAR-Treg cells are stimulated through the CAR. The plot demonstrates CAR-mediated induction of Treg activation / suppression markers CTLA4, LAP, CD71, GARP and LAG-3 at levels comparable to TCR stimulation. (Figure 3B) CAR stimulation induces production of the Treg-supportive cytokine IL-2 but not of pro-inflammatory cytokines: BBz and 28z CAR-expressing Treg cells were stimulated in vitro via CAR (K562-L) or endogenous TCR (CD3 / CD28 beads). Anti-OX40 antibodies were added to Treg / K562-L cultures to block potential signaling via the endogenous OX40L / OX40 pathway. The plot demonstrates CAR-mediated induction of IL-2 production, while production of the pro-inflammatory cytokines IFNg-γ, TNF-α and IL-17A remains unaffected. (Figure 3C) CAR stimulation improves the suppressive function of Treg cells in vitro: Control Treg cells and Treg cells expressing BBz or 28z CAR were co-cultured with CD3 / CD28 bead-activated conventional CD4 T cells (CD4 Tconv) in the presence or absence of OX40L-expressing K562 cells (K562-L and K562, respectively). The suppressive capacity of Treg cells was assessed by the relative inhibition of CD4 Tconv proliferation using flow cytometry. The plot depicts the increased suppressive capacity of CAR-Treg cells co-cultured with OX40L-expressing K562 cells. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] 1 shows the proposed suppressive mechanism for OX40L CAR-Treg. [Figure 5A] The effect of OX40L blockade in a mouse transplantation model is shown. (Fig. 5A) Schematic of the experimental design to evaluate the effect of anti-human OX40L (hOX40L) treatment on CD8+ T cell function. (Fig. 5B) Human CD8+ T cells were purified from mouse spleens. Specific lysis (%) was assessed by measuring the fluorescence intensity after 4 h incubation of fluorochrome-labeled CD8+ T cells and U937 cells. Error bars represent the mean ± SD (n = 5 mice / group) and data are representative of three independent experiments. Statistical significance was determined by Sidak's two-way analysis of variance (ANOVA) test. ns, not significant. (Fig. 5C) Clinical scores and survival of hPBMC mouse recipients after treatment with anti-hOX40L. [Figure 5B] See legend to Figure 5A. [Figure 6] We demonstrate that anti-OX40L / sirolimus prophylaxis results in long-term GVHD-free survival after allogeneic HCT. [Figure 7] Constructs encoding Treg-specific CARs or control constructs encoding neon green are outlined. [Figure 8A] Expression of OX40L-CAR constructs is shown. (FIG. 8A) Schematic of the generation of Ox40L CAR-Tregs. (FIG. 8B) Surface expression of FOXP3-driven OX40L-CAR and FOXP3-driven neon green control constructs. [Figure 8B] See legend to Figure 8A. [Figure 9A]Showing stable and selective expression of OX40L-CAR Tregs. (Figure 9A) Measurement of FOXP3 positive signal in CD4+ cells shows similar percentages in Tregs, Neon-Tregs and CAR-Tregs, demonstrating the stability of CAR-Tregs after 23 days of ex vivo expansion. (Figure 9B) Measurement of Neon Green positive signal in CAR-Tregs demonstrates stable and selective FOXP3 promoter activity in Tregs compared to CAR-Tcon. [Figure 9B] See legend to Figure 9A. [Figure 10A] The effect of OX40L CAR-Tregs on Treg inhibitory proteins and pro-inflammatory cytokines is shown. (Figure 10A) Schematic of OX40L-specific CAR-Treg activation. (Figure 10B) After viral transduction, OX40L CAR-Tregs and control Neon-Tregs were co-cultured with irradiated WT K562 cells or OX40L-expressing K562 cells. After 48-72 h, the expression of the indicated cytokines or inhibitory proteins was detected by flow cytometry. [Figure 10B] See legend to Figure 10A. [Figure 11A] Showing that CAR-Tregs suppress T cell expansion in vitro. (FIG. 11A) Schematic of the Treg suppression assay setup. (FIG. 11B) Measuring % CD4 suppression in response to different Treg:T responder ratios reveals that CAR-Tregs activated via OX40L engagement more potently suppress T cell expansion in vitro. [Figure 11B] See legend to Figure 11A. [Figure 12] Figures 12A-12B show that CAR-Tregs suppress T cell expansion in the MLR. (Figure 12A) Schematic of the MLR suppression assay. (Figure 12B) Measuring CD4 suppression (%) according to various Treg:T responder ratios reveals that CAR-Tregs suppress the MLR compared to Neon-Tregs. [Figure 13A]Effect of OX40L CAR-Treg on mouse GVHD model. (Figure 13A) Xenogeneic GVHD experimental setup. (Figure 13B) Human PBMCs isolated from healthy donors were injected retro-orbitally into sublethally irradiated NSG mice alone or in combination with control Neon-Treg or OX40L CAR-Treg. Mice were monitored twice weekly for weight loss, GvHD scores and survival endpoints. Both Neon-Treg and OX40L CAR-Treg delayed the onset of GvHD, while OX40L CAR-Treg showed significantly lower GvHD clinical scores and improved overall survival. ***p<0.001. [Figure 13B] See legend to Figure 13A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description Described herein is a chimeric antigen receptor (CAR) that is engineered in regulatory T cells (Treg) and specifically targets costimulatory molecules, such as OX40L. The technology described herein causes blocking of OX40-OX40L interaction and can be effective in many inflammatory diseases, autoimmune diseases, and graft-versus-host disease (GVHD). OX40L CAR-Treg restores immune tolerance and maintains the functional balance of conventional T cells (Tcon) and Treg, thereby providing additional, alternative, or superior therapeutic agents for the above diseases. Also described herein are polynucleotides and vectors encoding CAR polypeptides, cells expressing CAR polypeptides, pharmaceutical compositions comprising CAR polypeptides, and methods of using CAR polypeptides.

[0043] Thus, in one aspect of any of the embodiments, described herein is a method for producing a medicament comprising, from N-terminus to C-terminus, a. an extracellular recognition moiety that specifically binds to one or more costimulatory molecules, e.g., OX40L; b. a transmembrane segment; and c. Intracellular signal transduction part and wherein the chimeric antigen receptor (CAR) polypeptide comprises:

[0044] CAR is a receptor protein engineered to contain an exogenous recognition domain that specifically binds to a desired target, such as OX40L. Cells expressing or containing such CARs are thereby provided with the ability to target a desired target. These receptors are chimeric because they combine both antigen binding and immune cell activation functions in a single receptor in a combination that does not occur in nature, and / or because they combine antigen binding sequences and immune cell activation sequences of different genetic origins. In some embodiments of either aspect, the extracellular recognition portion of the CAR described herein does not naturally occur in the protein that contains the intracellular signaling portion described herein.

[0045] In some embodiments of any aspect, the CAR described herein comprises an extracellular recognition moiety. As used herein, "extracellular recognition moiety" refers to a polypeptide sequence that will be presented on the extracellular side of the cell membrane and bind to a target when the CAR is expressed in a cell. In some embodiments of any aspect, the extracellular recognition moiety comprises an antibody, an antibody reagent, an antigen-binding fragment thereof, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single domain antibody (sdAb). In some embodiments of any aspect, the extracellular recognition moiety is monovalent, bivalent, or multivalent. In some embodiments of any aspect, the extracellular recognition moiety comprises a human antibody or a human antibody reagent, a humanized antibody or a humanized antibody reagent, or a chimeric antibody or a chimeric antibody reagent.

[0046] The antibody reagent is specific for the targets and / or markers described herein, e.g., T cell costimulatory molecules. Such reagents are readily commercially available. In some embodiments of any aspect, the extracellular recognition moiety can be an antibody reagent that comprises one or more (e.g., one, two, three, four, five, or six) CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, the antibody reagent that is specific for the targets and / or markers described herein (e.g., specifically binds to the T cell costimulatory molecules described herein) can be an antibody reagent that comprises six CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, the antibody reagent that is specific for the targets and / or markers described herein (e.g., specifically binds to the T cell costimulatory molecules described herein) can be an antibody reagent that comprises three heavy chain CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., specifically binds to a T cell costimulatory molecule described herein) can be an antibody reagent comprising the three light chain CDRs of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., specifically binds to a T cell costimulatory molecule described herein) can be an antibody reagent comprising the VH and / or VL domains of any one of the antibodies described herein or known in the art. In some embodiments of any aspect, an antibody reagent specific for a target and / or marker described herein (e.g., specifically binds to a T cell costimulatory molecule described herein) can be an antibody reagent comprising the VH and VL domains of any one of the antibodies described herein or known in the art.Such antibody reagents are specifically contemplated for use in the methods and / or compositions described herein.

[0047] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule that contain the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. For the methods and compositions described herein, the amino acid positions assigned to the CDRs and FRs may be defined according to Rabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). The amino acid numbering of the antibody or antigen-binding fragment is also according to Rabat.

[0048] The terms "antigen-binding fragment" or "antigen-binding domain"; these are used interchangeably herein to refer to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest (e.g., specifically bind to a T cell costimulatory molecule described herein). Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fv fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH or VL domain (Ward et al., (1989) Nature 341:544-546; which is incorporated herein by reference in its entirety); and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. The sequences from the light and heavy chains can be provided in N-terminal to C-terminal order, respectively, or in reverse order.

[0049] Costimulatory molecules are a group of cell surface molecules that amplify or counteract the initial activation signal provided by the T cell receptor (TCR) to T cells after interaction with antigen / major histocompatibility complex (MHC). OX40L is an important costimulatory molecule, which is inducibly expressed on professional antigen-presenting cells (APCs) such as B cells, dendritic cells (DCs) and macrophages under inflammatory conditions. OX40L and its binding partner OX40 regulate cytokine production from T cells, antigen-presenting cells, NK cells, and NKT cells. Non-limiting examples of other T cell costimulatory molecules that can be targeted include CD28, CD80, CTLA-4, PDL-1, CD86, ICOS, ICOS-L, PDL-2, CD27, CD70, CD30, CD30L, CD40L, CD40, GITR, GITRL, TIM-1, TIM-2, TIM-3, and TIM-4.

[0050] In some embodiments of either aspect, the extracellular recognition moiety comprises an anti-OX40L antibody, antibody reagent or its antigen-binding portion; consists of an anti-OX40L antibody, antibody reagent or its antigen-binding portion; or consists essentially of an anti-OX40L antibody, antibody reagent or its antigen-binding portion.OX40L, also known as tumor necrosis factor ligand superfamily member 4, is a gene that encodes a cytokine of the tumor necrosis factor ligand family that mediates the adhesion of activated T cells to endothelial cells.The sequence of OX40L is known in the art for several species, including humans.See, for example, the NCBI database entry of Gene ID No: 7292.

[0051] Antibodies and antibody reagents that specifically bind to OX40L are known in the art and are commercially available. For example, the following anti-OX40L antibodies are commercially available: Ab263910 [EPR23155-317] from Abcam; Ab76130 [EP1168Y] from Abcam; LSBio's LS-B10561 [362CT19.3.3]; BioLegend's AB_2207272 / cat. no. 326307 [11C3.1]; MAD10541 [159403] from RnD Systems; MA5-37543 [362CT19.3.3] from Invitrogen; M02554 [OTI5D8] from BoosterBio; sc-71768 [2Q1716] from Santa Cruz Biotechnology; and Origene's AM01048PU-S [7D6].

