Butyrophilin a2 for inhibition of b cell activation and / or for prolonged humoral suppression or humoral tolerance

Abstract

Described herein are compositions and methods of use thereof for reducing inhibiting B cell activation, B cell proliferation, the production of pathologic, autoimmune or interfering antibodies; and / or for eliciting humoral suppression and / or B cell tolerance in a subject in need thereof by contacting immune cells of the subject or donor immune cells in vivo and / or ex vivo with a composition comprising a Butyrophilin, optionally Butyrophilin A2, or a conjugate or fusion protein comprising said Butyrophilin. The methods include the use of butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2- related isoform, or a BTN2A2-related isoform fragment, or conjugates, and fusion polypeptides containing, preferably which comprise an Fc region comprising one or more mutations which reduce FcR binding and / or which suppress FcRN mediated clearance.

Description

Attorney Docket No. 2969027.002013BUTYROPHILIN A2 AND RELATED ISOFORMS FOR INHIBITION OF B CELL ACTIVATION AND / OR FOR ELICITING PROLONGED HUMORAL SUPPRESSION OR HUMORAL TOLERANCECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application No. 63 / 688,964, filed on August 30, 2024, and to U.S. provisional patent application No. 63 / 699,876, filed on September 27, 2024, the contents of both of which are incorporated by reference in their entirety herein.

[0002] This application further relates to PCT Application No: PCT / US2024 / 018076 filed on March 1, 2024, which claims 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 both of which are hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing submitted as a computer readable form named "2969027o002013.xml", having a size in bytes of 71,146 bytes, and created on August 27, 2025. The information contained in this computer readable form is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0004] This invention relates to butyrophilin A2, its isoforms and fragments and conjugates thereof alone or in association with another moiety, e.g., an antigen, cell, tissue or organ or another therapeutic moiety, optionally one which inhibits antigen-specific B cell activation and / or B cell proliferation and the use thereof in the treatment of conditions, e.g., autoimmune, inflammatory, and allergic conditions involving pathologic antibodies and / or pathologic B cell activation and / or pathologic B cell proliferation.BACKGROUND

[0005] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of theAttorney Docket No. 2969027.002013 information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006] Currently treatment of autoimmunity is largely non-specific with steroids and drugs like cyclosporine and prograf. While there are some promise with newer agents such as CTLA4IG, there remains a need for additional drugs that can for example, synergize to improve autoimmunity and inflammation.

[0007] T cells recognize antigens presented through peptide:MHC complexes using surface expressed, heterodimeric abT cell receptors (TCRs). As the TCR lacks intrinsic kinase activity, signals initiated by TCR ligation involve recruitment of the Src family kinase Lek, which then phosphorylates immunoreceptor tyrosine-kinase-based motifs (ITAMs) within the TCR- associated CDS z-chain. Lek also phosphorylates subsequently recruited Zap70 kinase, thereby propagating requisite downstream signals required for full T cell activation. Studies performed over the past 30 years showed T cell activation is controlled in part by cell surface expressed CD45.

[0008] CD45 is a transmembrane glycoprotein that contains an intracellular tyrosine phosphatase domain capable of dephosphorylating multiple TCR immunoreceptor tyrosine activation (ITAM) motifs. Differential splicing results in expression of multiple CD45 isoforms (i.e. RA, RB, RC, RO). Following TCR stimulation, CD45 is initially recruited to the supramolecular activation cluster (SMAC) but is then expelled, segregating it from the TCR. Evidence suggests that this segregation of CD45's phosphatase activity from the TCR is essential for Lck-initiated signal propagation that results in full T-cell activation. Conversely, retention of CD45 within the T cell immune synapse (IS) regulates the strength and duration of TCR activation, and perturbations of CD45 activity contribute to development of autoimmune disease. In humans, aberrant CD45 expression has been reported in diseases including infantile cholestasis, haemophagocytic lymphohistiocytosis or erythrocytic haemophagocytosis, 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). Also, therapeutic modulation of CD45 function reportedly has direct clinical applicability in organ transplantation, treatment of autoimmune disease or microglial activation associated with Alzheimer disease (AD) (A Rheinlander et al., "CD45 in human physiology and clinical medicine", Immunology Letters, 196:22-32, April 2018). However, despite decades of work by multiple groups delineating these molecularAttorney Docket No. 2969027.002013 mechanisms, it is not known how CD45 segregation versus retention during TCR activation may be regulated, or if ligands co-presented by antigen-presenting cells (APCs) play a role.

[0009] Butyrophilins (BTNs) are glycoproteins enriched in breast milk that have imprecisely understood immune-regulatory effects and are implicated in maintaining maternal-fetal tolerance. The mRNAs encoding for BTN and BTN-like molecules are widely expressed in lymphoid and non-lymphoid tissues. Butyrophilin immunoglobulin domains exhibit structural similarities to the B7 family of co-receptors, including B7-1 / CD80, B7- 2 / CD86, ICOS-L and PD-L1.

