Butyrophilin A2 and related isoforms for the treatment of autoimmunity and inflammation

Abstract

Described herein are methods for reducing CD3-dependent T cell signaling in a subject in need thereof. Methods for increasing regulatory T (Treg) cells or decreasing type 17 helper T (Th17) cells are also described. These methods involve administering to a subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a conjugate or fusion polypeptide comprising any of the foregoing. These methods are beneficial for patients with autoimmune and inflammatory disorders, such as allergies, asthma, glomerulonephritis, inflammatory bowel disease, rheumatoid arthritis, autoimmune or inflammatory neurological diseases, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), psoriasis, myasthenia gravis, or HIV. Fusion proteins comprising BTN2A2 and an Fc domain are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 449,693, filed March 3, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in computer readable form, named "065472_000918WOPT_SequenceListing.xml," created on February 28, 2024, which is 55,203 bytes in size. The information contained in this computer readable form is incorporated herein by reference in its entirety.

[0003] The present invention relates to butyrophilin A2, its isoforms and fragments and conjugates thereof for the treatment of autoimmune and inflammatory disorders. [Background technology]

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor that any publication specifically or implicitly referenced is prior art.

[0005] Currently, treatment of autoimmunity is largely nonspecific, using steroids and drugs such as cyclosporine and Prograf. Although newer agents such as CTLA4IG show some promise, there remains a need for additional drugs that can, for example, act synergistically to ameliorate autoimmunity and inflammation.

[0006] T cells recognize antigens presented by peptide:MHC complexes using the heterodimeric abT cell receptor (TCR) expressed on their surface. Because the TCR lacks intrinsic kinase activity, signals initiated by TCR engagement involve the recruitment of the Src family kinase Lck, which then phosphorylates immunoreceptor tyrosine kinase base motifs (ITAMs) within the TCR-associated CD3 z-chain. Lck then phosphorylates the recruited Zap70 kinase, thereby transducing essential downstream signals required for full T cell activation. Multiple studies over the past 30 years have demonstrated that T cell activation is regulated in part by CD45 expressed on the cell surface.

[0007] CD45 is a transmembrane glycoprotein containing an intracellular tyrosine phosphatase domain capable of dephosphorylating multiple TCR immunoreceptor tyrosine-based activation (ITAM) motifs. Alternative splicing results in the expression of multiple CD45 isoforms (i.e., RA, RB, RC, and RO). Following TCR stimulation, CD45 is initially recruited to the supramolecular activation cluster (SMAC) but is subsequently excreted and dissociated from the TCR. Evidence suggests that this dissociation of CD45 phosphatase activity from the TCR is essential for Lck-initiated signaling, leading to full T cell activation. Conversely, CD45 retention within the T cell immune synapse (IS) controls the intensity and duration of TCR activation, and perturbations in CD45 activity contribute to the development of autoimmune diseases. In humans, abnormalities in CD45 expression have been reported in diseases including infantile cholestasis, hemophagocytic lymphohistiocytosis or erythrophagocytosis, nutritional disorders, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, and HIV (Tchilian et al., “Altered CD45 expression and disease,” TRENDS in Immunology 27(3):146-153, March 2006). Furthermore, therapeutic modulation of CD45 function has reportedly direct clinical applicability in organ transplantation, the treatment of autoimmune diseases, or microglial activation associated with Alzheimer's disease (AD) (A Rheinlaender et al., “CD45 in human physiology and clinical medicine,” Immunology Letters, 196:22-32, April 2018). However, despite decades of research by multiple groups uncovering these molecular mechanisms, it remains unclear how CD45 shedding or retention during TCR activation may be regulated, nor whether ligands co-presented by antigen-presenting cells (APCs) influence it.

[0008] Butyrophilin (BTN) is a glycoprotein enriched in breast milk that has poorly understood immunoregulatory functions and is involved in maintaining maternal-fetal immune tolerance. mRNA encoding BTN and BTN-like molecules is widely expressed in lymphoid and non-lymphoid tissues. The butyrophilin immunoglobulin domain shows structural similarity to the B7 family of co-receptors, including B7-1 / CD80, B7-2 / CD86, ICOS-L, and PD-L1. Butyrophilin 2A2 (BTN2A2) has previously been shown to be expressed by professional APCs, including B cells, macrophages, and dendritic cells (DCs). In vitro studies suggest that BTN2A2 can regulate T cell receptor (TCR) signaling and promote de novo Foxp3 expression. Mice genetically deficient in BTN2A2 exhibit impaired CD4+ regulatory T cell function, enhanced antitumor immunity, and enhanced clinical symptoms of experimental autoimmune encephalomyelitis, all of which are attributable to the loss of BTN2A2 in APCs. Although these accumulating findings implicate important immunoregulatory functions for BTN2A2, the precise molecular mechanisms underlying these effects remain unclear. Summary of the Invention

[0009] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0010] Various embodiments provide a method of reducing CD3-dependent T cell signaling in a subject in need thereof, comprising administering to the subject butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide comprising any of the foregoing.

[0011] Various embodiments provide methods for increasing regulatory T (Treg) cells or decreasing T helper type 17 (Th17) cells, or both, in a subject in need thereof, comprising administering to the subject butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide comprising any of the foregoing.

[0012] In various embodiments, BTN2A2, a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide can comprise human BTNA2 or a fragment or isoform thereof.

[0013] In various embodiments, the subject may be pregnant, and the method prevents or reduces the likelihood of fetal miscarriage by increasing Treg cells. In various embodiments, the subject may have renal impairment or a condition associated with renal impairment. In various embodiments, the subject may be an organ, tissue, or cell transplant recipient. In various embodiments, the method reduces the likelihood of organ, tissue, or cell transplant rejection, and / or an autoimmune or inflammatory response.

[0014] In various embodiments, the methods reduce the need for immunosuppressive therapy or reduce the amount of immunosuppressive therapy administered to the subject.

[0015] In various embodiments, the method may reduce or eliminate the toxic effects of immunosuppressive therapy in a subject that would otherwise be present.

[0016] In various embodiments, the subject can have an autoimmune or inflammatory disorder, or a disorder characterized by the accumulation of pathogenic Th1 / Th17 cells, eg, Th1 / Th17 polarized myeloid dendritic cells.

[0017] In various embodiments, the autoimmune or inflammatory disorder, or disorder characterized by the accumulation of pathogenic Th1 / Th17 cells, can include allergies, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological diseases, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV. In various embodiments, the autoimmune neurological disease can be multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody disease, or Alzheimer's disease.

[0018] Various embodiments provide a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0019] In various embodiments, the disease or condition can be an autoimmune or inflammatory disorder.

[0020] In various embodiments, the autoimmune or inflammatory disorder may include allergies, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological diseases, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV.

[0021] In various embodiments, the autoimmune neurological disease can be multiple sclerosis or myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0022] In various embodiments, the disease or condition can be associated with kidney damage.

[0023] In various embodiments, the disease or condition can be organ transplant rejection, and the method reduces the likelihood of organ transplant rejection.

[0024] In various embodiments, the disease or condition can be organ transplant rejection and the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy required by the subject.

[0025] In various embodiments, the toxic effects of immunosuppressive therapy may be reduced or eliminated in a subject.

[0026] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof can be linked to albumin, optionally human serum albumin.

[0027] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof can be linked or fused to an Fc domain or a fragment of an Fc domain (a "BTN2A2-Fc fusion protein"), or a BTN2A2-related isoform or a BTN2A2-related isoform fragment is linked or fused to an Fc domain or a fragment of an Fc domain (a "BTN2A2-related isoform-Fc fusion protein"), where the Fc domain is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, optionally mutated, and further optionally mutated to enhance or decrease Fc-associated effector function, and further optionally with any of the mutations shown in Table 3.

[0028] In various embodiments, BTN2A2 or a BTN2A2 fragment thereof can be linked by an Fc domain or a fragment of an Fc domain and a linker, optionally a peptide of 2 to 50 amino acids, or a BTN2A2-related isoform or a BTN2A2-related isoform fragment can be linked by an Fc domain or a fragment of an Fc domain and a linker, which can be G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO: 6), GGGGGSn (SEQ ID NO: 7), leucine zipper, aliphatic, or helical peptide.

[0029] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment, BTN2A2-related isoform, or BTN2A2-related isoform fragment thereof can be modified by glycosylation or PEGylation or lipidation, or by conjugation with cholesterol.

[0030] In various embodiments, the BTN2A2 fragment can include the extracellular domain of BTN2A2.

[0031] In various embodiments, the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0032] In various embodiments, the BTN2A2-related isoform can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

[0033] In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0034] Various embodiments provide a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or fragment of an Fc domain, which is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, and which may optionally be mutated, and which may further optionally be mutated to enhance or decrease Fc-associated effector function, and which may further optionally have any of the mutations shown in Table 3.

[0035] In various embodiments, the fusion polypeptide may further comprise a linker between the BTN2A2, BTN2A2 fragment, BTN2A2-related isoform, or BTN2A2-related isoform fragment and the Fc domain or fragment of an Fc domain, which is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, and which may optionally be mutated, and which may further optionally be mutated to enhance or decrease Fc-associated effector function, and which may further optionally be any of the mutations shown in Table 3.

[0036] In various embodiments, the Fc domain or a fragment of an Fc domain may be derived from an IgG1 antibody.

[0037] In various embodiments, the Fc domain or fragment of the Fc domain can include one or more mutations, hi various embodiments, the mutations can be Met208Leu, Asn214Ser, or both, relative to SEQ ID NO:4.

[0038] In various embodiments, the fusion polypeptide can further comprise a spacer peptide between the BTN2A2, BTN2A2 fragment, BTN2A2-related isoform, or BTN2A2-related isoform fragment and the Fc domain or fragment of an Fc domain.

[0039] In various embodiments, the spacer peptide may be selected from the group consisting of IEGRMDDISSTMVRS (SEQ ID NO: 56), IEGRMD (SEQ ID NO: 57), EEAEAAK (SEQ ID NO: 58), SIINFEKL (SEQ ID NO: 59), and GGGS (SEQ ID NO: 60).

[0040] In various embodiments, the BTN2A2 fragment can include the extracellular domain of BTN2A2.

[0041] In various embodiments, the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0042] In various embodiments, the BTN2A2-related isoform can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

[0043] In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0044] Various embodiments provide modified polypeptides comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, wherein the butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, is PEGylated or glycosylated.

[0045] In various embodiments, the BTN2A2 fragment can include the extracellular domain of BTN2A2.

[0046] In various embodiments, the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both. In various embodiments, the BTN2A2 fragment can bind to CD45, eg, CD45RO.

[0047] In various embodiments, the BTN2A2-related isoform can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0048] Various embodiments provide immunoassays comprising an anti-butyrophilin A2 (BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2 related isoform antibody, or an anti-BTN2A2 related isoform fragment antibody, or a combination thereof, immobilized on a solid support, and the solid support.

[0049] In various embodiments, the immunoassay may further include a biological sample obtained from the subject.

[0050] Various embodiments provide methods of using an assay of the invention for patient monitoring, comprising contacting a biological sample obtained from the patient with an assay of the invention and detecting a level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof.

[0051] In various embodiments, the method involves the production of (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or a fragment of an Fc domain; (iii) a modified polypeptide comprising a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or a fragment of an Fc domain; The method may further comprise selecting or administering a treatment selected from (i) a modified polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, which is PEGylated or glycosylated; or (ii) a non-BTN2A2-related treatment.

[0052] Various embodiments provide a method of using the assays of the invention for patient stratification, comprising contacting a biological sample obtained from the patient with an assay of the invention and detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and classifying the patient as having or having a level of (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (ii) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (iii) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (iv) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (v) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (vi ...i) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof, (viii) a fusion polypeptide comprising butyrophilin A2 (BTN2 (iii) a fusion polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or a fragment of an Fc domain; (iv) a modified polypeptide comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, which is PEGylated or glycosylated; or (v) stratifying for treatment with a non-BTN2A2-related treatment.

[0053] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

[0054] Exemplary embodiments are illustrated in the referenced drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative and not restrictive. [Brief explanation of the drawings]