[0052] In some embodiments, described herein is a CAR having an extracellular recognition portion that comprises an OX40L antibody reagent comprising a light chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 1-3 or a conservative substitution variant of such a nucleic acid sequence; consists of an OX40L antibody reagent comprising a light chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 1-3 or a conservative substitution variant of such a nucleic acid sequence; or consists essentially of an OX40L antibody reagent comprising a light chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 1-3 or a conservative substitution variant of such a nucleic acid sequence. In some embodiments, described herein is a CAR having an extracellular recognition portion that comprises an OX40L antibody reagent comprising a heavy chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 4-6 or a conservative substitution variant of such a nucleic acid sequence; consists of an OX40L antibody reagent comprising a heavy chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 4-6 or a conservative substitution variant of such a nucleic acid sequence; or consists essentially of an OX40L antibody reagent comprising a heavy chain CDR encoded by a nucleic acid sequence of SEQ ID Nos. 4-6 or a conservative substitution variant of such a nucleic acid sequence.

[0053] In some embodiments of any aspect, the extracellular recognition domain of a CAR described herein comprises a light chain CDR encoded by a nucleic acid sequence presented in SEQ ID NOs: 1-3 and a heavy chain CDR encoded by a nucleic acid sequence of SEQ ID NOs: 4-6, or those encoded by a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences of SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6, which maintain the same function. In some embodiments of any aspect, the CAR comprises at least one heavy or light chain complementarity determining region (CDR) encoded by a nucleic acid selected from the group consisting of: (a) a light chain CDR1 having the nucleic acid sequence of SEQ ID NO: 1; (b) a light chain CDR2 having the nucleic acid sequence of SEQ ID NO: 2; (c) a light chain CDR3 having the nucleic acid sequence of SEQ ID NO: 3; (d) a heavy chain CDR1 having the nucleic acid sequence of SEQ ID NO: 4; (e) a heavy chain CDR2 having the nucleic acid sequence of SEQ ID NO: 5; and (f) a heavy chain CDR3 having the nucleic acid sequence of SEQ ID NO: 6.

[0054] SEQ ID NO 1: is the nucleic acid sequence encoding the light chain CDR1. TIFF2025507727000002.tif4145

[0055] SEQ ID NO 2: the nucleic acid sequence encoding the light chain CDR2. TIFF2025507727000003.tif4145

[0056] SEQ ID NO 3: the nucleic acid sequence encoding the light chain CDR3. TIFF2025507727000004.tif4145

[0057] SEQ ID NO 4: is the nucleic acid sequence encoding heavy chain CDR1. TIFF2025507727000005.tif4145

[0058] SEQ ID NO 5: is the nucleic acid sequence encoding the heavy chain CDR2. TIFF2025507727000006.tif4145

[0059] SEQ ID NO 6: is the nucleic acid sequence encoding the heavy chain CDR3. TIFF2025507727000007.tif11145

[0060] In some embodiments, described herein are CARs with extracellular recognition moieties that comprise or consist essentially of an anti-OX40L antibody reagent comprising a light chain CDR of SEQ ID Nos. 7-9 or a conservative substitution variant of such sequences; an anti-OX40L antibody reagent comprising a light chain CDR of SEQ ID Nos. 7-9 or a conservative substitution variant of such sequences; or an anti-OX40L antibody reagent comprising a light chain CDR of SEQ ID Nos. 7-9 or a conservative substitution variant of such sequences. In some embodiments, described herein are CARs with extracellular recognition moieties that comprise or consist essentially of an anti-OX40L antibody reagent comprising a heavy chain CDR of SEQ ID Nos. 10-12 or a conservative substitution variant of such nucleic acid sequences; an anti-OX40L antibody reagent comprising a heavy chain CDR of SEQ ID Nos. 10-12 or a conservative substitution variant of such nucleic acid sequences; or an anti-OX40L antibody reagent comprising a heavy chain CDR of SEQ ID Nos. 10-12 or a conservative substitution variant of such nucleic acid sequences.

[0061] In some embodiments of any aspect, the extracellular recognition portion of a CAR described herein comprises a light chain CDR having an amino acid sequence as set forth in SEQ ID NOs: 7-9 and a heavy chain CDR having an amino acid sequence as set forth in SEQ ID NOs: 10-12, or an amino acid sequence that maintains the same function and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences of SEQ ID NOs: 7-9 and SEQ ID NOs: 10-12. In some embodiments of any aspect, the CAR comprises at least one heavy or light chain complementarity determining region (CDR) selected from the group consisting of: (g) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 7; (h) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 8; (i) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 9; (j) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 10; (k) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11; and (l) A heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0062] SEQ ID NO 7: Amino acid sequence of light chain CDR1. RASQSISSYLN 11

[0063] SEQ ID NO 8: Amino acid sequence of light chain CDR2. AASSLQS 7

[0064] SEQ ID NO 9: Amino acid sequence of the light chain CDR3. QQSHSVSFT 9

[0065] SEQ ID NO 10: the amino acid sequence of heavy chain CDR1. GFTFSNY 7

[0066] SEQ ID NO 11: The amino acid sequence of heavy chain CDR2. SGSGGA6

[0067] SEQ ID NO 12: The amino acid sequence of the heavy chain CDR3. TIFF2025507727000008.tif3128

[0068] The VH and VL domains of an antibody can be linked by a peptide linker to form a VH / VL single-chain antigen-binding domain (e.g., as an scFv). As used herein, the term "linker" refers to a chemical or peptide structure that covalently links two polypeptide moieties. The length of the linker can be varied to alter the ability of the linked domains to form, for example, intramolecular or intermolecular dimers. For example, diabodies contain a short linker peptide, usually 5 amino acids, between the VH and VL domains that does not allow the VH and VL domains to pair to form an antigen-binding domain; upon expression of two different VH-VL constructs with this short linker arrangement in one cell, the VH domain of the first VH-VL polypeptide chain is allowed to dimerize with the VL domain of the second VH-VL polypeptide chain and the corresponding VL domain of the first VH-VL polypeptide chain is allowed to dimerize with the VH domain of the second VH-VL polypeptide chain, thereby creating a bispecific construct. In contrast, when the VH and VL domains are separated by a longer peptide linker, most often 15-20 amino acids, the VH and VL domains on the same polypeptide chain can dimerize to form scFvs.

[0069] In some embodiments of any aspect, the CAR described herein comprises a linker domain between the VL and VH domains of the extracellular recognition moiety. In some embodiments of either aspect, the linker domain comprises a polypeptide encoded by SEQ ID NO:13, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:13; consists of a polypeptide encoded by SEQ ID NO:13, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:13; or consists of a polypeptide encoded by SEQ ID NO:13, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:13. The polypeptide may essentially consist of a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a polypeptide encoded by SEQ ID NO:13.

[0070] In some embodiments of either aspect, the linker domain is encoded by a nucleic acid sequence set forth in SEQ ID NO:13, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:13.

[0071] SEQ ID NO: 13 is a nucleic acid sequence encoding the CAR linker domain. TIFF2025507727000009.tif11145

[0072] In addition to scFv, non-antibody-based approaches have also been used to direct CAR specificity, usually utilizing ligand functional domains. As used herein, a "ligand functional domain" is a portion of a molecule that binds to another molecule. In some embodiments of any aspect, the polypeptides described herein can include cytokines, natural immune receptors, TNF receptors, growth factors, and structural proteins, all of which have been successfully used as CAR extracellular recognition moieties. In some embodiments of any aspect, the natural ligand functional domains that can direct CAR specificity in the polypeptides described herein include NKp30, NKG2D, DNAM-1, CD27, CD16, GM-CSF, adnectins, IL-13, IL-11, FSH, and T1E.

[0073] In some embodiments of either aspect, the CAR described herein further comprises a leader sequence on the N-terminus of the extracellular recognition moiety. As used herein, the term "leader sequence" refers to an amino-terminal sequence that includes or consists of a signal peptide. Signal peptides typically consist of 13-36 somewhat hydrophobic amino acids. Signal peptides have a common structure: a short, positively charged amino-terminal region (n-region); a central hydrophobic region (h-region); and a more polar carboxy-terminal region (c-region) that contains the site of cleavage by signal peptidase. At the ER lumen side, the signal peptide is cleaved off by signal peptidase. After successful folding of the nascent polypeptide by ER-resident chaperones and foldases, the protein is further directed to exit the ER. This process may be supported by the presence of an N-terminal pro-sequence.

[0074] In some embodiments of either aspect, the leader sequence encodes a CD8a signal peptide. In some embodiments, the leader sequence of a CAR described herein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 14, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14 that maintains the same function, or a codon-optimized version of SEQ ID NO: 14. In some embodiments of either aspect, the leader sequence comprises a polypeptide encoded by SEQ ID NO:14, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:14; consists of a polypeptide encoded by SEQ ID NO:14, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:14; or consists of a polypeptide encoded by SEQ ID NO:14, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:14. The polypeptide may essentially consist of a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a polypeptide encoded by SEQ ID NO:14.

[0075] SEQ ID NO: 14 is a nucleic acid sequence encoding the CAR leader sequence. TIFF2025507727000010.tif11145

[0076] The CAR polypeptide described herein comprises a transmembrane portion. The transmembrane portion is a structural component, typically comprising a hydrophobic alpha-helix that spans the cell membrane. It anchors the CAR to the cell membrane, bridging the extracellular hinge and / or recognition portion with the intracellular signaling portion. This domain promotes the stability of the receptor as a whole. Transmembrane structures and sequences are well known in the art, for example, for any transmembrane protein annotated in the PDB or NCBI database. With respect to the transmembrane portion, the CAR can be designed to include a transmembrane portion that is fused to the extracellular recognition portion of the CAR. In some embodiments of either aspect, a transmembrane portion that is naturally associated with one of the other portions in the CAR is used. In some cases, the transmembrane portion can be modified or selected by amino acid substitution to avoid binding of such a portion to the transmembrane portion of the same or different surface membrane protein, to minimize interaction with other members of the receptor complex.

[0077] In some embodiments of either aspect, the transmembrane portion can be derived from either a natural or synthetic source. If the source is natural, the transmembrane portion can be or be derived from the transmembrane portion of any membrane-bound or transmembrane protein. Particularly useful transmembrane portions for the CAR polypeptides described herein include (i.e., at least the transmembrane regions of) the α, β or ζ chain of the T cell receptor, CD28, CD23ζ, CD28, 4-1BB, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane portion can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine will be found at both ends of the synthetic transmembrane portion. Optionally, a short oligopeptide or polypeptide linker, preferably 2 to 10 amino acids in length, can form the link between the transmembrane portion of the CAR and the intracellular signaling portion. A glycine-serine doublet provides a particularly suitable linker. In some embodiments of either aspect, the transmembrane portion comprises the transmembrane portion of CD28. In some embodiments of either aspect, the transmembrane domain comprises the transmembrane portion of CD8.

[0078] The CAR may further comprise a hinge region between the extracellular recognition moiety and the transmembrane moiety. The term "hinge region" as used herein generally refers to any oligopeptide or polypeptide that functions to link the transmembrane moiety to the extracellular recognition moiety. In particular, the hinge region is used to provide greater flexibility and accessibility to the extracellular recognition moiety. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4 or CD28, or all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.

[0079] In some embodiments of any aspect, the hinge domain and transmembrane portion of a CAR described herein comprises a nucleic acid sequence as presented in SEQ ID NO: 15, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 that maintains the same function, or a polypeptide sequence encoded by a codon-optimized version of SEQ ID NO: 15; or consists essentially of a nucleic acid sequence as set forth in SEQ ID NO:15, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:15, or a polypeptide sequence encoded by a codon-optimized version of SEQ ID NO:15, which maintains the same function.