[0010] Butyrophilin 2A2 (BTN2A2) was previously shown to be expressed by professional APCs including B cells, macrophages, and dendritic cells (DCs). Results of in vitro studies suggest that BTN2A2 can modulate T cell receptor (TCR) signaling and promote de novo FoxpS expression. Mice genetically deficient in BTN2A2 exhibit impaired CD4+ regulatory T cell function, potentiated anti-tumor immunity, and augmented clinical manifestations of experimental autoimmune encephalomyelitis, all of which were attributable to deficiency of BTN2A2 in APCs. While these cumulative findings implicate a key immunoregulatory function for BTN2A2, the exact molecular mechanisms underlying these effects remain unclear.

[0011] Butyrophilins (BTNs) are glycoproteins enriched in breast milk that have imprecisely understood immune-regulatory effects and are implicated in maintaining maternal-fetal tolerance. The mRNAs encoding for BTN and BTN-like molecules are widely expressed in lymphoid and non-lymphoid tissues. Butyrophilin immunoglobulin domains exhibit structural similarities to the B7 family of co-receptors, including B7-1 / CD80, B7- 2 / CD86, ICOS-L and PD-L1.

[0012] Butyrophilin 2A2 (BTN2A2) was previously shown to be expressed by professional APCs including B cells, macrophages, and dendritic cells (DCs). Results of in vitro studies suggest that BTN2A2 can modulate T cell receptor (TCR) signaling and promote de novo Foxp3 expression. Mice genetically deficient in BTN2A2 exhibit impaired CD4+ regulatory T cell function, potentiated anti-tumor immunity, and augmented clinical manifestations of experimental autoimmune encephalomyelitis, all of which were attributable to deficiency of BTN2A2 in APCs.

[0013] Butyrophilin 2A2 (BTN2A2) Domain StructureAttorney Docket No. 2969027.002013

[0014] The BTN2A2 protein, also known as butyrophilin subfamily 2 member A2, is a member of the immunoglobulin superfamily and is characterized by its extracellular IgV and lgC2 domains, similar to B7 co-stimulatory molecules. It contains a transmembrane domain and an intracellular B30.2 domain. The B30.2 domain is known to fold into a 95 amino acid core with five alpha helices and three beta sheets, forming two hairpin structures.

[0015] Mature Murine BTN2A2 Protein

[0016] Mature mouse Butyrophilin 2A2 consists of 485 amino acid residues which consist of a 215 amino acid (aa) extracellular domain with two immunoglobulin-like domains, a 21 aa transmembrane segment, and a 249 aa cytoplasmic domain. Within the Ig-like domains, mouse Butyrophilin 2A2 shares 71% and 90% aa sequence identity with human and rat Butyrophilin 2A2, respectively.

[0017] Mature Human BTN2A2 Protein

[0018] The mature human Butyrophilin 2A2 (BTN2A2) protein sequence consists of 491 amino acids. lt is a type I transmembrane protein with an extracellular domain containing two immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic domain. Key features of the BTN2A2 protein sequence include an extracellular domain composed of 233 amino acids, includingtwo immunoglobulin-like domains, a transmembrane Domain comprising 21 amino acid residues which span the cell membrane and a cytoplasmic domain which is a 237 amino acid segment which extends into the cytoplasm. The gene encoding BTN2A2 can produce several isoforms through alternative splicing, resulting in variations in the extracellular and cytoplasmic domains.

[0019] BTN2A2 Extracellular Region

[0020] The BTN2A2 protein and its isoforms all comprise an extracellular region characterized by extracellular IgV and lgC2 domains. These IgV and lgC2 domains, similar to B7 family proteins, are believed to play a role in cell-cell interactions. The human BTN2A2 (Butyrophilin subfamily 2 member A2) extracellular region is composed of a 233 amino acid domain containing two immunoglobulin (Ig)-like domains. The extracellular domain of BTN2A2 has been demonstrated to bind to the CD45RO isoform expressed on activated T cell surfaces, which binding enhances CD45 phosphatase activity, leading to dampened T cell receptor (TCR) signaling.

[0021] BTN2A2 Transmembrane RegionAttorney Docket No. 2969027.002013

[0022] The BTN2A2 protein and its isoforms additionally all comprise a transmembrane domain the function of which is to anchor the protein in the cell membrane. The human BTN2A2 protein has a single-pass type I transmembrane region. This transmembrane region is a segment of 21 amino acids that anchors the protein to the cell membrane. BTN2A2 is classified as a Type I Transmembrane Protein, since its N-terminus is extracellular, and its C-terminus is intracellular. The transmembrane region is located between the extracellular and intracellular domains of the BTN2A2 protein.

[0023] BTN2A2 Intracellular Region

[0024] The BTN2A2 protein and its isoforms additionally all comprise an intracellular B30.2 Domain which is a globular domain known for its structural diversity and involvement in protein-protein interactions. This domain's structure includes five alpha helices and three beta sheets, forming two hairpin structures (A1 / A2 and A4 / A5). The human BTN2A2 intracellular region consists of a 237 amino acid cytoplasmic domain which domain includes a B30.2 (PRY-SPRY) domain. Alternative splicing can generate different isoforms of BTN2A2, some with variations or deletions in the cytoplasmic region.