[0055] [Figure 1](Panels A-D) show that BTN2A2-Fc enhances the interaction and colocalization of CD45 with TCR signaling proteins. (A) Jurkat cells were stimulated with immobilized anti-CD3 antibody (10 μg / ml) for 3 minutes in the presence or absence of BTN2A2-Fc (10 μg / ml). Cells were lysed in IP buffer, immunoprecipitated with anti-CD45 antibody, and immunoblotted for total Zap70 and CD3ζ. The right panel shows intensity plots shown as mean ± SD (N=3). (B) Immunostaining analysis shows the dissociation of CD45 from CD3ζ after Jurkat cell activation with plate-bound anti-CD3 antibody (10 mg / ml) in the presence or absence of recombinant BTN2A2-Fc protein (10 mg / ml) for 3 minutes. The right panel shows quantification of CD3ζ and CD45 colocalization from multiple fields in the presence or absence of BTN2A2-Fc protein. At least 50 cells from each group were included in the analysis shown in the right panel. (C) Coimmunoprecipitation experiments were performed using anti-CD3ε antibody in Jurkat cells under the same conditions as in (A), followed by immunoblotting with anti-CD45 antibody. (D) CD45-specific phosphatase activity was measured in immunoprecipitates from the samples in panel C using FDP (fluorescein diphosphate tetraammonium salt) substrate as described in the methods. Data are shown as mean ± SD (n = 3). One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 2](Panels A-H) CD45 phosphatase co-immunoprecipitated with BTN2A2. (A) Jurkat cells were immunoprecipitated with anti-BTN2A2 antibody or IgG and blotted with CD45 antibody either unstimulated or after 48 hours of activation with anti-CD3 (1 mg / ml). Input represents approximately 3% of the cell lysate. One representative experiment is shown. (B) Jurkat cells were treated with plate-bound anti-CD3 antibody (1 μg / ml) for 48 hours in the presence of recombinant BTN2A2-Fc or Fc-tagged protein. Cells were lysed in IP buffer, immunoprecipitated with protein G, and immunoblotted with anti-CD45 antibody. Input represents approximately 3% of the cell lysate. One representative experiment is shown. (C) Jurkat cells were treated with anti-CD3 antibody (1 μg / ml) in the presence of recombinant BTN2A2-Fc or Fc-tagged protein for 48 hours, crosslinked with BS3, lysed, immunoprecipitated with protein G, and immunoblotted with anti-CD45 antibody. Input was approximately 3% of the cell lysate. One representative blot is shown. (D) Ribbon diagram representation of the protein-protein interaction between BTN2A2 (pink) and CD45 (PTPRC) (white), showing amino acids (blue sticks) that may determine the interaction. Two critical glycosylation sites, N419 and N468, located in the fibronectin domain of CD45, which are crucial for the interaction, are shown as globular models. (E) Western blot demonstrating CD45 expression in wild-type Jurkat cells (lane 1) and Jurkat cells with CD45 knockdown (CD45-KD) (lane 2). CD45 expression in CD45-KD cells expressing wild-type CD45 or mutant CD45 (double mutant, i.e., N419A and N468A) is shown in lanes 3-4. GAPDH was the internal control used. (F) Coimmunoprecipitation of CD45 with BTN2A2 as described in panel B in CD45-deficient Jurkat cells expressing wild-type CD45 or mutant CD45 (double mutant, i.e., N419A and N468) and activated (1 mg / ml anti-CD3 antibody for 48 hours) in the presence of recombinant BTN2A2-Fc or Fc.(G) Western blot demonstrating expression of CD45 isoforms in Jurkat cells at baseline and 48 hours after stimulation with anti-CD3 antibody. (H) Western blot analysis of CD45 isoforms (CD45RO and RA) after immunoprecipitation with protein G agarose beads of lysates from activated Jurkat cells (1 mg / ml anti-CD3 antibody for 48 hours) in the presence of recombinant BTN2A2-Fc or Fc. One representative experiment is shown. [Figure 3](Panels A-D) Demonstration that BTN2A2-Fc enhances Treg and suppresses Th17 cell differentiation in an in vitro mixed lymphocyte reaction (MLR). (A) Flow cytometry analysis plots of primary CD4+ T cells (derived from the spleen and lymph nodes of Foxp3-GFP transgenic mice) incubated in an MLR with 1 μg / ml of conjugated anti-CD3 and in the presence or absence of 10 μg / ml of BTN2A2-Fc fusion protein for 7 days as described in the Methods and analyzed for CD4+CD25+Foxp3-GFP positive cell expression. As a positive control, the effect of TGF-β (1 ng / ml) under the same conditions is also shown. The right panel shows summary plots presented as the mean ± SD of three independent experiments. One-way ANOVA with Tukey's multiple comparison test; * p < 0.05. (B) Flow cytometry analysis of primary CD4+ T cells (isolated from mouse spleens and lymph nodes) incubated for 5 days in an MLR with anti-CD3 (0.5 mg / ml) antibody alone or with anti-CD3 + TGF-β (1.5 ng / ml) + IL-6 (10 ng / ml) + IL-1β (10 ng / ml) and / or recombinant BTN2A2-Fc (10 μg / ml) and analyzed for CD4+ RORgt-positive cells. The right panel shows summary plots shown as the mean ± SD of three independent experiments. One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*). (C) Flow cytometry analysis of the Foxp3-GFP-positive cell population (%) among total CD4+ T cells cocultured with dendritic cells (DCs) on day 7. Purified T cells were incubated ex vivo with DCs isolated from wild-type or BTN2A2- / - mice (at a ratio of 1:10) in RPMI on day 7. (D) Flow cytometry analysis of the Foxp3-GFP-positive cell population (%) among total CD4+ cells cocultured with B cells on day 7. Purified T cells were incubated ex vivo with B cells isolated from wild-type or BTN2A2- / - mice (at a ratio of 1:5) on day 7. Data from experiments in (C-D) are shown as mean ± SD (n = 3). Unpaired t-test; p < 0.05 (*), p < 0.001 (***). [Figure 4](Panels A–C) Inhibition of CD45 phosphatase activity in primary immune cells prevents BTN2A2-mediated Treg differentiation and Th17 suppression. (A) Immunoblot analysis of phosphorylated ZAP-70 (p-Zap70) and total Zap70 in Jurkat cells pretreated with CD45 phosphatase inhibitor for 1 hour and stimulated with plate-bound anti-CD3 antibody for 3 minutes in the presence or absence of recombinant BTN2A2-Fc (10 μg / ml). (B) Flow cytometry analysis plots of primary CD4+ T cells (derived from spleens and lymph nodes of Foxp3-GFP transgenic mice) incubated in an MLR with 1 μg / ml of conjugated anti-CD3 and / or 10 μg / ml of BTN2A2-Fc fusion protein in the absence or presence of a CD45 phosphatase inhibitor (125 nM) for 7 days and analyzed for CD4+CD25+Foxp3-GFP positive cell expression. The right panel shows summary plots shown as the mean ± SD of three independent experiments. (C) Flow cytometry analysis of primary CD4+ T cells (isolated from mouse spleens and lymph nodes) incubated in an MLR for 7 days with anti-CD3 (0.5 mg / ml) antibody alone, or with anti-CD3 + TGF-β (1.5 ng / ml) + IL-6 (10 ng / ml) + IL-1β (10 ng / ml) and / or recombinant BTN2A2-Fc (10 μg / ml) for 5 days in the absence or presence of a CD45 phosphatase inhibitor (125 nM), and analyzed for CD4+ RORgt-positive cells. The right panel shows summary plots presented as mean ± SD. One-way ANOVA with Tukey's multiple comparison test; p < 0.01 (**), p < 0.001 (***). [Figure 5A] (Panel A) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Schematic of the protocol and histological changes in the NTS-induced nephrotoxic glomerulonephritis model. GBM = glomerular basement membrane. [Figure 5B](Panel B) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Quantification of mean ± SEM proteinuria (day 7) in mice with NTS-induced glomerulonephritis treated with recombinant BTN2A2-Fc protein or vehicle control (control) (N = 12 / group); Mann-Whitney p < 0.001 (***). [Figure 5C] (Panel C) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Representative images of glomerular damage stained with PAS. The summary panel shows quantification of the % of glomeruli with crescents (mean ± SEM, N = 12 / group); Mann-Whitney p < 0.001 (***). [Figure 5D] (Panel D) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Representative images of glomerular damage stained with PAS. Crescent formation in mice with NTS-induced glomerulonephritis treated with recombinant BTN2A2-Fc protein or control. Scale bar 25 mm. [Figure 5E] (Panel E) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Relative Foxp3 expression compared to GAPDH as an internal control in CD4+ T cells purified from spleens and lymph nodes from mice with NTS-induced glomerulonephritis treated with BTN2A2-Fc protein or control. Data shown as mean ± SEM (N = 8 / group). Mann-Whitney test; * p < 0.05. [Figure 5F] (Panel F) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Relative RORgt expression compared to GAPDH as an internal control in CD4+ T cells purified from spleens and lymph nodes from mice with NTS-induced glomerulonephritis treated with BTN2A2-Fc protein or control. Data shown as mean ± SEM (N = 8 / group). Mann-Whitney test; * p < 0.05. [Figure 5G] (Panel G) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Quantitative PCR analysis shows CD5 expression in CD4+ T cells from mice with NTS-induced glomerulonephritis treated with BTN2A2-Fc protein or control. N=8 / group. [Figure 5H] (Panel H) Immunoblot analysis of IL17A in kidney tissue lysates from mice with glomerulonephritis treated with recombinant BTN2A2-Fc or control. [Figure 5I] (Panel I) BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). Immunoblot analysis of IL17A in kidney tissue lysates from mice with glomerulonephritis treated with recombinant BTN2A2-Fc or control. This panel shows quantification of Western blots. Data are presented as mean ± SEM. N = 4 per group. Unpaired t-test; p < 0.05 (*). [Figure 6](Panels A-H) BTN2A2(- / -) mice exhibiting exacerbation of crescentic glomerulonephritis. (A) Scatter plots show quantification of mean ± SEM proteinuria in wild-type and BTN2A2(- / -) mice with nephrotoxic glomerulonephritis on day 7 (N = 15). Mann-Whitney test; p<0.01 (**). (B, C) Representative images of glomerular damage by PAS staining. Wild-type mice exhibit minimal glomerular damage at low doses of nephrotoxic serum, while BTN2A2(- / -) mice exhibit severe glomerulonephritis (B). Scale bar is 25 mm. Summary data are shown as mean ± SEM of % glomerular crescents (C). N = 15 per group for all experiments. Mann-Whitney test; p<0.001 (***). (D-E) Relative Foxp3 mRNA (D) and RORgt mRNA (E) compared with GAPDH as an internal control in CD4+ T cells purified from spleen and lymph node cells from wild-type and BTN2A2 knockout mice with nephrotoxic serum-induced glomerulonephritis. (F) Quantitative PCR analysis shows CD5 expression in CD4+ T cells from wild-type and BTN2A2 knockout mice with nephrotoxic serum-induced glomerulonephritis. N=7 / group. (G-H) Immunoblotting for IL17A expression in kidney tissue lysates using anti-IL-17A antibody (G) and quantification (H) from wild-type and BTN2A2 null mice with nephrotoxic serum-induced glomerulonephritis. n≥4 / group. Unpaired t-test; p<0.01 (**), p<0.001 (***). [Figure 7A] (Panel A) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J × DBA / 2 pregnancy model. Pregnant CBA / J × DBA / 2 mice treated with BTN2A2 throughout gestation improved litter size compared to untreated mice. DBA / 2 × CBA / J mice were included as an additional control. Data are presented as mean ± SEM. N = 11 / group for all experiments. Kruskal-Wallis test with Dunn's test for multiple comparisons; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7B](Panel B) BTN2A2-Fc normalizes fetal resorption and increases litter size in a CBA / J × DBA / 2 gestational model. Pregnant CBA / J × DBA / 2 mice treated with BTN2A2 throughout gestation had reduced resorption compared to untreated mice. DBA / 2 × CBA / J mice were included as an additional control. Data are presented as mean ± SEM. N = 11 / group for all experiments. Kruskal-Wallis test with Dunn's test for multiple comparisons; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7C] (Panel C) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J × DBA / 2 pregnancy model. Quantitative PCR analysis demonstrates an increase in Foxp3-expressing CD4+ T cells in pregnant CBA / J × DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. N = 8 per group. Experimental data: One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7D] (Panel D) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J × DBA / 2 pregnancy model. Flow cytometry analysis shows an increase in Foxp3-expressing CD4+ T cells in pregnant CBA / J × DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. N = 8 per group. Experimental results: One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7E](Panel E) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J × DBA / 2 pregnancy model. Flow cytometry analysis (left panel) shows a decrease in the number of CD4+RORgt-positive cells in pregnant CBA / J × DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. The right panel shows quantification of summary data. N = 8 per group. Experimental data: One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7F] (Panel F) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J × DBA / 2 pregnancy model. Quantitative PCR analysis shows CD5 expression on CD4+ T cells in pregnant CBA / J × DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. N = 8 per group. Experimental, one-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 7G] (Panel G) BTN2A2-Fc normalizes fetal resorptions and increases litter size in the CBA / J × DBA / 2 pregnancy model. Immunoblot analysis of IL17A in placental tissue lysates from pregnant CBA / J × DBA / 2J mice treated with recombinant BTN2A2-Fc or control. Experimental results: One-way ANOVA with Tukey's multiple comparison test; p<0.05 (*), p<0.01 (**), p<0.001 (***). [Figure 7H] (Panel H) BTN2A2-Fc normalizes fetal resorptions and increases litter size in a CBA / J x DBA / 2 pregnancy model. This panel shows quantification of summary data. N=3 / group. [Figure 8A](Panel A) BTN2A2-Fc enhances Treg and suppresses Th17 cell differentiation in an in vitro human PBMC mixed lymphocyte reaction. Flow cytometry plots showing the percentage of Treg cells (CD4+CD25+Foxp3+ cells) in human PBMCs incubated for 7 days in the absence or presence of a CD45 phosphatase inhibitor (125 nM) with or without recombinant BTN2A2-Fc (10 mg / ml) in an MLR. Data are shown as the mean + / - SD, representing the percentage of Treg cells (CD4+CD25+Foxp3+ cells) among total CD4+ cells. N = 4 per group for all experiments. Unpaired two-tailed t-test; p < 0.001 (***). [Figure 8B] (Panel B) BTN2A2-Fc enhances Treg and suppresses Th17 cell differentiation in an in vitro human PBMC mixed lymphocyte reaction. Flow cytometry plots showing the percentage of Treg cells (CD4+CD25+Foxp3+ cells) in human PBMCs incubated for 7 days in the absence or presence of a CD45 phosphatase inhibitor (125 nM) with or without recombinant BTN2A2-Fc (10 mg / ml) in an MLR. Data are shown as the mean + / - SD, representing the percentage of Treg cells (CD4+CD25+Foxp3+ cells) among total CD4+ cells. N = 4 per group for all experiments. Unpaired two-tailed t-test; p < 0.001 (***). [Figure 8C](Panel C) Flow cytometry plots showing the percentage of CD4+RORgt+ (Th17) cells in human PBMCs incubated in an MLR for 7 days in the absence or presence of a CD45 phosphatase inhibitor (125 nM), or for 3 days with anti-CD3 antibody (0.5 mg / ml) alone or with recombinant BTN2A2-Fc (10 mg / ml) in the absence or presence of anti-CD3 antibody + TGF-β (1.5 ng / ml), IL-6 (10 ng / ml), and IL-1b (10 ng / ml). Data represent the mean ± standard deviation. N = 3 per group for all experiments. One-way ANOVA with Tukey's multiple comparison test; p < 0.01 (**). [Figure 8D] (Panel D) Flow cytometry plots showing the percentage of CD4+RORgt+ (Th17) cells in human PBMCs incubated in an MLR for 7 days in the absence or presence of a CD45 phosphatase inhibitor (125 nM), or for 3 days with anti-CD3 antibody (0.5 mg / ml) alone or with recombinant BTN2A2-Fc (10 mg / ml) in the absence or presence of anti-CD3 antibody + TGF-β (1.5 ng / ml), IL-6 (10 ng / ml), and IL-1b (10 ng / ml). Data represent the mean ± standard deviation. N = 3 per group for all experiments. One-way ANOVA with Tukey's multiple comparison test; p < 0.01 (**). [Figure 9] A representative schematic of a model in which BTN2A2 acts as a co-inhibitor of TCR signaling via CD45 phosphatase is shown. [Figure 10](Panels A-D) BTN2A2-Fc regulates T cell receptor signaling. (A) Schematic diagram showing recombinant human BTN2A2-Fc fusion. The recombinant fusion gene consists of two extracellular domains (IgV and IgC2) (green) of the BTN2A2 molecule cloned with the Fc region of a human IgG1 antibody (orange). This chimeric protein (BTN2A2-Fc) is separated by an IEGRMD (SEQ ID NO: 57) spacer polypeptide. (B) Coomassie blue staining of purified recombinant BTN2A2-Fc protein on a reducing (SDS) gel, with a protein ladder in the left column and a band corresponding to BTN2A2-Fc (approximately 55 kDa) in the two right columns. The bottom panel shows a Western blot of recombinant BTN2A2-Fc using a specific anti-human BTN2A2 antibody. (C) IL-2 secretion from Jurkat cells incubated for 24 hours with plate-immobilized anti-CD3 antibody (1 μg / ml) in the absence or presence of immobilized recombinant BTN2A2-Fc (10 μg / ml), 293 cell-derived human BTN2A2-Fc (R&D Systems), or mouse BTN2A2 (R&D Systems), and immobilized immunoglobulin Fc domain as a negative control (n=3 or 4). (D) Immunoblot analysis of phosphorylated proteins p-Zap70 and p-CD3-ζ, as well as total Zap70 and CD3-ζ, in Jurkat cells stimulated for 3 minutes with immobilized anti-CD3 antibody (10 μg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 μg / ml) or immunoglobulin Fc domain (10 μg / ml). The right panel shows relative intensity plots as mean ± SD (n=3). One-way ANOVA with Tukey's multiple comparison test; p<0.05(*), p<0.01(**), p<0.001(***). [Figure 11] Pervanadate studies are shown. Jurkat cells were treated with the permanent phosphatase inhibitor (pervanadate) ± BTN2A2-Fc or left untreated. After cell lysis, the phosphorylated proteins p-Zap70 and p-CD3-ζ were analyzed by Western blot. Total Zap70 and CD3-ζ served as controls. [Figure 12] Jurkat cells were stimulated with or without anti-CD3 antibody (1 μg / ml) for 48 h, and lysates were immunoblotted with anti-CD45 or anti-BTN2A2 antibody. GAPDH expression was used as a loading control. [Figure 13] (Panels A-B) CRISPR-Cas9 deletion of CD45 and site-directed mutagenesis of CD45. (A) Representative schematic of the CD45 sequence showing the location of gRNA sequence complementarity. The bottom panel shows an electropherogram of the sequence deleted from exon-1 upstream to exon-3 downstream. Nucleotide sequence: SEQ ID NO: 46; amino acid sequence: SEQ ID NO: 47. (B) Electropherogram showing the nucleotide changes at Asn419Ala from AAT to GCT and at Asn468Ala from AAT to GCT in the CD45 mutant gene. Top left: nucleotide sequence—SEQ ID NO: 48; amino acid sequence—SEQ ID NO: 49; top right: nucleotide sequence—SEQ ID NO: 50; amino acid sequence—SEQ ID NO: 51; bottom left: nucleotide sequence—SEQ ID NO: 52; amino acid sequence—SEQ ID NO: 53; bottom right: nucleotide sequence—SEQ ID NO: 54; amino acid sequence—SEQ ID NO: 55. [Figure 14A] (Panel A) BTN2A2-Fc inhibits proliferation, apoptosis, and IL-2 secretion of CD4+ T cells. Flow cytometry plots of CellTrace violet-stained purified mouse CD4+ cells on day 3 incubated with immobilized anti-CD3 and anti-CD28 antibodies (0.5 μg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 μg / ml) or immunoglobulin Fc domain (10 μg / ml). The right panel shows a bar graph of the percentage of non-proliferating cells (GO). Data are presented as mean ± SD (n = 4). Unpaired t-test; p < 0.001 (***). [Figure 14B](Panel B) BTN2A2-Fc inhibits CD4+ T cell proliferation, apoptosis, and IL2 secretion. Flow cytometry plots of Annexin-V stained purified mouse CD4+ cells on day 3 incubated with immobilized anti-CD3 and anti-CD28 antibodies (0.5 μg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 μg / ml) or immunoglobulin Fc domain (10 μg / ml). The right panel shows a bar graph of the percentage of apoptotic cells. Data are presented as mean ± SD (n = 4). Unpaired t-test; p < 0.001 (***). [Figure 14C] (Panel C) BTN2A2-Fc inhibits CD4+ T cell proliferation, apoptosis, and IL2 secretion. Flow cytometry plots of purified IL2-positive murine CD4+ cells incubated with immobilized anti-CD3 and anti-CD28 antibodies (0.5 μg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 μg / ml) or immunoglobulin Fc domain (10 μg / ml) on day 3. The right panel shows a bar graph of the percentage of IL2-positive cells. Data are presented as mean ± SD (n = 4). Unpaired t-test; p < 0.001 (***). [Figure 15] (Panels A-F) qPCR studies of candidate molecules in the T cell differentiation pathway. Quantitative PCR analysis of IL-2RB (A), Foxp3 (B), IL-21 (C), TBX21 (D), GATA3 (E), and Smad-3 (F) in primary CD4+ cells incubated for 1 and 5 days in the absence or presence of anti-CD3 antibody and / or recombinant BTN2A2-Fc. All data are presented as mean ± standard deviation. n = 3 / group for all experiments. One-way ANOVA with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). [Figure 16A](Panel A) Generation and characterization of BTN2A2 null mice. Btn2a2- / - mice were generated by CRISPR / Cas-mediated genome manipulation (cyagen.com). The Btn2a2 gene (NCBI reference sequence: NM_175938, Ensembl: ENSMUSG00000053216) is located on mouse chromosome 13. Eight exons were identified, with an ATG start codon in exon-2 and a TAG stop codon in exon-8 (transcript Btn2a2-203: ENSMUST00000110433). Exons 2 to 8 (encompassing 100.0% of the coding region) were selected as target sites. Cas9 and gRNA were co-injected into fertilized eggs to generate KO mice. The genotypes of the offspring were identified by PCR followed by sequencing analysis. The size of the effective KO region was approximately 10,500 bp. SEQ ID NOs for gRNA target sequences: gRNA1: SEQ ID NO: 39, gRNA2: SEQ ID NO: 40, gRNA3: SEQ ID NO: 41, gRNA4: SEQ ID NO: 42. SEQ ID NOs for genotyping primers: F1: SEQ ID NO: 43, F2: SEQ ID NO: 44, R1: SEQ ID NO: 45. [Figure 16B] (Panel B) Generation / characterization of BTN2A2 null mice. Agarose gel electrophoresis of genotyping PCR. Homozygous (Homo) mice show a single band at 700 bp, heterozygous (Het) mice have two bands at 700 bp and 500 bp, and wild-type (WT) mice have a single band at 500 bp. [Figure 17] (Panels A-B) BTN2A2 expression in antigen-presenting cells. qPCR analysis of BTN2A2 expression in magnetic bead-purified B cell (A) and dendritic cell (B) populations from the spleen and lymph nodes of wild-type and BTN2A2-KO mice. The left panel shows a flow cytometry plot of purified cells, and the right panel shows the fold change in RNA expression of BTN2A2 transcripts relative to GAPDH. [Figure 18] Includes a summary of the protocol for the miscarriage proneness model. [Figure 19]A schematic diagram showing the binding of BTN2A2 to myelin oligodendrocyte glycoprotein (MOG) is shown. Protein structure prediction software (Prime, www.schrodinger.com) was used to assess the binding of BTN2A2 to MOG, an antigen for multiple sclerosis and other MOG-related autoimmune diseases. The root mean square deviation (RMSD) of the atomic positions was less than 1A. DETAILED DESCRIPTION OF THE INVENTION

