Lactolipoprotein A2 and related isoforms for treatment of autoimmunity and inflammation

Abstract

Described herein are methods of reducing CD3-dependent T cell signaling in a subject in need thereof. Also described are methods of increasing T regulatory (Treg) cells or decreasing T helper 17 (Th17) cells. The methods involve administering to the subject a lactophilic lipoprotein A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a conjugate or fusion polypeptide comprising any of the foregoing. These methods are useful for treating diseases with autoimmune and inflammatory disorders such as allergy, asthma, glomerulonephritis, inflammatory bowel disease, rheumatoid arthritis, autoimmune or inflammatory nervous system disease, antibody-mediated transplant rejection, infant cholestasis, hemophagocytic lymphocythemia, erythrocytophagocythemia, malnutrition, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia, hyperplasia and hyperplasia. The traditional Chinese medicine composition is beneficial to patients suffering from systemic lupus erythematosus (lupus), psoriasis, myasthenia gravis or HIV. Further described are fusion proteins having BTN2A2 and an Fc domain.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application includes a claim of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 449,693, filed March 3, 2023, the entirety of which is hereby incorporated by reference.

[0003] REFERENCE TO SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in computer readable form and is herein incorporated by reference in its entirety. The name of the file containing this Sequence Listing is “065472_000918WOPT_SequenceListing.xml”. The file is 55,203 bytes and was created on February 28, 2024. The information contained in the computer readable form is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0005] The present invention relates to butyrophilin A2, isoforms thereof, and fragments and conjugates thereof, for use in the treatment of autoimmune and inflammatory disorders. BACKGROUND

[0006] 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 description herein includes information that can be useful in understanding the present application. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed application, or that any publication specifically or implicitly referenced is prior art.

[0007] Currently, treatment of autoimmunity is largely non-specific, using steroids and drugs like cyclosporine and prograf. While newer agents such as CTLA4IG have some promise, there remains a need for additional drugs that can, for example, act synergistically to improve autoimmunity and inflammation.

[0008] T cells recognize antigens presented through peptide:MHC complexes using a surface-expressed heterodimeric abT cell receptor (TCR). Since the TCR lacks intrinsic kinase activity, signals initiated by TCR ligation involve recruitment of the Src family kinase Lck, which then phosphorylates immunoreceptor tyrosine-based motif (ITAM)-based motifs within the TCR-associated CD3 z chain. Lck also phosphorylates the subsequently recruited Zap70 kinase, propagating the necessary downstream signals required for full T cell activation. Studies conducted over the past 30 years have shown that T cell activation is in part controlled by CD45, which is expressed on the cell surface.

[0009] CD45 is a transmembrane glycoprotein containing an intracellular tyrosine phosphatase domain capable of dephosphorylating multiple TCR immune receptor tyrosine activation (ITAM) motifs. Differential splicing leads to the expression of multiple CD45 isoforms (RA, RB, RC, RO). Upon TCR stimulation, CD45 is initially recruited to the supramolecular activation cluster (SMAC) but is subsequently expelled, dissociating it from the TCR. Evidence suggests that this dissociation of CD45 phosphatase activity from the TCR is crucial for Lck-initiated signaling propagation, which leads to full T cell activation. Conversely, CD45 retention within the T cell immune synapse (IS) regulates the intensity and duration of TCR activation, and perturbations of CD45 activity can contribute to the development of autoimmune diseases. In humans, abnormal CD45 expression has been reported in diseases including infantile cholestasis, hemophagocytic lymphohistiocytosis or erythrocytic hemophagocytosis, malnutrition, 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 been reported to have direct clinical applications in organ transplantation, treatment of autoimmune diseases, or microglial activation associated with Alzheimer's disease (AD) (A (e.g., "CD45 in human physiology and clinical medicine", Immunology Letters, 196:22-32, April 2018). However, despite decades of elucidation of these molecular mechanisms by multiple groups, it remains unclear how the dissociation and retention of CD45 during TCR activation may be regulated, or whether ligands co-presented by antigen-presenting cells (APCs) play a role.

[0010] Lactolipoprotein (BTN) is a glycoprotein abundant in breast milk with an incompletely understood immunomodulatory role and is involved in maintaining maternal-fetal tolerance. The mRNA encoding BTN and BTN-like molecules is widely expressed in lymphoid and non-lymphoid tissues. The BTN immunoglobulin domain exhibits structural similarity to B7 family co-receptors (including B7-1 / CD80, B7-2 / CD86, ICOS-L, and PD-L1), and BTN2A2 (BTN2A2) has previously been shown to be expressed by specialized APCs (including B cells, macrophages, and dendritic cells (DCs)). In vitro studies have shown that BTN2A2 can regulate T cell receptor (TCR) signaling and promote de novo Foxp3 expression. Mice lacking BTN2A2 exhibit impaired CD4+ regulated T cell function, enhanced antitumor immunity, and enhanced clinical manifestations of experimental autoimmune encephalomyelitis, all attributed to the lack of BTN2A2 in APCs. Although these accumulated findings suggest that BTN2A2 has a key immunoregulatory function, the exact molecular mechanisms behind these effects remain unclear. Summary of the Invention

[0011] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods, which are intended to be exemplary and illustrative and not to limit the scope.

[0012] Various implementation schemes provide a method for reducing CD3-dependent T cell signaling in a subject in need, the method comprising administering to the subject lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-associated isoform, a fragment thereof, or a fusion peptide comprising any of the foregoing.

[0013] Various implementations provide a method for increasing T regulatory (Treg) cells or decreasing T helper 17 (Th17) cells or both in a subject in need, comprising administering to the subject lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-associated isoform, a BTN2A2-associated isoform fragment, or a fusion polypeptide comprising any of the foregoing.

[0014] In various implementations, BTN2A2, its fragments, BTN2A2-related isoforms, BTN2A2-related isoform fragments, or fusion peptides may include human BTNA2 or fragments or isoforms thereof.

[0015] In various embodiments, the subject may be pregnant, and the method suppresses or reduces the likelihood of miscarriage by increasing Treg cells. In various embodiments, the subject may have kidney injury or a kidney injury-related condition. 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 autoimmune or inflammatory reactions.

[0016] In various implementations, this method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to subjects.

[0017] In various implementation schemes, this method can reduce or eliminate the toxic effects of immunosuppressive therapy in subjects.

[0018] In various implementation schemes, subjects may have an autoimmune or inflammatory condition or a condition characterized by the accumulation of pathological Th1 / Th17 cells (e.g., Th1 / Th17 cell-polarized myeloid dendritic cells).

[0019] In various implementations, the autoimmune or inflammatory condition, or condition characterized by pathological Th1 / Th17 cell accumulation, may include allergies, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disorders, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, malnutrition, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV. In various implementations, the autoimmune neurological disorder may be multiple sclerosis or myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0020] Various implementation schemes provide a method for treating a disease or ailment in a subject in need, the method comprising administering to the subject lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment.

[0021] In various implementation schemes, the disease or ailment can be an autoimmune condition or an inflammatory condition.

[0022] In various implementations, the autoimmune or inflammatory condition may include allergies, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disorders, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, malnutrition, systemic lupus erythematosus (lupus), sarcoidosis, psoriasis, myasthenia gravis, or HIV.

[0023] In various implementation schemes, autoimmune neurological disorders can be multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody diseases, or Alzheimer's disease.

[0024] In various implementations, the disease or ailment can be associated with kidney damage.

[0025] In various implementation schemes, the disease or ailment can be organ transplant rejection, and this method reduces the likelihood of organ transplant rejection.

[0026] In various implementations, the disease or ailment may be organ transplant rejection, and the method reduces the need for immunosuppressive therapy or the amount of immunosuppressive therapy required by the subject.

[0027] In various implementation schemes, the toxic effects of immunosuppressive therapy can be reduced or eliminated in subjects.

[0028] In various embodiments, lactolipoprotein A2 (BTN2A2) or a fragment thereof may be linked to albumin, optionally human serum albumin.

[0029] In various embodiments, lactolipoprotein A2 (BTN2A2) or a BTN2A2 fragment thereof may be linked or fused to an Fc domain or an Fc domain fragment (“BTN2A2-Fc fusion protein”), or wherein a BTN2A2-related isoform or a BTN2A2-related isoform fragment is linked or fused to an Fc domain or an Fc domain fragment (“BTN2A2-related isoform-Fc fusion protein”), optionally IgG1, IgG2, IgG3, or IgG4 Fc, the Fc domain or Fc domain fragment optionally being mutated, further optionally mutated to enhance or reduce Fc-related effector function, further optionally any of the mutations shown in Table 3.

[0030] In various embodiments, BTN2A2 or a BTN2A2 fragment thereof may be linked to an Fc domain or an Fc domain fragment via a linker, optionally a peptide of 2-50 amino acids, or a BTN2A2-related isoform or a BTN2A2-related isoform fragment may be linked to an Fc domain or an Fc domain fragment via a linker, and the linker may be G, polyserine, polyglycine, glycine-serine, GGGGSn(SEQ ID NO:6)n, GGGGGSn(SEQ ID NO:7)n, leucine zipper, aliphatic compound, or helical peptide.

[0031] In various implementations, lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment may be modified by glycosylation, polyethylene glycolation, lipoylation, or by attachment to cholesterol.

[0032] In various implementations, the BTN2A2 fragment may contain the extracellular domain of BTN2A2.

[0033] In various implementation schemes, the BTN2A2 fragment does not contain a transmembrane domain, a cytoplasmic domain, or both.

[0034] In various implementation schemes, the related isoforms of BTN2A2 can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

[0035] In various implementation schemes, the BTN2A2-related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0036] Various embodiments provide a fusion peptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform or a BTN2A2-related isoform fragment, or a combination thereof; and an Fc domain or an Fc domain fragment, optionally IgG1, IgG2, IgG3 or IgG4 Fc, which may optionally be mutated, further optionally mutated to enhance or reduce Fc-related effector function, further optionally any of the mutations shown in Table 3.

[0037] In various embodiments, the fusion peptide may further include a linker located between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, a BTN2A2-related isoform fragment and an Fc domain or Fc domain fragment, optionally between IgG1, IgG2, IgG3 or IgG4 Fc, the Fc domain or Fc domain fragment optionally being mutated, further optionally mutated to enhance or reduce Fc-related effector function, further optionally any of the mutations shown in Table 3.

[0038] In various implementations, the Fc domain or Fc domain fragment may be derived from the IgG1 antibody.

[0039] In various embodiments, the Fc domain or Fc domain segment may contain one or more mutations. In various embodiments, the mutation may be Met208Leu, Asn214Ser, or both, as referenced in SEQ ID NO:4.

[0040] In various embodiments, the fusion peptide may further comprise a spacer peptide located between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform and an Fc domain or an Fc domain fragment.

[0041] In various embodiments, the spacer peptide can be 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).

[0042] In various implementations, the BTN2A2 fragment may contain the extracellular domain of BTN2A2.

[0043] In various implementation schemes, the BTN2A2 fragment does not contain a transmembrane domain, a cytoplasmic domain, or both.

[0044] In various implementation schemes, the related isoforms of BTN2A2 can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3.

[0045] In various implementation schemes, the BTN2A2-related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0046] Various embodiments provide a modified polypeptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactolipoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform or a BTN2A2 related isoform fragment, or a combination thereof is polyethylene glycol-modified or glycosylated.

[0047] In various implementations, the BTN2A2 fragment may contain the extracellular domain of BTN2A2.

[0048] In various embodiments, the BTN2A2 fragment does not contain a transmembrane domain, a cytoplasmic domain, or both. In various embodiments, the BTN2A2 fragment may bind to CD45 (e.g., CD45RO).

[0049] In various implementations, the BTN2A2 related isoforms can be BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various implementations, the BTN2A2 related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0050] Various implementations provide an immunoassay comprising: an anti-lactolipoprotein A2 (BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2 related isoform antibody, or an anti-BTN2A2 related isoform antibody, or a combination thereof, immobilized on a solid support; and a solid support.

[0051] In various implementations, the immunoassay may further include a biological sample obtained from the subject.

[0052] Various embodiments provide a method for monitoring a patient using the assay of the present invention, the method comprising: contacting a biological sample obtained from a patient with the assay of the present invention; and detecting the levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms or BTN2A2-related isoforms, or combinations thereof.

[0053] In various embodiments, the method may further include selecting or administering a treatment selected from: (i) lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; (ii) a fusion polypeptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; and an Fc domain or Fc knot. (iii) a modified polypeptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform or a BTN2A2-related isoform fragment, or a combination thereof, wherein the lactolipoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform or a BTN2A2-related isoform fragment, or a combination thereof is PEGylated or glycosylated; or (iv) a non-BTN2A2-related treatment.

[0054] Various embodiments provide a method for patient stratification using the assay of the present invention, the method comprising: contacting a biological sample obtained from a patient with the assay of the present invention; detecting levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments, or combinations thereof; and stratifying the patient for treatment with: (i) lactolipoprotein A2 (BTN2A2), its BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments; and (ii) a fusion polypeptide comprising: lactolipoprotein A2 (BTN2A2). (iii) A BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; and an Fc domain or an Fc domain fragment; (iv) a modified polypeptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof, wherein the lactolipoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or a combination thereof is PEGylated or glycosylated; or (iv) a non-BTN2A2-related treatment.

[0055] 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. Attached Figure Description

[0056] Exemplary embodiments are illustrated in the referenced figures. The embodiments and figures disclosed herein are intended to be illustrative rather than restrictive.