[0025] The sequence of the human BTN2A2 intracellular region is set forth below:

[0026] LVSGLPPGSDQEPGPGPGPRDPLSQPRSLGLTQPELGLTSPLELGLTLPELGLTQLGLTSP QALGLTSPEPSLGLTQPALGLTSPELGLASPQELGLTSPEPSLGLTQPALGLASPEPSLGLTQPALGLASPEP SLGLTQPALGLASPQELGLTSLELGLSSPERLGLGDSGQEPLGPDSQVEPGLGDSGLQELGLGDSGLEEPL GPDRSPLELGLTDSGWEPGLGDSQSEPLGTDSGLEELGLGDASEVELGLGDSGPEEPLGLD (SEQ ID NO: 62).

[0027] BTN2A2 IgV Domain

[0028] As is noted above, BTN2A2 proteins all comprises an IgV domain located in the extracellular region or domain. The IgV domain of mouse BTN2A2 reportedly spans residues D28 to A143 (BTNL2D28 A143) or Q30 to M 142. The domain is located at the C-terminus of the signal peptide which comprises the N-terminal 27 residues of BTN2A2. This signal peptide is generally removed during post-translational processing.

[0029] The N-terminal IgV domain of human BTN L2 reportedly spans residues 34 to 145 or residues 34-147 of the human BTN L2 protein. The human BTNL2 signal peptide again is at the N-terminus of the IgV domain and comprises residues 1-32, which residues are generally removed during post-translational processing.

[0030] Human BTN2A2 IgV Domain Sequence:Attorney Docket No. 2969027.002013

[0031] FTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTE EQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLV Residues 34-145 (SEQ ID NO: 63)

[0032] Human BTN2A2 IgV Domain Sequence:

[0033] FTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVA 147 Residues 34- 147 (SEQ ID NO: 64)

[0034] Human BTN2A2 Signal Sequence:

[0035] MEPAAALHFSLPASLLLLLLLLLLSLCALVSA Residues 1-32 (SEQ ID NO: 65)

[0036] A Q residue intervenes the human BTNL2 signal peptide and IgV domain.

[0037] BTN2A2 IgC Domain

[0038] As is noted above, BTN2A2 proteins all comprises an IgC domain located in the extracellular region or extracellular domain.

[0039] Murine BTN2A2 IgC Domain:

[0040] The IgC domain of mouse BTN2A2 reportedly spans residues 150-232. The sequence is shown below.

[0041] PLIKMKTLEDGSILLECTSEGWYPEPRAVWRDPYDEVVPALEEEYTADREGLFTVTMTIIIRDCSVRNMTCSVNNTLLSQEVE (residues 150-232) (SEQ ID NO: 66)

[0042] Human BTN2A2 IgC Domain:

[0043] The human BTNL2 Immunoglobulin C (IgC) domain reportedly corresponds to residues 149-235 of the mature protein.

[0044] Human BTN2A2 IgC Domain Sequence:

[0045] PLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEK (Residues 149-235) (SEQ ID NO: 67)

[0046] In our prior PCT Application, PCT / US2024 / 018076 filed on March 1, 2024, incorporated by reference herein, we showed that BTN2 fusion proteins, e.g., BTN2-Fc and BTN 2-al bum in fusion proteins inhibited T-cell effector functions (Thl7, IL-17 and cell proliferation) while inducing the production of regulatory T-cells (T-regs) that inhibit deleterious and inflammatory reactions. The inventors also showed in several animal models that the treatment of glomerulonephritis inhibits crescent formation (injury markers) and reduces proteinuria, reduces Thl7, IL-17, and elicits an increase in the number of Tregs. We also showed in humans that BTN2A2 elicits an increase in Tregs and inhibits T-effector cells.Attorney Docket No. 2969027.002013

[0047] Notwithstanding the foregoing, there is a need for a further understanding as to the actions and effects of Butyrophilins (BTNs) on immunity and on specific immune cells as this potentially will facilitate novel therapies using Butyrophilins and fragments, variants, conjugates, fusions and modified forms thereof. Also, there is a need for improved BTN2A2 fusion proteins. Particularly there is a need for improved human BTN2A2-Fc fusion proteins which when expressed in recombinant cells, e.g., mammalian cells, optionally CHO or insect cells, e.g., Spodoptera frugiperda (Sf9) cells, and isolated / purified retain BTN2A2 and CD45RO binding and BTN2A2-mediated biologic activities and which preferably when expressed are not substantially prone to aggregation.SUMMARY OF THE INVENTION

[0048] The present invention achieves these objectives by providing a greater understanding as to the effects of Butyrophilins (BTNs), e.g., Butyrophilin A2 or BTN2A2 on immunity, particularly the discovery that Butyrophilins (BTNs), e.g., Butyrophilin A2 elicit suppressive effects on B cells, and based thereon provides novel therapies using Butyrophilins and fragments, variants, conjugates and modified forms thereof.