[0056] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0057] As used herein, unless otherwise specifically specified herein, the term "about" when used in conjunction with a referenced numerical indication means adding or subtracting up to 5% of the referenced numerical indication to the referenced numerical indication. For example, the expression "about 50%" encompasses a range of 45% to 55%. In various embodiments, the term "about" when used in conjunction with a referenced numerical indication, when specifically specified in the claims, can mean adding or subtracting up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication to the referenced numerical indication.

[0058] The sequence identity percentage (%) with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, and does not consider any conservative substitutions as part of the sequence identity.The alignment for determining the amino acid sequence identity percentage can be achieved by various known means, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve the maximum alignment over the entire length of the sequence to be compared.

[0059] "Linked," as used herein in the context of linking peptides, polypeptides, or proteins, refers to being "connected" either directly or indirectly. Indirect linkages can be mediated by polypeptide linkers, such as polyglycine or glycine-serine polypeptides, e.g., (GGGGS)n (SEQ ID NO:6)n or (GGGGGS)n (SEQ ID NO:7)n, where n is an integer. In various embodiments, n is an integer between 1 and 10, 10 and 20, 20 and 30, 30 and 40, or 50 and 100. Other such linkers are known in the art and are considered to be encompassed by this term.

[0060] Herein, we demonstrate that BTN2A2 functions as a ligand for CD45, binding both to the TCR complex and the CD45RO isoform on the T cell surface, resulting in the retention of CD45 phosphatase activity in the vicinity of the TCR complex. Consequently, enhanced phosphatase activity reduces downstream TCR signaling, thereby promoting the expansion of regulatory T cells (Tregs) and suppressing the differentiation of effector T cells. We further demonstrate the significant immunomodulatory effects of BTN2A2 in a mouse model of autoimmune glomerulonephritis, showing that BTN2A2 gene deletion exacerbated renal damage, whereas administration of recombinant BTN2A2 limited the severity of the disease. Furthermore, in a second mouse model of immune-mediated spontaneous abortion, treatment of pregnant dams with recombinant BTN2A2 helped animals tolerate the pregnancy and reduced fetal loss.

[0061] The data contained herein provide evidence from multiple perspectives demonstrating that BTN2A2 functions as a ligand for CD45, preferentially binding to the CD45RO isoform, and that BTN2A2 functions to prevent CD45 dissociation from the TCR / CD3 complex, resulting in the maintenance of CD45 phosphatase activity within the immune synapse, impairing downstream signaling cascades (see Figure 9). Consequently, BTN2A2 promotes the differentiation of regulatory T cells while suppressing the proliferation of effector Th17 cells, and reduces the severity of NTS-induced glomerulonephritis and immune-mediated pregnancy loss in a mouse model of immunological disease. Our findings suggest that during corresponding T cell / APC interactions, APCs produce BTN2A2, which couples T cell-expressed CD45RO to the TCR, functioning as a molecular brake upon activation of the TCR complex by peptide / MHC complexes. Our data suggest that in CD45RO+ T cells, BTN2A2 binds both CD45 and CD3e, likely maintaining them in close proximity, allowing CD45 phosphatase activity to persist and dephosphorylate the CD3 ITAM domain and Zap70, limiting downstream TCR signals required for full T cell activation.

[0062] Our data also indicate that BTN2A2 modulates, but does not completely block, TCR signaling by enhancing the phosphatase activity of CD45 in the TCR complex, thereby suppressing the differentiation and expansion of effector T cells while enabling the expansion of Tregs. Previous studies have shown that transient targeting of CD45 induced potent antigen-specific regulatory T cells and induced transplantation tolerance. Our data suggest that BTN2A2, expressed on APCs and DCs, is likely an endogenous ligand for CD45 on T cells. CD45 is expressed as multiple splicing products, and CD45RA and CD45RB have been thought to be replaced by CD45RO after T cell activation. These studies suggest that BTN2A2 primarily binds to the CD45RO isoform expressed on activated T cells, demonstrating the specificity of its action. Other studies have shown that several human glycoproteins, including galectin-1 and CD22, bind to CD45, but there is no clear evidence that these putative ligands regulate CD45 phosphatase activity. Other identified CD45 ligands include pUL11, a viral protein expressed by cytomegalovirus-infected cells, and the E3 / 49K protein expressed by adenovirus-infected cells. Both of these viral proteins may play a role in inhibiting antiviral immunity by blocking TCR signaling, although the exact mechanism remains unclear.

[0063] We show that BTN2A2-Fc limits the clinical symptoms of glomerular crescent formation and proteinuria in murine NTS glomerulonephritis and reduces miscarriage in a separate orthogonal mouse model, both of which are associated with the expansion of Tregs and the reduction of Th17 cell populations. CD5, a marker of T cell activation, was attenuated by BTN2A2-Fc. Our data further demonstrate that BTN2A2-Fc is a potentially useful immunomodulator with potential clinical applications in various immune-mediated diseases and transplantation. Our observations represent an important step forward in our understanding of how naturally occurring proteins can alter inflammatory events by interacting with CD45 to maintain TCR complex phosphatase activity, increasing Tregs, and reducing Th17 cell populations.

[0064] Because CD45 is also expressed on B cells, it is possible that some of the beneficial effects of BTN2A2 in our animal studies may result from its action on B cells.

[0065] Our findings suggest that binding of CD45 phosphatase to the TCR complex by BTN2A2 leads to suppression of TCR signaling, resulting in the expansion of Tregs and the suppression of Th17 cells. While we demonstrate the beneficial effects of BTN2A2 in two orthogonal models of autoimmunity / immune tolerance, our data suggest that the BTN2A2 / CD45 signaling pathway may be targeted in a wide variety of immune-mediated diseases, including inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, and transplant rejection.

[0066] Accordingly, various embodiments of the present invention are based, at least in part, on these findings.

[0067] Various embodiments provide a method of reducing CD3-dependent T cell signaling in a subject in need thereof, the method comprising administering to the subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0068] Various embodiments provide a method for increasing regulatory T (Treg) cells or decreasing T helper 17 (Th17) cells, or both, in a subject in need thereof, comprising administering to the subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0069] Based on the phylogenetic tree, BTN1A1, B2N2A1, BTN2A2, BTN3A1, BTN3A2, and BTN3A3 are closely related, with similar structures, particularly their extracellular domains. Thus, all of these isoforms, particularly their extracellular domains, are expected to have similar functions to BTN2A2 in their ability to modulate the balance between Treg and Th17 cells, and be used in accordance with various embodiments of the present invention.

[0070] In various embodiments, the subject is pregnant and the method prevents or reduces the likelihood of miscarriage of the fetus by increasing Treg cells.