[0080] SEQ ID NO: 15 is a nucleic acid sequence encoding the CAR hinge and transmembrane portion. TIFF2025507727000011.tif48151

[0081] In some embodiments of any aspect, the CAR described herein comprises an intracellular signaling portion. As used herein, "intracellular signaling portion" refers to a polypeptide sequence that comprises one or more TCR signaling domains and is displayed in the intracellular space of the cell membrane when the CAR is expressed in a cell. After an antigen binds to the extracellular recognition portion, the CAR receptors cluster together to transmit an activation signal, and the intracellular signaling portion of the receptor sustains the signaling in the T cell. Regular T cell activation by TCR signaling relies on phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the intracellular domain of CD3-zeta. To mimic this process, the intracellular domain of CD3-zeta is commonly used as part of the intracellular signaling portion of the CAR described herein. In some embodiments of any aspect, any ITAM-containing domain can be used in the intracellular signaling portion of the CAR polypeptide described herein. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the CAR polypeptides described herein include those derived from TCRζ (also referred to as ζ chain, CD3ζ, or CD247), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. The cytoplasmic signaling molecule in the CAR polypeptide can include CD3ζ or a cytoplasmic signaling sequence derived therefrom.

[0082] In addition to CD3 signaling, T cell persistence after activation is promoted by costimulatory molecules. Thus, the intercellular signaling portion of the CAR described herein can further include one or more co-signaling domains from costimulatory proteins. The engagement of these co-signaling domains improves T cell proliferation, cytokine secretion, resistance to apoptosis, and persistence in vivo. In some embodiments of either aspect, co-signaling domains from a wide variety of costimulatory molecules can be used in the polypeptides described herein, including CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like. The costimulatory domains of various TCR complex proteins are known in the art, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as annotated in the NCBI database entries. In some aspects, described herein are CAR polypeptides that include at least one intracellular signaling moiety.

[0083] In some embodiments of either aspect, the intracellular portion comprises a signaling domain of CD3-zeta and a signaling domain of CD28. In some embodiments of either aspect, the intracellular portion comprises a signaling domain of CD3-zeta and a signaling domain of 4-1BB. In some embodiments of either aspect, the intracellular domain comprises one or more co-signaling domains from a CD28 domain, a 4-1BB domain, an IL2Rα or IL2Rβ JAK3 / STAT3 complex docking site, and a TGFβ-R SMAD2 / 3 docking site.

[0084] In one embodiment of either aspect, the intracellular signaling moiety is a signaling domain from a protein selected from the group consisting of: TCRC; FcRy; FcRp; CD3zeta; CD3y; CD35; CD3s; CD3C; CD22; CD79a; CD79b; CD66d; CARD11; CD2; CD7; CD27; CD28; CD30; CD40; CD54 (ICAM); CD83; CD134 (OX40); CD137 (4-1BB); CD150 (SLAMF1); CD152 (CTLA4); CD223 (LAG3); CD270 (HVEM); CD273 (PD-L2); CD274 (PD-Ll); CD278 (ICOS); DAP10; LAT; KD2C SLP76; TRIM; ZAP70; and 41BB.

[0085] In some embodiments of any aspect, the CAR polypeptide described herein comprises the intracellular signaling domains of CD28 and CD3-zeta encoded by the nucleic acid sequence presented as SEQ ID NO: 16, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16 that maintains the same function, or a codon-optimized version of SEQ ID NO: 16.

[0086] SEQ ID NO: 16 is a nucleic acid sequence encoding the CAR intracellular signaling portion of CD28 and CD3-zeta. TIFF2025507727000012.tif134155

[0087] In some embodiments of any aspect, the CAR polypeptide described herein comprises the intracellular signaling portions of 4-1BB and CD3-zeta encoded by a nucleic acid sequence comprising SEQ ID NO: 17, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 that maintains the same function, or a codon-optimized version of SEQ ID NO: 17.

[0088] SEQ ID NO: 17 is a nucleic acid sequence encoding the CAR intracellular signaling portion of 4-1BB and CD3-zeta. TIFF2025507727000013.tif113155

[0089] In some aspects, described herein is a CAR polypeptide that comprises at least one detectable marker. As used herein, the term "detectable marker" refers to a moiety that, when attached to a CAR polypeptide, confers detectability to the polypeptide or another molecule to which the polypeptide binds. In some embodiments of either aspect, the CAR polypeptide comprises one detectable marker and / or detectable polypeptide. In some embodiments of either aspect, the CAR polypeptide comprises a detectable marker or detectable polypeptide at the C-terminus of the intracellular signaling portion.

[0090] In some embodiments of either aspect, fluorescent moieties can be used as detectable markers, but detectable markers also include, for example, isotopes, fluorescent proteins and peptides, enzymes, components of specific binding pairs, chromophores, affinity tags as defined herein, antibodies, colloidal metals (i.e., gold) and quantum dots.Detectable markers can be directly or indirectly detectable.Directly detectable markers do not require additional reagents or substrates to generate detectable signals.Examples include isotopes and fluorophores.Indirectly detectable markers require the presence or action of one or more cofactors or substrates. Examples include enzymes such as β-galactosidase, detectable by the production of a colored reaction product upon cleavage of a substrate such as the chromogen X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), horseradish peroxidase, detectable by the production of a colored reaction product in the presence of the substrate diaminobenzidine, alkaline phosphatase, detectable by the production of a colored reaction product in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, and affinity tags. Non-limiting examples of affinity tags include Strep tags, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), FLAG tags, HA tags, Myc tags, poly(His) tags, and derivatives thereof. In some embodiments of either aspect, the detectable marker is selected from GFP, V5, HA1, Myc, VSV-G, HSV, FLAG, HIS, mCherry, AU1, and biotin.

[0091] In some embodiments of any aspect, the detectable marker can be located anywhere in the CAR polypeptide described herein. In some embodiments of any aspect, the detectable marker is located between any portion of the CAR polypeptide described herein, but is not found within a functional domain or portion or does not disrupt the function of the domain or portion. In some embodiments of any aspect, the detectable marker is located at the C-terminus of the polypeptide. Such a marker can be used to detect intracellular expression of the CAR polypeptide. In some embodiments of any aspect, the detectable marker located at the C-terminus of the polypeptide comprises neon green or another marker described herein.

[0092] In some embodiments of any aspect, a detectable marker of a CAR polypeptide described herein is encoded by a nucleic acid sequence comprising SEQ ID NO: 18, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 18 that maintains the same function, or a codon-optimized version of one of SEQ ID NOs: 18.

[0093] SEQ ID NO: 18 is a nucleic acid sequence encoding the CAR neon green detectable marker. TIFF2025507727000014.tif157155

[0094] In some embodiments of any aspect, the CAR polypeptides described herein, particularly those administered to a subject or that are part of a pharmaceutical composition, do not contain detectable markers that are immunogenic. In some embodiments of any aspect, the CAR polypeptides described herein do not contain GFP, mCherry, HA1, or any other immunogenic markers.

[0095] In some embodiments of either aspect, the CAR further comprises a cleavage site between the intracellular signaling moiety and the detectable marker. In some embodiments of either aspect, the cleavage site is a short amino acid sequence (e.g., a peptide of approximately 18-22 aa in length) that can catalyze its own cleavage. In some embodiments of either aspect, the cleavage site belongs to the 2A peptide family. Non-limiting examples of 2A peptides include P2A, E2A, F2A, and T2A. F2A is derived from foot and mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; and T2A is derived from thosea asigna virus 2A. The cleavage sites of these 2A peptides are known in the art. In some embodiments of either aspect, the N-terminus of the 2A peptide comprises the sequence "GSG" (Gly-Ser-Gly). In some embodiments of either aspect, the N-terminus of the 2A peptide does not include the sequence "GSG" (Gly-Ser-Gly).

[0096] Cleavage via 2A peptides begins after protein translation. It is triggered by cleavage of the peptide bond between proline (P) and glycine (G) at the C-terminus of the 2A peptide. The molecular mechanism of 2A peptide-mediated cleavage does not involve true proteolytic cleavage but involves ribosomal "skipping" of the glycyl-prolyl peptide bond formation. Different 2A peptides exhibit different efficiencies of autocleavage, with P2A being the most efficient and F2A being the least efficient. Therefore, up to 50% of F2A-bound protein may remain in the cell as uncleaved protein.

[0097] In some embodiments of any aspect, the cleavage site of a CAR described herein is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 19, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 19 that maintains the same function, or a codon-optimized version of one of SEQ ID NOs: 19. In some embodiments of either aspect, the cleavage site comprises a polypeptide encoded by SEQ ID NO:2, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:2; consists of a polypeptide encoded by SEQ ID NO:2, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:2; or consists of a polypeptide encoded by SEQ ID NO:2, or a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polypeptide encoded by SEQ ID NO:2. The polypeptide may essentially consist of a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a polypeptide encoded by HLA-1, HLA-2, HLA-3, HLA-4, HLA-5, HLA-6, HLA-7, HLA-8, HLA-9, HLA-10, HLA-11, HLA-12, HLA-13, HLA-14, HLA-15, HLA-16, HLA-17, HLA-18, HLA-19, HLA-20, HLA-21, HLA-22, HLA-23, HLA-24, HLA-25, HLA-26, HLA-27, HLA-31, HLA-32, HLA-33, HLA-34, HLA-35, HLA-36, HLA-37, HLA-38, HLA-39, HLA-40, HLA-41, HLA-42, HLA-43, HLA-44, HLA-45, HLA-46, HLA-47, HLA-48, HLA-49, HLA-49, HLA-49, HLA-41, HLA-42, HLA-43, HLA-44, HLA-45, HLA-45, HLA-46, HLA-47, HLA-48 ...

[0098] SEQ ID NO: 19 is a nucleic acid sequence encoding the CAR cleavage site. TIFF2025507727000015.tif11145

[0099] In some embodiments of any aspect, the T cell or T cell population described herein comprises, consists of, or consists essentially of regulatory T cells (Treg). As used herein, "regulatory T cells" or "Treg" refers to T cells (lymphocytes) that have immunoregulatory properties and the ability to suppress the proliferation and / or effector function of other T cell populations. As described herein below, several cell surface molecules are used to characterize and define Treg cells. Regulatory T cells or Treg cells have been shown to play an important role in maintaining immune tolerance by suppressing the action of autoreactive effector cells, and are critically involved in preventing the onset of autoimmune reactions.

[0100] In some embodiments of any aspect, the Treg cells can be T cells expressing one or more markers selected from the group consisting of CTLA4; PDL1; FOXP3; LAP; GARP; CD25; CD4; and CD27. In some embodiments of any aspect, the Treg cells can be T cells expressing detectable levels of one or more markers selected from the group consisting of CTLA4; PDL1; FOXP3; LAP; GARP; CD25; CD4; and CD27. In some embodiments of any aspect, the Treg cells can be T cells positive for one or more markers selected from the group consisting of CTLA4; PDL1; FOXP3; LAP; GARP; CD25; CD4; and CD27. In some embodiments of any aspect, the Treg cells can be CD8- (e.g., NCBI gene ID: 925) CD4+ (e.g., NCBI gene ID: 920) CD3+ cells. In some embodiments of any aspect, the Treg cells express Foxp3. In some embodiments of either aspect, the Treg cells express CD4 and CD25.

[0101] Where "X" is a cell surface marker, "X+" indicates that the marker is present on the indicated cell, while "X-" indicates that the marker is absent. Those skilled in the art will be able to use standard techniques to assess the molecules present on the cells, for example, using immunofluorescence to detect commercially available antibodies bound to the marker molecules. Such identifiers are often used when sorting or identifying cells by FACS, where gates can be established to divide cells based on the expression level of the marker.