[0049] More particularly the invention is based on the surprising discovery that Butyrophilins and conjugates thereof specifically act on B cells and thereby inhibit B cell activation, proliferation and antibody production and that based thereon that Butyrophilins and fragments, variants, conjugates and modified forms thereof may be used to treat or prevent conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production and / or for eliciting prolonged humoral suppression or humoral tolerance in subjects with conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production, e.g., autoimmune, allergic and inflammatory conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production.

[0050] Related to the foregoing, the following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0051] In particular the present invention relates to the discovery that Butyrophilin A2 and Butyrophilin A2 conjugates inhibit B cell activation and / or B cell proliferation and antibody production. Based on this discovery the present invention specifically relates to theAttorney Docket No. 2969027.002013 use of Butyrophilin A2, related isoforms, fragments, and conjugates or modified forms of any of the foregoing for inhibiting B cell activation and / or B cell proliferation and / or antibody production and / or for eliciting acute or prolonged antigen-specific humoral suppression and / or tolerance in vitro or in vivo in a subject in need thereof.

[0052] In specific embodiments the invention provides a method of inhibiting B cell activation, B cell proliferation, antibody production, and / or for eliciting humoral suppression and / or B cell tolerance optionally against a target antigen in a subject in need thereof by contacting immune cells of the subject or donor immune cells with a Butyrophilin or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising any of the foregoing, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising said Butyrophilin or fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, optionally wherein said Butyrophilin, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to at least one halflife extending moiety, optionally an albumin, an Fc protein, polyethylene glycol, a lipid, PASylation (attachment to poly-aspartate and serine) polymers, glycosylation, albumin binding moieties, or a combination of any of the foregoing, further optionally wherein said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which optionally comprises one or more mutations which impair or promote an Fc effector function; further optionally wherein said Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which optionally comprises one or more mutations which impair or promote an Fc effector function which (i) enhances FcRN binding, (ii) impairs or eliminate binding to one or more FcRs and / or (iii) impairs or eliminates binding to complement proteins such as Clq, further optionally wherein the Fc comprises one or more of the mutations identified in TABLE 1; further optionally wherein said Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which Fc comprises the following mutations: L234A, L235A, and P329G (LALAPG); furtherAttorney Docket No. 2969027.002013 optionally wherein said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, possesses at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the corresponding native Butyrophilin, optionally human Butyrophilin A2 or the corresponding fragment thereof, wherein said fragment optionally comprises the extracellular domain of the Butyrophilin, optionally human Butyrophilin A2 and / orthe Butyrophilin fragment, optionally Butyrophilin A2 fragment does not comprise the transmembrane domain, cytoplasmic domain, or both of the corresponding native Butyrophilin, optionally Butyrophilin A2; further optionally wherein the Butyrophilin or a fragment, variant or modified form thereof comprises BTN1A1, B2N2A1, B2N2A2, BTN3A1, BTN3A2 or BTN3A3 or a fragment of any of the foregoing and is preferably human BTN1A1, B2N2A1, B2N2A2, BTN3A1, BTN3A2 or BTN3A3 or a fragment thereof.

[0053] In any of the foregoing methods said contacting may occur before, proximate to, or after the subject is to be or has been exposed to an antigen against which humoral suppression and / or B cell tolerance is to be elicited.

[0054] In any of the foregoing methods the contacting may induce antigen-specific humoral suppression and / or B cell tolerance.

[0055] In any of the foregoing methods the contacting may induce prolonged antigenspecific humoral suppression and / or B cell tolerance.

[0056] In any of the foregoing methods the contacting may induce acute antigenspecific humoral suppression and / or B cell tolerance.

[0057] In any of the foregoing methods the contacting may be effected ex vivo.

[0058] In any of the foregoing methods the contacting may be effected in vivo.

[0059] In any of the foregoing methods the contacting may be effected ex vivo and in vivo.

[0060] In any of the foregoing methods the immune cells comprise bone marrow, peripheral blood cells, B cells, B cell progenitors, stem cells, Tregs, Bregs, hematopoietic stem cells or any combination of the foregoing.

[0061] In any of the foregoing methods, the method may further comprise contacting said immune cells with a desired antigen to which humoral suppression and / or B cell tolerance is to be induced.Attorney Docket No. 2969027.002013

[0062] In any of the foregoing methods, the method may further comprise contacting said immune cells with a soluble antigen to which humoral suppression and / or B cell tolerance is to be induced.

[0063] In any of the foregoing methods, the method may further comprise contacting said immune cells with an antigen expressed on a cell which humoral suppression and / or B cell tolerance is to be induced.

[0064] In any of the foregoing methods, the method may further comprise contacting said immune cells with a nucleic acid, optionally an mRNA, further optionally comprised in a lipid nanoparticle, viral vector or plasmid which encodes an antigen to which humoral suppression and / or B cell tolerance is to be induced.

[0065] In any of the foregoing methods, the antigen may comprise an autoantigen, allergen, therapeutic agent, parasitic antigen, or an antigen expressed by donor cells.