[0071] In various embodiments, the subject has renal impairment.

[0072] In various embodiments, the subject is an organ, tissue, or cell therapy transplant recipient. In various embodiments, the method reduces the likelihood of organ, tissue, or cell transplant rejection. In various embodiments, the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to the subject. In various embodiments, the method reduces the toxic effects of immunosuppressive therapy on the subject.

[0073] In various embodiments, the subject has an autoimmune disorder or an inflammatory disorder. In various embodiments, the autoimmune disorder or an inflammatory disorder comprises allergy, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disease, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), psoriasis, sarcoidosis, myasthenia gravis, or HIV. In various embodiments, the autoimmune neurological disease is multiple sclerosis. In various embodiments, the autoimmune neurological disease is myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0074] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof is fused to an Fc domain or a fragment of an Fc domain (a "BTN2A2-Fc fusion protein") and / or another half-life extending moiety, e.g., PEG, albumin, lipidation, cholesterol, or glycosylation. In various embodiments, a BTN2A2-related isoform or a BTN2A2-related isoform fragment is fused to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of the Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease certain Fc-associated effector functions (a "BTN2A2-related isoform-Fc fusion protein").

[0075] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof is linked to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions (a "BTN2A2-Fc fusion protein"). In various embodiments, a BTN2A2-related isoform or a BTN2A2-related isoform fragment is linked to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions (a "BTN2A2-related isoform-Fc fusion protein").

[0076] In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO: 6)n, GGGGGSn (SEQ ID NO: 7)n, leucine zipper, r aliphatic, or helical peptide.

[0077] In various embodiments, a spacer peptide is present between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and the Fc domain or a fragment of the Fc domain. That is, a spacer is present between the BTN2A2 portion and the Fc domain portion. For example, a fusion protein includes a BTN2A2-spacer-Fc domain, or a fragment of a BTN2A2-spacer-Fc domain, or a BTN2A2 fragment-spacer-Fc domain, or a fragment of a BTN2A2 fragment-spacer-Fc domain. These examples are not intended to limit the possible combinations, but rather to provide clarity.

[0078] Examples of spacers include, but are not limited to, IEGRMDDISSTMVRS (SEQ ID NO: 56), IEGRMD (SEQ ID NO: 57), EAEAEAK (SEQ ID NO: 58), SIINFEKL (SEQ ID NO: 59), and GGGS (SEQ ID NO: 60).

[0079] In various embodiments, the Fc domain includes a mutation selected from Met208Leu, Asn214Ser, or both, according to SEQ ID NO: 4. The mutation can extend the half-life of B2N2A2Fc. (See, e.g., Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," Front Immunol. 2019;10:1296.)

[0080] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0081] In various embodiments, the BTN2A2 fragment comprises the amino acid sequence of SEQ ID NO:3.

[0082] In various embodiments, a fusion protein having SEQ ID NO: 1 or SEQ ID NO: 2 is administered according to the embodiments described herein. In various embodiments, a fusion protein having SEQ ID NO: 1 or SEQ ID NO: 2 in which the spacer IEGRMDDISSTMVRS (SEQ ID NO: 56) is replaced with IEGRM (SEQ ID NO: 57) is administered according to the embodiments described herein.

[0083] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment is modified by glycosylation or PEGylation, which modifications can increase the half-life of butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0084] Modification by PEGylation can extend the half-life of butyrophilin A2 (BTN2A2) or its BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies," Biodrugs 2008;22(5):315-329, incorporated herein by reference as if fully set forth. Thus, for example, PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include, but are not limited to, 1-5 kDa, 5 kDa, 5-10 kDa, 10-15 kDa, 15-20 kDa, 20-25 kDa, 25-30 kDa, 30-40 kDa, 40-50 kDa, 50 kDa, 50-60 kDa, and 60-75 kDa.

[0085] Another approach to improving the pharmacokinetic (PK) properties of BTN2A2 or a fragment thereof is to conjugate it with albumin, such as human serum albumin (HSA), thereby preventing proteolysis and rapid renal filtration (due to the relatively large size of HSA, approximately 66 kDa). Yet another approach to improving the pharmacokinetic (PK) properties of BTN2A2 or a fragment thereof is glycosylation. Yet another approach to improving the pharmacokinetic (PK) properties of BTN2A2 or a fragment thereof is to conjugate it with lipids or cholesterol.

[0086] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise the cytoplasmic domain. In various embodiments, the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0087] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3, or a fragment or fusion polypeptide comprising any of the foregoing, optionally an Fc-fusion polypeptide, typically an Fc-fusion polypeptide of human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease certain Fc-associated effector functions.

[0088] In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both. In various embodiments, the BTN2A2-related isoform fragment binds to CD45, eg, CD45RO.

[0089] Various embodiments of the present invention provide methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a fusion polypeptide comprising any of the foregoing, optionally an Fc-fusion polypeptide.

[0090] In various embodiments, the disease or condition is an autoimmune disorder or a disorder associated with inflammation.

[0091] In various embodiments, the autoimmune or inflammatory disorder is glomerulonephritis, inflammatory bowel disease, rheumatoid arthritis, autoimmune neurological disease, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus, myasthenia gravis, or HIV.

[0092] In various embodiments, the autoimmune neurological disease is multiple sclerosis. In various embodiments, the autoimmune neurological disease is myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0093] In various embodiments, the disease or condition is renal failure.

[0094] In various embodiments, the disease or condition is organ, tissue, or cell transplant rejection and the method reduces the likelihood of organ, tissue, or cell transplant rejection. In various embodiments, the disease or condition is organ, tissue, or cell transplant rejection and the method reduces the need for immunosuppressive therapy or a reduction in the amount of immunosuppressive therapy required by the subject. In various embodiments, treatment results in a reduction or elimination in the subject of the toxic effects of immunosuppressive therapy that would occur in the absence of the method.

[0095] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof is fused to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions (a "BTN2A2-Fc fusion protein"). In various embodiments, a BTN2A2-related isoform or a BTN2A2-related isoform fragment is fused to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions (a "BTN2A2-related isoform-Fc fusion protein").

[0096] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof is linked to an Fc domain or a fragment of an Fc domain, typically the Fc domain or a fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions ("BTN2A2-Fc fusion protein"). In various embodiments, a BTN2A2-related isoform or a BTN2A2-related isoform fragment is linked to an Fc domain or a fragment of an Fc domain ("BTN2A2-related isoform-Fc fusion protein"). In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO: 6), GGGGGSn (SEQ ID NO: 7), leucine zipper, aliphatic, or helical peptide.

[0097] In various embodiments, a spacer peptide, optionally 2 to 50 amino acids in length, is present between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and an Fc domain or fragment of an Fc domain, typically an Fc domain or fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-associated effector functions. That is, a spacer is present between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein may include a BTN2A2-spacer-Fc domain, or a fragment of a BTN2A2-spacer-Fc domain, or a BTN2A2 fragment-spacer-Fc domain, or a fragment of a BTN2A2 fragment-spacer-Fc domain. These examples are not intended to limit the possible combinations but rather to provide clarity.

[0098] Examples of spacers include, but are not limited to, IEGRMDDISSTMVRS (SEQ ID NO: 56), IEGRMD (SEQ ID NO: 57), EAEAEAK (SEQ ID NO: 58), SIINFEKL (SEQ ID NO: 59), and GGGS (SEQ ID NO: 60).

[0099] In various embodiments, the Fc domain, e.g., an IgG1 Fc domain, includes a mutation selected from Met208Leu, Asn214Ser, or both, according to SEQ ID NO: 4. The mutation can extend the half-life of B2N2A2Fc. (See, e.g., Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," Front Immunol. 2019;10:1296.)

[0100] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0101] In various embodiments, a fusion protein having SEQ ID NO: 1 or SEQ ID NO: 2 is administered according to the embodiments described herein. In various embodiments, a fusion protein having SEQ ID NO: 1 or SEQ ID NO: 2 in which the spacer IEGRMDDISSTMVRS (SEQ ID NO: 56) is replaced with IEGRM (SEQ ID NO: 57) is administered according to the embodiments described herein.

[0102] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment, BTN2A2-related isoform, or BTN2A2-related isoform fragment thereof is modified by binding to albumin, eg, human serum albumin (HAS).

[0103] In various embodiments, butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment is modified by glycosylation or PEGylation, which modifications can increase the half-life of butyrophilin A2 (BTN2A2) or a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0104] Modification by PEGylation can extend the half-life of butyrophilin A2 (BTN2A2) or its BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies," Biodrugs 2008;22(5):315-329, incorporated herein by reference as if fully set forth. Thus, for example, PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include, but are not limited to, 1-5 kDa, 5 kDa, 5-10 kDa, 10-15 kDa, 15-20 kDa, 20-25 kDa, 25-30 kDa, 30-40 kDa, 40-50 kDa, 50 kDa, 50-60 kDa, and 60-75 kDa.

[0105] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise the cytoplasmic domain. In various embodiments, the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0106] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0107] Various embodiments of the present invention provide fusion proteins comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, and an Fc domain or fragment of an Fc domain, typically an Fc domain or fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease certain Fc-associated effector functions.

[0108] In various embodiments, the fusion protein is not a fusion protein having SEQ ID NO: 1. In various embodiments, the fusion protein is not a fusion protein having SEQ ID NO: 1, where the spacer IEGRMDDISSTMVRS (SEQ ID NO: 56) is replaced with IEGRM (SEQ ID NO: 57).

[0109] In various embodiments, the Fc domain or fragment of an Fc domain is derived from an IgG1 antibody.

[0110] In various embodiments, the Fc domain or fragment of the Fc domain, typically the Fc domain or fragment of the Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, may optionally be modified to increase or decrease certain Fc-associated effector functions and may contain one or more mutations. In various embodiments, the mutation is Met208Leu, Asn214Ser, or both, according to SEQ ID NO: 4. The mutation to the Fc domain results in an extended half-life of the fusion protein.

[0111] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. Alternatively, BTN2A2 can comprise an Fc variant selected from any of the following:

[0112] Alternative Fc variants

[0113] As described above, the subject BTN2A2 polypeptides and fragments may comprise an Fc region that may optionally be mutated to modulate Fc-associated effector function. In some embodiments, one or more amino acid modifications are introduced into the Fc region, thereby generating an Fc region variant or Fc variant. The Fc region may comprise the C-terminal region of an immunoglobulin heavy chain, including the hinge region, CH2 domain, CH3 domain, or any combination thereof. As used herein, Fc region includes native-sequence Fc regions and variant Fc regions. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution, addition, or deletion) at one or more amino acid positions.

[0114] In some embodiments, the variant Fc region comprises at least one amino acid modification in the Fc region. Combinations of amino acid modifications are also useful. For example, the variant Fc region may comprise two, three, four, five, etc. substitutions at specific Fc region positions identified herein.

[0115] In some embodiments, the altered effector function is a decreased effector function. In some embodiments, the altered effector function is an increased effector function. Effector function generally refers to biological events that result from the interaction of an antibody Fc region with an Fc receptor or ligand. Non-limiting effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some instances, antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (e.g., natural killer cells, neutrophils, macrophages) recognize antibodies bound to target cells and subsequently cause lysis of the target cells. In some cases, complement-dependent cytotoxicity (CDC) refers to the lysis of target cells in the presence of complement, where the complement pathway is initiated by the binding of C1q to target-bound antibodies.

[0116] In certain cases, reducing effector function is beneficial. In some examples, modifications in the Fc region generate Fc variants with (a) reduced antibody-dependent cell-mediated cytotoxicity (ADCC), (b) reduced complement-mediated cytotoxicity (CDC), and / or (c) reduced affinity for C1q. In some embodiments, the Fc region is modified to reduce antibody-dependent cell-mediated cytotoxicity (ADCC), reduce antibody-dependent cell-mediated phagocytosis (ADCP), reduce complement-mediated cytotoxicity (CDC), and / or reduce affinity for C1q by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 29, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 (EU numbering).

[0117] [Table 1-1] [Table 1-2]

[0118] In vitro assays for assessing the ADCC activity of a molecule of interest are well known in the art. Non-limiting examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Useful effector cells for use in such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0119] In some embodiments, the variant Fc region exhibits reduced effector function compared to wild-type human IgG1. Non-limiting examples of Fc mutations in IgG1 that in certain instances reduce ADCC and / or CDC include substitutions at one or more of the following positions in IgG1: 231, 232, 234, 235, 236, 237, 238, 239, 264, 265, 267, 269, 270, 297, 299, 318, 320, 322, 325, 327, 328, 329, 330, and 331, where the numbering system for the constant region is that of the EU index as described by Kabat.

[0120] In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an N297A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an N297Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an N297D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a D265A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an S228P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L235A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L237A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L234A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an E233P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L234V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a C236 deletion according to the Kabat numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a P238A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an A327Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a P329A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a P329G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L235E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a P331S substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising an L234F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 235G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 235Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 235R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 235S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 236F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 236R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 237E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 237K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 237N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 237R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 2381 substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238V substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 238Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 248A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 2541 substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254T substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 254V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 255N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 256H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 256K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 256R substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 256V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 264S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 265H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 265K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 265S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 265Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 267G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 267H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 2671 substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 267K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 268K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 269N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 269Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 270A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 270G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 270M substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 270N substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 271T substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 272N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 279F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 279K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 279L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 292E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 292F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 292G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 292L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 293S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 301W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 304E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 31IE substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 311G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 311S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 316F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 327T substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 328V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 329Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 330R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 339E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 339L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 3431 substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 343V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 373A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 373G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 373S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 376E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 376W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 376Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 380D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 382D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 382P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 385P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 424H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 424M substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 424V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 434L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 438G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 439E substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 439H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 439Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440M substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440T Fc region substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 440V substitution according to the EU numbering system.