[0057] Figure 1 (subplot AD) illustrates the enhanced interaction and co-localization of CD45 with TCR signaling proteins by BTN2A2-Fc. (A) Jurkat cells were stimulated for 3 min with immobilized anti-CD3 antibody (10 μg / ml) in the presence or absence of BTN2A2-Fc (10 μg / ml). Cells were lysed in IP buffer, immunoprecipitated with anti-CD45 antibody, and immunoblotted against total Zap70 and CD3ζ. The right subplot shows an intensity map depicted as mean ± SD (N = 3). (B) Immunostaining analysis showed that CD45 and CD3ζ dissociated 3 min after activation of Jurkat cells with plate-bound anti-CD3 antibody (10 mg / ml) in the presence or absence of recombinant BTN2A2-Fc protein (10 mg / ml). The right subplot shows the quantification of co-localization of CD3ζ and CD45 in the presence or absence of BTN2A2-Fc protein from multiple fields of view. At least 50 cells per group were included in the analysis shown in the right-hand subplot. (C) Co-immunoprecipitation was performed in Jurkat cells using anti-CD3ε antibody under the same conditions as in (A), followed by Western blotting with anti-CD45 antibody. (D) CD45-specific phosphatase activity in immunoprecipitates from samples in subplot C was measured using FDP (fluorescein diphosphate tetraammonium) substrate as described in this method. Data are plotted as mean ± SD (N = 3). One-way ANOVA was performed with Tukey's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

[0058] Figure 2 (subplot AH) shows co-immunoprecipitation of CD45 phosphatase with BTN2A2. (A) Jurkat cells were immunoprecipitated with anti-BTN2A2 antibody or IgG and blotted with CD45 antibody in an unstimulated state or 48 hours after activation with anti-CD3 (1 mg / ml). The input volume was approximately 3% of the cell lysate. A representative experiment is depicted. (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 blotted with anti-CD45 antibody. The input volume was approximately 3% of the cell lysate. A representative experiment is depicted. (C) Jurkat cells were treated with anti-CD3 antibody (1 μg / ml) for 48 hours in the presence of recombinant BTN2A2-Fc or Fc-tagged protein, cross-linked with BS3, lysed, immunoprecipitated with protein G, and blotted with anti-CD45 antibody. The input was approximately 3% of the cell lysate. A representative blot was drawn. (D) A banded representation of the protein-protein interaction between BTN2A2 (pink) and CD45 (PTPRC) (white), showing the potential amino acids (blue bars) that determine the interaction between BTN2A2 and CD45. Two residues, N419 and N468, located in the fibronectin domain of CD45 and crucial to this interaction (which are key glycosylation sites) are indicated in the spherical model. (E) A proteomic blot showing CD45 expression in wild-type Jurkat cells (lane 1) and CD45 knockdown Jurkat cells (CD45-KD) (lane 2); lanes 3-4 depict CD45 expression in CD45-KD cells in which wild-type CD45 or mutant CD45 (double mutants - N419A and N468A) are expressed. GAPDH was used as an internal control. (F) Co-immunoprecipitation of CD45 with BTN2A2 in CD45-deficient Jurkat cells, as depicted in subfigure B. In these CD45-deficient Jurkat cells, wild-type CD45 or mutant CD45 (double mutants - N419A and N468) were expressed and activated in the presence of recombinant BTN2A2-Fc or Fc (1 mg / ml anti-CD3 antibody, 48 h). (G) Western blot showing the expression of CD45 isoforms in Jurkat cells at baseline and 48 h after stimulation with anti-CD3 antibody. (H) Western blot analysis of CD45 isoforms (CD45RO and RA) after immunoprecipitation of lysates obtained from Jurkat cells activated in the presence of recombinant BTN2A2-Fc or Fc (1 mg / ml anti-CD3 antibody, 48 h) using Protein G agar beads. A representative experiment is depicted.

[0059] Figure 3 (subplot AD) shows that BTN2A2-Fc enhances Treg and inhibits Th17 cell differentiation in an in vitro mixed lymphocyte reaction (MLR). (A) Flow cytometry analysis of primary CD4+ T cells (spleen and lymph nodes from Foxp3-GFP transgenic mice) incubated for 7 days in MLR with 1 μg / ml anti-CD3 bound as described in the Methods section, in the presence or absence of 10 μg / ml BTN2A2-Fc fusion protein, and CD4+CD25+Foxp3-GFP+ve cell expression was analyzed. The effect of TGF-b (1 ng / ml) under the same conditions was also depicted as a positive control. The right subplot shows a summary plot depicting the mean ± SD of three independent experiments. One-way ANOVA combined with plot-based multiple comparison test; p < 0.05 (*). (B) Flow cytometry analysis of primary CD4+ T cells (isolated from mouse spleen and lymph nodes) incubated for 5 days in an MLR with either anti-CD3 (0.5 mg / ml) antibody alone or anti-CD3+TGF-b (1.5 ng / ml) + IL6 (10 ng / ml) + IL-1β (10 ng / ml) and / or recombinant BTN2A2-Fc (10 μg / ml) and analyzed as CD4+RORgt+ve cells. The right-hand subplot shows a summary plot depicting the mean ± SD of 3 independent experiments. One-way ANOVA combined with plot-based multiple comparison test; p < 0.05 (*). (C) Flow cytometry analysis of the percentage of Foxp3-GFP+ve cells in the total CD4+ T cell population co-cultured with dendritic cells (DC) on day 7. Purified T cells were incubated in RPMI with DC cells (1:10 ratio) isolated from wild-type or BTN2A2- / - mice on day 7. (D) Flow cytometry analysis of the percentage of Foxp3-GFP+ve cells in total CD4+ cells co-cultured with B cells on day 7. Purified T cells were incubated in RPMI with B cells (1:5 ratio) isolated from wild-type or BTN2A2- / - mice on day 7. Experimental data in (CD) are plotted as mean ± SD (n = 3). Unpaired t-test; p < 0.05 (*), p < 0.001 (***).

[0060] Figure 4 (subplot AC) shows that inhibiting CD45 phosphatase activity in primary immune cells blocks BTN2A2-mediated Treg differentiation and Th17 inhibition. (A) Immunoblot analysis of phosphorylated ZAP-70 (p-Zap70) and total Zap70 in Jurkat cells pretreated with a CD45 phosphatase inhibitor for 1 hour and stimulated for 3 minutes with plate-bound anti-CD3 (10 μg / ml) antibody in or without recombinant BTN2A2-Fc (10 μg / ml). (B) Flow cytometry analysis of primary CD4+ T cells (spleen and lymph nodes from Foxp3-GFP transgenic mice) incubated for 7 days in MLR with 1 μg / ml anti-CD3 and / or 10 μg / ml BTN2A2-Fc fusion protein in the absence or presence of a CD45 phosphatase inhibitor (125 nM), and expression in CD4+CD25+Foxp3-GFP+ cells was analyzed. The right-hand subplot shows a summary plot depicting mean ± SD of three independent experiments. (μC) Flow cytometry analysis of primary CD4+ T cells (isolated from mouse spleen and lymph nodes) incubated for 5 days in an MLR for 7 days with either anti-CD3 (0.5 mg / ml) antibody alone or anti-CD3+TGF-b (1.5 ng / ml) + IL6 (10 ng / ml) + IL-1β (10 ng / ml) and / or recombinant BTN2A2-Fc (10 g / ml) in the absence or presence of a CD45 phosphatase inhibitor (125 nM), and CD4+RORgt+ve cells were analyzed. The right-hand subplot shows a summary plot depicting mean ± SD. One-way ANOVA with multiple comparison test; p < 0.01 (**), p < 0.001 (***).

[0061] Figure 5 (sub-figure AI) shows that BTN2A2-Fc improved nephrotoxic serum (NTS)-induced crescentic glomerulonephritis in mice. (A) Schematic diagram and histological changes of the NTS-induced nephrotoxic glomerulonephritis model. GBM = glomerular basement membrane. (B) Quantification of mean ± SEM proteinuria (on day 7) in mice (N=12 / group) with NTS-induced glomerulonephritis treated with recombinant BTN2A2-Fc protein or the mediator (control); Mann-Whitney test p<0.001 (***). (CD) Representative images of glomerular damage based on PAS staining. Crescent formation in mice with NTS-induced glomerulonephritis treated with recombinant BTN2A2-Fc protein or the control (D). Scale bar: 25 mm. Summary subplot (C) depicts the quantification of glomerular percentage with crescents (mean ± SEM, N = 12 / group); Mann-Whitney p < 0.001 (***). (EF) Relative Foxp3 (D) and RORgt (E) expression in CD4+ T cells purified from the spleen and lymph nodes of mice with NTS-induced glomerulonephritis treated with BTN2A2-Fc protein or control, compared with GAPDH as an internal control. Data are depicted as mean ± SEM (N = 8 / group). Mann-Whitney test; p < 0.05 (*). (G) 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. (HI) Immunoblot analysis of IL17A in renal tissue lysates from mice with glomerulonephritis treated with recombinant BTN2A2-Fc or control (H). Subfigure I shows the quantification of the protein blot. Data are presented as mean ± SEM. N = 4 per group. Unpaired t-test; p < 0.05 (*).

[0062] Figure 6 (subplot AH) depicts BTN2A2(- / -) mice exhibiting exacerbated crescentic glomerulonephritis. (A) Scatter plot shows the mean ± SEM quantification of 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 based on PAS staining. Wild-type mice showed minimal glomerular damage when treated with low doses of nephrotoxic serum, while BTN2A2(- / -) mice showed severe glomerulonephritis (B). Scale bar is 25 mm. Summary data are depicted as mean ± SEM of glomerular crescent % (C). N = 15 per group for all experiments. Mann-Whitney test; p < 0.001 (***). (DE) Relative Foxp3 mRNA (D) and RORgt mRNA (E) in CD4+ve T cells purified from spleen and lymph node cells of wild-type and BTN2A2 knockout mice with nephrotoxic serum-induced glomerulonephritis, compared with GAPDH as an internal control. (F) Quantitative PCR analysis showing CD5 expression in CD4+ T cells from wild-type and BTN2A2 knockout mice with nephrotoxic serum-induced glomerulonephritis. N = 7 / group. (GH) Immunoblotting (G) of IL17A expression in kidney tissue lysates using anti-IL-17A antibody, and quantification (H) from wild-type and BTN2A2 knockout mice with nephrotoxic serum-induced glomerulonephritis. n ≥ 4 per group. Unpaired t-test; p < 0.01 (**), p < 0.001 (***).

[0063] Figure 7 (subplot AH) shows that in the CBA / J x DBA / 2 pregnancy model, BTN2A2-Fc rescued fetal resorption and increased litter size (AB). Pregnant CBA / J x DBA / 2 mice treated with BTN2A2 throughout pregnancy had increased litter size (A) and reduced resorption (B) compared to untreated mice. DBA / 2x CBA / J mice were included as an additional control. Data (A, B) are presented as mean ± SEM. N = 11 per group for all experiments. Kruskal-Wallis test combined with Dunn's multiple comparison test; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). (CD) Quantitative PCR (C) and flow cytometry (D) analyses show an increase in Foxp3-expressing CD4+ T cells in pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. N=8 / group. (E) Flow cytometry analysis (left subplot) shows a decrease in the number of CD4+RORgt+ve cells in pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. The right subplot shows the quantification of the pooled data. N=8 / group. (F) Quantitative PCR analysis shows CD5 expression in CD4+ T cells of pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc compared to control mice. N=8 / group. (G, H) Immunoblot analysis of IL17A in placental tissue lysates from pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc or control (left subplot). The right-hand subplot shows the quantification of the summary data. N=3 for each group. One-way ANOVA was used for the experiments in CG, combined with a plot-based multiple comparison test; p<0.05(*), p<0.01(**), p<0.001(***).

[0064] Figure 8 (subplot AD) shows that BTN2A2-Fc enhances Treg cells and inhibits Th17 cell differentiation in a mixed lymphocyte reaction of human PBMCs in vitro. (AB). Flow cytometry plots show the percentage of Treg cells (CD4+CD25+Foxp3+ve cells) in human PBMCs incubated for 7 days in MLR with / without recombinant BTN2A2-Fc (10 mg / ml) in the absence or presence of a CD45 phosphatase inhibitor (125 nM). Data are plotted as mean + / - SD, showing the percentage of Treg cells (CD4+CD25+Foxp3+ve cells) in total CD4+ cells. N=4 per group for all experiments. Unpaired two-tailed t-test; p<0.001 (***). (CD). Flow cytometry plot showing the percentage of CD4+RORgt+ve (Th17 cells) in human PBMCs incubated for 3 days in a 7-day MLR with either anti-CD3 antibody alone (0.5 mg / ml) or anti-CD3 antibody + TGF-β (1.5 ng / ml) + IL-6 (10 ng / ml) + IL-1β (10 ng / ml) in the absence or presence of recombinant BTN2A2-Fc (10 mg / ml) in the absence or presence of CD45 phosphatase inhibitor (125 nM). Data are presented as mean ± standard deviation. N = 3 per group for all experiments. One-way ANOVA test and combined plot multiple comparison test were performed; p < 0.01 (**).

[0065] Figure 9 A representative schematic diagram of a model in which BTN2A2 acts as a co-inhibitor of TCR signaling via CD45 phosphatase is shown.

[0066] Figure 10 (subfigure AD) shows the regulation of T cell receptor signaling by BTN2A2-Fc. (A) shows a schematic diagram of the recombinant human BTN2A2-Fc fusion. This recombinant fusion gene consists of two extracellular domains (IgV and IgC2) (green) of the cloned BTN2A2 molecule and the Fc region (orange) of the human IgG1 antibody. The chimeric protein (BTN2A2-Fc) is separated by the IEGRMD (SEQ ID NO: 57) spacer polypeptide. (B) Coomassie brilliant blue staining of purified recombinant BTN2A2-Fc protein on a reducing (SDS) gel. The left column shows the protein ladder, and the two right columns show the bands corresponding to BTN2A2-Fc (approximately 55 kDa). The subfigure below shows Western blotting of recombinant BTN2A2-Fc using a specific anti-human BTN2A2 antibody. (C) Depicts IL-2 secretion from Jurkat cells, 293 cell-derived human BTN2A2-Fc (R&D systems), or mouse BTN2A2 (R&D systems) in the absence or presence of immobilized recombinant BTN2A2-Fc (10 μg / ml), along with plate-immobilized anti-CD3 antibody (1 μg / ml) as a negative control (N=3 or 4). (D) Immunoblot analysis of phosphorylated proteins, p-Zap70, and p-CD3-ζ, and total Zap70 and CD3-ζ, in Jurkat cells stimulated for 3 min 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-hand subplot shows relative intensity plots as mean ± SD (N=3). One-way ANOVA combined with Tugi's multiple comparison test; p<0.05(*), p<0.01(**), p<0.001(***).