[0066] In any of the foregoing methods, the antigen may comprise a food allergen, an aeroallergen, an animal product, a plant derived allergen, a microbial allergen, optionally a mite, fungus, yeast, etc., a drug, a hormone, a therapeutic enzyme, a therapeutic antibody, a blood or clotting factor, insect venom, or latex.

[0067] In any of the foregoing methods, the antigen may be a food allergen selected from a peanut allergen, a tree nut allergen, a dairy allergen, a wheat allergen, a soy allergen, an egg allergen, a shellfish allergen, a meat allergen, a sesame allergen, and a corn allergen.

[0068] In any of the foregoing methods, said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or fragment thereof, optionally Butyrophilin A2 or fragment thereof, and optionally said antigen are administered in the same or different compositions, and further optionally are administered by one or more of injection, e.g., intravenous, intramuscular, subcutaneous, intrathecal, or injection into the bone marrow; mucosal administration, intranasal administration, oral administration, topical administration, or any combination of the foregoing.

[0069] In any of the foregoing methods, the immune cells or a graft, tissue or organ or composition comprising immune cells of a subject or donor may be contacted ex vivo with Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2, optionally human BTN2A2, or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment, variant or modified form thereof,Attorney Docket No. 2969027.002013 optionally Butyrophilin A2 or a fragment, variant or modified form thereof and an antigen, the immune cells are optionally expanded in culture, and after contacting and optionally expansion are infused into the subject.

[0070] In any of the foregoing methods, the subject may be afflicted with a B cell mediated inflammatory, autoimmune or allergic disorder and / or a condition.

[0071] In any of the foregoing methods the subject may be afflicted with a neurological disorder, SLE, multiple sclerosis, an autoimmune kidney disease (optionally IgA nephropathy, ANCA vasculitis, or membranous glomerulonephritis), Sjogren's Syndrome, B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, advanced stage follicular lymphoma, chronic lymphocytic leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, Pulmonary arterial hypertension (PAH), Systemic sclerosis, Refractory hypertension, Autoimmune hypoglycemia, and / or Anti-NMDA receptor encephalitis.

[0072] In any of the foregoing methods, the subject may have received a transplanted cell, tissue or organ and shows signs and / or has a history of antibody mediated transplant rejection.

[0073] In any of the foregoing methods, the subject may be one who has or is to receive cell therapy, or a tissue or organ transplant, optionally one or more of kidney, heart, lung, pancreas, bone marrow, and liver.

[0074] In any of the foregoing methods, the subject may be one who is afflicted with a cytopathy mediated by allo- & auto-antibodies and / or pathogenic B-cells.

[0075] In any of the foregoing methods, the subject may be one who is afflicted with a condition selected from immune thrombocytopenic purpura (ITP), hemolysis, hemolytic anemia, and / or a condition characterized by antibodies to lymphocytes and / or antibodies against other lymphoid and myeloid cellular elements.

[0076] In any of the foregoing methods, the method may comprise administering butyrophilin A2 (BTN2A2), fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment or a fusion polypeptide comprising any of the foregoing, and an antigen to which antigen-specific humoral suppression and / or B cell tolerance is to be elicited in the subject.

[0077] In any of the foregoing methods, the subject may be afflicted with multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease and the method may further comprise contacting said immune cells ex vivo or in vivoAttorney Docket No. 2969027.002013 with myelin oligodendrocyte glycoprotein (MOG) or amyloid protein proximate to when the immune cells are contacted with Buty rophil i n or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof.

[0078] In any of the foregoing methods, the subject may be afflicted with a chronic inflammatory, autoimmune or allergic disorder involving pathologic antibodies or pathologic B cell expansion, e.g., one that remits and relapses, optionally multiple sclerosis, optionally Relapsing-Remitting Multiple Sclerosis (RRMS), vasculitis, optionally ANCA-Associated Vasculitis, rheumatoid arthritis, or lupus, optionally relapsing focal or diffuse lupus nephritis, and the Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin, optionally Butyrophilin A2 is administered during active bouts of the disease.

[0079] In any of the foregoing methods, said Butyrophilin or fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment or conjugate comprising alleviates or reduces the expression pathologic antibodies or pathologic B cell expansion and / or results in longer periods or disease remission.

[0080] In any of the foregoing methods, the Butyrophilin or a fragment thereof, optionally Butyrophilin A2 (BTN2A2) or a BTN2A2 fragment may be linked to an albumin, optionally human serum albumin.

[0081] In any of the foregoing methods, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof may be linked or fused to a Fc domain or an Fc fragment ("BTN2A2-Fc fusion protein") or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2-related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally any of the mutations shown in Table 1.

[0082] In any of the foregoing methods, the BTN2A2 or the BTN2A2 fragment thereof may be linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, and theAttorney Docket No. 2969027.002013 linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID N0:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, or helical peptides.

[0083] In any of the foregoing methods, the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoform fragment may be modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol.

[0084] In any of the foregoing methods, the BTN2A2 fragment may comprise an extracellular domain of BTN2A2.

[0085] In any of the foregoing methods, the BTN2A2 fragment may not comprise a transmembrane domain, a cytoplasmic domain, or both.