[0121] In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235E, G237A, A330S, and / or P331S according to EU numbering. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising E233P according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P and L235E. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L235E according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A and L235A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and G237A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and P329G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234F, L235E, and P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235E, and G237A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235E, G237A, and P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, G237A, P238S, H268A, A330S, and P331S (IgG1) according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and P329A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising G236R and L328R according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising G237A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising F241A according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising V264A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising D265A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising D265A and N297A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising D265A and N297G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising D270A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising N297A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising N297G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising N297D according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising N297Q according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising P329A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising P329G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising P329R according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising A330L according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising P331A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region. In some embodiments, the variant Fc region comprises an IgG4 Fc region. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P, F234A, and L235A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2-IgG4 cross-subclass (IgG2 / G4) Fc region. In some embodiments, the variant Fc region comprises an IgG2-IgG3 cross-subclass Fc region. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising H268Q, V309L, A330S, and P33IS according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising V234A, G237A, P238S, H268A, V309L, A330S, and P33IS according to the EU numbering system. In some embodiments, the antibody comprises an Fc region comprising high mannose glycosylation.

[0122] In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235A, and P329G (according to EU numbering). In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235E, G237A, A330S, and P331S (according to EU numbering). In some embodiments, the one or more mutations relative to the wild-type Fc region are selected from the group consisting of N297A / Q / G, L235A / G237A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, L234A / L235A / P329G, V234A / G237A / P238S / H268A / V309L / A330S / P331S, and L234F / L235E / D265A. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising an S228P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising an A330S substitution according to the EU numbering system, hi some embodiments, the variant Fc region comprises an IgG4 Fc region comprising a P331S substitution according to the EU numbering system.

[0123] In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising an A330S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising a P331S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising a 234A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising a 237A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region, and the one or more mutations are, according to EU numbering, (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y, or 329R, or (k) 331 S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238L, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 2541, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267L, or 267K, (u) 268K, (v ) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292L, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343L or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, ( ll) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 4341, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A, and G237A, (xx) L234A, L235A, and P329G, (yy) L234F, L235E,and P331S, (zz) L234A, L235E, and G237A, (aaa), L234A, L235E, G237A, and P33IS, (bbb) L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc) L234A, L235A, and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (uu).

[0124] In various embodiments, the fusion protein further comprises a spacer peptide, optionally of 2 to 50 amino acids, between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and the Fc domain or fragment of the Fc domain. That is, there is a spacer between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein includes a BTN2A2-spacer-Fc domain, a fragment of a BTN2A2-spacer-Fc domain, a BTN2A2 fragment-spacer-Fc domain, or a fragment of a BTN2A2 fragment-spacer-Fc domain. These examples are not intended to limit the possible combinations but rather to provide clarity.

[0125] Examples of spacers include, but are not limited to, IEGRMDDISSTMVRS (SEQ ID NO: 56), IEGRMD (SEQ ID NO: 57), EAEAEAK (SEQ ID NO: 58), SIINFEKL (SEQ ID NO: 59), and GGGS (SEQ ID NO: 60).

[0126] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise the cytoplasmic domain. In various embodiments, the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0127] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0128] In various embodiments, the fusion protein has the sequence of SEQ ID NO:2.

[0129] Various embodiments provide modified polypeptides comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, wherein the butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, is PEGylated, lipidated, or glycosylated.

[0130] These modifications can increase the half-life of butyrophilin A2 (BTN2A2) or a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof.

[0131] Modification by PEGylation can extend the half-life of butyrophilin A2 (BTN2A2) or its BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies," Biodrugs 2008;22(5):315-329, incorporated herein by reference as if fully set forth. Thus, for example, PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include, but are not limited to, 1-5 kDa, 5 kDa, 5-10 kDa, 10-15 kDa, 15-20 kDa, 20-25 kDa, 25-30 kDa, 30-40 kDa, 40-50 kDa, 50 kDa, 50-60 kDa, and 60-75 kDa.

[0132] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both. In various embodiments, the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0133] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

[0134] Various embodiments provide immunoassays that detect butyrophilin A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments, or combinations thereof.

[0135] In various embodiments, the immunoassay is an ELISA.

[0136] In various embodiments, the immunoassay comprises an anti-butyrophilin A2 (BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2 related isoform antibody, or an anti-BTN2A2 related isoform fragment antibody, or a combination thereof, immobilized on a solid support, and the solid support.

[0137] In various embodiments, the immunoassay further comprises a biological sample obtained from a subject. The subject may be a subject for whom information regarding levels of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment is desired.

[0138] Various embodiments provide methods of using the immunoassays of the invention for patient monitoring, comprising contacting a biological sample obtained from the patient with the immunoassay of the invention and detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof.

[0139] In various embodiments, the method further comprises selecting or performing a treatment of the invention described herein. (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, with albumin, e.g., HSA, or an Fc domain or fragment of an Fc domain, e.g., an Fc domain or fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease certain Fc-associated effector functions. a fusion polypeptide comprising (iii) a modified polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, which is PEGylated, lipidated, or glycosylated. or a modified polypeptide comprising (iv) non-BTN2A2-related treatment The method further includes selecting or performing one of:

[0140] Various embodiments provide methods of using the immunoassays of the invention for patient stratification, comprising contacting a biological sample obtained from a patient with an immunoassay of the invention, detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and stratifying the patient for treatment with a treatment of the invention described herein.

[0141] Various embodiments provide a method of using the immunoassay of the invention for patient stratification, comprising contacting a biological sample obtained from the patient with the immunoassay of the invention, detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and determining whether the patient has received one of the following treatments: (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or fragment of an Fc domain, typically an Fc domain or fragment of an Fc domain of a human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease certain Fc-associated effector functions. a fusion polypeptide comprising (iii) a modified polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, which is PEGylated, lipidated, or glycosylated. or a modified polypeptide comprising (iv) non-BTN2A2-related treatment and stratifying for treatment with one of: Wild-type amino acid sequence (SEQ ID NO: 1) (237aa BTN2A2 EC domain (bold and italic) + 15aa Xa and vector cloning sequence + 227aa Fc region (bold)) [ka] Mutant amino acid sequence with long-acting protein (SEQ ID NO: 2) (237aa BTN2A2 EC domain (bold and italic) + 2aa vector cloning sequence + 227aa Fc region (bold), underlined aa substitutions) [ka] SEQ ID NO: 3 (BTN2A2 EC domain) MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEEQMEEYRGRITFVSKDINRGSVALVIHN VTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETV SEQ ID NO: 4 (Fc region (wt)) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5 (Fc region (mutant)) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0142] In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient in combination with a therapeutically effective amount of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, fusion protein, or modified polypeptide described herein. A "pharmaceutically acceptable excipient" refers to an excipient that is generally safe, non-toxic, and desirable and useful in preparing pharmaceutical compositions, including excipients acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0143] In certain embodiments, compounds of the present invention may contain one or more acidic functional groups and, therefore, may form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts, esters, amides, and prodrugs," as used herein, refers to carboxylic acid salts, amino acid addition salts, esters, amides, and prodrugs of compounds of the present invention that are, within the scope of sound medical judgment, suitable for use in contact with patient tissues without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and that are effective for the intended uses of the compounds of the present invention. The term "salts" refers to relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in free base form with a suitable organic or inorganic acid and isolating the salt so formed. These may include cations based on alkali and alkaline earth metals, e.g., sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. (See, e.g., Berge SM, et al. (1977) J. Pharm. Sci. 66, 1, incorporated herein by reference).

[0144] The term "pharmaceutically acceptable esters" refers to the relatively non-toxic esterification products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the purified compounds in their free acid or hydroxyl forms with a suitable esterifying agent. Carboxylic acids can be converted to esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of solvation under physiological conditions, such as alkyl esters, methyl, ethyl, and propyl esters.

[0145] As used herein, a "pharmaceutically acceptable salt or prodrug" is a salt or prodrug that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or the like, and that is effective for its intended use, commensurate with a reasonable benefit / risk ratio.

[0146] The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce a functionally active peptide or peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to a biologically, pharmaceutically, or therapeutically active form of the compound. Prodrugs of one or more peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, can be designed to alter the metabolic stability or transport characteristics of one or more peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, mask side effects or toxicity, improve the taste of the compound, or modify other characteristics or properties of the compound. Given knowledge of in vivo pharmacodynamic processes and drug metabolism to yield a pharmaceutically active form of one or more peptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, those skilled in the pharmaceutical arts generally can design prodrugs of the compounds. Suitable examples of prodrugs include methyl, ethyl, and glycerol esters of the corresponding acids.

[0147] In various embodiments, pharmaceutical compositions according to the present invention can be formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal, or parenteral. "Transdermal" administration can be achieved using topical creams or ointments or by transdermal patches. "Parenteral" generally refers to routes of administration associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal. When administered via the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder. For enteral administration, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, liquids, powders, granules, emulsions, microspheres or nanospheres, or lipid or polymer vesicles that allow controlled release. For parenteral administration, the composition may be in the form of a solution or suspension for infusion or injection. For topical administration, pharmaceutical compositions based on the compounds according to the present invention may be formulated for treating the skin and mucous membranes and may be in the form of ointments, creams, emulsions, plasters, powders, impregnated pads, liquids, gels, sprays, lotions, or suspensions. They may also be in the form of microspheres or nanospheres, lipid or polymer vesicles, or polymer patches and hydrogels that allow controlled release. These topical compositions may be in either anhydrous or aqueous form depending on the clinical indication. For ocular administration, they may be in the form of eye drops.

[0148] The pharmaceutical composition according to the present invention may also contain any pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other components of the formulation. Each component of the carrier must also be suitable for use in contact with any tissue or organ it may come into contact with, meaning that it must not pose a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0149] The pharmaceutical compositions of the present invention can also be encapsulated, tableted, or prepared into emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the compositions or to facilitate their preparation. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. Carriers can also include sustained-release materials such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0150] Pharmaceutical preparations are prepared according to conventional techniques in pharmacy, including crushing, mixing, granulating, and optionally compressing for tablet formulations, or crushing, mixing, and filling for hard gelatin capsule formulations.When a liquid carrier is used, the preparation is in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension.Such liquid preparations can be administered directly or filled into soft gelatin capsules.

[0151] The pharmaceutical compositions according to the present invention can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that will produce the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, disease type and stage, general health, response to a given dosage, and type of medication), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 2012). th edition, Williams & Wilkins PA, USA (2000).

[0152] kit The present invention is also directed to kits. Thus, in some embodiments, the kit contains a composition comprising butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, a fusion protein, or a modified polypeptide described herein.

[0153] The exact nature of the components comprised in the kits of the invention will depend on their intended purpose. In one embodiment, the kit is configured specifically for the treatment of mammalian subjects. In another embodiment, the kit is configured specifically for the treatment of human subjects. In a further embodiment, the kit is configured for veterinary use to treat subjects, including, but not limited to, livestock animals, domestic animals, and laboratory animals. In another embodiment, the kit is configured for the detection of butyrophilin A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments.

[0154] Instructions for use may be included in the kit. "Instructions for use" typically include specific language describing techniques to be used in using the components of the kit to achieve a desired result. Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful equipment readily recognized by those skilled in the art.

[0155] The materials or components provided in the kit can be stored and provided to the practitioner in any convenient and suitable manner that preserves their operability and usefulness. For example, the components can be in dissolved, dehydrated, or lyophilized form, and they can be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging material(s). As used herein, the phrase "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the present invention. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of holding the individual components of the kit. Thus, for example, the package can be a glass vial used to contain a suitable amount of a composition of the present invention containing butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, a fusion protein, or a modified polypeptide described herein. In another example, the package can be a glass vial used to contain a suitable amount of a composition of the invention containing an anti-butyrophilin A2 (BTN2A2) antibody, anti-BTN2A2 fragment antibody, anti-BTN2A2-related isoform antibody, or anti-BTN2A2-related isoform fragment antibody described herein. The packaging material generally has an exterior label indicating the contents and / or purpose of the kit and / or its components. [Example]

[0156] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0157] Example 1 Materials and Methods Generation of BTN2A2-Fc construct and purification of recombinant protein Soluble recombinant BTN2A2-Fc protein was produced using a baculovirus-infected insect cell system, a scalable method that has been successfully used for the production of recombinant proteins in the immune system. A 711-bp upstream region of the human BTN2A2 gene (NP_008926.2), containing the signal peptide and two extracellular domains (IgV and IgC2), was amplified from cDNA derived from 293T cells using specific primers (see Table 1). The amplified region was first cloned into the pFUSE-hIgG1-Fc1 vector (InvivoGen, catalog no. pfuse-hg1fc1) using the Age1 and EcoRV restriction sites. The BTN2A2 region with the Fc region was then amplified using specific primers (Table 1) and cloned into the pBacPak8 vector using the Xba1 and Sac1 restriction sites. Paired-end sequencing of the clones was performed by Sanger sequencing to confirm the sequence. Primers were synthesized by Integrated DNA Technologies, Inc. (IDT), and Phusion® High-Fidelity PCR Master Mix containing HF buffer was used for all PCR amplification reactions (New England Biolabs). Baculovirus was generated by co-transfecting the pBacPak8-BTN2A2-Fc clone (TakaraBio, catalog no. 631401) with linearized baculovirus DNA into Sf9 insect cells according to the manufacturer's protocol. Baculovirus passages (P0, P1, P2) were prepared as described in the manufacturer's protocol (TakaraBio, catalog no. 631402). The P3 baculovirus stock was used for large-scale protein purification using Hi5 cells grown in serum-free medium (Express Five™ SFM, ThermoFisher, catalog no. 10486025). A Pierce™ Protein G agarose column (ThermoFisher Scientific, catalog number 20398) was used to purify the secreted soluble BTN2A2-Fc protein from the supernatant. Finally, the purified protein was suspended in 1× phosphate-buffered saline (1× PBS).Protein purity was confirmed by SDS-PAGE followed by Coomassie blue staining. The specificity of the purified protein was confirmed by Western blot analysis using a specific BTN2A2 antibody. Commercially available recombinant 293 cell-derived human BTN2A2-Fc (catalog no. 8918-BT, R&D systems) and mouse BTN2A2 (catalog no. 8997-BT-050, R&D systems) were used for comparative studies with baculovirus-produced recombinant B2N2A2-Fc. [Table 2]

[0158] T cell activation and IL-2 secretion assays In vitro T cell activation was performed. Briefly, 96-well plates were coated overnight at 4°C with anti-CD3 mAb (OKT3, clone x) at a concentration of 1 μg / ml in PBS in the absence or presence of recombinant BTN2A2-Fc (10 μg / ml) or Fc-tagged protein (Cat. No. 10702-HNAH, Sino Biological Inc.), or 293 cell-derived human BTN2A2-Fc or mouse BTN2A2. A total of 2.5 × 10 5 Jurkat cells / well were added to pre-coated flat-bottom 96-well plates. Cells were incubated for 24 hours in a 37°C incubator with 5% CO2. A total of 100 μl of medium was used to measure IL-2 production using an IL-2 ELISA kit from Millipore-Sigma (catalog no. RAB0286-1KT) according to the manufacturer's protocol.