[0067] Figure 11 The pervanadate study was described. Jurkat cells were treated with a permanent phosphatase inhibitor (pervanadate) or untreated, with or without BTN2A2-Fc. After cell lysis, phosphorylated proteins p-Zap70 and p-CD3-ζ were analyzed by Western blotting. Total Zap70 and CD3-ζ served as controls.

[0068] Figure 12 Jurkat cells were stimulated for 48 hours with / without anti-CD3 antibody (1 μg / ml), and the lysates were immunoblotted with anti-CD45 or anti-BTN2A2 antibody. GAPDH expression was used as a loading control.

[0069] Figure 13 (sub-figures AB) depicts the deletion and site-directed mutagenesis of CD45 by CRISPR-Cas9. (A) A representative schematic diagram of the CD45 sequence showing the position of gRNA sequence complementation. The lower sub-figure shows an electrophoresis diagram of the deleted sequence from upstream of exon 1 to downstream of exon 3. Nucleotide sequence: SEQ ID NO:46; Amino acid sequence: SEQ ID NO:47. (B) An electrophoresis diagram showing the nucleotide changes from AAT to GCT at Asn419Ala and from AA to GCT at Asn468Ala 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.

[0070] Figure 14 (subplot AC) shows the inhibition of CD4+ve T cell proliferation, apoptosis, and IL2 secretion by BTN2A2-Fc. (A) Flow cytometry plot of purified mouse CD4+ve cells stained with CellTrace violet and 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 subplot shows a bar chart of the percentage of non-proliferating cells (G0). (B) Flow cytometry plot of purified mouse CD4+ve cells stained with Annexin-V and 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-hand subplot shows a bar chart of the percentage of apoptotic cells. (C) Flow cytometry plot of purified IL2-positive mouse CD4+ve cells incubated on day 3 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-hand subplot shows a bar chart of the percentage of IL2-positive cells. Data are plotted as mean ± SD (n = 4). Unpaired t-test; p < 0.001 (***).

[0071] Figure 15 (sub-figure AF) illustrates the qPCR study 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) was performed in primary CD4 cells incubated for 1 and 5 days with or without anti-CD3 antibody and / or recombinant BTN2A2-Fc. All data are presented as mean ± standard deviation. n = 3 for each experiment. One-way ANOVA combined with Tuki's multiple comparison test was used; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

[0072] Figure 16 (sub-figures AB) illustrates the generation / characterization of BTN2A2 gene knockout mice. (A) Btn2a2- / - mice constructed via CRISPR / Cas-mediated genome engineering (cyagen.com). The Btn2a2 gene (NCBI reference sequence: NM_175938; Ensembl: ENSMUSG00000053216) is located on mouse chromosome 13. Eight exons were identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (transcript Btn2a2-203: ENSMUST00000110433). Exons 2–8 (covering 100.0% of the coding region) were selected as target sites. Cas9 and gRNA were co-injected into fertilized eggs to generate KO mice. Genotyping of pups was performed by PCR followed by sequencing analysis. Size of the effective KO region: approximately 10500 bp. gRNA target sequences SEQ ID NO: gRNA1: SEQ ID NO: 39; gRNA2: SEQ ID NO: 40; gRNA3: SEQ ID NO: 41; gRNA4: SEQ ID NO: 42. Genotyping primers SEQ ID NO: F1: SEQ ID NO: 43; F2: SEQ ID NO: 44; R1: SEQ ID NO: 45. (B) Agarose gel electrophoresis image of genotyping PCR. Homozygous mice show a single band at 700 bp, heterozygous mice show two bands at 700 bp and 500 bp, and wild-type mice (WT) show a single band at 500 bp.

[0073] Figure 17 (subplots AB) shows BTN2A2 expression in antigen-presenting cells. qPCR analysis of BTN2A2 expression in B cell (A) and dendritic cell (B) populations purified from the spleen and lymph nodes of wild-type and BTN2A2-KO mice using magnetic beads. The left subplot shows a flow cytometry plot of the purified cells, and the right subplot shows the fold change in BTN2A2 transcript RNA expression compared to GAPDH.

[0074] Figure 18 A schematic scheme containing a spontaneous abortion model.

[0075] Figure 19 A schematic diagram illustrating the binding of BTN2A2 to myelin oligodendrocyte glycoprotein (MOG) is shown. The binding of BTN2A2 to MOG antigens associated with multiple sclerosis and other MOG-related autoimmune diseases was evaluated using protein structure prediction software (Prime, www.schrodinger.com). The root mean square (RMSD) of atomic positions is <1 Å.

[0076] Invention Description

[0077] All references cited herein are incorporated herein by way of citation in their entirety, as if fully explained. Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Those skilled in the art will recognize many methods and materials similar to or equivalent to those described herein that can be used to practice this invention. In fact, this invention is by no means limited to the methods and materials described. For the purposes of this invention, the following terms are defined.

[0078] As used herein, the term "about" when used in conjunction with a referenced numerical indication means that the referenced numerical indication is added to or subtracted from that numerical indication by a maximum of 5%, unless otherwise expressly stated herein. For example, the expression "about 50%" covers a range of 45% to 55%. In various embodiments, if specifically stated in the claims, the term "about" when used in conjunction with a referenced numerical indication may mean that the referenced numerical indication is added to or subtracted from that numerical indication by a maximum of 4%, 3%, 2%, 1%, 0.5%, or 0.25%.

[0079] The percentage of sequence identity (%) of the reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for sequence alignment can be determined, including the algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0080] As used herein in the context of linked peptides, polypeptides, or proteins, "linked" means "directly or indirectly linked." Indirect linking can be mediated by polypeptide linkers (such as polyglycine or glycine-serine polypeptides), for example (GGGGS)n (SEQ ID NO:6)n or (GGGGG S)n (SEQ ID NO:7)n (where n is an integer). In various embodiments, n is an integer from 1 to 10, 10 to 20, 20 to 30, 30 to 40, or 50 to 100. Other such linkers are known in the art and are considered to be covered by this terminology.

[0081] This paper describes our demonstration that BTN2A2 acts as a ligand for CD45, binding to both the TCR complex and the CD45RO isoform on the surface of T cells, thereby preserving CD45 phosphatase activity near the TCR complex. This enhanced phosphatase activity thus reduces downstream TCR signaling, promoting regulatory T cell (Treg) proliferation and inhibiting T effector cell differentiation. We further demonstrate the significant immunomodulatory role of BTN2A2 in a mouse model of autoimmune glomerulonephritis, showing that BTN2A2 gene deficiency exacerbates kidney damage, while administration of recombinant BTN2A2 limits disease severity. Furthermore, in a second immune-mediated spontaneous abortion mouse model, treatment of pregnant mice with recombinant BTN2A2 helped the animals tolerate their pregnancies, thereby reducing fetal loss.

[0082] The data contained in this article provide multiple pieces of evidence that BTN2A2 functions as a ligand for CD45, preferentially binding to the CD45RO isoform and preventing CD45 dissociation from the TCR / CD3 complex. This results in persistent CD45 phosphatase activity within the immune synapse, thereby reducing downstream signaling cascades (see [link to article]). Figure 9Therefore, BTN2A2 reduces the proliferation of effector Th17 cells while promoting the differentiation of regulatory T cells and decreasing the severity of NTS-induced glomerulonephritis and immune-mediated pregnancy loss in an immune disease mouse model. Our results suggest that during homologous T cell / APC interactions, APCs produce BTN2A2, which links T cell-expressed CD45RO to the TCR, thus acting as a molecular brake on the activation of the TCR complex by the peptide / MHC complex. Our data indicate that in CD45RO+ T cells, BTN2A2 binds to both CD45 and CD3e, potentially maintaining them in close proximity, allowing for the persistence of CD45 phosphatase activity, dephosphorylation of the CD3ITAM domain and Zap70, and restriction of downstream TCR signaling required for full T cell activation.

[0083] Our data also indicate that BTN2A2 enhances CD45 phosphatase activity within the TCR complex, thereby modulating TCR signaling but not completely blocking it, and thus allowing Treg expansion while reducing effector T cell differentiation / expansion. In previous studies, transient targeting of CD45 induced potent antigen-specific regulatory T cells and fostered transplant tolerance. Our data suggest that BTN2A2 expressed on APCs and DCs may be an endogenous ligand for CD45 on T cells. CD45 is expressed as a multiplexed splice product, and CD45RA and CD45RB are thought to be replaced by CD45RO upon T cell activation. These studies suggest that BTN2A2 primarily binds to the CD45RO isoform expressed on activated T cells, implying that BTN2A2's role is specific. Although others have demonstrated that various human glycoproteins, including galactolectin-1 and CD22, bind to CD45, there is no clear evidence that these putative ligands modulate CD45 phosphatase activity. Other identified ligands of CD45 include pUL11 (a viral protein expressed by cells infected with cytomegalovirus) and the E3 / 49K protein expressed by cells infected with adenovirus; both viral proteins may play a role in suppressing antiviral immunity by inhibiting TCR signaling, although the exact mechanism is unclear.

[0084] We demonstrate that BTN2A2-Fc limits the clinical manifestations of glomerular crescent formation and proteinuria in mouse NTS glomerulonephritis and reduces abortion in a unique orthogonal mouse model, both of which are associated with increased Tregs and decreased Th17 cell populations. The T cell activation marker CD5 was attenuated by BTN2A2-Fc. Our data further suggest that BTN2A2-Fc is a potentially useful immunomodulator with translational potential in a variety of immune-mediated diseases and transplantation. Our observations represent an important step towards understanding how a naturally occurring protein alters inflammatory events by interacting with CD45 to maintain TCR complex phosphatase activity, increase Tregs, and decrease the Th17 cell population.

[0085] Since CD45 is also expressed on B cells, some of the beneficial effects of BTN2A2 in our animal studies may derive from its effects on B cells.

[0086] Our results indicate that BTN2A2 linking CD45 phosphatase to the TCR complex leads to attenuated TCR signaling, resulting in Treg amplification and Th17 cell suppression. While we demonstrated the beneficial effects of BTN2A2 in two orthogonal autoimmune / immune tolerance models, our data suggest that the BTN2A2 / CD45 signaling pathway can be targeted in a variety of immune-mediated diseases, such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, and transplant rejection.

[0087] Therefore, various embodiments of the present invention are based at least in part on these research results.

[0088] Various implementation schemes provide a method for reducing CD3-dependent T cell signaling in subjects in need, the method comprising administering to the subject lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-associated isoform, or a fragment thereof.

[0089] Various implementation schemes provide a method for increasing T regulatory (Treg) cells or decreasing T helper 17 (Th17) cells or both in a subject in need, comprising administering to the subject lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-associated isoform, or a fragment thereof.

[0090] Based on the phylogenetic tree, BTN1A1, B2N2A1, BTN2A2, BTN3A1, BTN3A2, and BTN3A3 have similar structures, particularly their extracellular domains, and are closely related. Therefore, all these isoforms, especially their extracellular domains, are expected to have similar functions to BTN2A2 in regulating Treg and Th17 cell homeostasis and can be used according to various embodiments of the present invention.

[0091] In various implementation schemes, the subjects are pregnant, and the method inhibits or reduces the likelihood of miscarriage by increasing Treg cells.

[0092] In all implementation schemes, the subjects had kidney damage.

[0093] In various implementations, the subject is an organ, tissue, or cell therapy transplant recipient. In various implementations, the method reduces the likelihood of organ, tissue, or cell transplant rejection. In various implementations, the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to the subject. In various implementations, the method reduces the toxicity of immunosuppressive therapy to the subject.

[0094] In various implementation schemes, the subjects suffer from an autoimmune or inflammatory condition. In various implementation schemes, this autoimmune or inflammatory condition includes allergies, asthma, glomerulonephritis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, autoimmune neurological disorders, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, malnutrition, systemic lupus erythematosus (lupus), psoriasis, sarcoidosis, myasthenia gravis, or HIV. In various implementation schemes, the autoimmune neurological disorder is multiple sclerosis. In various implementation schemes, the autoimmune neurological disorder is myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0095] In various embodiments, lactolipoprotein A2 (BTN2A2) or a fragment thereof is fused to an Fc domain or an Fc domain fragment (“BTN2A2-Fc fusion protein”) and / or another portion that extends half-life (such as PEG, albumin, lipidation, cholesterol, or glycosylation). In various embodiments, a BTN2A2-related isoform or a fragment thereof is fused to an Fc domain or an Fc domain fragment (“BTN2A2-related isoform-Fc fusion protein”), typically the Fc domain or an Fc domain fragment of human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0096] In various embodiments, a lactolipoprotein A2 (BTN2A2) or a fragment thereof is linked to an Fc domain or an Fc domain fragment (“BTN2A2-Fc fusion protein”), typically human IgG, such as the Fc domain or an Fc domain fragment of human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions. In various embodiments, a BTN2A2-related isoform or a fragment thereof is linked to an Fc domain or an Fc domain fragment (“BTN2A2-related isoform-Fc fusion protein”), typically human IgG, such as the Fc domain or an Fc domain fragment of human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0097] In various implementations, the connector is G, polyserine, polyglycine, glycine-serine, GGGGSn(SEQ ID NO:6)n, GGGGGSn(SEQ ID NO:7)n, leucine zipper, aliphatic compound, or helical peptide.

[0098] In various embodiments, the spacer peptide is located between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and an Fc domain or an Fc domain fragment. That is, a spacer region exists between the BTN2A2 portion and the Fc domain portion. For example, fusion proteins include a BTN2A2-spacer-Fc domain, or a BTN2A2-spacer-Fc domain fragment, or a BTN2A2 fragment-spacer-Fc domain, or a BTN2A2 fragment-spacer-Fc domain fragment. These examples are not intended to limit possible combinations, but rather to provide clarity.