[0086] In any of the foregoing methods, the BTN2A2-related isoform may be one of BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3.

[0087] In any of the foregoing methods, the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0088] In any of the foregoing methods, a Butyrophilin A2 (BTN2A2)-Fc fusion protein is administered wherein (i) the BTN2A2 moiety of the fusion protein comprises at least one BTN2A2 polypeptide possessing at least 90% sequence identity to the IgV domain of human or murine BTN2A2 and which BTN2A2 polypeptide lacks or only comprises a fragment of the BTN2A2 IgC domain; (ii) an Fc region, and optionally (iii) a peptide linker which intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG, or the BTNA2 moiety in the BTN2A2-Fc fusion protein comprises or consists of a polypeptide possessing at least 95-99% sequence identity to the IgV domain of BTN2A2, optionally human BTN2A2 IgV domain; optionally wherein the BTN2A2-Fc fusion protein comprises two of said BTN2A2 polypeptides, wherein optionally a peptide linker intervenes said BTN2A2 polypeptides, optionally a 1-50, 2-25, 2- 15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG; or the BTN2A2-Fc fusion protein comprises a human Fc region, optionally a human IgGl, lgG2, lgG3 or lgG4 Fc region; or the BTN2A2-Fc fusion protein comprises a human IgGl or lgG2 Fc region, optionally one comprising one or more mutations which impair or enhance one or more effectorAttorney Docket No. 2969027.002013 functions, e.g., FcR binding, complement binding, FcRN binding, further optionally wherein said one or more mutations comprise the Fc mutations identified in Table 1; or the BTN2A2- Fc fusion protein comprises a mutated IgGl Fc region wherein the mutations comprise or consist of (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum halflife, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G substitution mutation and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues, and further optionally the linker is GGSGG; or the BTN2A2-Fc fusion protein comprises a BTN2A2 polypeptide or CD45 binding fragment thereof and a mutated IgGl Fc region, wherein the mutated IgGl Fc region comprises (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum halflife, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G mutation, and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, further optionally a 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues, and still further wherein the linker comprises or consists of GGSGG or a multimer thereof; or the BTN2A2-Fc fusion protein comprises at its N-terminus a signal peptide, further optionally the endogenous BTN2A2 signal peptide or an immunoglobulin signal peptide; or the BTN2A2-Fc fusion protein comprises a flexible linker intervening the BTN2A2 and Fc moiety which comprises or consists of GGSGG; or the BTN2A2-Fc fusion protein comprises the amino acid sequence contained in Figure 24 (SEQ ID NO: 61), and contains or does not contain the endogenous BTN2A2 signal peptide or another signal peptide, e.g., an immunoglobulin signal peptide; or any combination of the foregoing.

[0089] In any of the foregoing methods, the method may further comprise the use or inclusion of one or more of other actives which comprise one or more of a steroid, optionally a glucocorticoid, further optionally prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone; methotrexate, azathioprine mycophenolate, cyclophosphamide, a TNF inhibitor, optionally HUMIRA, etanercept, infliximab, adalimumab,Attorney Docket No. 2969027.002013 certolizumab pegol, or golimumab, an IL-6 antagonist, optionally an antagonist anti-IL-6 antibody, an anti-IL-17 antagonist, optionally Secukinumab, Ixekizumab, or Brodalumab, or another B cell suppressant or B cell depleting agent, optionally a CD40 antagonist, further optionally an anti-CD40 antagonist antibody, a CD154 antagonist, further optionally an anti- CD154 antagonist antibody, a B cell depleting antibody, optionally an anti-CD20 antibody, further optionally rituximab, ibritumomab, tiuxetan, or tositumomab; an anti-CD22 antibody, an anti-CD19 antibody, an anti-CD38 antibody, an anti-BAFF receptor antibody, folic acid, or any other small molecule B cell suppressants; optionally wherein the administration of the other active or actives is effected before, at the same time, or after administration of butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment or a fusion polypeptide comprising any of the foregoing.

[0090] In any of the foregoing methods, the method may result in acute or prolonged suppression of B cell activation and / or proliferation.

[0091] In any of the foregoing methods, the method may result in acute or prolonged antigen specific B cell tolerance.

[0092] In any of the foregoing methods, the method may be used to desensitize a subject who is to / or has received HLA incompatible cells, tissue and / or organ.

[0093] In any of the foregoing methods, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof may be linked or fused to a Fc domain or an Fc fragment ("BTN2A2-Fc fusion protein") or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2-related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally wherein the Fc, optionally an IgGl Fc comprises one or more of the mutations shown in TABLE 1 and / or comprises any one, two, three, four or all five of M252Y, S254T, T256E, and / or H433K and N434F, and / or is the variant IgGl Fc contained in SEQ ID NO: 6, further optionally wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment comprises the BTN2A2 polypeptide in SEQ ID NO: 6 or a variant thereof wherein the RS linker peptide intervening the BTNA2 polypeptide and the IgGl Fc variant optionally is replaced by another peptide linker, preferably a non-cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine,Attorney Docket No. 2969027.002013 glycine-serine, GGGGSn (SEQ ID N0:6)n, GGGGGSn (SEQ ID N0:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

[0094] In any of the foregoing methods, the BTN2A2 or the BTN2A2 fragment thereof may be linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, preferably a non-cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

[0095] The method of any one of the previous claims, wherein the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoform fragment is modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol.