[0159] In vitro TCR stimulation and signaling analysis For short-term activation of Jurkat cells, 48-well tissue culture plates were coated with anti-CD3 mAb (OKT3, 10 μg / mL) and BTN2A2-Fc (10 μg / mL) or Fc tag (10 μg / mL). The plates were incubated overnight at 4°C, and each well was washed twice with PBS. A total of 2 × 10 cells were then added to 75 μl of 1 × PBS. 6 Cells were added to each well for 3 minutes at 37°C. The reaction was then stopped with ice-cold PBS. Pervanadate was prepared by incubating vanadate (200 mM) and HO (200 mM) at a 1:2 ratio for 15 minutes at room temperature. Jurkat cells in 1x PBS were treated with pervanadate at a final concentration of 0.1 mM for 5 minutes in an incubator (5% CO and 37°C). The cells were then harvested and lysed in lysis buffer (RIPA or IP lysis buffer) before immunoprecipitation or immunoblotting.

[0160] Immunoprecipitation and immunoblotting Jurkat cells were activated with plate-bound anti-CD3 BTN2A2-Fc protein or plate-bound anti-CD3 Fc-tagged protein as a control, or left unactivated. Cells were harvested, washed in PBS, resuspended in IP lysis buffer (10 mM HEPES pH 7.5, 0.5 mM EDTA, 0.5% NP-40, 250 mM NaCl, 1x phosSTOP, and protease inhibitors), vortexed, incubated on ice for 30 minutes, and clarified by centrifugation at 15,000 g for 10 minutes. An aliquot of the protein lysate was used for Western blotting, and the remainder was incubated with specific antibodies or IgG controls for 4 hours at 4°C. Protein A / G agarose (ThermoFisher Scientific) was added to the lysate and incubated for an additional 2 hours. Beads were washed once in IP lysis buffer containing 0.5% NP-40 and twice in IP lysis buffer containing 0.2% NP-40. Proteins were eluted with SDS sample buffer supplemented with DTT by heating at 70°C for 5–10 min. For Western blot analysis, elution fractions from non-activated cells, cells co-activated with plate-bound anti-CD3 antibody and BTN2A2-Fc, or cells co-activated with anti-CD3 antibody and Fc tag control, and their respective input controls were blotted as described below. Approximately 12 × 10 6Jurkat cells were used for each condition and lysed in 300 ml of IP lysis buffer. For immunoprecipitation, lysates were incubated with 1 mg of antibody against the specific protein or the respective IgG control. Protein A / G agarose beads were used to pull down the complexes, which were eluted in 100 μl of sample buffer. Western blots were probed with anti-phospho-Zap70 and anti-phospho-CD3z antibodies, as well as antibodies against total Zap70 and CD3z proteins (see Table 2). For phosphatase activity, the immunoprecipitated material was directly mixed into fluorescein diphosphate tetraammonium salt substrate (FDP, catalog number F2999, Thermofisher Scientific), and activity was measured using a Molecular Devices SpectraMax® M2 plate reader as recommended by the manufacturer's protocol. [Table 3-1] [Table 3-2]

[0161] Extracellular crosslinking with BS3 (bis[sulfosuccinimidyl]suberate) was performed according to the user manual (Thermo Scientific MAN0011240). Jurkat cells were washed with PBS and then incubated with 10 μg / mL BTN2A2-Fc and Fc-tagged proteins on ice for 1 h. To induce crosslinking, BS3 (ThermoFisher) was added to a final concentration of 5 mM and incubated at room temperature for 30 min. The reaction was stopped using a final concentration of 20 mM Tris HCl, pH 7.5, for 15 min at room temperature. Cells were then washed extensively in PBS, resuspended in IP lysis buffer, and clarified by centrifugation at 14,000 g for 10 min. Protein G agarose beads were used to pull down BTN2A2-Fc and Fc-tagged proteins. The beads were washed and eluted with SDS sample buffer supplemented with dithiothreitol. Elution samples were analyzed by Western blot detection with anti-CD45 antibody.

[0162] For Western blot analysis, Jurkat cells were harvested and centrifuged. The cell pellet was resuspended in 2.5 volumes of RIPA buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.5% SDS, containing protease and phosphatase inhibitors). Total protein concentration in the lysates was determined by measuring absorbance at 595 nm in a microplate reader (Bio-Rad) using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). A total of 40–80 μg of each protein sample was used for Western blot analysis. Protein lysates prepared after Jurkat cell treatment and / or immunoprecipitation were separated on 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes. The membranes were blocked for 1 hour in blocking solution (5% BSA in PBS containing 0.1% Tween [PBST]). The membranes were incubated with primary antibodies overnight at 4°C, washed three times with PBST (10 min each), incubated with secondary antibodies at room temperature for 1 hour, and finally washed three times with PBST (10 min each). Blots were visualized using an ECL assay (ThermoFisher Scientific). The antibodies used for immunoblotting analysis are listed in Table 1.

[0163] Molecular modeling of BTN2A2 and PTPRC A molecular model of BTN2A2 was constructed using homology modeling. A search for homologous structures in the extracellular domain of BTN2A2 revealed that BTN3A2 shares approximately 47% sequence identity with BTN2A2. Using the crystal structure of BTN3A2 as a template, a three-dimensional model of BTN2A2 was constructed using the SWISS-MODEL workspace. The molecular structure of BTN2A2 was then subjected to a short 1.2-ns molecular dynamics simulation using Desmond (Schrodinger, Inc., San Diego, CA). The three-dimensional structure of PTPRC was retrieved from the Protein Data Bank (PDB code: 5FN7). RosettaDock 4.0 was used to determine the interaction between BTN2A2 and PTPRC proteins. The top 10 predicted models were then subjected to a 1.2-ns molecular simulation using Desmond, followed by minimization. The energetically most stable docking model was then used to identify potential amino acids for mutation.

[0164] CRISPR-Cas9 deletion of the CD45 gene and site-directed mutagenesis of CD45 CD45 knockdown cell lines were generated using the CRISPR-Cas9 system. Based on the CD45 protospacer adjacent motif (PAM) sequence (Table 2), two different guide RNAs complementary to exon-1 upstream and exon-3 downstream of CD45 were designed. These guide RNA sequences were cloned into the lenti-Crispr v2 blast vector, which contains the Cas9-encoding gene and a blasticidin resistance gene. Lentivirus was generated using envelope and packaging plasmids. Jurkat cells were then transduced with the lentivirus expressing the guide RNA and Cas9 gene, and blasticidin-resistant cells were selected. Single-cell cloning was performed to isolate independent clones. Deletion of exons 1 to 3 was confirmed by direct Sanger sequencing (Figure 13A).

[0165] CD45 (PTPRC) (NM_002838) human tagged ORF clone was purchased from origene technologies, Inc. Site-directed mutagenesis was performed using the QuickChange mutagenesis kit (Invitrogen) to introduce N419A and N468A mutations. The introduction of the mutations was confirmed by direct sequencing (Figure 13B). Lentivirus was generated using envelope and packaging plasmids. Jurkat cells lacking the CD45 gene were then transduced with lentivirus expressing the mutant CD45 gene.

[0166] In vitro Treg and Th17 differentiation by mixed lymphocyte reaction (MLR) For Treg differentiation, fresh spleens and lymph nodes (inguinal, brachial, and axillary) were harvested from mice and washed with 1x RPMI medium. Spleens were dissected into small pieces, and single cells were prepared by disrupting the tissue through a 70-micron strainer using the plunger end of a syringe. The cells were then suspended in 5 ml of RPMI medium. Cells were stimulated with plate-bound anti-CD3 (1 μg / ml) with or without BTN2A2-Fc fusion protein (10 μg / ml) and cocultured with irradiated CD1 mouse splenocytes for 7 days. Cells were labeled with immunofluorescent antibodies and analyzed for CD4+CD25+Foxp3-GFP coexpression using flow cytometry. CD4+ cells on days 1 and 5 incubated with anti-CD3 antibody in the absence or presence of recombinant BTN2A2-Fc were used to extract RNA for qPCR analysis. Following the protocols recommended by the manufacturers, CD4-positive T cells were isolated using the EasySep™ Mouse CD4+ T Cell Isolation Kit (Stem Cell Tech), B cells were isolated using the EasySep™ Mouse B Cell Isolation Kit (Stem Cell Tech), pan-DC cells were isolated using the EasySep™ Mouse Pan-DC Cell Enrichment Kit II (Stem Cell Tech), and T cells were isolated using the EasySep™ Mouse T Cell Isolation Kit (Stem Cell Tech).

[0167] To explore the effect of BTN2A2 on Th17 differentiation, 1 × 10 6 Spleen cells were stimulated with soluble anti-CD3 antibody (0.5 mg / ml) and irradiated CD1 mouse splenocytes (1:1 ratio) with or without recombinant BTN2A2-Fc (10 μg / ml), as well as a Th17 differentiation cytokine cocktail (TGF-b1 1.5 ng / ml, IL-6 10 ng / ml, IL-1b 10 ng / ml). Cells were cocultured for 5 days and subjected to intracellular immunofluorescence staining of RORgt (a Th17 cell marker). Cells were analyzed for CD4+RORgt coexpression using flow cytometry. A CD45 phosphatase inhibitor (compound 211, catalog no. 530197, Millipore Sigma), previously characterized as an irreversible and selective blocker of the allosteric pocket at the D1-D2 domain interface, distant from the substrate binding / catalytic site, was used at a concentration of 125 nM during T cell differentiation studies.

[0168] Flow cytometry analysis Immune cells were freshly collected from mouse spleens and lymph nodes (inguinal, brachial, and axillary). Cells were counted using a Hemavet 950FS hematology analyzer (Drew Scientific, Miami Lake, FL). Cell immunostaining and flow cytometry analysis were performed as previously described (Li et al., 2018). Briefly, cells isolated from mice or in vitro-treated cultures were harvested and stained with Ghost Dye™ Red 780 viability dye (Cell Signaling). Cells were washed extensively with 0.5% BSA in 1x PBS, resuspended in staining buffer (FBS), and further stained with fluorescent dye-conjugated antibodies against cell surface markers: anti-CD3, anti-CD4, and anti-CD25. Cells were washed with staining buffer to remove excess antibodies and then fixed / permeabilized using Mouse Foxp3 Buffer Set (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Fixed / permeabilized cells were incubated in rat serum for 15 minutes and stained with intracellular markers (Foxp3 and / or RORgt). Cells harvested from Foxp3-GFP transgenic mice were stained with anti-CD3, anti-CD4, and anti-CD25 antibodies, washed, and suspended in a buffer containing DAPI. Cells were directly analyzed for CD4+CD25+Foxp3-GFP or CD4+RORgt expression. Stained cells were analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA). Details of the commercial antibodies used in this study are listed in Table 1.

[0169] Cell proliferation and apoptosis assays CD4+ cells were isolated from freshly harvested spleens of wild-type B6 mice as described above. Cells were cultured at 10 6Cells were stained with CellTrace™ Violet dye at a concentration of 5 mM dye per ml of dilution according to the manufacturer's protocol (Invitrogen). 2.5 x 106 CellTrace™ Violet-stained cells were incubated with BTN2A2-Fc fusion protein (10 μg / ml) or Fc control (10 μg / ml) on conjugated anti-CD3 (0.5 μg / ml) and anti-CD28 (0.5 μg / ml). Cells were cultured in RPMI medium supplemented with 10% FBS and 1% Pen-Strp at 37°C and 5% CO2 for 3 days. The unstimulated parental generation (G0) is shown as the brightest peak on the far right of the histogram (Figure 14). Cells were stained with Annexin V-FITC conjugate for the apoptotic cell assay. For intracellular staining of IL2, cells were fixed, permeabilized, stained with IL2 antibody, and analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA).

[0170] Immunofluorescence studies Immunofluorescence staining for CD45 and CD3ζ was performed. Anti-CD3 antibody (10 mg / ml) and recombinant BTN2A2-Fc (10 mg / ml) were coated onto glass poly-L-lysine coverslips in PBS overnight at 4°C. 1 x 10 cells / ml in 75 μl of 1 x PBS were used. 6Jurkat cells were exposed to the coated coverslips for 3 minutes at 37°C and fixed using a Cytofix / Cytoperm fixation / permeabilization kit (BD Biosciences). The coverslips were then gently washed twice with 1x PBS. The cells were blocked with blocking buffer (5% goat / mouse serum in 0.3% Triton-X100 in 1x PBS) and stained with mouse anti-human CD45 (Abcam, catalog no. ab8216) and rabbit anti-human CD3ζ (Abcam, catalog no. Ab226263). The coverslips were then gently washed three times with 1x PBS for 10 minutes each and stained with donkey anti-mouse IgG antibody Alexa Fluor™ 555 and goat anti-rabbit IgG antibody Alexa Fluor™ 488 (ThermoFisher Scientific). The coverslips were then gently washed three times with 1x PBS for 10 minutes each, incubated in DAPI for 5 minutes, and mounted using ProLong™ Diamond Antifade Mountant (ThermoFisher Scientific). The glass slides were then imaged using a Zeiss LSM780 confocal microscope at 63x resolution using oil immersion. Images were analyzed as 8-bit images, and the intensity of the red and green channels was measured in grayscale fluorescence units ranging from 0 to 255. Colocalization analysis was performed using the Image-J plugin "Colocalization Finder." At least seven fields were analyzed per experimental condition, with an average of 50 T cells analyzed to obtain the average amount of colocalization.

[0171] Quantitative polymerase chain reaction (QPCR) For gene expression analysis, total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific). cDNA was synthesized from 500 ng of total RNA using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific) on a QuantStudio Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as an internal control. Gene expression was compared using the ΔΔCt method. Primer details are listed in (Table 2).

[0172] Animal models All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols used in this study were approved by the Animal Care and Use Committee of Cedars-Sinai Medical Center.