[0102] In one embodiment, the method for preparing the engineered cells described herein includes obtaining a cell population and enriching CD25+ T regulatory cells, for example, by using an antibody specific for CD25. Methods for enriching CD25+ T regulatory cells from a cell population are clear to those skilled in the art and can include the use of methods such as FACS or affinity purification. In some embodiments, the population of Treg enriched cells contains less than 30%, 20%, 10%, 5% or less of non-Treg cells. In some embodiments, a vector encoding a CAR described herein is transfected into the Treg enriched cells. Treg cells expressing a CAR, for example, Treg enriched cells, can be used to induce tolerance to an antigen targeted or specifically bound by the CAR.

[0103] In CAR-T cell therapy, T cells engineered with CARs are used for treatment. The premise of CAR-T immunotherapy is to modify T cells to recognize specific molecules or cells and more effectively target, block, or destroy them. After T cells are harvested and genetically modified, the resulting CAR-T cells are infused into patients to treat them. CAR-T cells may be derived from T cells in the patient's own blood (autologous) or from the T cells of another healthy donor (allogeneic). Once isolated, these T cells are genetically engineered to express a specific CAR and programmed to target an antigen.

[0104] Further discussion of CARs can be found, for example, in Maus et al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 2012 97:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 2012 18:6436-6445, each of which is incorporated by reference in its entirety.

[0105] In one aspect of any of the embodiments, described herein is a nucleic acid molecule encoding a CAR described herein. In one aspect of any of the embodiments, described herein is a vector, e.g., an expression vector, that includes a nucleic acid molecule encoding a CAR described herein.

[0106] In some embodiments of any embodiment, the vector described herein is an expression vector or a plasmid, for example, a recombinant vector or a plasmid.As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a transgene into a host cell.The term "vector" includes plasmid, minichromosome, phage, naked DNA, and the like.See, for example, U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and 5,919,670, and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).One type of vector is "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, in which an additional DNA segment is ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, since plasmids are the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0107] A cloning vector is one that can autonomously replicate or integrate into a genome in a host cell; and is further characterized by one or more endonuclease restriction sites into which a desired DNA sequence can be ligated, so that the vector can be cut in a determinable manner and the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, the replication of the desired sequence can occur many times as the plasmid increases in copy number in a host cell, such as a host bacterium, or it can occur only once per host before the host replicates by mitosis. In the case of a phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0108] An expression vector is one into which a desired DNA sequence is inserted by restriction and ligation so that it can be operably linked to regulatory sequences and expressed as an RNA transcript. A vector can further include one or more marker sequences suitable for use in identifying cells that have been transformed or transfected with the vector or cells that have not been transformed or transfected with the vector. Markers include, for example, genes that code for proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that code for enzymes whose activity is detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably connected.

[0109] As used herein, a coding sequence and a regulatory sequence are said to be "functionally" connected when they are covalently linked such that the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. If it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be functionally connected when induction of a promoter in the 5'regulatory sequence results in transcription of the coding sequence, and when the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to induce transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is said to be functionally connected to a coding sequence if it can effect transcription of that DNA sequence such that the resulting transcript can be translated into a desired protein or polypeptide.

[0110] When the nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, various transcription control sequences (e.g., promoter / enhancer sequences) can be used to induce its expression. The promoter can be the native promoter, i.e., the promoter of the gene in endogenous relationship, which provides the normal regulation of the expression of the gene. In some embodiments, the promoter can be constitutive, i.e., the promoter is not regulated and the gene linked to it is continuously transcribed. Various conditional promoters can also be used, such as promoters that are controlled by the presence or absence of a molecule.

[0111] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally may include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, as necessary, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region, including a promoter sequence for transcriptional control of an operably connected gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. Vectors of the invention may optionally include 5' leader or signal sequences. The selection and design of appropriate vectors is within the ability and discretion of one of ordinary skill in the art.

[0112] In some embodiments of either aspect, the promoter is a eukaryotic or human constitutive promoter, including but not limited to, FoxP3 promoter, Fox P3 and IKZF2 / Helios hybrid promoter, MND promoter, etc. In some embodiments of either aspect, the vector comprises the human FoxP3 promoter (e.g., SEQ ID NO: 20), a constitutive promoter of human origin that can be used to drive gene expression in regulatory T cells in in vitro and in vivo settings.

[0113] In some embodiments of any aspect, the FoxP3 promoter of a CAR described herein is encoded by a nucleic acid sequence as set forth in SEQ ID NO:20, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 that maintains the same function, or a codon-optimized version of SEQ ID NO:20.

[0114] SEQ ID NO:20 is a nucleic acid sequence encoding the FoxP3 promoter. TIFF2025507727000016.tif112145TIFF2025507727000017.tif241137TIFF2025507727000018.tif40128

[0115] In some embodiments of either aspect, the promoter is a promoter provided in Accession No. MK012431, e.g., nucleotide numbers 4650-6041 and 7338-8213 of Accession No. MK012431, i.e., the version of the sequence available as of February 28, 2023.

[0116] Expression vectors that contain all the elements necessary for expression are commercially available and known to those skilled in the art.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.Cells are genetically engineered by introducing heterologous DNA (RNA) into the cell.The heterologous DNA (RNA) is placed under the operable control of transcriptional elements to allow expression of the heterologous DNA in the host cell.

[0117] In some embodiments, the vector comprises a selectable marker, for example, for selectively amplifying the vector in bacteria. Non-limiting examples of selectable marker genes for use in bacteria include antibiotic resistance genes that confer resistance to ampicillin, tetracycline, and kanamycin. Tetracycline (tet) and ampicillin (amp) resistance marker genes can be obtained from any of several commercially available vectors, including pBR322 (available from New England BioLabs, Beverly, Mass., catalog number 303-3s). The tet coding sequence is contained within nucleotide numbers 86-476; the amp gene is contained within nucleotide numbers 3295-4155. The nucleotide sequence of the kanamycin (kan) gene is available from New England BioLabs vector pACYC 177, catalog number 401-L, GenBank accession number X06402.

[0118] In some embodiments of any aspect, one or more of the nucleic acids encoding a CAR described herein can be integrated into the genome of the cell.

[0119] In some embodiments of either aspect, immune cells (e.g., Tregs) comprising a CAR polypeptide can be used to treat inflammatory conditions or autoimmune diseases. In some embodiments, immune cells (e.g., T cells) comprising a CAR polypeptide directed against an autoimmune disease-specific antigen can be used to treat autoimmune diseases. "Autoimmune disease" refers to a group of diseases in which a subject's own antibodies react with host tissues, or in which immune effector T cells react with endogenous self-peptides, causing tissue destruction. Thus, an immune response is initiated against a subject's own antigens, referred to as autoantigens. As used herein, "autoantigens" refers to antigens of normal host tissues. Normal host tissues do not contain neoplastic cells.

[0120] As used herein, an autoantigen is an endogenous protein or a fragment thereof that triggers this pathogenic immune response. An autoantigen can be any substance or part thereof that is normally found in a mammal that is the first (or primary) target of attack by the immune system in an autoimmune disease. This term also includes antigenic substances that induce a condition with the characteristics of an autoimmune disease when administered to a mammal. In addition, this term includes a peptic subclass that essentially consists of an immunodominant epitope or immunodominant epitope region of an autoantigen. An immunodominant epitope or region in an induced autoimmune condition is a fragment of an autoantigen that can be used instead of the entire autoantigen to induce disease. In humans with autoimmune disease, an immunodominant epitope or region is a fragment of an antigen that is specific to the tissue or organ that is under autoimmune attack and is recognized by a significant proportion (e.g., the majority, but not necessarily the absolute majority) of autoimmune attacking T cells.

[0121] Autoantigens known to be associated with autoimmune diseases include myelin proteins associated with demyelinating diseases such as multiple sclerosis and experimental autoimmune myelitis; collagen, and rheumatoid arthritis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65); islet cell antigens (ICA512; ICA12) associated with insulin-dependent diabetes mellitus.

[0122] A common feature in several autoimmune-related diseases and inflammatory conditions is the involvement of proinflammatory CD4+ T cells. These T cells are responsible for the release of inflammatory Th1-type cytokines. Cytokines characterized as Th1-type include interleukin 2 (IL-2), gamma-interferon, TNFα, and IL-12. Such proinflammatory cytokines act to stimulate immune responses, often resulting in the destruction of self-tissues. Cytokines associated with suppressing T cell responses are Th2-type and include IL-10, IL-4, and TGF-β. It has been shown that Th1-type and Th2-type T cells may use the same antigen receptor in response to immunogens, with the former generating stimulatory responses and the latter generating inhibitory responses.