[0099] Examples of spacer regions 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).

[0100] In various embodiments, the Fc domain contains a mutation selected from Met208Leu, Asn214Ser, or both of reference SEQ ID NO:4. This mutation can extend the half-life of B2N2A2Fc. (See, for example, Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life”, Front Immunol. 2019; 10:1296)

[0101] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0102] In various embodiments, the BTN2A2 fragment contains the amino acid sequence of SEQ ID NO:3.

[0103] 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 is administered according to the embodiments described herein, wherein the spacer region IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced by IEGRMD (SEQ ID NO:57).

[0104] In various embodiments, lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment are modified by glycosylation or polyethylene glycol modification. These modifications can prolong the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment.

[0105] Modification by polyethylene glycolation can extend the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment. Examples of polyethylene glycolation include Veronese and Mero, “The Impact of PEGylation on Biological Therapies”, Biodrugs 2008; 22(5):315-329, which is incorporated herein by reference as described in its entirety. Thus, PEG or branched PEG (PEG2) can be used, for example. 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.

[0106] Another way to improve the pharmacokinetic (PK) properties of BTN2A2 or its fragments is to attach it to albumin, such as human serum albumin (HSA), which prevents proteolytic degradation and rapid renal filtration (due to the relatively large size of HSA, approximately 66 kDa). Yet another way to improve the pharmacokinetic (PK) properties of BTN2A2 or its fragments is through glycosylation. Still another way to improve the pharmacokinetic (PK) properties of BTN2A2 or its fragments is through attachment to lipids or cholesterol.

[0107] In various embodiments, the BTN2A2 fragment includes an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not include a transmembrane domain. In various embodiments, the BTN2A2 fragment does not include a cytoplasmic domain. In various embodiments, the BTNA2 fragment binds to CD45 (e.g., CD45RO).

[0108] In various embodiments, the BTN2A2 related isoforms are BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3, or fragments or fusion peptides containing any of the foregoing, optionally Fc fusion peptides, typically human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0109] In various embodiments, the BTN2A2-related isoform fragment does not contain a transmembrane domain, a cytoplasmic domain, or both. In various embodiments, the BTN2A2-related isoform fragment binds to CD45 (e.g., CD45RO).

[0110] Various embodiments of the present invention provide a method for treating a disease or ailment in a subject in need, the method comprising: administering to the subject lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform or a BTN2A2-related isoform fragment thereof, or a fusion polypeptide comprising any of the foregoing, optionally an Fc fusion polypeptide.

[0111] In various implementation schemes, the disease or ailment is an autoimmune condition or an inflammation-related condition.

[0112] In various implementation schemes, the autoimmune or inflammatory condition is glomerulonephritis, inflammatory bowel disease, rheumatoid arthritis, autoimmune neurological disorders, antibody-mediated transplant rejection, infantile cholestasis, hemophagocytic lymphohistiocytosis, erythrophagocytosis, malnutrition, systemic lupus erythematosus, myasthenia gravis, or HIV.

[0113] In various implementations, the autoimmune neurological disease is multiple sclerosis. In various implementations, the autoimmune neurological disease is myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0114] In various implementations, the disease or ailment is kidney damage.

[0115] In various embodiments, the disease or ailment 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 ailment is organ, tissue, or cell transplant rejection, and the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy required by the subject. In various embodiments, the treatment results in a reduction or elimination of other aspects of the toxicity of immunosuppressive therapy in the subject.

[0116] In various embodiments, a fragment of lactolipoprotein A2 (BTN2A2) or a BTN2A2 fragment thereof is fused to an Fc domain or an Fc domain fragment (“BTN2A2-Fc fusion protein”), typically human IgG, such as the Fc domain or an Fc domain fragment of human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions. In various embodiments, a BTN2A2-related isoform or a BTN2A2-related isoform fragment is fused to an Fc domain or an Fc domain fragment (“BTN2A2-related isoform-Fc fusion protein”), typically human IgG, such as the Fc domain or an Fc domain fragment of human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0117] In various embodiments, a lactolipoprotein A2 (BTN2A2) or a fragment thereof is linked to an Fc domain or an Fc domain fragment (“BTN2A2-Fc fusion protein”), typically human IgG, such as the Fc domain or an Fc domain fragment of human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions. In various embodiments, a BTN2A2-related isoform or a fragment thereof is linked to an Fc domain or an Fc domain fragment (“BTN2A2-related isoform-Fc fusion protein”). In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, a leucine zipper, an aliphatic compound, or a helical peptide.

[0118] In various embodiments, the spacer peptide (optionally derived from 2-50 amino acids) is located between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and an Fc domain or Fc domain fragment, typically between the Fc domain or Fc domain fragment of human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions. That is, a spacer region exists between the BTN2A2 portion and the Fc domain portion. For example, fusion proteins include a BTN2A2-spacer region-Fc domain, or a BTN2A2-spacer region-Fc domain fragment, or a BTN2A2 fragment-spacer region-Fc domain, or a BTN2A2 fragment-spacer region-Fc domain fragment. These examples are not intended to limit possible combinations, but rather to provide clarity.

[0119] Examples of spacer regions 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).

[0120] In various implementations, the Fc domain, such as the IgG1 Fc domain, contains a mutation selected from Met 208Leu, Asn214Ser, or both, of reference SEQ ID NO:4. This mutation can prolong the half-life of B2N2A2Fc. (See, for example, Saunders, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life”, Front Immunol. 2019; 10:1296)

[0121] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

[0122] 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 is administered according to the embodiments described herein, wherein the spacer region IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced by IEGRMD (SEQ ID NO:57).

[0123] In various embodiments, lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment is modified by attachment to albumin, such as human serum albumin (HAS).

[0124] In various embodiments, lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment are modified by glycosylation or polyethylene glycol modification. These modifications can prolong the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment.

[0125] Modification by polyethylene glycolation can extend the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment. Examples of polyethylene glycolation include Veronese and Mero, “The Impact of PEGylation on Biological Therapies”, Biodrugs 2008; 22(5):315-329, which is incorporated herein by reference as described in its entirety. Thus, PEG or branched PEG (PEG2) can be used, for example. 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.

[0126] In various embodiments, the BTN2A2 fragment includes an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not include a transmembrane domain. In various embodiments, the BTN2A2 fragment does not include a cytoplasmic domain. In various embodiments, the BTN2A2 fragment binds to CD45 (e.g., CD45RO).

[0127] In various implementations, the BTN2A2 related isoforms are BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various implementations, the BTN2A2 related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0128] Various embodiments of the present invention provide a fusion protein comprising: a lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; and an Fc domain or Fc domain fragment, typically the Fc domain or Fc domain fragment of human IgG, such as human IgG1, IgG2, IgG3, or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0129] In various embodiments, the fusion protein is not the fusion protein having SEQ ID NO:1. In various embodiments, the fusion protein is not the fusion protein having SEQ ID NO:1, wherein the spacer region IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced by IEGRMD (SEQ ID NO:57).

[0130] In various implementations, the Fc domain or Fc domain fragment is derived from the IgG1 antibody.

[0131] In various embodiments, the Fc domain or Fc domain fragment, typically human IgG, such as human IgG1, IgG2, IgG3, or IgG4, may optionally be modified to increase or decrease specific Fc-related effector functions and may contain one or more mutations. In various embodiments, the mutation is Met208Leu, Asn214Ser, or both, as referenced in SEQ ID NO:4. Mutations to the Fc domain prolong the half-life of the fusion protein.

[0132] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. Alternatively, BTN2A2 may comprise an Fc variant selected from any of the following:

[0133] Alternative Fc variants

[0134] As mentioned above, the subject BTN2A2 peptide and fragment may include an Fc region, which may optionally be mutagenized to modulate Fc-related effector functions. In some embodiments, one or more amino acid modifications are introduced into the Fc region, thereby producing an Fc region variant or Fc variant. The Fc region may include a C-terminal region of an immunoglobulin heavy chain comprising a hinge region, a CH2 domain, a CH3 domain, or any combination thereof. As used herein, the Fc region includes native sequence Fc regions and variant Fc regions. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid modifications (e.g., substitutions, additions, or deletions) at one or more amino acid positions.

[0135] In some embodiments, the variant Fc region includes at least one amino acid modification located within the Fc region. Combining amino acid modifications is also useful. For example, the variant Fc region may include two, three, four, five, or other substitutions located at specific Fc region positions identified herein.

[0136] In some implementations, the altered effector function is a reduced effector function. In some implementations, the altered effector function is an increased effector function. An effector function generally refers to a biological event resulting from the interaction of the antibody's Fc region with an Fc receptor or ligand. Non-restricted effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by antigen-presenting cells mediated by immune complexes, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some cases, antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which non-specific cytotoxic cells expressing Fc receptors (e.g., natural killer cells, neutrophils, macrophages) recognize bound antibodies on target cells, subsequently leading to target cell lysis. 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 a target-bound antibody.

[0137] In some cases, reduced effector function is beneficial. In some cases, modifications in the Fc region produce Fc variants characterized by: (a) reduced antibody-dependent cell-mediated cytotoxicity (ADCC), (b) reduced complement-mediated cytotoxicity (CDC), and / or (c) reduced affinity for Cl q. In some implementations, the Fc region is modified by modifying one or more amino acids at the following positions to reduce antibody-dependent cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-mediated cytotoxicity (CDC), and / or affinity for C1q: 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, 329, 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 number). In some implementations, the variant Fc region is selected from Table 3.

[0138] Table 3

[0139]

[0140]

[0141]

[0142] In vitro assays for evaluating the ADCC activity of target molecules are well known in the art. Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Useful effector cells used in such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0143] In some implementations, the variant Fc region exhibits reduced effector function compared to wild-type human IgG1. Non-limiting examples of Fc mutations in IgG1 that reduce ADCC and / or CDC in certain cases include substitutions at one or more of the following locations: 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 in IgG1, wherein the constant region numbering system is the EU index numbering system proposed by Kabat.

[0144] In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with N297A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with N297Q according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with N297D according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with D265A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with S228P according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with L235A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with L237A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with L234A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with E233P according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising L234 V substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising C236 deletion according to the Kabat numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P238 A substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising A327Q substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P329 A substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P329G substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising L235E substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P331S substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising L234F substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 235G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 235Q according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 235R according to the EU numbering system.In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 235S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 236F according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 236R according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 237E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 237K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 237N according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 237R according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 2381 according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238W according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 238Y according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 248A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254D according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 2541 according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254N according to the EU numbering system.In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254P according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254Q according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254T according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 254V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 255N according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 256H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 256K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 256R according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 256V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 264S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 265H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 265K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 265S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 265Y according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 267G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 267H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 2671 according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 267K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 268K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 269N according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 269Q according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 270A according to the EU numbering system.In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 270G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 270M according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 270N according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 271T according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 272N according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 279F according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 279K according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 279L according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 292E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 292F according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 292G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 292L according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 293S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 301W according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 304E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 31IE according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 311G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 311S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 316F according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 327T according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 328V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 329Y according to the EU numbering system.In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 330R according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 339E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 339L according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 3431 according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 343V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 373A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 373G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 373S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 376E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 376W according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 376Y according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 380D according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 382D according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 382P according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 385P according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 424H according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 424M according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 424V according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 434L according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 438G according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 439E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 439H according to the EU numbering system.In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 439Q according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440D according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440E according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440F according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440M according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440T according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region substituted with 440V according to the EU numbering system.

[0145] In some embodiments, the variant Fc region includes IgG1 Fc regions L234A, L235E, G237A, A330S, and / or P331S according to EU numbering. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising E233P according to the EU numbering system. In some embodiments, the variant Fc region includes an Ig G4 Fc region comprising S228P and L235E. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising L235E according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A and L235A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235A, and G237A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235A, and P329G according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234F, L235E, and P331S according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235E, and G237A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235E, G237A, and P331S according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235A, G237A, P238S, H268A, A330S, and P331S (IgG1) according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising L234A, L235A, and P329A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising G236R and L328R according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising G237A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising F241A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising V264A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising D265A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising D265A and N297A according to the EU numbering system.In some embodiments, the variant Fc region includes IgG1 Fc regions comprising D265A and N297G according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising D270A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising N297A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising N297G according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising N297D according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising N297Q according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising P329A according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising P329G according to the EU numbering system. In some embodiments, the variant Fc region includes IgG1 Fc regions comprising P329R according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising A330L according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P331A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG1 Fc region comprising P331S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG2 Fc region. In some embodiments, the variant Fc region includes an IgG4 Fc region. In some embodiments, the variant Fc region includes an IgG4 Fc region comprising S228P according to the EU numbering system. In some embodiments, the variant Fc region includes IgG4 Fc regions comprising S228P, F234A, and L235A according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG2-IgG4 cross-subclass (IgG2 / G4) Fc region. In some embodiments, the variant Fc region includes an IgG2-IgG3 cross-subclass Fc region. In some embodiments, the variant Fc region includes IgG2 Fc regions comprising H268Q, V309L, A330S, and P33IS according to the EU numbering system. In some embodiments, the variant Fc region includes IgG2 Fc regions comprising V234A, G237A, P238S, H268A, V309L, A330S, and P33IS according to the EU numbering system. In some embodiments, the antibody comprises an Fc region containing highly mannose-glycosylated Fc regions.

[0146] 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 designations. In some embodiments, the one or more mutations relative to the wild-type Fc region include L234A, L235E, G237A, A330S, and P331S according to EU designations, or consist of L234A, L235E, G237A, A330S, and P331S according to EU designations. 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 includes an IgG4 Fc region containing an S228P substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG4 Fc region substituted with A330S according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG4 Fc region substituted with P331S according to the EU numbering system.