[0173] Foxp3EGFP (C.Cg-Foxp3 tm2Tch / J (strain number 006769) mice: Foxp3EGF mice, which co-express EGFP and the regulatory T cell-specific transcription factor Foxp3 under the control of an endogenous promoter, were obtained from the Jackson Laboratory.

[0174] BTN2A2 knockout mice (- / -): BTN2A2(- / -) mice (strain: C57BL / 6J-Btn2a2<em>1< / em>cyagen) were generated by deleting a 10,460 base pair region of the btn2a2 gene, including exons 2 to 8, using CRISPR-Cas9 technology at a commercial facility (Cyagen Biosciences) (Figure 16). A set of three PCR primers (Table 1) was used to identify the genotype of the mice using polymerase chain reaction. In agarose gel electrophoresis, wild-type mice showed a single 500 bp band, heterozygotes showed two bands of 700 bp and 500 bp, and homozygotes showed a single 500 bp band (Figure 16).

[0175] Glomerulonephritis Model: The details of the protocol for inducing crescentic glomerulonephritis (GN) have been previously described. Nephrotoxic serum was generated in rabbits by repeated immunization with purified glomeruli in complete and incomplete Freund's adjuvant. Mice were pre-immunized with normal rabbit IgG and complete Freund's adjuvant 5 days before administration of nephrotoxic serum. Nephrotoxic serum nephritis was induced on day 0 by intravenous injection of 20 ml or 10 ml of nephrotoxic serum. Four doses of 25 mg BTN2A2-Fc fusion protein or vehicle (control) were injected i.p. on days 0, 2, 4, and 6 after nephrotoxic serum injection. Mice were sacrificed on day 7, and tissues, urine, blood cells, and plasma were collected. Immune cells were collected from fresh whole spleens and lymph nodes (inguinal, brachial, and axillary), and CD4+ T cells were isolated using the EasySep™ Mouse CD4+ T Cell Isolation Kit (STEM CELL TECH). Protein lysates of flash-frozen tissue in liquid nitrogen were prepared by homogenizing 5 mg of tissue in 500 μl of ice-cold RIPA lysis buffer. The lysates were stirred at 4°C for 2 hours, centrifuged at 16,000 g for 20 minutes at 4°C, and the supernatant was collected. Approximately 80 mg of tissue lysate was used to detect IL17A expression using Western blot.

[0176] To obtain the urinary protein-to-creatinine ratio (mg / mg) for assessment of proteinuria, urinary protein was measured with a protein assay dye (No. 500-0006, Bio-Rad, Hercules, CA), and urinary creatinine was measured using a creatinine assay kit (No. DICT-500, BioAssay Systems, Hayward, CA). Fibrinoid necrosis (a precursor lesion to crescents, defined by findings of GBM rupture, fibrin deposition, and karyorrhexis) and crescents were assessed in all glomeruli on one paraffin section from each mouse using periodic acid-methenamine silver and PAS staining, respectively. Representative images were captured using a light microscope (Nikon Eclipse 50i, Nikon, Tokyo, Japan). The histopathological diagnosis of GN was evaluated by a board-certified renal pathologist (MY).

[0177] Abortion model: DBA / 2 and CBA / J mice were purchased from Jackson Laboratory. We mated male DBA / 2 mice with female CBA / J mice to record immunologically mediated pregnancy loss as described elsewhere (Zenclussen et al., 2005). As a control, we mated female DBA / 2 mice with male CBA / J mice and evaluated litter size, pup weight, and fetal resorption rate. To study the therapeutic effect of BTN2A2, four doses of 25 mg of BTN2A2-Fc fusion protein were intraperitoneally injected into CBA / J female mice mated with male DBA / 2 mice on days 6, 9, 12, and 15 of pregnancy. The animals were sacrificed on day 18 of pregnancy, and blood / tissues were collected for molecular analysis.

[0178] statistics Data were graphed and statistics were performed using GraphPad Prism version 9.2. Data are expressed as mean ± standard deviation (SD) as indicated. Data were tested for normality using the Shapiro-Wilk test. An unpaired t-test was used to compare data between two groups that passed the normality test; otherwise, a Mann-Whitney test was used. For data from three or more groups, a one-way ANOVA test with Tukey's test for multiple comparisons was used for data that passed the normality test; for data that did not, a Kruskal-Wallis test followed by Dunn's test for multiple comparisons was used for comparison. Statistical significance was defined as p<0.05 (*), p<0.01 (**), or p<0.001 (***). The number of experiments per experiment is indicated in the figure legends.

[0179] Example 2 Human studies This study was approved by the Institutional Review Board at Cedars-Sinai Medical Center.

[0180] Treg assay: Whole blood was collected from healthy, unrelated individuals, and peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll-Hypaque gradient centrifugation. Stimulator PBMCs were irradiated and then exposed to responder PBMCs at a 1:1 ratio (1 × 10) in the absence or presence of 0 or 10 mg / ml BTN2A2-Fc. 6 The MLR mixture was then incubated for 7 days for Treg measurement. First, the MLR mixture was stained with antibodies against CD45, CD3, CD4, CD25, and CD127. After permeabilization, the cells were stained with antibodies against Foxp3. After cell acquisition by flow cytometry, lymphocytes separated from CD45+ leukocytes were plotted against CD4. CD4+ cells were plotted as CD25 vs. CD127, and then CD25+CD127 was plotted. 低 / - Cells were plotted against Foxp3. CD4+ / CD25+ / CD127 低 / - / Foxp3+ cells were designated as Treg cells. Treg cell levels were expressed as % Treg cells among CD4+ T cells.

[0181] Th17 assay: Human PBMCs (responder cells) isolated by Ficoll-Hypaque gradient centrifugation were cultured at a 1:1 ratio (1 × 106 / ml of each PBMC) on irradiated stimulator cells with a Th17 differentiation cytokine cocktail (anti-CD3 antibody 0.5 μg / ml, TGFb 1.5 ng / ml, IL-6 10 ng / ml, IL-1b 10 ng / ml) for 5 days, with or without recombinant BTN2A2-Fc (10 mg / ml). Cells were stained for surface markers CD3 and CD4. After permeabilization, cells were stained with anti-RORgt antibody and analyzed for CD4+RORgt co-expression using flow cytometry. Th17 cells were expressed as the % of RORgt-positive cells among CD4 T cells. A CD45 phosphatase inhibitor was used at a concentration of 125 nM during T cell differentiation studies.

[0182] Example 3 result BTN2A2 blocks CD3-dependent signaling in Jurkat cells To elucidate the mechanism underlying BTN2A2's ability to regulate T cell immunity (Ammann et al., 2013; Sarter et al., 2016), we generated a soluble recombinant human BTN2A2-Fc fusion protein using a baculovirus expression system. Consistent with previous reports in murine systems (Ammann et al., 2013), recombinant human BTN2A2-Fc, but not recombinant Fc control protein, blocked anti-CD3-induced IL-2 production by Jurkat cells (Figures 10A–10C).

[0183] Next, when we evaluated signaling components downstream of the TCR, we observed that BTN2A2-Fc reduced anti-CD3-induced phosphorylation of Zap70 and CD3z in Jurkat cells (Figure 10D). Addition of the phosphatase inhibitor pervanadate resulted in hyperphosphorylation of Zap70 and CD3z at baseline (unstimulated), and in contrast to the findings in the absence of pervanadate, addition of BTN2A2-Fc to the cultures had no effect on the hyperphosphorylated proteins (Figure 11). Together, these data suggest that BTN2A2-Fc activates phosphatases that downregulate TCR signaling.

[0184] BTN2A2 blocks TCR signaling by binding to and enhancing CD45 phosphatase Based on known molecular mechanisms linking CD45 phosphatase activity to TCR signaling (Jung et al., 2021; Leupin et al., 2000), we tested the hypothesis that the inhibitory effect of BTN2A2 on TCR activation is mediated through its interaction with CD45. Western blotting of CD45-associated proteins after co-immunoprecipitation (co-IP) of CD45 in unstimulated Jurkat cells demonstrated that in the absence of TCR activation, CD45 normally associates with several components of the TCR complex (Zap70 and CD3z). However, activation of the TCR complex with an anti-CD3 antibody resulted in the dissociation of CD45 from Zap70 and CD3z. In contrast, TCR activation in the presence of BTNT2A2 left CD45 associated with the TCR complex (Figure 1A), limiting T cell activation. Immunocytochemistry confirmed the colocalization of CD45 and CD3z in untreated cells at baseline, the segregation of CD45 after anti-CD3-induced TCR activation, and the maintenance of CD45 and CD3z colocalization when cells were stimulated in the presence of BTN2A2, suggesting that BTN2A2 prevents the exclusion of CD45 from the immune synapse during the early stages of T cell activation (Figure 1B).

[0185] Co-IP experiments further demonstrated that, compared with unstimulated cells, anti-CD3 activation reduced the association of CD3e with CD45, whereas TCR activation in the presence of BTN2A2 amplified the interaction of CD45 with CD3e (Figure 1C). Functional experiments observing TCR-associated CD45 phosphatase activity showed that when TCR complexes were isolated by co-IP with anti-CD3e antibodies, CD45 phosphatase activity was detectable at baseline, decreased after TCR activation, and maintained when cells were activated in the presence of BTN2A2, suggesting dissociation of CD45 from the TCR complex after activation and its retention in the presence of BTN2A2 (Jung et al., 2021; Leupin et al., 2000) (Figure 1D). Given previous work showing that activation of CD45 phosphatase on lipid microdomains on the surface of T cells results in decreased sensitivity of TCR-mediated signaling (He et al., 2002), our new data demonstrate that BTN2A2 suppresses TCR signaling by activating CD45 phosphatase activity at the TCR complex, thereby preventing crucial phosphorylation events downstream of the TCR.

[0186] BTN2A2 interacts with CD45 phosphatase Next, we used a co-IP experimental strategy using Jurkat cells to verify the direct interaction between CD45 and endogenously expressed BTN2A2 (Figure 2A). These assays showed that CD45 co-immunoprecipitated with endogenous BTN2A2 in unstimulated Jurkat cells, and binding was significantly enhanced in activated cells. Anti-CD3 activation of Jurkat cells did not alter endogenous CD45 or BTN2A2 expression levels (Figure 12). Complementation experiments using cells treated with exogenous BTN2A2-Fc protein or an Fc protein control confirmed that exogenous BTN2A2-Fc, but not the control Fc-tagged protein, bound to CD45 in activated T cells (Figure 2B-C). We then performed molecular modeling to identify regions in the extracellular domain of CD45 that could interact with BTN2A2. Protein homology modeling revealed that BTN2A2 interacts with the extracellular fibronectin domain of the CD45 protein, and that amino acids Asn-419 and Asn-468 (putative N-glycosylation sites based on their location within the NXS / T consensus motif) are crucial for this interaction (Figure 2D). We then performed site-directed mutagenesis of asparagine to alanine and confirmed that the interaction of mutant CD45 with BTN2A2 was significantly attenuated (Figure 13 and Figures 2E-F).

[0187] Based on our observation that CD45 binds to endogenous BTN2A2 after activation, we hypothesized that BTN2A2 preferentially bound the CD45RO isoform, since CD45RO is predominantly expressed on activated T cells (Hermiston et al., 2003) and is upregulated after Jurkat cell activation (Figure 2G). Taking this into consideration, we repeated the pull-down assay using isoform-specific antibodies (Figure 2H). These experiments confirmed that BTN2A2 preferentially bound the CD45RO isoform, which is the predominant isoform in activated T cells.

[0188] BTN2A2 enhances regulatory T cell expansion and suppresses Th17 cell populations in primary murine T cells Activated CD4+ T cells differentiate into several effector cell subsets based on the activation events and cytokine environment present during activation. Because BTN2A2 has previously been reported to induce Foxp3 expression in CD4+ T cells (Ammann et al., 2013), in short-term culture studies, we examined whether T cells activated in the presence of BTN2A2 would increase the Treg population in splenocytes from Foxp3-EGFP mice, which co-express EGFP when expressing Foxp3. We cultured mouse T cells (Foxp3+ reporter mice—BALB / cJ strain) in the presence of irradiated allogeneic mouse splenocytes (CD1 strain) with or without recombinant BTN2A2-Fc. These assays demonstrated a 50% increase in CD4+CD25+Foxp3+ T cells in BTN2A2-Fc-treated cells compared to controls, a magnitude comparable to that observed in positive control cultures using exogenous TGF-β (Figure 3A). Interestingly, BTN2A2-Fc activated CD4 + Short-term exposure to T cells resulted in reduced proliferation and improved survival, with multiple phenotypes observed in a prodifferentiated state, comparable to those reported for TGF-β (McKarns and Schwartz, 2005) (Figure S14). Parallel assays showed that recombinant BTN2A2 blocked IL-6 / TGF-β / IL-1β-induced production of RORgt, a signature transcription factor for Th17 cells (Figure 3B).

[0189] To further explore the short-term effects of BTN2A2 on T cell subset expression patterns in mixed lymphocyte reaction (MLR) culture conditions, we assessed gene expression patterns of molecules related to Th1, Th2, Treg, and Th17 cells by RT-PCR. BTN2A2 upregulated IL2Rbeta and Foxp3 and downregulated IL-21, consistent with our in vitro findings of Treg expansion and Th17 cell suppression (Figure 15A-C). The assay further demonstrated downregulation of transcription factors controlling the Th1 (TBX21) and Th2 (GATA3) differentiation pathways at day 5 (Figure 15D-F).

[0190] To explore the role of endogenous BTN2A2, we genetically knocked out mouse BTN2A2 using a CRISPR-Cas9 strategy (Figure 16). We confirmed the absence of BTN2A2 expression in professional APCs (CD11c+ dendritic cells and CD19+ B cells) in knockout mice compared with wild-type littermates (Figure 17). Interestingly, when APCs isolated from BTN2A2- / - animals were cocultured with mouse T cells from Foxp3+ reporter mice, the absence of BTN2A2 in both isolated dendritic cells and isolated B cells from BTN2A2- / - animals reduced the differentiation of mouse T cells into Foxp3+ Tregs compared with dendritic cells and B cells from wild-type littermates (Figure 3C-D).