[0123] Described herein are methods of treating an autoimmune disease, comprising administering an effective amount of a CAR or CAR-T composition to a patient in need thereof. In some embodiments of any one of the described methods, the autoimmune disorder is thyroiditis, type 1 diabetes, Hashimoto's thyroiditis, Graves' disease, celiac disease, multiple sclerosis, Guillain-Barré syndrome, Addison's disease, and Raynaud's phenomenon, Goodpasture's disease, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, osteoarthritis, collagen II-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile onset rheumatoid arthritis, progressive chronic arthritis (arthritis chronica progrediente), osteoarthritis, primary chronic polyarthritis (polyarthritis chronica). primaria), reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis, atopy, e.g., atopic diseases, e.g., hay fever and Job's syndrome, dermatitis, e.g., contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria, e.g., chronic allergic urticaria and chronic idiopathic urticaria, e.g., chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic sclerosis), sclerosis, e.g. systemic sclerosis, multiple sclerosis (MS), e.g. spino-ophthalmic MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g. Crohn's disease, autoimmune-mediated gastrointestinal diseases), colitis, e.g. ulcerative colitis, colonic ulcer, microscopic colitis, collagenous colitis, polypoid colitispolyposa), necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes, such as adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti), autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmadsen encephalitis and limbic and / or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, such as chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), Idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN), including types I and II, and rapidly progressive GN, proliferative nephritis, autoimmune polyendocrine deficiency, balanitis, e.g., plasma cell balanitis, balanoposthitis, erythema annulare centrifugally, erythema dyschromicus perstans, erythema multiforme, granuloma annulare, lichen sclerosus atrophicus, lichen simplex chronicus, lichen spinous, lichen planus, ichthyosis phyllodes, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema, e.g., allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma, e.g., asthma bronchitis,bronchiale), bronchial asthma and autoimmune asthma, conditions with infiltration of T cells and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood groups during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, e.g., lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus, discoid lupus erythematosus, alopecia, systemic lupus erythematosus (SLE), e.g., cutaneous SLE, or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus erythematosus, juvenile-onset (type I) diabetes mellitus, e.g., childhood insulin-dependent diabetes mellitus (IDDM), and adult-onset diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic aortopathy, mediated by cytokines and T lymphocytes. immune responses associated with acute and delayed hypersensitivity due to sarcoidosis, granulomatosis, e.g., lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytopenia, vasculitis, e.g., vasculitis, large vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis CNS vasculitis, dermatomyositis, Cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, e.g., systemic necrotizing vasculitis, and ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vasculitis, temporal arteritis, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia, or immune-mediated hemolytic anemia, e.g., autoimmune hemolytic anemia (AIHA), pernicious anemia (pernicious anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, immune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, CNS inflammatory disorders, multiple organ injury syndromes e.g. secondary to sepsis, trauma or hemorrhage, antigen-antibody complex mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid e.g. bullous pemphigoid and cutaneous pemphigoid, pemphigus (pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous membrane pemphigoid)pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex disorders, e.g. immune complex nephritis, antibody mediated nephritis, polyneuropathy, chronic neuropathy, e.g. IgM polyneuropathy or IgM mediated neuropathy, and autoimmune or immune mediated thrombocytopenia, e.g. idiopathic thrombocytopenic purpura (ITP), e.g. chronic or acute ITP, scleritis, e.g. idiopathic keratoscleritis, episcleritis, autoimmune diseases of the testes and ovaries, e.g. autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine disorders, e.g. thyroiditis, e.g. autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, idiopathic hypothyroidism, polyglandular syndromes, e.g. autoimmune thyroiditis, Autoimmune polyglandular syndromes (or polyendocrinopathy syndromes), paraneoplastic syndromes, e.g. neurological paraneoplastic syndromes, e.g. Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis, e.g. allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis, e.g. thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, lupoid hepatitis, giant cell hepatitis, autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-graft) vs.NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatitis, transient acantholytic dermatosis, cirrhosis, e.g., primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune enteropathy, Infectious ear diseases, e.g. autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, e.g. refractory or relapsing or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-associated pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis, e.g. monoclonal B-cell lymphocytosis (e.g. benign monoclonal hypergammaglobulinemia and monoclonal gammopathy of undetermined significance)inflammatory myopathy, focal or segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune liver disorders, fibromyalgia, polyendocrine deficiency, Schmidt's syndrome, adrenalitis, gastrotrophy, presenile dementia, demyelinating diseases, e.g. autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia), male and female encephalomyelopathy, e.g. antisperm antibodies and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird breeder's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, alveolitis, e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, Sumpter's syndrome, Caplan's syndrome, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema tenuifolia, erythroblastosis fetalis, eosinophilic fasciitis, Charmant's syndrome, Felty's syndrome, cyclitis, e.g. chronic cyclitis, heterochronic cyclitis cyclitis, iridocyclitis (acute or chronic), or Fuchs' cyclitis, Henoch-Schönlein purpura, SCID, sepsis, endotoxemia, post-vaccination syndrome, Evans' syndrome, autoimmune dysgonadism, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgiapolymyalgia), chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, aphthous stomatitis, arteriosclerotic disorders, azoospermia, autoimmune hemolysis, Beck's disease, allergic enterocolitis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, regional ileitis, leukopenia, transverse myelitis, primary idiopathic myxedema, sympathetic ophthalmia, acute polyradiculitis, pyoderma gangrenosum, acquired splenic atrophy, vitiligo, toxic shock syndrome, conditions with T cell infiltration, leukocyte adhesion deficiency, acute and delayed hypersensitivity-related immune responses mediated by cytokines and T lymphocytes , diseases with leukocyte extravasation, multi-organ injury syndrome, antigen-antibody complex-mediated disease, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), myocarditis, nephrotic syndrome, primary sclerosing cholangitis, acute or chronic sinusitis, ethmoid, frontal, maxillary or sphenoid sinusitis, eosinophilic-related diseases, e.g. eosinophilia, pulmonary infiltrative eosinophilia, eosinophilia-myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, eosinophil-containing granulomas, seronegative The disease is selected from the group consisting of spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, Bratton syndrome, transient infantile hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, vascular ectasia, autoimmune disorders associated with collagen diseases, rheumatism, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, lymphomatous bronchitis, inflammatory skin diseases, skin diseases with an acute inflammatory component, and autoimmune uveoretinitis (AUR).

[0124] Described herein are methods for treating an autoimmune or inflammatory condition, comprising administering to a patient in need thereof an effective amount of a CAR or CAR-T composition. In some embodiments of any one of the described methods, the autoimmune or inflammatory condition is selected from the group consisting of allograft rejection, xenograft rejection, and graft-versus-host disease.

[0125] In some embodiments, the methods described herein relate to the treatment or prevention of transplant rejection or GVHD in a subject having a tissue transplant, organ transplant, or hematopoietic stem cell transplant (HCT) using one or more compositions, CARs, or cells described herein. In some embodiments of any aspect, the transplant rejection is allograft rejection or xenograft rejection. In another embodiment, the methods described herein relate to the treatment or prevention of graft-versus-host disease (GVHD) in a subject having a tissue transplant, organ transplant, or HCT using one or more compositions, CARs, or cells described herein.

[0126] As used herein, "GVHD" refers to a disease characterized by an active process of donor cells attacking the recipient's own cells. GVHD may develop soon after transplantation, e.g., within weeks or months (acute GVHD), or may develop much later after transplantation, e.g., at least 3-6 months (chronic GVHD). Symptoms of acute GVHD include, but are not limited to, skin rash or blisters, abdominal pain or discomfort, diarrhea, jaundice, and edema. Symptoms of chronic GVHD include, but are not limited to, changes in skin or nail texture, hair loss or thinning, muscle pain or weakness, blurred vision, mouth sores, shortness of breath, persistent cough, abdominal pain or discomfort, and diarrhea.

[0127] A subject can be identified as having or at risk of having GVHD by a skilled clinician. Diagnostic tests useful for identifying a subject with GVHD are known in the art and will vary based on the type of transplant the subject has received. Diagnosis of GVHD is made, for example, by physical examination for signs and symptoms of GVHD known in the art, serological tests for liver, gallbladder, kidney, and hematopoietic cell dysfunction, histological analysis of biopsies obtained from the affected organ, and radiological imaging of the affected organ. In one embodiment, the method further comprises administering at least a second therapeutic agent. In one embodiment, the composition, CAR or cells described herein are administered in combination with abatacept (Orencia®) or belatacept (Nulojix®). Abatacept and belatacept, developed by Bristol-Meyers Squibb, are fusion proteins composed of the Fc region of immunoglobulin IgG1 fused to the extracellular domain of CTLA-4. Abatacept is currently FDA-approved for the treatment of rheumatoid arthritis. Belatacept, which differs from abatacept by only two amino acids, is an immunosuppressant intended to prevent rejection after kidney transplants.

[0128] In some embodiments of any aspect, the transplant is vascularized composite tissue transplant (VCA).In another embodiment, the transplant is any type of transplantation procedure, for example, any heart transplant, any lung transplant, any liver transplant, any pancreas transplant, any cornea transplant, any trachea transplant, any kidney transplant, any skin transplant, or any autograft (for example, tissue transplant).In one embodiment of any aspect, the transplant is any allograft or xenograft.A skilled practitioner will be able to use standard procedure protocols to perform transplantation or identify the subject that has received the transplant.

[0129] The compositions and methods described herein can be administered to a subject to treat or prevent autoimmune disease, inflammatory conditions, or transplant rejection. In some embodiments, the methods described herein include administering to a subject an effective amount of the compositions, CARs, or cells described herein to alleviate symptoms of inflammatory conditions, autoimmune diseases, or transplant rejection. As used herein, "alleviating symptoms of inflammatory conditions, autoimmune diseases, or transplant rejection" refers to ameliorating any condition or symptom associated with inflammatory conditions, autoimmune diseases, or transplant rejection. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, as measured by any standard technique. A variety of means for administering the compositions described herein to a subject are known to those skilled in the art. Such methods can include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, dermal, topical, injection, or intratumoral administration. Administration can be local or systemic.

[0130] The administration of the compositions contemplated herein can be carried out by any convenient method, including aerosol inhalation, injection, ingestion, infusion, implantation or transplantation.In a preferred embodiment, the compositions are administered parenterally.The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal and intrazygomatic injection and infusion.In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into tumor, lymph node or site of infection.

[0131] A pharmaceutical composition comprising the cells described herein, e.g., T cells or CAR cells, can be administered in the form of 1 to 10 10 / kg body weight, preferably 10 cells 3 ~10 8 It can be stated that the cells can be administered in a dosage of 10 cells / kg body weight, including all integer values ​​within those ranges. The number of cells depends on the intended end use of the composition, as well as the cell type contained therein. For the uses described herein, the cells are generally in a volume of 1 liter or less, and can be 500 mL or less, 250 mL or even 100 mL or less. Thus, the desired cell density is typically about 10 cells / kg body weight. 6 cells / ml, typically 10 7 cells / ml, typically 10 8 A clinically relevant number of immune cells is estimated at 10 cells / ml or greater. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In some aspects of the invention, particularly since all fused cells are redirected to a specific target antigen, the number of fusion cells can be more than 10. 6 Pieces / kilogram (10 per patient) 6 ~10 11 Lower numbers of cells may be administered, ranging from about 1×10 cells to about 1×10 cells. The CAR-expressing cell composition may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing treatment. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance induction of an immune response. In some embodiments, the dosage is about 1×10 cells. 2 pieces~cells approx. 1×10 10 In some embodiments, the dosage is about 1×10 cells / kg body weight. 5pieces~cells approx. 1×10 8 In some embodiments, the dosage is about 1×10 cells / kg body weight. 6 pieces~cells approx. 1×10 7 In some embodiments, the dosage is about 1×10 cells / kg body weight. 6 The dose of cells can be administered in units per kg body weight. In some embodiments, a single dose of cells can be administered. In some embodiments, the dose of cells can be repeated, for example, once, twice or more. In some embodiments, the dose of cells can be administered, for example, on a daily, weekly or monthly basis.

[0132] The dosage range of the agents, e.g., CARs, cells or compositions described herein depends on potency and includes amounts large enough to produce the desired effect, e.g., prevention of transplant rejection, reduction of inflammation, etc. The dosage should not be so large as to cause unacceptable adverse side effects. In general, dosages vary according to the age, condition and sex of the patient and can be determined by one skilled in the art. Dosages can also be adjusted by the individual physician in the event of any complications. In some embodiments, dosages range from 0.001 mg / kg body weight to 0.5 mg / kg body weight. In some embodiments, the dosage range is from 5 μg / kg body weight to 100 μg / kg body weight. Alternatively, the dosage range can be titrated to maintain serum levels of 1 μg / mL to 1000 μg / mL. For systemic administration, a therapeutic amount, such as, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or more, can be administered to a subject.

[0133] The administration of the above dose can be repeated.In some embodiments, the dose is given once a day or multiple times a day, for example, but not limited to, three times a day.In some embodiments, the above dose is given daily for several weeks or months.The duration of treatment depends on the clinical progress of the subject and its responsiveness to treatment.

[0134] In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg. In some embodiments, the dose can be about 2 mg / kg. In some embodiments, the dose can be about 4 mg / kg. In some embodiments, the dose can be about 5 mg / kg. In some embodiments, the dose can be about 6 mg / kg. In some embodiments, the dose can be about 8 mg / kg. In some embodiments, the dose can be about 10 mg / kg. In some embodiments, the dose can be about 15 mg / kg. In some embodiments, the dose can be about 100 mg / kg. 2 ~about 700 mg / m 2 In some embodiments, the dose can be about 250 mg / m 2 In some embodiments, the dose can be about 375 mg / m 2 In some embodiments, the dose can be about 400 mg / m 2 In some embodiments, the dose can be about 500 mg / m 2 It can be.

[0135] In some embodiments, the dose can be administered intravenously. In some embodiments, the intravenous administration can be an infusion administered over a period of about 10 minutes to about 3 hours. In some embodiments, the intravenous administration can be an infusion administered over a period of about 30 minutes to about 90 minutes.