[0147] In some embodiments, the variant Fc region includes an IgG2 Fc region comprising an A330S substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG2 Fc region comprising a P331S substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG2 Fc region comprising a 234A substitution according to the EU numbering system. In some embodiments, the variant Fc region includes an IgG2 Fc region comprising a 237A substitution according to the EU numbering system.

[0148] In some implementations, the variant Fc region includes the IgG1 Fc region, and wherein the one or more mutations include (a) 297A, 297Q, 297G, or 297D according to EU numbers; (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, or 328F. 9Y or 329R(k)331S; (1)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) (a) 267G, 267H, 267L or 267K; (b) 268K; (c) 269N or 269Q; (d) 270A, 270G, 270M or 270N; (e) 271T; (f) 272N; (g) 292E, 292F, 292G or 292L; (c) 293S; (d) 301W; (c) 304E; (d) 311E, 311G or 311S; (e) 316F; (f) 328V; (g) 330R; (hh) 339E or 339L; (i) 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 P33 IS(bbb)L234A, L235A, G237A, P238S, H268A, A330S, andP331S; (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 any combination of (ppp)(a)-(uu).

[0149] In various embodiments, the fusion protein further comprises a spacer peptide located between BTN2A2, a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment and an Fc domain or Fc domain fragment, the spacer peptide optionally being derived from 2-50 amino acids. That is, a spacer region exists between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein includes a BTN2A2-spacer region-Fc domain, or a BTN2A2-spacer region-Fc domain fragment, or a BTN2A2 fragment-spacer region-Fc domain, or a BTN2A2 fragment-spacer region-Fc domain fragment. These examples are not intended to limit possible combinations, but rather to provide clarity.

[0150] Examples of spacer regions 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).

[0151] In various embodiments, the BTN2A2 fragment includes an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not include a transmembrane domain. In various embodiments, the BTN2A2 fragment does not include a cytoplasmic domain. In various embodiments, the BTN2A2 fragment binds to CD45 (e.g., CD45RO).

[0152] In various implementations, the BTN2A2 related isoforms are BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various implementations, the BTN2A2 related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0153] In various implementations, the fusion protein has the sequence SEQ ID NO:2.

[0154] Various embodiments provide a modified polypeptide comprising: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactolipoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform or a BTN2A2 related isoform fragment, or a combination thereof is PEGylated, lipoylated, or glycosylated.

[0155] These modifications can extend the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment.

[0156] Modification by polyethylene glycolation can extend the half-life of lactolipoprotein A2 (BTN2A2) or its BTN2A2 fragment, BTN2A2 related isoform, or BTN2A2 related isoform fragment. Examples of polyethylene glycolation include Veronese and Mero, “The Impact of PEGylation on Biological Therapies”, Biodrugs 2008; 22(5):315-329, which is incorporated herein by reference as described in its entirety. Thus, PEG or branched PEG (PEG2) can be used, for example. 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.

[0157] In various embodiments, the BTN2A2 fragment includes an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not include a transmembrane domain, a cytoplasmic domain, or both. In various embodiments, the BTN2A2 fragment binds to CD45 (e.g., CD45RO).

[0158] In various implementations, the BTN2A2 related isoforms are BTN1A1, B2N2A1, BTN3A1, BTN3A2, or BTN3A3. In various implementations, the BTN2A2 related isoform fragments do not contain a transmembrane domain, a cytoplasmic domain, or both.

[0159] Various implementations provide an immunoassay for detecting lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments, or combinations thereof.

[0160] In various implementation schemes, the immunoassay is ELISA.

[0161] In various embodiments, the immunoassay includes an anti-lactolipoprotein A2 (BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2 related isoform antibody, or an anti-BTN2A2 related isoform antibody, or a combination thereof, immobilized on a solid support; and the solid support.

[0162] In various implementations, the immunoassay further includes a biological sample obtained from a subject. The subject may be someone who wants to know information about the levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments.

[0163] Various embodiments provide a method for monitoring a patient using the immunoassay of the present invention, the method comprising: contacting a biological sample obtained from a patient with the bioassay of the present invention; and detecting the levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms or BTN2A2-related isoforms, or combinations thereof.

[0164] In various embodiments, the method further includes selecting or applying the treatment of the present invention as described herein. In various embodiments, the method further includes selecting or applying one of the following treatments:

[0165] (i) lactolipoprotein A2 (BTN2A2), its BTN2A2 fragment, BTN2A2 related isoforms or BTN2A2 related isoforms;

[0166] (ii) a fusion polypeptide comprising:

[0167] The following are included: lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms or fragments of BTN2A2 related isoforms, or combinations thereof; and albumin, such as HSA or Fc domains or Fc domain fragments, such as human IgG, such as human IgG1, IgG2, IgG3 or IgG4, the Fc domains or Fc domain fragments of which may optionally be modified to increase or decrease specific Fc-related effector functions;

[0168] (iii) A modified polypeptide comprising:

[0169] Lactophoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform, or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactophoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform, or a combination thereof is PEGylated, lipidated, or glycosylated; or

[0170] (iv) Non-BTN2A2 related treatments.

[0171] Various embodiments provide a method for patient stratification using the immunoassay of the present invention, the method comprising: contacting a biological sample obtained from a patient with the bioassay of the present invention; detecting levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoforms, or combinations thereof; and stratifying the patient for treatment with the treatment of the present invention as described herein.

[0172] Various embodiments provide a method for patient stratification using the immunoassay of the present invention, the method comprising: contacting a biological sample obtained from a patient with the bioassay of the present invention; detecting levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoforms, or combinations thereof; and stratifying the patient for treatment with one of the following:

[0173] (i) lactolipoprotein A2 (BTN2A2), its BTN2A2 fragment, BTN2A2 related isoforms or BTN2A2 related isoforms;

[0174] (ii) a fusion polypeptide comprising:

[0175] The following are included: lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms or fragments thereof; and Fc domains or fragments thereof, typically human IgG, such as human IgG1, IgG2, IgG3 or IgG4, which may optionally be modified to increase or decrease specific Fc-related effector functions.

[0176] (iii) A modified polypeptide comprising:

[0177] Lactophoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform, or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactophoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform, or a combination thereof is PEGylated, lipidated, or glycosylated; or

[0178] (iv) Non-BTN2A2 related treatments.

[0179] Wild-type amino acid sequence (SEQ ID NO:1)

[0180] (237aa BTN2A2 EC domain (bold and italic) + 15aa Xa and vector clone sequence + 227aa Fc region (bold))

[0181] MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQEN GIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIEGRMDDISST MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK has a mutated amino acid sequence of a long-acting protein (SEQ ID NO:2).

[0182] (237aa BTN2A2 EC domain (bold and italic) + 2aa vector cloning sequence + 227aa Fc region (bold), underlined to indicate aa substitution)

[0183] MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV L HEALH S HYTQKSLSLSPGK

[0184] SEQ ID NO:3 (BTN2A2 EC domain)

[0185] MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETV

[0186] SEQ ID NO:4 (Fc region (wt))

[0187] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0188] SEQ ID NO:5 (Fc region (mutant))

[0189] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0190] In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of the lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, a fusion protein, or a modified polypeptide as described herein. "Pharmaceutically acceptable excipient" means an excipient that can be used to prepare generally safe, non-toxic, and desirable pharmaceutical compositions, and includes excipients suitable for veterinary use and for human use. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0191] In some embodiments, the compounds of the present invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. As used herein, the terms "pharmaceutically acceptable salts, esters, amides, and prodrugs" refer to those carboxylates, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention that, within reasonable medical judgment, are suitable for use in contact with a patient's tissues without excessive toxicity, irritation, allergic reactions, etc., have a reasonable benefit / risk ratio, and are effective for the intended use of the compounds of the present invention. The term "salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds of the present invention. These salts may be prepared in situ during the final separation and purification of the compounds, or by reacting the purified compound, in its free base form, with a suitable organic or inorganic acid alone and separating the resulting salt. These salts may include cations based on alkali metals and alkaline earth metals, such as 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, for example, Berge SM et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0192] The term "pharmaceutically acceptable ester" 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 by reacting the purified compounds in their free acid form or 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 being solvated under physiological conditions, such as alkyl esters, methyl esters, ethyl esters, and propyl esters.

[0193] As used in this article, a “pharmaceutically acceptable salt or prodrug” is a salt or prodrug that, within reasonable medical judgment, is suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic reactions, etc., has a reasonable benefit / risk ratio, and is effective for its intended use.

[0194] The term "prodrug" refers to a compound that is rapidly converted in vivo to produce one or more functionally active peptides, mutants, variants, analogs, or derivatives thereof, as disclosed herein. As used herein, a prodrug is a compound that, upon administration in vivo, is metabolized or otherwise converted into its biologically, pharmaceutically, or therapeutically active form. Prodrugs of one or more peptides, mutants, variants, analogs, or derivatives thereof, as disclosed herein, can be designed to alter the metabolic stability or transport properties of the peptides, mutants, variants, analogs, or derivatives thereof, to mask side effects or toxicity, to improve the taste of the compound, or to alter other properties or characteristics of the compound. Those skilled in the pharmaceutical art can typically design prodrugs of a compound once they have obtained the pharmaceutically active form of the one or more peptides, mutants, variants, analogs, or derivatives thereof, as disclosed herein, based on their understanding of pharmacokinetic processes and in vivo drug metabolism. Suitable examples of prodrugs include methyl, ethyl, and glycerol esters of the corresponding acids.

[0195] In various embodiments, the pharmaceutical compositions according to the 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, mucosal, dermal, or parenteral administration. "Percutaneous" administration can be accomplished using topical creams or ointments, or by means of percutaneous patches. "Parenteral" refers to routes of administration generally associated with injection, including intraorbital, infusion, intra-arterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal injection. By the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder. By the enteral route, the pharmaceutical composition can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres, liposomes, or polymeric vesicles that allow controlled release. via the parenteral route, the compositions can be in the form of solutions or suspensions for infusion or injection. via the topical route, pharmaceutical compositions based on the compounds according to the invention can be formulated for the treatment of skin and mucous membranes and are in the form of ointments, creams, milk products, oils, powders, dip pads, solutions, gels, sprays, lotions, or suspensions. They can also be in the form of microspheres or nanospheres, liposomes, polymer vesicles, polymer patches, and hydrogels that allow controlled release. These topical compositions can be in anhydrous or aqueous forms, depending on the clinical indication. When applied via the ocular route, they can be in the form of eye drops.

[0196] Pharmaceutical compositions according to the invention may also contain any pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium that participates in carrying or transporting a target compound from one tissue, organ, or part of the body to another. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable," meaning it must be compatible with the other components of the formulation. It must also be suitable for use in contact with any tissue or organ it may come into contact with, meaning it must not pose a risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that seriously outweigh its therapeutic benefits.

[0197] The pharmaceutical compositions according to the invention can also be formulated into capsules, tablets, or prepared as emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the composition, or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. Carriers may also include sustained-release materials, such as glyceryl monostearate or glyceryl distearate alone or with waxes.

[0198] Pharmaceutical formulations are prepared using standard pharmaceutical techniques, including grinding, mixing, granulation, and tableting (where necessary) to form tablets; or grinding, mixing, and filling to form hard gelatin capsules. When a liquid carrier is used, the formulation will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations can be administered directly orally or filled into soft gelatin capsules.

[0199] The pharmaceutical compositions according to the invention can be delivered in a therapeutically effective amount. A precise therapeutically effective amount is the amount of composition that will produce the most effective results in terms of therapeutic efficacy 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 patient's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through routine experiments, such as by monitoring the subject's response to the compound and adjusting the dose accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (edited by Gennaro, 20th edition, Williams & Wilkins PA, USA) (2000).

[0200] Reagent test kit

[0201] The present invention also relates to a kit. Therefore, in some embodiments, the kit contains a composition comprising: lactolipoprotein A2 (BTN2A2) as described herein, a BTN2A2 fragment, a BTN2A2-related isoform or a BTN2A2-related isoform fragment, a fusion protein, or a modified polypeptide.

[0202] The exact properties of the components configured in the kit of the present invention depend on its intended purpose. In one embodiment, the kit is specifically configured for treating mammalian subjects. In another embodiment, the kit is specifically configured for treating human subjects. In a further embodiment, the kit is configured for veterinary applications, treating subjects such as, but not limited to, livestock, domestic animals, and laboratory animals. In another embodiment, the kit is configured for detecting lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments.

[0203] Instructions for use may be included in the kit. These instructions typically contain a tangible expression describing the techniques to be used to achieve the desired results when using the components of the kit. Optionally, the kit may also contain other useful components such as diluents, buffers, pharmaceutically acceptable carriers, syringes, tubing, applicators, aspiration or measuring tools, bandaging materials, or other useful instruments that will be readily recognized by those skilled in the art.

[0204] Materials or components assembled in the kit may be provided to the operator in any convenient and suitable manner to maintain its operability and usability. For example, these components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperatures. These components are typically contained in one or more suitable packaging materials. 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 containing a single kit component. Thus, for example, the package may be a glass vial for containing an appropriate amount of the compositions of the present invention containing: lactolipoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, a fusion protein, or a modified polypeptide, as described herein. In another example, the packaging material may be a glass vial for containing an appropriate amount of the composition of the present invention, namely, the anti-lactolipoprotein A2 (BTN2A2) antibody, anti-BTN2A2 fragment antibody, anti-BTN2A2 related isoform antibody, or anti-BTN2A2 related isoform antibody as described herein. The packaging material typically has an outer label indicating the contents and / or intended use of the kit and / or its components. Example

[0205] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Specific materials mentioned are merely illustrative and not intended to limit the scope of the invention. Those skilled in the art can develop equivalent means or reactants without exercising inventive capabilities and without departing from the scope of the invention.