[0096] The method of any one of the previous claims, wherein overall antibody levels and / or antibodies specific to one or more target antigens are assayed in one or more samples from the subject after and / or during treatment to assess the efficacy of the treatment method.

[0097] In specific embodiments the invention further provides compositions for inhibiting B cell activation, B cell proliferation, antibody production, and / or for eliciting humoral suppression and / or B cell tolerance A composition comprising (i) a butyrophilin fusion polypeptide or modified butyrophilin polypeptide, comprising (a) a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof; and (b) optionally an albumin or Fc domain or Fc fragment, optionally an IgGl, lgG2, lgG3 or lgG4 Fc or Fc fragment, preferably an IgGl, which optionally may be mutated, which further is optionally mutated in order to enhance or reduce one or more Fc-associated effector functions, further optionally any of the mutations shown in Table 1, and / or is modified to enhance half-life such as by glycosylation or PEGylation or lipidation or by attachment to cholesterol, and wherein (a) and (b) are directly or indirectly linked, optionally by a linker peptide; and (ii) an antigen against which humoral suppression and / or B cell tolerance is to be elicited, or a nucleic acid encoding said antigen, optionally comprised in a viral vector, plasmid or lipid nanoparticle.Attorney Docket No. 2969027.002013

[0098] In some embodiments the composition comprises a butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises an Fc domain, optionally an IgGl Fc domain, which Fc comprises one or more mutations, optionally wherein the mutations comprise Met208Leu, Asn214Ser, or both, in reference to SEQ ID NO:4.

[0099] In some embodiments the composition comprises a e butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises a spacer peptide between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the albumin or Fc domain or the Fc fragment, optionally an IgGl Fc.

[0100] In some embodiments the composition comprises a butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises a spacer peptide between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the albumin or Fc domain or the Fc fragment, optionally an IgGl Fc 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 NQ:60).

[0101] In some embodiments the composition comprises a BTN2A2 fragment which comprises the extracellular domain of BTN2A2.

[0102] In some embodiments the composition comprises a BTN2A2 fragment which does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0103] In some embodiments the composition comprises a BTN2A2-related isoform selected from BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3.

[0104] In some embodiments the composition comprises a BTN2A2-related isoform fragment which does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0105] In some embodiments the composition comprises a butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or BTN2A2-related isoform fragment, which is PEGylated or glycosylated.

[0106] In some embodiments the composition comprises a BTN2A2 fragment which binds to CD45.

[0107] In some embodiments the composition further comprises immune cells of the subject who is to be treated with the composition, and / or the composition comprises donor antigens and / or donor cells, of a living or non-living donor from which the subject is to be administered or transplanted cells, a tissue and / or an organ therefrom.Attorney Docket No. 2969027.002013

[0108] In some embodiments the composition comprises an antigen selected from an autoantigen, allergen, therapeutic agent, parasitic antigen, or an antigen expressed by donor cells, optionally a food allergen, an aeroallergen, an animal product, a plant derived allergen, a microbial allergen, optionally a mite, fungus, yeast, etc., a drug, a hormone, a therapeutic enzyme, a therapeutic antibody, a blood or clotting factor, insect venom, or latex, further optionally a peanut allergen, a tree nut allergen, a dairy allergen, a wheat allergen, a soy allergen, an egg allergen, a shellfish allergen, a meat allergen, a sesame allergen, and a corn allergen.

[0109] In some embodiments the composition further comprises one or more of a further comprises the use or inclusion of one or more of a which further comprises the use or inclusion of one or more of a steroid, optionally a glucocorticoid, further optionally prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone; methotrexate, azathioprine mycophenolate, cyclophosphamide, a TNF inhibitor, optionally HUMIRA, etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab, an IL-6 antagonist, optionally an antagonist anti-IL-6 antibody, an anti-IL-17 antagonist, optionally Secukinumab, Ixekizumab, or Brodalumab, or another B cell suppressant or B cell depleting agent, optionally a CD40 antagonist, further optionally an anti-CD40 antagonist antibody, a CD154 antagonist, further optionally an anti-CD154 antagonist antibody, a B cell depleting antibody, optionally an anti-CD20 antibody, further optionally rituximab, ibritumomab, tiuxetan, or tositumomab; an anti-CD22 antibody, an anti-CD19 antibody, an anti-CD38 antibody, an anti-BAFF receptor antibody, folic acid, or any other small molecule B cell suppressants.

[0110] In some embodiments the composition when administered to a subject in need thereof results in acute or prolonged suppression of B cell activation and / or proliferation.

[0111] In some embodiments the composition when administered to a subject in need thereof results in, or provides for, antigen specific B cell tolerance.