[0136] In some embodiments, the dose can be administered about every week. In some embodiments, the dose can be administered weekly. In some embodiments, the dose can be administered weekly for about 12 weeks to about 18 weeks. In some embodiments, the dose can be administered about every two weeks. In some embodiments, the dose can be administered about every three weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered about every two weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered about every three weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered intravenously about every two weeks. In some embodiments, the dose can be about 2 mg / kg to about 15 mg / kg administered intravenously about every three weeks. In some embodiments, the dose can be about 200 mg / m2 to about 400 mg / m2 administered intravenously about every week. In some embodiments, the dose can be about 200 mg / m2 to about 400 mg / m2 administered intravenously about every two weeks. In some embodiments, the dose can be about 200 mg / m2 to about 400 mg / m2 administered intravenously about every 3 weeks. In some embodiments, a total of about 2 to about 10 doses are administered. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, administration occurs over a total of about 4 weeks to about 12 weeks. In some embodiments, administration occurs over a total of about 6 weeks. In some embodiments, administration occurs over a total of about 8 weeks. In some embodiments, administration occurs over a total of about 12 weeks. In some embodiments, the initial dose can be about 1.5 to about 2.5 times larger than subsequent doses.

[0137] In some embodiments, the dose can be about 1 mg to about 2000 mg. In some embodiments, the dose can be about 3 mg. In some embodiments, the dose can be about 10 mg. In some embodiments, the dose can be about 30 mg. In some embodiments, the dose can be about 1000 mg. In some embodiments, the dose can be about 2000 mg. In some embodiments, the dose can be about 3 mg administered daily by intravenous infusion. In some embodiments, the dose can be about 10 mg administered daily by intravenous infusion. In some embodiments, the dose can be about 30 mg administered three times a week by intravenous infusion.

[0138] The therapeutically effective amount is the amount of drug that is sufficient to produce a statistically significant, measurable change in inflammatory condition, autoimmune disease, transplant rejection or GVHD, or to prevent the occurrence of inflammatory condition, autoimmune disease, transplant rejection or GVHD.Such effective amount can be accurately measured in clinical trials and animal tests.

[0139] Drugs can be administered intravenously by injection or slow infusion over time.For example, with the appropriate formulation for a given route, drugs useful in the methods and compositions described herein can be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and if desired, can be delivered by peristaltic means or other means known to those skilled in the art.The compounds used herein are preferably administered orally, intravenously or intramuscularly.Local administration, for example directly to the site of organ or tissue transplantation, is also specifically contemplated.

[0140] Therapeutic compositions containing at least one agent can be conveniently administered, for example, in unit doses. The term "unit dose" when used in relation to therapeutic compositions refers to physically discrete units as unitary dosages appropriate for subjects, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0141] Compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount and timing of administration will depend on the subject being treated, capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired.

[0142] The exact amount of active ingredient that needs to be administered depends on the judgment of the physician and is specific to each individual.However, the dosage range suitable for systemic application is disclosed herein and depends on the route of administration.The dosage regime suitable for administration also varies, but is represented by an initial administration, followed by repeated doses at intervals of one or more hours by subsequent injections or other administrations.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration within the range specified for in vivo therapy is contemplated.

[0143] In some embodiments, the method further includes administering the composition, CAR, or cells described herein with one or more additional inflammatory, autoimmune, GVHD or transplant rejection agents, biologics, drugs or treatments as part of a combination therapy. Exemplary treatments for transplant rejection or GVHD include immunosuppressants such as cyclosporine (Neoral, Sandimmune, Gengraf, and Restasis), tacrolimus (Prograf, Protopic, Astagraf XL, and Envalsus XR), methotrexate (Trexol, Lasbo, Rheumatrex, and Otrexap (PF)), sirolimus (Rapamune), mycophenolic acid (Myfortic and Cellcept), rituximab (Rituxan), etanercept (Enbrel), pentostatin (Nipent), ruxolitinib (Jakafi); chemotherapy such as methotrexate (Trexol, Lasbo, Rheumatrex, and Otrexap (PF)), antithymocyte globulin (Atgam, Thymoglobulin); Steroids, such as prednisone (Deltasone, Rayos and Prednisone Intensor), methylprednisolone (Medrol, Sol-Medrol and Depo-Medrol), budesonide (Entocort EC, Uceris); antifungals, such as posaconazole (Noxafil); antivirals, such as acyclovir (Zovirax and Sitavig), valacyclovir (Valtrex); and antibiotics, such as sulfamethoxazole / trimethoprim (Bactrim, Sulfatrim and Bactrim DS); protease inhibitors, such as alpha 1-protease inhibitors (Zemira); extracorporeal photochemotherapy; Monoclonal antibodies (daclizumab (Zimbryta), basiliximab (Simulect)), brentuximab vedotin (Adcetris), alemtuzumab (Campas, Lemtrada), tocilizumab (Actemra); including but not limited to infusion of mesenchymal stromal cells.Exemplary treatments for autoimmune diseases include insulin, such as insulin glulisine (Apidra and Apidra Solostar), insulin detemir (Levemir and Levemir FlexTouch), insulin aspart (Novolog, Novolog FlexPen and Novolog Penfill), insulin lispro (Humalog and Humalog QuickPen), insulin, insulin glargine (Lantus, Lantus Solostar and Touzio Solostar); dietary supplements, such as glucose tablets; and hormones, such as glucagon (Glucagen and Glucagon Emergency Kit (Human)), antidiabetics (metformin (D-Care DM2, Fortamet, Glucophage, Glucophage XR, Glumetza, Riomet), glucagon-like peptide-1 (GLP-1) receptor agonists (liraglutide (Saxenda; Victoza) or semaglutide (Ozempic) or sodium-glucose cotransporter 2 (SGLT2) inhibitors: Empagliflozin (Jardiance), canagliflozin (Invocana); Sulfonylureas: Glipizide (Glipizide XL, Glucotrol, Glucotrol XL); Meglitinide analogues: Repaglinide (Pranzin); Thiazolidinediones: Pioglitazone (Actos); Dipeptidyl peptidase-4 [DPP-4] inhibitors: including, but not limited to, sitagliptin (Januvia), saxagliptin (Onglyza), linagliptin (Tradienta), alogliptin (Nesina).

[0144] The efficacy of a given treatment, for example for inflammatory conditions, autoimmune diseases, transplant rejection or GVHD, can be determined by a skilled clinician. However, if any one or all of the signs or symptoms are changed in a beneficial way, or if other clinically recognized symptoms are improved or even improved by at least 10% after treatment with an agent described herein, as the term "effective treatment" is used herein, the treatment is considered to be "effective treatment". Efficacy can also be measured by the individual's lack of deterioration as assessed by hospitalization or the need for medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or are described herein.

[0145] An effective amount for treating a disease means an amount sufficient to provide effective treatment as defined herein for the disease when administered to a mammal in need thereof. The efficacy of an agent can be determined by, for example, evaluating physical indicators of autoimmune disease (e.g., ANA results), transplant rejection (e.g., high fever, tenderness at the transplant site, etc.), or GVHD (e.g., redness, pain, or other symptoms at the transplant site). Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Dosage can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Also, the dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of active ingredient that achieves half-maximal inhibition of symptoms) determined in cell culture or in a suitable animal model. The level in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays, for example, assays that evaluate post-transplantation response, levels of inflammation, ANA measurements, among others. The dosage can be determined by a physician and adjusted as necessary to suit the observed effects of treatment.

[0146] Efficacy can also be measured by the individual not getting worse, as assessed by hospitalization, or the need for medical intervention (i.e., progression of the disease is halted). Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals), including: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An amount effective for treating a disease means an amount sufficient to provide effective treatment, as defined herein, for the disease when administered to a subject in need thereof. The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response (e.g., reduced inflammation, etc.). It is well within the capabilities of one of skill in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be evaluated in the treatment of animal models of conditions described herein, such as inflammatory conditions, autoimmune diseases, transplant rejection or GVHD.When using experimental animal models, the efficacy of treatment is demonstrated when statistically significant changes in markers such as inflammation are observed.

[0147] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising the CAR described herein and, optionally, a pharma- ceutically acceptable carrier. In some embodiments, the active ingredient of the pharmaceutical composition comprises the CAR described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of the CAR described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of the CAR described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials which can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol, ... Licorice; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms "excipient," "carrier," "pharmaceutical acceptable carrier," and the like are used interchangeably herein. In some embodiments, the carrier inhibits degradation of the active agents described herein.

[0148] In some embodiments, the pharmaceutical composition comprising the CAR described herein can be in a parenteral dosage form. Because administration of a parenteral dosage form typically avoids the patient's natural defense against contaminants, the parenteral dosage form is preferably sterile or can be sterilized before administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharma- ceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled release parenteral dosage forms can be prepared for administration to a patient, including, but not limited to, DUROS®-type dosage forms and dose dumping.

[0149] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosed CAR are well known to those skilled in the art. Examples include, but are not limited to: water for injection USP; saline; glucose solution; aqueous vehicles, such as but not limited to sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles, such as but not limited to ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles, such as but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that change or modify the solubility of the pharma- ceutically acceptable salt of the active ingredient can also be incorporated into the parenteral dosage forms of the present disclosure, including conventional and controlled release parenteral dosage forms.

[0150] Pharmaceutical compositions can also be formulated to be suitable for oral administration, for example, as a discrete dosage form, such as, but not limited to, a tablet (including, but not limited to, a scored or coated tablet), a pill, a caplet, a capsule, a chewable tablet, a powder packet, a cachet, a troche, a wafer, an aerosol spray, or a liquid, such as, but not limited to, a syrup, an elixir, a solution, or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of a pharma- ceutically acceptable salt of the disclosed compounds and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0151] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms may result in large variations in the concentration of the drug in the blood and other tissues of the patient. These variations may affect a number of parameters, such as frequency of administration, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, etc. Advantageously, controlled release formulations can be used to control the onset of action of the drug, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled or extended release dosage forms or formulations can be used to ensure that maximum efficacy of the drug is achieved while minimizing potential adverse effects and safety concerns that can arise from both administering an underdose of the drug (i.e., below the minimum therapeutic level) and exceeding the toxic level of the drug. In some embodiments, the composition can be administered in a sustained release formulation.

[0152] Controlled release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of optimally designed controlled release preparations in medical treatments is characterized by the use of a minimum of drug substance to cure or control a condition in a minimum amount of time. Advantages of controlled release formulations include: 1) extended activity of the drug; 2) reduced dosing frequency; 3) improved patient compliance; 4) less total drug use; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved efficacy of treatment; 9) enhanced or reduced loss of drug activity; and 10) improved rate of disease or condition control. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0153] Most controlled release formulations are designed to initially release a certain amount of drug (active ingredient) that rapidly produces the desired therapeutic effect, and then gradually and continuously release different amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions, including, but not limited to, pH, ionic strength, osmolality, temperature, enzymes, water, and other physiological conditions or compounds.

[0154] Various known controlled release or extended release dosage forms, formulations and devices can be adapted for use with the salt and composition of the present disclosure.Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (e.g., OROS® (Alza Corporation, Mountain View, Calif. USA)), or combinations thereof, which provide the desired release profile in various ratios.

[0155] For convenience, the meanings of some terms and phrases used in the specification, examples, and the appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases include the meanings provided below. Since the scope of the present invention is limited only by the appended claims, the definitions are provided to aid in the description of specific embodiments and are not intended to limit the invention described in the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the event of an apparent discrepancy between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0156] For convenience, certain terms used herein in the specification, examples, and appended claims are collected here.