[0206] Example 1

[0207] Materials and methods

[0208] BTN2A2-Fc construct generation and recombinant protein purification

[0209] Soluble recombinant BTN2A2-Fc protein was generated using an insect cell system infected with baculovirus. This method is scalable and has been successfully used to generate recombinant proteins in the immune system. A 711 bp upstream region of the human BTN2A2 gene (NP_008926.2) containing a 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 number pfuse-hg1fc1) using Age1 and EcoRV restriction sites. Subsequently, the BTN2A2 region adjacent to the Fc region was amplified using specific primers (Table 1), and this region was cloned into the pBacPak8 vector using Xba1 and Sac1 restriction sites. The clones were sequenced end-to-end using Sanger sequencing to confirm the sequences. Primers were synthesized by Integrated DNA Technologies, Inc. (IDT), and all PCR amplification reactions used primers containing HF buffer. High-Fidelity PCR Master Mix (New England Biolabs). Baculovirus was generated by co-transfecting the pBacPak8-BTN2A2-Fc clone with linearized baculovirus DNA (TakaraBio catalog number 631401) 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 number 631402). The P3 baculovirus stock solution was used in serum-free medium (Express Five). TM Large-scale protein purification was performed using Hi5 cells grown in SFM (ThermoFisher, catalog number 10486025). Pierce was used... TM Soluble BTN2A2-Fc protein secreted from the supernatant was purified using a Protein G agarose column (ThermoFisher Scientific, catalog number 20398). The purified protein was finally resuspended in 1x phosphate-buffered saline (1x PBS). Protein purity was checked by SDS-PAGE followed by Coomassie brilliant blue staining. The specificity of the purified protein was confirmed by Western blot analysis using a specific BTN2A2 antibody. Comparative studies were conducted using commercially available recombinant 293 cell-derived human BTN2A2-Fc (catalog number 8918-BT; R&D systems) and mouse BTN2A2 (catalog number 8997-BT-050; R&D systems) with baculovirus-derived recombinant B2N2A2-Fc.

[0210] Table 1. Primer sequences

[0211]

[0212]

[0213] T cell activation and IL-2 secretion assay

[0214] In vitro T cell activation was performed. Briefly, 96-well plates were coated overnight at 4°C with 1 μg / ml anti-CD3 mAb (OKT3, clone x) in PBS, with or without recombinant BTN2A2-Fc (10 μg / ml) or Fc-tagged protein (Cat#10702-HNAH, Sino Biological Inc), or 293 cell-derived human BTN2A2-Fc, or mouse BTN2A2, with or without it. A total of 2.5 x 10⁻⁶ cells were then used. 5 One Jurkat cell / well was added to a pre-coated flat-bottomed 96-well plate. Cells were incubated for 24 hours in a 37°C incubator containing 5% CO2. IL-2 production was measured using a total of 100 μl of culture medium with the IL-2 ELISA kit from Millipore-Sigma (catalog number RAB0286-1KT) according to the manufacturer's protocol.

[0215] In vitro TCR stimulation and signal transduction analysis

[0216] To short-term activate Jurkat cells, 48-well tissue culture plates were coated with anti-CD3 mAb (OKT3, 10 μg / mL) and either 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. Then, the cells were incubated at 37°C with a total of 2x10⁻⁵ cells in 75 μl of 1xPBS. 6 One cell was added to each well and held for 3 min. The reaction was then terminated with ice-cold PBS. Pervanadate was prepared by incubating vanadate (200 mM) and H2O2 (200 mM) at a 1:2 ratio at room temperature for 15 min. Jurkat cells in 1xPBS were treated with a final concentration of 0.1 mM pervanadate for 5 min in an incubator (5% CO2 and 37°C). The cells were then collected, lysed in lysis buffer (RIPA or IP lysis buffer), and subsequently subjected to immunoprecipitation or immunoblotting.

[0217] Immunoprecipitation and immunoblotting

[0218] Jurkat cells were activated with plate-bound anti-CD3 BTN2A2-Fc or Fc-tagged proteins as a control or left unactivated. Cells were collected, washed in PBS, and 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 inhibitor), vortexed on ice for 30 min, and clarified by centrifugation at 15,000 g for 10 min. Aliquots of the protein lysates were used for Western blot analysis, while the remainder was incubated with a specific antibody or IgG control at 4 °C for 4 h. Protein A / G agarose (ThermoFisher Scientific) was added to the lysates and incubated for another 2 h. 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 using SDS sample buffer supplemented with DTT by heating at 70°C for 5–10 min. For Western blot analysis, eluted fractions from unactivated cells, cells co-activated with plate-bound anti-CD3 antibody and BTN2A2-Fc, or cells co-activated with anti-CD3 antibody and Fc-tagged controls, along with their respective input controls, were blotted as described below. Approximately 12 x 10⁻⁶ ppm was used under each condition. 6 Jurkat cells were lysed in 300 ml IP lysis buffer. For immunoprecipitation, the lysate was incubated with 1 mg of antibody against a specific protein or the corresponding IgG control. The complex was pulled down using protein A / G agarose beads and eluted in 100 μL sample buffer. The protein blot was probed with antiphosphorylated Zap70 and antiphosphorylated CD3z antibodies, as well as antibodies against total Zap70 and CD3z proteins (see Table 2). For phosphatase activity, the immunoprecipitate was directly mixed with fluorescein diphosphate tetraammonium salt substrate (FDP, catalog number F2999, Thermofisher Scientific) and Molecular Devices was used as recommended by the manufacturer's included protocol. The M2 plate reader measures activity.

[0219] Table 2. Antibody List

[0220]

[0221]

[0222] Extracellular crosslinking was performed using BS3 (bis[sulfosuccinimide] octanoate) according to the user manual (Thermo Scientific MAN0011240). Jurkat cells were washed with PBS and then incubated on ice for 1 hour with 10 μg / mL BTN2A2-Fc and Fc-tagged proteins. 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 terminated at room temperature with 20 mM Tris HCl (pH 7.5) for 15 min. The cells were then thoroughly washed with 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. The eluted samples were analyzed by Western blot analysis using anti-CD45 antibody detection.

[0223] For Western blot analysis, Jurkat cells were collected 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). Pierce was used. TM The BCA Protein Assay Kit (ThermoFisher Scientific) determines the total protein concentration in the lysates by measuring absorbance at 595 nm on a Bio-Rad microplate reader. Western blot analysis was performed using a total of 40–80 μg of each protein sample. Protein lysates prepared after Jurkat cell treatment and / or immunoprecipitation were separated in 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 [PBST] containing 0.1% Tween). The membranes were incubated overnight with primary antibody at 4°C, washed three times with PBST (10 min each), incubated with secondary antibody at room temperature for 1 hour, and finally washed three times with PBST (10 min each). The blots were visualized using ECL assay (ThermoFisher Scientific). Antibodies used for immunoblotting analysis are listed in Table 1.

[0224] Molecular modeling of BTN2A2 and PTPRC

[0225] A molecular model of BTN2A2 was generated using homology modeling. A search for homologous structures of the extracellular domains of BTN2A2 revealed that BTN3A2 shares approximately 47% sequence homology with BTN2A2. A three-dimensional model of BTN2A2 was generated using the SWISS-MODEL working region, based on the crystal structure of BTN3A2. Subsequently, a short 1.2 ns molecular dynamics simulation of the BTN2A2 molecular structure was performed using Desmond (Schrodinger, Inc., San Diego, CA). The three-dimensional structure of PTPRC was retrieved from the protein database (PDB code: 5FN7). RosettaDock 4.0 was used to determine the interaction between BTN2A2 and PTPRC proteins. A 1.2 ns molecular simulation was then performed on the top 10 predicted models, followed by minimization using Desmond. Finally, the most energy-stable docking model was used to identify potential amino acids for mutation.

[0226] CRISPR-Cas9 Deletion of CD45 Gene and Site-Directed Mutagenesis of CD45

[0227] CD45 knockdown cell lines were generated using the CRISPR-Cas9 system. Based on the adjacent protospacer motif (PAM) sequences of CD45 exons 1 and 3, two different guide RNAs were designed to be complementary to the upstream of CD45 exon 1 and the downstream of CD45 exon 3 (Table 2). These guide RNA sequences were cloned into the lenti-Crispr v2 blast vector containing the Cas9 encoding gene and the blast fungicide resistance gene. Lentiviral viruses were generated using envelope and packaging plasmids. Jurkat cells were then transduced with the lentivirus expressing the guide RNA and the Cas9 gene, and blast fungicide resistant cells were selected. Single-cell cloning was performed to isolate independent clones. The deletion of exons 1 through 3 was confirmed by direct Sanger sequencing. Figure 13A ).

[0228] The CD45(PTPRC)(NM_002838) human-marked ORF clone was purchased from origene technologies, Inc. Site-directed mutagenesis was performed using the QuickChange mutagenesis kit (Invitrogen) to introduce the N419A and N468A mutations. The introduction of these mutations was confirmed by direct sequencing. Figure 13B Lentivirals were generated using envelope and packaging plasmids. Jurkat cells with the CD45 gene deleted were then transduced with lentiviruses expressing the mutant CD45 gene.

[0229] In vitro Treg and Th17 differentiation via mixed lymphocyte response (MLR)

[0230] To facilitate Treg differentiation, fresh spleens and lymph nodes (groin, arm, and axilla) were harvested from mice and washed with 1x RPMI medium. The spleens were cut into small pieces, and single cells were prepared by homogenizing the tissue with the plunger end of a syringe and passing it through a 70-micron coarse filter. 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 co-cultured with irradiated spleen cells from CD1 mice for 7 days. Cells were labeled with immunofluorescence antibodies, and co-expression of CD4+CD25+Foxp3-GFP+ve cells was analyzed by flow cytometry. RNA was extracted from CD4+ve cells incubated with anti-CD3 antibody on days 1 and 5 in the absence or presence of recombinant BTN2A2-Fc for qPCR analysis. EasySep was used according to the manufacturer's recommended protocol. TM Mouse CD4+ T cell isolation kit (Stem Cell Tech) was used to isolate CD4+ VE T cells; EasySep was used. TM Mouse B cell isolation kit (Stem Cell Tech) was used to isolate B cells; EasySep was used. TM Pan-DC cells were isolated using the Mouse Pan-DC Cell Enrichment Kit II (Stem Cell Tech); EasySep was used to isolate the cells. TM Mouse T cell isolation kit (StemCell Tech) for isolating T cells.

[0231] To investigate the effect of BTN2A2 on Th17 differentiation, 1 x 102 cells were stimulated with soluble anti-CD3 antibody (0.5 mg / ml) and irradiated spleen cells from CD1 mice (ratio 1:1) and a mixture of Th17 differentiation cytokines (TGF-β1 1.5 ng / ml, IL-6 10 ng / ml, IL-1β 10 ng / ml) with or without recombinant BTN2A2-Fc (10 μg / ml). 6 Spleen cells were cultured for 5 days and subjected to intracellular immunofluorescence staining for RORgt (a Th17 cell marker). CD4+RORgt+ve co-expression was analyzed by flow cytometry. During T cell differentiation studies, a CD45 phosphatase inhibitor (compound 211, catalog number 530197; Millipore Sigma) was used at a concentration of 125 nM. This inhibitor has previously been described as irreversible and is a selective blocker of the allosteric pocket at the D1-D2 domain interface, far from the substrate-binding / catalytic site.

[0232] Flow cytometry analysis

[0233] Immune cells were freshly harvested from the spleen and lymph nodes (groin, armpit, and axilla) of mice. Cell counting was performed using a Hemavet 950FS hematology analyzer (Drew Scientific, Miami Lake, FL). Immunostaining and flow cytometry analysis of the cells were performed as previously described (Li et al., 2018). In short, cells isolated from mice or from treated in vitro cultures were harvested and analyzed using Ghost Dye. TM Red 780 Viability Dye (Cell Signaling) staining. Cells were thoroughly washed with 0.5% BSA in 1xPBS and resuspended in staining buffer (FBS); they were then further stained with fluorescently conjugated antibodies against cell surface markers anti-CD3, anti-CD4, and anti-CD25. Cells were washed with staining buffer to remove excess antibody and then fixed / permeabilized using a mouse Foxp3 buffer kit (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 in DAPI-containing buffer, and resuspended in DAPI-containing buffer. Direct analysis of CD4+CD25+Foxp3-GFP or CD4+RORgt expression in cells was performed. The stained cells were analyzed using BD LSRFortessa (BD Biosciences, San Jose, CA). Details of the commercial antibodies used in this study are provided in Table 1.

[0234] Cell proliferation and apoptosis assay

[0235] CD4+ cells were isolated from the spleen of freshly harvested wild-type B6 mice as described above. The cells were processed using CellTrace according to the manufacturer's protocol (Invitrogen). TM Violet dye at a concentration of 5 mM and 10 6 Staining was performed at a dilution of 1.5 x 10⁶ cells / mL. TMViolet-stained cells were incubated on a mixture of anti-CD3 (0.5 μg / ml) and anti-CD28 (0.5 μg / ml) containing either BTN2A2-Fc fusion protein (10 μg / ml) or an Fc control (10 μg / ml). Cells were cultured for 3 days in RPMI medium supplemented with 10% FBS and 1% Pen-Strp at 37°C and 5% CO2. Unstimulated parental generations (G0) are represented by the brightest peak on the far right of the histogram (Fig. 14). Cells were stained with Annexin V-FITC conjugates for apoptosis assays. For intracellular IL2 staining, cells were fixed, permeabilized, stained with IL2 antibody, and analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA).