[0191] The improvement in Treg / Th17 balance induced by BTN2A2 depends on CD45 phosphatase activity. To test whether CD45 phosphatase activity is essential for the effects of BTN2A2 on TCR signaling and Treg expansion, we used a previously validated CD45-specific phosphatase inhibitor (Perron et al., 2014). Addition of BTN2A2-Fc to activated Jurkat T cells reduced ZAP70 kinase phosphorylation, but inhibition of CD45 phosphatase activity with this small molecule inhibitor (Perron et al., 2014) abolished this effect (Figure 4A). When CD45 phosphatase activity was inhibited during BTN2A2-Fc treatment, we also observed a complete abolition of the effects of BTN2A2-Fc on Treg expansion and Th17 population reduction (Figure 4B-C). Taken together with the other experiments described above, these studies link the effects of BTN2A2 on CD45 phosphatase activity within the TCR complex and immune synapse to the balance of Treg and Th17 cells.

[0192] BTN2A2-Fc therapy exhibits immunoregulatory functions in vivo To examine whether and how the immunoregulatory effects of BTN2A2 observed in vitro correlate with in vivo findings, we used two distinct mouse model systems. Consistent with previous reports (Saito et al., 2022; Tipping and Holdsworth, 2006), injection of nephrotoxic serum (NTS) into wild-type B6 mice induced crescentic glomerulonephritis accompanied by significant proteinuria (Figures 5A–D). Administration of BTN2A2-Fc for several days after NTS administration reduced proteinuria and glomerular crescent formation. BTN2A2-Fc administration also increased CD4+ Foxp3 gene expression (Figure 5E), decreased CD4+ RORgt gene expression (Figure 5F), and attenuated the level of the T cell activation marker CD5 (Figure 5G) in CD4+ T cells purified from spleen and lymph nodes. Furthermore, BTN2A2-Fc reduced IL17A protein expression in the kidney compared to controls (Figure 5G-I).

[0193] Next, we administered a lower dose of NTS (50% less than the amount used in the above experiments) to BTN2A2- / - mice (Figure 16) and wild-type littermate controls (Figure 6). These experiments demonstrated that the lower dose of NTS induced relatively mild crescentic glomerulonephritis and proteinuria in wild-type control animals, whereas in BTN2A2- / - animals, we observed exacerbated crescentic glomerulonephritis and severe proteinuria (Figures 6A-C). We also observed reduced Foxp3 gene expression and enhanced RORgt gene expression in spleen / lymph node CD4+ T cells from BTN2A2- / - mice compared with controls (Figures 6D-E). CD5 expression was enhanced in CD4+ T cells from BTN2A2- / - mice compared with wild-type mice (Figure 6F). Analysis of kidney tissue 1 week after administration of nephrotoxic serum also showed increased IL17A protein expression in the kidneys of BTN2A2 − / − compared with control animals ( Figure 6G-H ).

[0194] To assess the generalizability of the role of BTN2A2 in immune regulation, we studied the effects of recombinant BTN2A2-Fc in DBA / 2 mice crossed with CBA / J mice, a model of immunologically mediated abortion (Clark et al., 2008). We observed that DBA / 2 males × CBA / J females had reduced litter size and higher spontaneous resorption rates compared with CBA / J males × DBA / 2 females. Administration of BTN2A2-Fc improved litter size and normalized the excessive abortion rate observed in DBA / 2 males × CBA / J females (Figures 7A-B and 18). The beneficial effects of BTN2A2-Fc were associated with increased frequencies of spleen / lymph node Foxp3+ Tregs, decreased frequencies of spleen / lymph node Th17 cells, and relative attenuation of CD5 expression, consistent with our findings in the autoimmune GN model (Figures 7D-F). In addition, BTN2A2-Fc protein also reduced IL-17 protein expression in the placenta, which correlated with improved litter size (FIGS. 7G-H).

[0195] BTN2A2 enhances Treg cell expansion and suppresses Th17 cell populations in human PBMCs To assess whether the effects of recombinant BTN2A2 apply to human T cells, we analyzed human PBMCs activated by mixed lymphocyte reaction (MLR, using allogeneic stimulator cells) with and without recombinant BTN2A2-Fc, and after 7 days, the CD4+CD25+CD127 低 / - The number of Foxp3+ Treg cells was quantified. These cocultures showed that BTN2A2-Fc induced a two-fold increase in Tregs compared with controls under MLR conditions, but BTN2A2-Fc failed to induce Tregs in the presence of a CD45 inhibitor (Figure 8A-B). Parallel experiments revealed that recombinant BTN2A2-Fc potently blocked the expansion of Th17 cells induced by TGFβ-IL6-IL1b (Figure 8C-D). Interestingly, BTN2A2-Fc failed to block cytokine-induced Th17 cells in the presence of a CD45 inhibitor. Taken together, these observations suggest that CD45 phosphatase activity is required for BTN2A2 action in humans.

[0196] Various embodiments of the present invention have been described above in the detailed description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive of modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included therein. Unless specifically stated otherwise, it is the inventors' intention that the words and phrases in this specification and claims be given the ordinary and familiar meaning to those skilled in the relevant technical field(s).

[0197] The foregoing description of various embodiments of the present invention known to the applicant at the time of filing this application has been presented for purposes of illustration and description, and is intended to be so. The description is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described embodiments serve to illustrate the principles of the present invention and its practical application, and to enable those skilled in the art to utilize the invention in various embodiments and in various modifications suitable for the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0198] While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the present invention and its broader aspects, and therefore, the appended claims are intended to encompass within their scope all such changes and modifications as are within the true spirit and scope of the present invention. As used herein, the terms "comprising" or "comprises" are used in the context of compositions, methods, and their respective component(s) that are useful in certain embodiments, but are open-ended with respect to the inclusion of any unspecified elements, whether useful or not. In general, it will be understood by those skilled in the art that the terms used herein are generally intended as "open-ended" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). The open-ended term "comprising," as a synonym for terms such as including, containing, or having, is used herein to describe and claim the invention, although the invention or embodiments thereof may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0199] Unless otherwise stated, the terms "a," "an," and "the," and similar references, when used in the context of describing particular embodiments of the present application (particularly in the context of the claims), can be construed to encompass both the singular and the plural. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples provided with respect to certain embodiments herein, or the use of illustrative language (e.g., "such as"), are intended merely to better clarify the application and do not impose limitations on the scope of the application as otherwise claimed. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0200] "Optional" or "optionally" means that the thing described thereafter may or may not occur, and thus the description includes cases where the thing occurs and cases where it does not occur.

[0201] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limiting. The members of each group may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for convenience and / or patentability. When any such inclusion or deletion is made, the specification is herein deemed to contain the modified group and, therefore, fulfills all Markush group descriptions used in the appended claims.

Claims

1. 1. A method of reducing CD3-dependent T cell signaling in a subject in need thereof, comprising: administering to said subject butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide comprising any of the foregoing. The method comprising:

2. 1. A method for increasing regulatory T (Treg) cells or decreasing T helper type 17 (Th17) cells, or both, in a subject in need thereof, comprising: administering to said subject butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide comprising any of the foregoing. The method comprising:

3. 3. The method of claim 1 or 2, wherein the BTN2A2, the fragment thereof, the BTN2A2-related isoform, the BTN2A2-related isoform fragment, or the fusion polypeptide comprises human BTNA2 or a fragment or isoform thereof.

4. 10. The method of any one of the preceding claims, wherein the subject is pregnant and the method prevents or reduces the likelihood of miscarriage of the fetus by increasing Treg cells.

5. 10. The method of any one of the preceding claims, wherein the subject has renal impairment or a condition associated with renal impairment.

6. 10. The method of any one of the preceding claims, wherein the subject is an organ, tissue or cell transplant recipient.

7. 7. The method of claim 6, wherein the method reduces the likelihood of organ, tissue or cell transplant rejection, and / or an autoimmune or inflammatory response.

8. 10. The method of any one of the preceding claims, which reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to said subject.

9. 10. The method of claim 8, wherein the toxic effects of the immunosuppressive therapy in the subject are reduced or eliminated in the absence of the method.

10. 10. The method of any one of the preceding claims, wherein the subject has an autoimmune or inflammatory disorder, or a disorder characterized by the accumulation of pathogenic Th1 / Th17 cells.

11. 11. The method of claim 10, wherein the autoimmune disorder or the inflammatory disorder, or the disorder characterized by the accumulation of pathogenic Th1 / Th17 cells comprises allergy, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disease, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV.

12. 12. The method of claim 11, wherein the autoimmune neurological disease is multiple sclerosis or myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

13. 1. A method of treating a disease or condition in a subject in need thereof, comprising: administering to said subject butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment thereof. The method comprising:

14. 14. The method of claim 13, wherein the disease or condition is an autoimmune or inflammatory disorder.

15. 15. The method of claim 14, wherein the autoimmune disorder or the inflammatory disorder comprises allergy, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disease, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, nutritional disorders, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV.

16. 16. The method of claim 15, wherein the autoimmune neurological disease is multiple sclerosis or myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

17. 14. The method of claim 13, wherein the disease or condition is associated with renal damage.

18. 14. The method of claim 13, wherein the disease or condition is organ transplant rejection and the method reduces the likelihood of said organ transplant rejection.

19. 14. The method of claim 13, wherein the disease or condition is organ transplant rejection and the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy required by the subject.

20. 20. The method of claim 19, wherein the toxic effects of the immunosuppressive therapy are reduced or eliminated in the subject.

21. 10. The method of any one of the preceding claims, wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked to albumin, optionally human serum albumin.

22. 10. The method of any one of the preceding claims, wherein the Butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked or fused to an Fc domain or a fragment of the Fc domain (a "BTN2A2-Fc fusion protein"), or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or a fragment of the Fc domain (a "BTN2A2-related isoform-Fc fusion protein"), wherein the Fc domain is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, optionally mutated, and further optionally mutated to enhance or decrease Fc-associated effector function, and further optionally mutated to any of the mutations shown in Table 3.

23. 23. The method of claim 22, wherein the BTN2A2 or the BTN2A2 fragment thereof is linked to an Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2 to 50 amino acids, or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, wherein the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO: 6)n, GGGGGSn (SEQ ID NO: 7)n, leucine zipper, r-aliphatic, or helical peptide.

24. 10. The method of any one of the preceding claims, wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is modified by glycosylation or PEGylation or lipidation, or by conjugation with cholesterol.

25. 10. The method of any one of the preceding claims, wherein the BTN2A2 fragment comprises the extracellular domain of BTN2A2.

26. 10. The method of any one of the preceding claims, wherein the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

27. 10. The method of any one of the preceding claims, wherein the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

28. 10. The method of any one of the preceding claims, wherein the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

29. Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; an Fc domain or a fragment of said Fc domain, which is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, and which is optionally mutated, and further optionally mutated to enhance or decrease Fc-associated effector function, and further optionally with any of the mutations shown in Table 3; A fusion polypeptide comprising:

30. 30. The fusion polypeptide of claim 29, further comprising a linker between the BTN2A2, the BTN2A2 fragment, the BTN2A2-related isoform, the BTN2A2-related isoform fragment, and the Fc domain or the fragment of the Fc domain, which is optionally an IgG1, IgG2, IgG3, or IgG4 Fc, and which is optionally mutated and further optionally mutated to enhance or decrease Fc-associated effector function, and further optionally has any of the mutations shown in Table 3.

31. 31. The fusion protein of claim 30, wherein the Fc domain or the fragment of the Fc domain is derived from an IgG1 antibody.

32. 32. The fusion protein of claim 29 or 31, wherein the Fc domain or the fragment of the Fc domain comprises one or more mutations.

33. 33. The fusion protein of claim 32, wherein the mutation is Met208Leu, Asn214Ser, or both, based on SEQ ID NO:

4.

34. 34. The fusion protein of any one of claims 29 to 33, further comprising a spacer peptide between the BTN2A2, the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment and the Fc domain or the fragment of the Fc domain.

35. 35. The fusion protein of any one of claims 29 to 34, wherein the spacer peptide is selected from the group consisting of IEGRMDDISSTMVRS (SEQ ID NO: 56), IEGRMD (SEQ ID NO: 57), EAEAEAK (SEQ ID NO: 58), SIINFEKL (SEQ ID NO: 59), and GGGS (SEQ ID NO: 60).

36. 36. The fusion protein of any one of claims 29 to 35, wherein the BTN2A2 fragment comprises the extracellular domain of BTN2A2.

37. 34. The fusion protein of any one of claims 29 to 33, wherein the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

38. 36. The fusion protein of any one of claims 29 to 35, wherein the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

39. 39. The fusion protein of any one of claims 29 to 35 and 38, wherein the BTN2A2-related isoform fragment does not include a transmembrane domain, a cytoplasmic domain, or both.

40. Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof Including, the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or the combination thereof, is PEGylated or glycosylated; Modified polypeptides.

41. 41. The modified polypeptide of claim 40, wherein the BTN2A2 fragment comprises the extracellular domain of BTN2A2.

42. 42. The modified polypeptide of claim 40 or 41, wherein the BTN2A2 fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

43. 43. The modified polypeptide of claim 41 or 42, wherein the BTN2A2 fragment binds to CD45.

44. The modified polypeptide of claim 4040, wherein the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

45. 45. The modified polypeptide of claim 40 or 44, wherein the BTN2A2-related isoform fragment does not include the transmembrane domain, the cytoplasmic domain, or both.

46. an anti-butyrophilin A2 (BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2 related isoform antibody, or an anti-BTN2A2 related isoform fragment antibody, or a combination thereof, immobilized on a solid support; the solid support; An immunoassay comprising:

47. 47. The assay of claim 46, further comprising a biological sample obtained from a subject.

48. 44. A method of using the assay of claim 43 for patient monitoring, comprising: contacting a biological sample obtained from a patient with the assay of claim 43; detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; The method comprising:

49. (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or a fragment of the Fc domain. the fusion polypeptide comprising (iii) a modified polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or a combination thereof, is PEGylated or glycosylated. or the modified polypeptide comprising (iv) non-BTN2A2-related treatment 49. The method of claim 48, further comprising selecting or performing a treatment selected from:

50. 47. A method of using the assay of claim 46 for patient stratification, comprising: contacting a biological sample obtained from a patient with the assay of claim 43; detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; The patient (i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, and an Fc domain or a fragment of the Fc domain. the fusion polypeptide comprising (iii) a modified polypeptide, Butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or a combination thereof, is PEGylated or glycosylated. or the modified polypeptide comprising (iv) non-BTN2A2-related treatment and stratifying for treatment with The method comprising:

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