[0157] The terms "reduce", "reduced", "reduction" or "inhibit" are all used herein to mean a reduction by a statistically significant amount. In some embodiments, "reduce", "reduction", "reduce" or "inhibit" typically means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is recognized as being within the normal range for individuals without the given disorder.

[0158] The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% and any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or condition, an "increase" is a statistically significant increase in such level.

[0159] The term "ameliorate" or "improvement," when applied to a score on a standardized scale or assessment, e.g., of symptoms or severity, means a statistically significant, favorable change in the scale or rating on that scale.

[0160] As used herein, "reference level" refers to a level or value for a given parameter to which the level or value in a given sample or situation is compared to determine whether the level or value has changed in a meaningful way. A reference level can be a level or a level from a sample that has not been treated to change the parameter. A reference level can alternatively be a level or a level from a normal or otherwise unaffected sample. A reference level can alternatively be a level or a level from a sample obtained from a subject at a previous time point, for example, before a given treatment.

[0161] As used herein, "subject" refers to a human or animal. Usually, animals are vertebrates, such as primates, rodents, livestock or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as house cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient" and "subject" are used interchangeably herein.

[0162] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. A non-human mammal can be advantageously used as a subject that represents an animal model of an inflammatory condition, an autoimmune disease, a transplant rejection, or GVHD. The subject can be male or female.

[0163] The subject may have previously been diagnosed or identified as suffering from or having a condition requiring treatment (e.g., an inflammatory condition, an autoimmune disease, transplant rejection or GVHD) or one or more complications associated with such a condition, and may optionally have already been treated for an inflammatory condition, an autoimmune disease, transplant rejection or GVHD, or one or more complications associated with an inflammatory condition, an autoimmune disease, transplant rejection or GVHD. Alternatively, the subject may also not have previously been diagnosed as having an inflammatory condition, an autoimmune disease, transplant rejection or GVHD, or one or more complications associated with an inflammatory condition, an autoimmune disease, transplant rejection or GVHD. For example, the subject may be one that exhibits one or more risk factors for an inflammatory condition, an autoimmune disease, transplant rejection or GVHD, or one or more complications associated with an inflammatory condition, an autoimmune disease, transplant rejection or GVHD, or one or more complications associated with an inflammatory condition, an autoimmune disease, transplant rejection or GVHD, or one that does not exhibit risk factors.

[0164] A "subject in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk for developing the condition, such as an inflammatory condition, an autoimmune disease, transplant rejection, or GVHD.

[0165] As used herein, a "suitable control" refers to an untreated, otherwise identical cell or population (e.g., a subject that has not been administered an agent described herein, or a subject that has been administered only a subset of the agents described herein, compared to a non-control cell).

[0166] In some embodiments, the nucleic acid encoding the CAR described herein is contained by a vector. In some aspects described herein, the nucleic acid sequence encoding the CAR described herein, or any module thereof, is operably linked to another sequence, for example, a promoter in a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when associated with appropriate control elements and can transfer a gene sequence to a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0167] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The sequences to be expressed are often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, in human cells for expression, and in prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins, and, where appropriate, secreting proteins, including, but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0168] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle.A viral vector can contain the nucleic acid encoding a CAR described herein instead of a non-essential viral gene.The vector and / or particle can be utilized to transfer any nucleic acid into cells, either in vitro or in vivo.Many forms of viral vectors are known in the art.

[0169] "Recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It should be understood that the vector described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in the subject in the state of high copy number extrachromosomal DNA, thereby eliminating the potential effects of integration into chromosome.

[0170] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymer, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acid can be either single-stranded or double-stranded. Single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[0171] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues connected to each other by peptide bonds between the α-amino group and the carboxy group of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified (e.g., phosphorylated, glycated, glycosylated, etc.) amino acids and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used for relatively large polypeptides, while the term "peptide" is often used for small polypeptides, the usage of these terms in the art overlaps. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, natural proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the above.

[0172] As used herein, "antibody" refers to the following: IgG, IgM, IgA, IgD or IgE molecules, or antigen-specific antibody fragments thereof (including, but not limited to, Fab, F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, closed conformation multispecific antibodies, disulfide-linked scfv, diabodies), which may be isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria, which may be derived from a species which naturally produces the antibody or which may be produced by recombinant DNA technology.

[0173] As used herein, an "antigen" is a molecule to which a binding site on an antibody drug binds. Typically, an antigen is bound by an antibody ligand and can generate an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or part thereof. The term "antigenic determinant" refers to an epitope on an antigen that is recognized by an antigen-binding molecule, more specifically, by the antigen-binding site of the molecule.

[0174] As used herein, the term "antibody reagent" refers to a polypeptide that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. An antibody reagent can include an antibody or a polypeptide that contains an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can include a monoclonal antibody or a polypeptide that contains an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39, which is incorporated herein by reference in its entirety)) as well as complete antibodies. Antibodies can have structural characteristics of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primates (human and non-human primates), and can be primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[0175] The VH and VL regions can be further subdivided into hypervariable regions termed "complementarity determining regions" ("CDRs"), which are interspersed with more conserved regions termed "framework regions" ("FRs"). The extent of the framework regions and CDRs has been precisely defined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference in their entirety). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0176] The term "antigen-binding fragment" or "antigen-binding domain" is used interchangeably herein to refer to one or more fragments of a full-length antibody that retains the ability to specifically bind to a target of interest. Examples of binding fragments that are included in the term "antigen-binding fragment" of a full-length antibody include: (i) Fab fragment, i.e., a monovalent fragment that consists of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, i.e., a bivalent fragment that comprises two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment that consists of VH and CH1 domains; (iv) Fv fragment that consists of the VL and VH domains of a single arm of an antibody; (v) dAb fragment that consists of VH or VL domain (Ward et al., (1989) Nature 341:544-546; this is incorporated herein by reference in its entirety); and (vi) isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.

[0177] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells and / or particles, in which a first entity binds to a second, target entity with higher specificity and affinity than it binds to a third, non-target entity.In some embodiments, specific binding can refer to the affinity of a first entity to a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or higher than its affinity to a third, non-target entity.A reagent specific for a given target is one that exhibits specific binding to that target under the conditions of the assay used.

[0178] Furthermore, and as described herein, recombinant humanized antibodies can be further optimized for human therapy to reduce potential immunogenicity while maintaining functional activity. In this regard, functional activity refers to a polypeptide that can exhibit one or more known functional activities associated with the recombinant antibody or antibody reagent described herein. Such functional activities include, for example, the ability to bind to a target.

[0179] As used herein, the terms "treat", "treatment", "treating" or "amelioration" refer to therapeutic treatments whose purpose is to reverse, alleviate, improve, inhibit, slow or prevent the progression or severity of a condition associated with a disease or disorder, such as an inflammatory response, an autoimmune disease, a transplant rejection, or GVHD. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition associated with a disease or disorder, such as an inflammatory response, an autoimmune disease, a transplant rejection, or GVHD. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, a stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or palliation of the disease state, remission (whether partial or total), and / or a reduction in mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0180] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0181] As used herein, the term "administering" refers to the placement of an agent, such as a CAR, composition, or cell disclosed herein, into a subject by a method or route that results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions containing the compounds disclosed herein can be administered by any suitable route that results in an effective treatment in the subject.

[0182] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0183] Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," when used in connection with percentages, can mean ±1%.

[0184] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and each component thereof that is essential to the method or composition, but also embrace the inclusion of non-specified elements, whether essential or not.

[0185] The term "consisting of" refers to compositions, methods, and each component described herein, excluding any element not recited in that description of the embodiment.

[0186] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment.

[0187] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to denote non-limiting examples. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0188] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meaning commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the appended claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the entire contents of which are incorporated herein by reference in their entirety.

[0189] Other terms are defined herein within the description of various aspects of the invention.

[0190] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications cited throughout this application, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0191] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Specific embodiments of the present disclosure and examples thereof are described herein for illustrative purposes, but as one skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide still further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, some modifications can be made to protein structures without affecting biological or chemical action in type or amount, due to considerations of biological functional equivalence. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0192] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements in other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0193] In some embodiments, the technology may be defined in any of the following numbered paragraphs: 1. From the N-terminus to the C-terminus a) an extracellular recognition moiety that specifically binds to OX40L; b) a transmembrane segment; and c) Intracellular signal transduction A chimeric antigen receptor (CAR) polypeptide comprising: 2. The CAR of item 1, further comprising a detectable polypeptide at the C-terminus of the intracellular signaling portion. 3. The CAR of item 2, wherein the detectable polypeptide is a fluorescent polypeptide. 4. Item 4. The CAR of item 3, wherein the fluorescent polypeptide is Neon Green. 5. Item 5. The CAR according to any one of Items 2 to 4, further comprising a cleavage site between the intracellular signaling moiety and the detectable polypeptide. 6. The CAR of paragraph 5, wherein the cleavage site is a cleavable T2A site or a tandem P2A-T2A site. 7. The CAR of any of the preceding paragraphs, wherein the recognition moiety is an antibody reagent or a ligand functional domain. 8. The CAR described in item 7, wherein the antibody reagent is an scFV. 9. Item 9. The CAR according to any one of Items 7 to 8, wherein the antibody reagent is an anti-OX40L antibody reagent. 10. The antibody reagent is CDR sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the six CDRs of SEQ ID NOs: 1-6 The CAR of item 9, comprising: 11. The antibody reagent is A sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the amino acid sequence of SEQ ID NOs: 7-12. The CAR of item 9, comprising: 12. 12. The CAR of any of paragraphs 1 to 11, wherein the intracellular signaling portion comprises one or more of a CD28 co-signaling domain, a 41BB co-signaling domain, an IL2Rα JAK3 and IL2Rβ STAT5 complex docking site, a TGFβ-R SMAD2 / 3 docking site, and a CD3ζ signaling domain. 13. 12. The CAR of any one of items 1 to 11, wherein the intracellular signaling portion comprises a CD28 co-signaling domain and a CD3 zeta domain. 14. 12. The CAR of any one of items 1 to 11, wherein the intracellular signaling portion comprises a 41BB co-signaling domain and a CD3ζ domain. 15. A nucleic acid molecule encoding a CAR described in any of the preceding paragraphs. 16. The nucleic acid molecule of paragraph 15, wherein expression of the CAR is controlled by a Treg-specific promoter or an MND promoter. 17. 17. The nucleic acid molecule of claim 16, wherein the Treg-specific promoter comprises a FoxP3 promoter or a FoxP3 and IKZF2 / Helios hybrid promoter. 18. A vector comprising the nucleic acid molecule according to any one of items 15 to 17. 19. A cell comprising the CAR according to any one of items 1 to 14, or the nucleic acid molecule or vector according to any one of items 15 to 18. 20. The cell of paragraph 19, which is a Treg. twenty one. The cell of paragraph 20, wherein the Treg expresses Foxp3. twenty two. The cell of paragraph 20, wherein the Treg expresses CD4 and CD25. twenty three. A cell population, at least 80% of which are the cells of paragraph 19. twenty four. 24. A method of treating an autoimmune or inflammatory condition in a subject in need thereof, comprising administering to the subject the cell or cell population according to any one of items 19 to 23. twenty five. 25. The method of claim 24, wherein the autoimmune or inflammatory condition comprises allograft rejection, or xenograft rejection, or graft-versus-host disease (GVHD). 26. 25. The method of claim 24, wherein the autoimmune or inflammatory condition is selected from the group consisting of inflammatory bowel disease; rheumatoid arthritis; type I diabetes or autoimmune insulitis; multiple sclerosis; autoimmune thyroiditis; autoimmune gastritis; autoimmune uveitis or uveoretinitis; autoimmune orchitis; autoimmune oophoritis; psoriasis; vitiligo; autoimmune prostatitis; any unwanted immune response; tissue rejection; and inflammatory conditions. 27. The method of paragraphs 24 to 26, wherein the population of Tregs is autologous to the subject. 28. The method of any one of paragraphs 24 to 26, wherein the population of Tregs is allogeneic to the subject. 29. A method of treating an autoimmune or inflammatory condition in a subject in need thereof comprising administering to the subject a cell or population of cells. 24. The cell or cell population according to any one of items 19 to 23, for use in a method for treating a disease. 30. 25. The cell or cell population of paragraph 24, wherein said autoimmune or inflammatory condition comprises allograft or xenograft rejection, or graft-versus-host disease (GVHD). 31. 25. The cell or cell population of paragraph 24, wherein the autoimmune or inflammatory condition is selected from the group consisting of inflammatory bowel disease; rheumatoid arthritis; type I diabetes or autoimmune insulitis; multiple sclerosis; autoimmune thyroiditis; autoimmune gastritis; autoimmune uveitis or uveoretinitis; autoimmune orchitis; autoimmune oophoritis; psoriasis; vitiligo; autoimmune prostatitis; any unwanted immune response; tissue rejection; and an inflammatory condition. 32. 27. The cell or cell population of paragraphs 24 to 26, wherein the population of Tregs is autologous to the subject. 33. 27. The cell or cell population of paragraphs 24 to 26, wherein the population of Tregs is allogeneic to the subject. EXAMPLES