[0236] Immunofluorescence studies

[0237] Immunofluorescence staining targeting 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 and incubated overnight at 4°C. Immunofluorescence staining was then performed on 1x10 μL poly-L-lysine coverslips in 75 μL of 1xPBS at 37°C. 6 Jurkat cells were exposed to coated coverslips for 3 min and fixed using the Cytofix / Cytoperm-Fixation / Permeabilization Kit (BD Biosciences). The coverslips were then gently washed twice with 1x PBS. Cells were blocked with blocking buffer (5% goat / mouse serum in 1x PBS containing 0.3% Triton-X100) and stained with mouse anti-human CD45 (abcam catalog number ab8216) and rabbit anti-human CD3ζ (abcam catalog number Ab226263). The coverslips were then gently washed three times with 1x PBS for 10 min each time and stained with donkey anti-mouse IgG antibody Alexa Fluor. TM 555 and goat anti-rabbit IgG antibody Alexa Fluor TM 488 (ThermoFisher Scientific) staining. Then, gently wash the coverslip three times with 1x PBS for 10 minutes each time, incubate in DAPI for 5 minutes, and use ProLong... TMA Diamond Antifade Mountant (ThermoFisher Scientific) was mounted on a glass slide. The glass slide was then imaged at 63x resolution via oil immersion using a Zeiss LSM 780 confocal microscope. Images were analyzed as 8-bit images, and the intensities of the red and green channels were measured in gray-scale fluorescence units from 0 to 255. Colocalization analysis was performed using the Image-J plugin “Colocalization Finder.” At least seven fields of view were analyzed for each experimental condition, with an average of 50 T cells analyzed to derive the average colocalization amount.

[0238] Quantitative polymerase chain reaction (QPCR)

[0239] To perform gene expression analysis, TRIzol was used. TM Total RNA was extracted using reagents (Thermo Fisher Scientific). SuperScript was used. TM IV VILO TM Master Mix (Thermo Fisher Scientific) synthesized cDNA from 500 ng of total RNA. PowerUp was used. TM SYBR TM Quantitative real-time PCR was performed using Green Master Mix (Thermo Fisher Scientific) on the QuantSt udio 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 provided in (Table 2).

[0240] animal models

[0241] All animal experiments were conducted 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 Cedars-Sinai Medical Center Institutional Animal Care and Use Committee.

[0242] Foxp3 EGFP (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 Jackson Laboratory.

[0243] BTN2A2 knockout mice (- / -): BTN2A2 (- / -) Mice (strain: C57BL / 6J-Btn2a2em1cyagen) were generated by commercially available companies (Cyagen Biosciences) using CRISPR-Cas9 technology to delete a 10460-base-pair region of the btn2a2 gene containing exons 2–8 (Figure 16). Genotyping of the mice was performed using polymerase chain reaction (PCR) with three sets of primers (Table 1). Wild-type mice showed a single 500 bp band on agarose gel electrophoresis, heterozygous mice showed two bands (700 bp and 500 bp), and homozygous mice showed a single 500 bp band (Figure 16).

[0244] Model of glomerulonephritis: A detailed protocol for inducing crescentic glomerulonephritis (GN) has been previously described. Nephrotoxic serum was generated in rabbits through repeated immunization with purified glomeruli in both complete and incomplete Freund's adjuvants. Five days prior to administration of the nephrotoxic serum, mice were pre-immunized with normal rabbit IgG and complete Freund's adjuvant. Nephrotoxic serum nephritis was induced by intravenous injection of 20 ml or 10 ml of the nephrotoxic serum on day 0. Four doses of 25 mg BTN2A2-Fc fusion protein or a mediator (control) were administered intraperitoneally on days 0, 2, 4, and 6 after the nephrotoxic serum injection. Mice were sacrificed on day 7 to collect tissues, urine, blood cells, and plasma. Immune cells were harvested from fresh whole spleens and lymph nodes (groin, armpit, and axilla) and processed using EasySep. TM CD4+ T cells were isolated using a mouse CD4+ T cell isolation kit (STEM CELL TECH). Protein lysates of the tissue 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 16000 g for 20 min at 4°C, and the supernatant was collected. IL17A expression was detected using Western blotting with approximately 80 mg of the tissue lysate.

[0245] To obtain the protein-to-creatinine ratio (mg / mg) in urine for evaluating proteinuria, urinary protein was measured using a protein assay dye (No. 500-0006, Bio-Rad, 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 of crescents, defined by the presence of GBM rupture, fibrin deposition, and nucleoid fragmentation) and crescents in all glomeruli were evaluated using periodate-silver hexamine and PAS staining on a paraffin section from each mouse. Representative images were captured using an optical microscope (Nikon Eclipse 50i, Nikon, Tokyo, Japan). The histopathological diagnosis of GN was evaluated by a licensed nephrologist (MY).

[0246] Model of miscarriage: DBA / 2 and CBA / J mice were purchased from Jackson Laboratory. Male DBA / 2 mice were crossed with female CBA / J mice to document immune-mediated pregnancy loss, as described elsewhere (Zenclussen et al., 2005). As a control, female DBA / 2 mice were crossed with male CBA / J strains, and we evaluated litter size, pup weight, and litter reabsorption rate. To investigate the therapeutic effect of BTN2A2, four doses of 25 mg BTN2A2-Fc fusion protein were administered intraperitoneally to female CBA / J mice crossed with male DBA / 2 mice on days 6, 9, 12, and 15 of gestation. Animals were sacrificed on day 18 of gestation, and blood / tissue was harvested for molecular analysis.

[0247] statistics

[0248] Data were plotted and statistically analyzed using GraphPad Prism version 9.2. Data are expressed as mean ± standard deviation (SD) as indicated. The Shapiro-Wilk test was used to test the normality of the data. Unpaired t-tests were used to compare data from two groups that passed the normality test; otherwise, the Mann-Whitney test was used. For data with three or more groups, one-way ANOVA combined with the Tukey multiple comparison test was used for data that passed the normality test; otherwise, the Kruskal-Wollis test was used for comparison, followed by the Dunn multiple comparison test. Statistically significant differences were defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). The legend indicates the number of trials for each experiment.

[0249] Example 2

[0250] Human Research

[0251] This study has been approved by the Institutional Review Board at the Cedars-Sinai Medical Center.

[0252] Treg assay: Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood drawn from unrelated healthy individuals using Ficoll-Hypaque gradient centrifugation. After irradiation with stimulator PBMCs, they were centrifuged at a 1:1 ratio (1 × 10⁻¹¹ cells per mL) in the absence or presence of 0 or 10 mg / ml BTN2A2-Fc. 6 Each type of PBMC was exposed to responder PBMCs and then incubated for 7 days to measure Tregs. The MLR mixture was first stained with antibodies against CD45, CD3, CD4, CD25, and CD127. After permeabilization, the cells were stained with an antibody against Foxp3. After obtaining cells by flow cytometry, lymphocytes isolated from CD45+ leukocytes were mapped against CD4. CD4+ cells were mapped against CD127 using CD25, and then against CD25+CD127. 低 / - Cell mapping against Foxp3. CD4+ / CD25+ / CD127 低 / - / Foxp3+ cells were designated as Treg cells. Treg cell levels were expressed as the percentage of Treg cells among CD4+ T cells.

[0253] Thl7 assay: Human PBMCs (responder cells) isolated by Ficoll–Hypaque gradient centrifugation, with or without recombinant BTN2A2-Fc (10 mg / ml), were cultured at a 1:1 ratio (1 × 10⁶ cells per ml) in irradiated stimulated progeny cells for 5 days, along with a mixture of Th17 differentiation cytokines (anti-CD3 antibody 0.5 μg / ml, TGFb 1.5 ng / ml, IL-6 10 ng / ml, IL-1b 10 ng / ml). Cells were stained with surface markers CD3 and CD4. After permeabilization, cells were stained with anti-RORgt antibody, and CD4+RORgt+ve co-expression was analyzed by flow cytometry. Th17 cells are expressed as the percentage of RORgt+ve cells in CD4 T cells. A CD45 phosphatase inhibitor at a concentration of 125 nM was used during T cell differentiation studies.

[0254] Example 3

[0255] result

[0256] BTN2A2 blocks CD3-dependent signaling in Jurkat cells.

[0257] To elucidate the potential mechanisms by which BTN2A2 regulates 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 mouse systems (Ammann et al., 2013), recombinant human BTN2A2-Fc, rather than the recombinant Fc control protein, blocked the production of IL-2 (…) in anti-CD3-induced Jurkat cells. Figures 10A-10C ).

[0258] When we next evaluated the downstream signaling components of the TCR, we observed that BTN2A2-Fc reduced anti-CD3-induced phosphorylation of Zap70 and CD3z in Jurkat cells. Figure 10D The addition of the phosphatase inhibitor pervanadate led to hyperphosphorylation of Zap70 and CD3z at baseline (unstimulated), and contrary to the results in the absence of pervanadate, the addition of BTN2A2-Fc to the culture had no effect on the hyperphosphorylated proteins. Figure 11 These data collectively indicate that BTN2A2-Fc activates phosphatases that downregulate TCR signaling.

[0259] BTN2A2 blocks TCR signaling by binding to and enhancing CD45 phosphatase.

[0260] Based on the 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 blot analysis of CD45-related proteins following co-immunoprecipitation (co-IP) in unstimulated Jurkat cells showed 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. Conversely, TCR activation in the presence of BTN2A2 resulted in CD45 maintaining association with the TCR complex. Figure 1AThis limits T cell activation. Immunocytochemistry confirmed that CD45 and CD3z colocalized in untreated cells at baseline, CD45 dissociated after anti-CD3-induced TCR activation, and the colocalization of CD45 and CD3z was maintained when cells were stimulated in the presence of BTN2A2. This suggests that BTN2A2 prevents CD45 from being excluded from the immune synapse during the early stages of T cell activation. Figure 1B ).

[0261] Co-IP experiments further showed that, compared with unstimulated cells, anti-CD3 activation reduced the association between CD3e and CD45, while TCR activation in the presence of BTN2A2 enhanced the interaction between CD45 and CD3e. Figure 1C Functional experiments investigating TCR-related CD45 phosphatase activity showed that when the TCR complex was separated from the anti-CD3e antibody via co-IP, CD45 phosphatase activity was detectable at baseline, decreased after TCR activation, and was maintained when cells were activated in the presence of BTN2A2. This indicates that CD45 is dissociated from the TCR complex after activation but is retained in the presence of BTN2A2 (Jung et al., 2021; Leupin et al., 2000). Figure 1D Building on previous research showing that activation of CD45 phosphatase on the lipid microstructure domains on the T cell surface leads to reduced sensitivity of TCR-mediated signaling (He et al., 2002), our new data suggest that BTN2A2 attenuates TCR signaling by activating CD45 phosphatase activity in the TCR complex, thereby blocking crucial downstream phosphorylation events of the TCR.

[0262] BTN2A2 interacts with CD45 phosphatase

[0263] Next, we employed a co-IP experimental strategy using Jurkat cells to test the direct interaction between CD45 and endogenously expressed BTN2A2. Figure 2A These assays showed that CD45 co-immunoprecipitates with endogenous BTN2A2 in unstimulated Jurkat cells, while binding is significantly enhanced in activated cells. Anti-CD3 activation of Jurkat cells did not alter the expression levels of endogenous CD45 and BTN2A2. Figure 12 Supplemental experiments using cells treated with exogenous BTN2A2-Fc protein or a control Fc protein confirmed that exogenous BTN2A2-Fc, rather than the control Fc tag protein, binds to CD45 in activated T cells. Figure 2B-C). We then performed molecular modeling to identify regions of CD45's extracellular domain that may interact with BTN2A2. Protein homology modeling revealed that BTN2A2 interacts with the extracellular fibronectin domain of CD45, and that amino acids -Asn-419 and Asn-468 (N-glycosylation sites inferred from their positions within the NXS / T consensus motif) are crucial for this interaction. Figure 2D Then, we performed site-directed mutagenesis of asparagine to alanine and confirmed that the interaction between mutant CD45 and BTN2A2 was significantly weakened (Figure 13 and 14). Figure 2E -F).

[0264] Based on our observations, CD45 binds to endogenous BTN2A2 upon activation. We suspect that BTN2A2 preferentially binds to the CD45RO isoform, as CD45RO is primarily expressed in activated T cells (Hermiston et al., 2003) and is upregulated after Jurkat cell activation. Figure 2G With this in mind, we repeated the pull-down assay using isotype-specific antibodies. Figure 2H These experiments confirm that BTN2A2 preferentially binds to the CD45RO isoform, which is the major isoform in activated T cells.

[0265] BTN2A2 enhances the proliferation of regulatory T cells and inhibits the Th17 cell population in primary mouse T cells.

[0266] Activated CD4+ T cells differentiate into several effector cell subsets based on the activation event and the cytokine environment present during activation. Given previous reports that BTN2A2 induces Foxp3 expression in CD4+ T cells in short-term culture studies (Ammann et al., 2013), we tested whether T cells activated in the presence of BTN2A2 increased the Treg population in spleen cells of Foxp3-EGFP mice co-expressing EGFP when Foxp3 was expressed. We cultured mouse T cells (Foxp3+ reporter mice - BALB / cJ strain) in the presence of irradiated allogeneic mouse spleen cells (CD1 strain) with or without recombinant BTN2A2-Fc. These assays showed that BTN2A2-Fc-treated cells had a 50% increase in CD4+CD25+Foxp3+ T cells compared to the control, similar to the magnitude observed in the positive control culture using exogenous TGF-β. Figure 3A Interestingly, BTN2A2 is exposed to activated CD4 for short periods. +T cell proliferation was reduced and survival increased, with a pro-differentiation phenotype noted and similar to reports on TGF-β (McKarns and Schwartz, 2005) (Figure 14). Parallel assays showed that recombinant BTN2A2 blocked IL-6 / TGF-β / IL-1β-induced RORgt (the signature transcription factor of Th17 cells) production. Figure 3B ).

[0267] To further explore the short-term effects of BTN2A2 on T cell subset expression patterns under mixed lymphocyte response (MLR) culture conditions, we assessed the gene expression patterns of molecules associated with Th1, Th2, Treg, and Th17 cells using RT-PCR. BTN2A2 upregulated IL2Rbeta and Foxp3 and downregulated IL-21, consistent with our in vitro studies of Treg amplification and Th17 cell suppression. Figure 15A -C). This assay further showed that on day 5, transcription factors regulating the differentiation pathways of Th1 (TBX21) and Th2 (GATA3) were downregulated ( Figure 15D -F).

[0268] To explore the role of endogenous BTN2A2, we knocked out the mouse BTN2A2 gene using a CRISPR-Cas9 strategy (Figure 16) and confirmed that BTN2A2 expression was absent in the professional APCs (CD11c+ dendritic cells and CD19+ B cells) of knockout mice compared to wild-type littermates (Figure 17). Interestingly, when isolated APCs from BTN2A2- / - animals were co-cultured with mouse T cells from Foxp3+ reporter mice, the lack of BTN2A2 in the isolated dendritic cells and isolated B cells from BTN2A2- / - animals impaired the differentiation of mouse T cells into Foxp3+ Tregs compared to dendritic cells and B cells from wild-type littermates. Figure 3C -D).

[0269] The enhancement of the BTN2A2-induced Treg / Th17 balance depends on CD45 phosphatase activity.

[0270] To test whether CD45 phosphatase activity is crucial for the role of BTN2A2 in TCR signaling and Treg amplification, we used a previously validated CD45-specific phosphatase inhibitor (Perron et al., 2014). Adding BTN2A2-Fc to activated JurkatT cells reduced ZAP70 kinase phosphorylation, but inhibiting CD45 phosphatase activity with this small molecule inhibitor (Perron et al., 2014) eliminated this effect. Figure 4AWhen we inhibited CD45 phosphatase activity during BTN2A2-Fc treatment, we also observed that the effects of BTN2A2-Fc on Treg increase and Th17 decrease populations were completely eliminated. Figure 4B -C). Combined with other experiments described above, these studies link the effects of BTN2A2 on the activity of CD45 phosphatase within the TCR complex and immune synapses, as well as on Treg and Th17 cell homeostasis.

[0271] BTN2A2-Fc therapy exhibits immunomodulatory functions in vivo.

[0272] To test whether and how the in vitro observed immunomodulatory effects of BTN2A2 correlated with in vivo findings, we used two different mouse model systems. Consistent with previous reports (Saito et al., 2022; Tipping and Holdsworth, 2006), injection of nephrotoxic serum (NTS) in wild-type B6 mice induced crescentic glomerulonephritis with severe proteinuria. Figure 5A -D). Administration of BTN2A2-Fc reduced proteinuria and glomerular crescent formation over several days following NTS administration. Administration of BTN2A2-Fc also increased CD4+Foxp3 gene expression in CD4+ T cells purified from the spleen and lymph nodes. Figure 5E This reduced the expression of the CD4+RORgt gene in the cell. Figure 5F This reduced the level of CD5, a T cell activation marker, in the cell. Figure 5G Furthermore, compared to the control, BTN2A2-Fc reduced IL17A protein expression in the kidneys. Figure 5G -I).

[0273] Next, we administered lower doses of NTS (50% less than the amount used in the previous experiments) to BTN2A2- / - mice (Fig. 16) and wild-type littermate controls (Fig. 6). These experiments showed that lower doses of NTS induced relatively mild crescentic glomerulonephritis and proteinuria in wild-type controls; however, in BTN2A2- / - animals, we observed exacerbated crescentic glomerulonephritis and severe proteinuria. Figure 6A We also observed decreased Foxp3 gene expression and increased RORgt gene expression in spleen / lymph node CD4+ T cells of BTN2A2- / - mice compared to controls. Figure 6D -E). CD5 in CD4+ T cells of BTN2A2- / - mice is enhanced compared to wild-type mice ( Figure 6FAnalysis of kidney tissue one week after administration of nephrotoxic serum also showed that IL17A protein expression in the kidneys of BTN2A2- / - animals was increased compared to control animals. Figure 6G -H).

[0274] To evaluate the generalizability of the role of BTN2A2 in immune regulation, we investigated the effects of recombinant BTN2A2-Fc in DBA / 2 mice mated with CBA / J mice, a model of immune-mediated abortion (Clark et al., 2008). We demonstrated that DBA / 2 males x CBA / J females had reduced litter size and higher spontaneous aspiration rates compared to CBA / J males x DBA / 2 females. Administration of BTN2A2-Fc increased litter size and rescued the excessive abortion rate observed in DBA / 2 males x CBA / J females. Figure 7A -B and Figure 18 The beneficial effects of BTN2A2-Fc were associated with increased frequency of Foxp3+ Treg cells in the spleen / lymph nodes, decreased frequency of Th17 cells in the spleen / lymph nodes, and relatively weakened CD5, which is consistent with our findings in an autoimmune GN model. Figure 7D -F). Furthermore, BTN2A2-Fc protein also reduces placental IL-17 protein expression, which is associated with increased litter size (-F). Figure 7G -H).

[0275] BTN2A2 enhances Treg cell proliferation and suppresses Th17 cell populations in human PBMCs.

[0276] To evaluate whether the effects of recombinant BTN2A2 are applicable to human T cells, we analyzed human PBMCs activated by a mixed lymphocyte response (MLR, using allogeneic stimulatory daughter cells) with and without recombinant BTN2A2-Fc, and quantified CD4+CD25+CD127 after 7 days. 低 / - The number of Foxp3+ Treg cells was determined. These co-cultures showed that BTN2A2-Fc induced a 2-fold increase in Tregs under MLR conditions compared to the control; however, BTN2A2-Fc failed to induce Treg expansion in the presence of a CD45 inhibitor. Figure 8A -B). Parallel experiments showed that recombinant BTN2A2-Fc strongly blocked the TGFb-IL6-IL1b-induced increase in Th17 cells (-B). Figure 8C Interestingly, BTN2A2-Fc failed to block cytokine-induced Th17 cell activity in the presence of CD45 inhibitors. Taken together, these observations suggest that CD45 phosphatase activity is also necessary for the function of BTN2A2 in humans.

[0277] Various embodiments of the invention have been described above in a detailed description. While these descriptions directly depict the above embodiments, it should be understood that modifications and / or variations to the specific embodiments shown and described herein can be conceived by those skilled in the art. Any such modifications or variations falling within the scope of this specification are also intended to be included within its scope. Unless specifically stated otherwise, it is the inventors' intention to provide the common and customary meaning of the words and phrases in the specification and claims for those skilled in the art of application.

[0278] The foregoing description of various embodiments of the invention known to the applicant at the time of filing this application has been given and is intended for illustrative and descriptive purposes. This specification is not intended to be exhaustive, nor is it intended to limit the invention to the precise forms disclosed, and many modifications and variations are possible in accordance with the foregoing teachings. The described embodiments are intended to explain the principles of the invention and its practical application, and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications suitable for the particular intended use. Therefore, the invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention.

[0279] While specific embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made based on the teachings herein without departing from the invention and its broader aspects, and therefore, the appended claims will cover all such changes and modifications falling within the true spirit and scope of the invention. As used herein, the terms “comprising” or “including” are used to refer to compositions, methods, and one or more corresponding components thereof that are useful to embodiments but may contain unspecified elements (whether useful or not). Those skilled in the art will understand that, in general, the terms used herein are often intended as “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “including” should be interpreted as “including but not limited to,” etc.). Although the open-ended term “comprising” is used herein as a synonym for terms such as including, containing, or having, alternative terms such as “consisting of” or “essentially consisting of” may also be used to describe the invention or embodiments thereof.

[0280] Unless otherwise stated, the terms “a,” “an,” and “the,” and similar expressions used in the context of describing a particular embodiment of the application (particularly in the context of the claims) are to be interpreted as encompassing both singular and plural. The enumeration of numerical ranges herein is intended only as a way of individually referring to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such as”) provided for certain embodiments herein is intended only to better clarify the application and is not intended to limit the scope of the application as otherwise claimed. The abbreviation “e.g.” derives from the Latin word for example (exempli gratia) and is used herein to indicate non-limiting examples. Therefore, the abbreviation “e.g.” is synonymous with the term “for example.” The language in the specification should not be interpreted as indicating any unclaimed element essential to the practice of this application.

[0281] "Optional" or "optionally" means that the situation described below may or may not occur, such that the description includes both the scenario in which the situation occurs and the scenario in which the situation does not occur.

[0282] The groups of alternative elements or embodiments disclosed herein should not be construed as limiting. Each group member may be mentioned and claimed individually or in any combination with other members of the group or other elements present herein. For convenience and / or patentability, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to contain the group as modified, and thus conform to the written description of all Markush groups as used in the appended claims.

Claims

1. A method for reducing CD3-dependent T cell signaling in a subject in need, comprising: The subject was given lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide containing any of the foregoing.

2. A method for increasing T regulatory (Treg) cells or decreasing T helper 17 (Th17) cells in a subject in need, or both, comprising: The subject was given lactolipoprotein A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment, or a fusion polypeptide containing any of the foregoing.

3. The method of claim 1 or 2, wherein the BTN2A2, its fragments, BTN2A2 related isoforms, BTN2A2 related isoform fragments or fusion peptides comprise human BTNA2 or fragments or isoforms thereof.

4. The method as claimed in any of the preceding claims, wherein the subject is pregnant, and the method inhibits or reduces the likelihood of miscarriage by increasing Treg cells.

5. The method as described in any of the preceding claims, wherein the subject suffers from kidney injury or a kidney injury-related disorder.

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

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

8. The method as described in any of the preceding claims, wherein the method reduces the need for immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to the subject.

9. The method of claim 8, wherein the method reduces or eliminates other aspects of the toxicity of the immunosuppressive therapy in the subject.

10. The method of any of the preceding claims, wherein the subject suffers from an autoimmune or inflammatory condition or a condition characterized by pathological Th1 / Th17 cell accumulation.

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

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

13. A method for treating a disease or ailment of a subject in need, comprising: The subjects were given lactolipoprotein A2 (BTN2A2), its BTN2A2 fragment, BTN2A2 related isoforms, or BTN2A2 related isoforms.

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

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

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

17. The method of claim 13, wherein the disease or ailment is related to kidney injury.

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

19. The method of claim 13, wherein the disease or ailment 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. The method of claim 19, wherein the toxic effects of the immunosuppressive therapy are reduced or eliminated in the subject.

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

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

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

24. The method of any of the preceding claims, wherein the lactolipoprotein A2 (BTN2A2) or a fragment thereof, the BTN2A2 related isoform, or the fragment thereof is modified by glycosylation, polyethylene glycolation, lipoylation, or attachment to cholesterol.

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

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

27. The method as claimed in any of the preceding claims, wherein the related isoform of BTN2A2 is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3.

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

29. A fusion polypeptide comprising: Lactophoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms or BTN2A2-related isoforms or combinations thereof; and Fc domains or Fc domain fragments, optionally IgG1, IgG2, IgG3, or IgG4Fc, wherein the Fc domains or Fc domain fragments may optionally be mutated, further optionally mutated to enhance or reduce Fc-related effector function, further optionally any of the mutations shown in Table 3.

30. The fusion polypeptide of claim 29, further comprising a linker located between the BTN2A2, the BTN2A2 fragment, the BTN2A2 related isoform, the BTN2A2 related isoform fragment and the Fc domain or the Fc domain fragment, optionally IgG1, IgG2, IgG3 or IgG4 Fc, wherein the Fc domain or the Fc domain fragment may optionally be mutated, further optionally mutated to enhance or reduce Fc-related effector function, further optionally any of the mutations shown in Table 3.

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

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

4.

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

35. The fusion protein according to any one of claims 29-34, 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. The fusion protein of any one of claims 29-35, wherein the BTN2A2 fragment comprises the extracellular domain of the BTN2A2.

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

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

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

40. A modified polypeptide comprising: Lactophoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms, or BTN2A2 related isoforms, or combinations thereof. The lactolipoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform, or the BTN2A2 related isoform fragment, or a combination thereof, is PEGylated or glycosylated.

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

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

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. The modified polypeptide of claims 40 and 44, wherein the BTN2A2-related isoform fragment does not contain the transmembrane domain, the cytoplasmic domain, or both.

46. ​​An immunoassay, comprising: Anti-lactolipoprotein A2 (BTN2A2) antibodies, anti-BTN2A2 fragment antibodies, anti-BTN2A2-related isoform antibodies, or anti-BTN2A2-related isoform antibodies, or combinations thereof, immobilized on a solid support; and The solid support.

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

48. A method for monitoring a patient using the assay method of claim 43, comprising: Contact the biological sample obtained from the patient with the assay method of claim 43; as well as Detect the levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms or BTN2A2 related isoforms, or combinations thereof.

49. The method of claim 48, further comprising selecting or administering a treatment selected from: (i) lactolipoprotein A2 (BTN2A2), its BTN2A2 fragment, BTN2A2 related isoforms or BTN2A2 related isoforms; (ii) a fusion polypeptide, wherein the fusion polypeptide comprises: The lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms or BTN2A2 related isoforms or combinations thereof; and the Fc domain or Fc domain fragments; (iii) A modified polypeptide, said modified polypeptide comprising: Lactophoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform, or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactophoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform, or the BTN2A2 related isoform fragment, or a combination thereof, is PEGylated or glycosylated; or (iv) Non-BTN2A2 related treatments.

50. A method for patient stratification using the assay of claim 46, comprising: Contact the biological sample obtained from the patient with the assay method of claim 43; Detecting the levels of lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or combinations thereof; and Patients are stratified for the following treatments: (i) lactolipoprotein A2 (BTN2A2), its BTN2A2 fragment, BTN2A2 related isoforms or BTN2A2 related isoforms; (ii) a fusion polypeptide, wherein the fusion polypeptide comprises: The lactolipoprotein A2 (BTN2A2), BTN2A2 fragments, BTN2A2 related isoforms or BTN2A2 related isoforms or combinations thereof; and the Fc domain or Fc domain fragments; (iii) A modified polypeptide, said modified polypeptide comprising: Lactophoprotein A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2 related isoform, or a BTN2A2 related isoform fragment, or a combination thereof, wherein the lactophoprotein A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2 related isoform, or the BTN2A2 related isoform fragment, or a combination thereof, is PEGylated or glycosylated; or (iv) Non-BTN2A2 related treatments.

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