[0194] Example 1 We have designed and constructed several chimeric antigen receptor (CAR) constructs, such as linking single-chain fragment variant antigen-binding fragments of anti-OX40L antibody with intracellular CD3ζ and 4-1BB or CD28 signaling domains. We have also designed constructs that are further linked to fluorescent protein reporter neon green to visualize expression. In some of the constructs described herein, the expression of the construct is controlled by linking a Treg-specific promoter or by linking a non-specific MND promoter. These OX40L-CAR constructs have been successfully transduced into Jurkat lymphoid cells and into human FACS-purified CD25+CD127- Treg cells (Figures 1A-1B and 7). The OX40L-CAR construct is specifically expressed in Tregs, and CAR expression does not prevent ex vivo expansion of Tregs (Figures 1C-1D, 2A-2D, 8A-8B, and 9A-9B). Stimulation of Treg cells transduced with the OX40L-CAR construct induces Treg activation markers at levels comparable to TCR stimulation (Figure 3A). Furthermore, stimulation induces the production of inhibitory proteins such as IL-2, CTLA4, LAG-3, GARP, and LAP without inducing the production of pro-inflammatory cytokines such as IL17A, TNFa, and IFNg (Figures 3B and 10A-10B). The OX40L-CAR construct can successfully mediate T suppressive activity (Figure 3C). These data demonstrate that CAR-Treg cells remain functional and have T suppressive function. This functionality is illustrated in the model shown in Figure 4.

[0195] Blocking the interaction between OX40L and OX40 with anti-hOX40L antibodies can effectively prevent and treat aGVHD by preventing the cytolytic activity of CD8+ T cells (Figures 5A-5B and 6). As demonstrated herein, OX40L-CAR-Tregs suppress T cell proliferation in vitro compared to Neon-Tregs. Furthermore, OX40L-CAR-Tregs demonstrated a higher ability to inhibit the activation of monocyte-derived dendritic cells, highlighting the superior suppressive activity of these OX40L-CAR-Tregs compared to non-targeted Neon-Tregs (Figures 11A-11B and 12A-12B). Importantly, sublethally irradiated NOD.Cg-Prkdc scid I12rg tm1Wjl In a human xenograft model of GvHD induced by transplantation of human PBMCs into .SzJ (NSG) mice, OX40L CAR-Tregs demonstrated superior GvHD disease control compared to both PBMCs alone and Neon-Tregs (Figures 13A-13B). Clinical scores were assessed by a combined system including five parameters: weight loss, posture, mobility, skin and fur condition, and curves were compared using a two-way analysis of variance (ANOVA) mixed effects analysis, which demonstrated an improvement in clinical xenograft GvHD with OX40L CAR-Tregs > Neon-Tregs > PBMCs alone over the entire analysis period (p <0.001). An example of a significant improvement in clinical scores at Day +14 is as follows: Combined GvHD clinical score = PBMC 6.5 vs. Neon-Tregs 2.5 vs. OX40L CAR-Tregs 0.15, p <0.0001 for all comparisons. This control of clinical GvHD resulted in extended recipient survival when compared to both PBMCs alone and control Neon-Tregs (MST = 34 days for OX40L-CAR-Tregs, 20 days for Neon-Tregs, 14 days for PBMCs alone, p<0.001).

[0196] Collectively, these results demonstrate a novel and effective approach to enhance Treg suppressive function and control GvHD using OX40L-CAR Tregs. Because OX40L is upregulated on APCs in inflammatory environments and its expression is restricted to APCs and activated endothelial cells, this approach provides a unique strategy for the control of alloimmunity after HCT. Furthermore, these OX40L-CAR Tregs may be broadly applicable beyond HCT to control alloimmunity after solid organ transplantation and to suppress T cell activation in autoimmune diseases.

[0197] Example 2 FOXP3 regulatory elements used in CAR-Tregs: The endogenous FOXP3 promoter contains at least four highly conserved regulatory elements located across a broad region of the FOXP3 gene [1]. These elements have been identified as the FOXP3 core promoter sequence, and three other conserved non-coding sequences (CNS 1-3). CNS1 is also known as the TGF-β sensor / enhancer, CNS2 is also known as the Treg cell-specific demethylation region (TSDR), and CNS3 is known as the FOXP3 pioneer element. These four regulatory elements play unique roles in stabilizing and tightly controlling FOXP3 expression in regulatory T (Treg) cells. When Treg cells receive signals from TCR and CD28 stimulation, these signals induce the activation of several transcription factors, including NFAT, c-Rel, and AP-1, which bind to both the FOXP3 core promoter sequence and CNS3 of the FOXP3 gene to initiate FOXP3 transcription. In contrast, CNS2 plays an essential role in maintaining FOXP3 expression. CNS2 contains a CpG island that is highly demethylated by the AML1 / RUNX1 complex, which promotes DNA demethylation in this region, and therefore this region is also known as TSDR. The demethylated TSDR further recruits STAT5 from IL-2 signaling and other transcription factors induced by TCR / CD28 stimulation to stabilize FOXP3 transcription. TGF-β-induced SMAD2 / 3 gene expression, which is also actively involved in FOXP3 gene expression, occurs through TGF-β binding to CNS1.

[0198] Because the native FOXP3 promoter is too large to be directly utilized in lentiviral vectors as part of a gene expression cassette, we employed the human synthetic FOXP3 promoter to selectively drive expression of the OX40L CAR gene in Treg cells. We designed this promoter using the same design previously published to restore lineage-specific FoxP3 expression in mouse HSCs [2]. Finally, we cloned all three CNS enhancer elements and placed them in tandem upstream of the FOXP3 core promoter sequence. In addition, we included FOXP3 5' and 3' untranslated regions (or UTRs) before and after the CAR gene cassette to maintain the possibility of post-transcriptional regulation of the FOXP3 transcript. With the control of the synthetic human FOXP3 promoter, the OX40L CAR protein is more selectively expressed on Treg cells rather than on rare conventional T (Tcon) cells that may be expanded in small numbers during the Treg purification process.

[0199] References: TIFF2025507727000019.tif48153

Claims

1. From the N-terminus to the C-terminus a) an extracellular recognition moiety that specifically binds to OX40L; b) a transmembrane segment; and c) Intracellular signaling A chimeric antigen receptor (CAR) polypeptide comprising:

2. The CAR polypeptide of claim 1, further comprising a detectable polypeptide at the C-terminus of the intracellular signaling moiety.

3. The CAR polypeptide of claim 2, wherein the detectable polypeptide is a fluorescent polypeptide.

4. The CAR polypeptide of claim 3, wherein the fluorescent polypeptide is Neon Green.

5. 3. The CAR polypeptide of claim 2, further comprising a cleavage site between the intracellular signaling moiety and the detectable polypeptide.

6. The CAR polypeptide of claim 5, wherein the cleavage site is a cleavable T2A site or a tandem P2A-T2A site.

7. The CAR polypeptide of claim 1, wherein the recognition moiety is an antibody reagent or a ligand functional domain.

8. The CAR polypeptide of claim 7, wherein the antibody reagent is an scFV.

9. The CAR polypeptide of claim 7, wherein the antibody reagent is an anti-OX40L antibody reagent.

10. The antibody reagent is CDR sequences at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the six CDRs of SEQ ID NOs: 1-6 10. The CAR polypeptide of claim 9, comprising:

11. The antibody reagent is A sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the amino acid sequence of SEQ ID NOs: 7-12 10. The CAR polypeptide of claim 9, comprising:

12. 2. The CAR polypeptide of claim 1, wherein the intracellular signaling portion comprises one or more of a CD28 co-signaling domain, a 41BB co-signaling domain, an IL2Rα JAK3 and IL2Rβ STAT5 complex docking site, a TGFβ-R SMAD2 / 3 docking site, and a CD3ζ signaling domain.

13. The CAR polypeptide of claim 1, wherein the intracellular signaling portion comprises a CD28 co-signaling domain and a CD3ζ domain.

14. The CAR polypeptide of claim 1, wherein the intracellular signaling portion comprises a 41BB co-signaling domain and a CD3ζ domain.

15. A nucleic acid molecule encoding a CAR polypeptide described in any one of claims 1 to 14.

16. The nucleic acid molecule of claim 15, wherein expression of the CAR polypeptide is controlled by a Treg-specific promoter or an MND promoter.

17. 17. The nucleic acid molecule of claim 16, wherein the Treg-specific promoter comprises a FoxP3 promoter or a FoxP3 and IKZF2 / Helios hybrid promoter.

18. A vector comprising the nucleic acid molecule of claim 15.

19. A cell comprising: (i) a CAR polypeptide described in any one of claims 1 to 14, (ii) a nucleic acid molecule encoding the CAR polypeptide, or (iii) a vector containing the nucleic acid molecule.

20. The cell of claim 19, which is a Treg.

21. The cell of claim 20, wherein the Treg expresses Foxp3.

22. The cell of claim 20, wherein the Treg expresses CD4 and CD25.

23. 20. A cell population, at least 80% of which are cells of claim 19.

24. A pharmaceutical composition for treating an autoimmune or inflammatory condition in a subject in need thereof, comprising a cell according to claim 19 or a population of cells according to claim 19.

25. 25. The pharmaceutical composition of claim 24, wherein the autoimmune or inflammatory condition comprises allograft or xenograft rejection, or graft-versus-host disease (GVHD).

26. 25. The pharmaceutical composition of claim 24, wherein the autoimmune or inflammatory condition is selected from the group consisting of inflammatory bowel disease; rheumatoid arthritis; type 1 diabetes or autoimmune insulitis; multiple sclerosis; autoimmune thyroiditis; autoimmune gastritis; autoimmune uveitis or uveoretinitis; autoimmune orchitis; autoimmune oophoritis; psoriasis; vitiligo; autoimmune prostatitis; any unwanted immune response; tissue rejection; and an inflammatory condition.

27. 25. The pharmaceutical composition of claim 24, wherein the population of Tregs is autologous to the subject.

28. 25. The pharmaceutical composition of claim 24, wherein the population of Tregs is allogeneic to the subject.

Images