[0112] In some embodiments the composition when administered to a subject who is to / or has received HLA incompatible cells, tissue and / or organ desensitizes the subject, i.e., the subject does not elicit or elicits a reduced humoral response against the HLA incompatible cells, tissue and / or organ.Attorney Docket No. 2969027.002013

[0113] In some embodiments the composition comprises a butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked or fused to a Fc domain or an Fc fragment ("BTN2A2- Fc fusion protein") or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2-related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally wherein the Fc, optionally an IgGl Fc comprises one or more of the mutations shown in TABLE 1 and / or comprises any one, two, three, four or all five of M252Y, S254T, T256E, and / or H433K and N434F, and / or is the variant IgGl Fc contained in SEQ ID NO: 6, further optionally wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment comprises the BTN2A2 polypeptide in SEQ ID NO: 6 or a variant thereof wherein the RS linker peptide intervening the BTNA2 polypeptide and the IgGl Fc variant optionally is replaced by another peptide linker, preferably a non-cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

[0114] In some embodiments the composition comprises a butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, wherein the BTN2A2 or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform orthe BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, preferably a non- cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

[0115] In some embodiments the composition comprises a butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, wherein the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoform fragment is modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol.Attorney Docket No. 2969027.002013

[0116] In some embodiments the composition comprises a butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, wherein the BTN2A2 fragment comprises an extracellular domain of BTN2A2.

[0117] In some embodiments the composition comprises a BTN2A2 fragment which does not comprise a transmembrane domain, a cytoplasmic domain, or both.

[0118] In some embodiments the composition comprises a BTN2A2-related isoform selected from BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3.

[0119] In some embodiments the composition comprises a BTN2A2-related isoform or fragment which does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0120] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein wherein (i) the BTN2A2 moiety of the fusion protein comprises at least one BTN2A2 polypeptide possessing at least 90% sequence identity to the IgV domain of human or murine BTN2A2 and which BTN2A2 polypeptide lacks or only comprises a fragment of the BTN2A2 IgC domain; (ii) an Fc region, and optionally (iii) a peptide linker which intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG.

[0121] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein wherein the BTNA2 moiety in the BTN2A2-Fc fusion protein comprises or consists of a polypeptide possessing at least 95-99% sequence identity to the IgV domain of BTN2A2, optionally human BTN2A2 IgV domain.

[0122] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises two or more of said BTN2A2 polypeptides or fragments, optionally a IgV domain, wherein optionally a peptide linker intervenes said BTN2A2 polypeptides, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG.Attorney Docket No. 2969027.002013

[0123] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises a human Fc region, optionally a human IgGl, lgG2, lgG3 or lgG4 Fc region.

[0124] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises a human IgGl or lgG2 Fc region, optionally one comprising one or more mutations which impair or enhance one or more effector functions, e.g., FcR binding, complement binding, FcRN binding, further optionally wherein said one or more mutations comprise the Fc mutations identified in Table 1.

[0125] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises a mutated IgGl Fc region wherein the mutations comprise or consist of (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G substitution mutation and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues, and further optionally the linker is GGSGG.

[0126] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises a BTN2A2 polypeptide or CD45 binding fragment thereof and a mutated IgGl Fc region, wherein the mutated IgGl Fc region comprises (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G mutation, and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, further optionally a 2-25, 2-15, 2- 10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues, and still further wherein the linker comprises or consists of GGSGG or a multimer thereof.

[0127] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises at its N-terminus a signal peptide, further optionally the endogenous BTN2A2 signal peptide or an immunoglobulin signal peptide.Attorney Docket No. 2969027.002013

[0128] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises a flexible linker intervening the BTN2A2 and Fc moiety which comprises or consists of GGSGG.

[0129] In some embodiments the composition comprises a Butyrophilin A2 (BTN2A2)- Fc fusion protein which comprises the amino acid sequence contained in Figure 24, which contains or does not contain the endogenous BTN2A2 signal peptide.BRIEF DESCRIPTION OF THE FIGURES

[0130] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0131] Figure 1A-1D (panels A-D) shows that BTN2A2-Fc enhances interaction and colocalization of CD45 with TCR signaling proteins. (A) Jurkat cells were stimulated for 3 min with immobilized anti-CD3 antibody (10 pg / ml) in the presence or absence of BTN2A2-Fc (10 g / rril). Cells were lysed in IP buffer, immunoprecipitated with anti-CD45 antibody and immunoblotted for total Zap70 and CD3 . Right panel show intensity plots depicted as mean±SD (N=3) (B) Immunostaining analysis shows segregation of CD45 from CD3 in the presence or absence of recombinant BTN2A2-Fc protein (10 mg / ml) after Jurkat cells activation by plate-bound anti-CD3 antibody (10 mg / ml) for 3 min. Right panel shows quantification of CD3^ and CD45 colocalization in the presence or absence of BTN2A2-Fc proteins from multiple fields. At least 50 cells from each group were included in the analysis shown in the right panel. (C) Co-immunoprecipitation experiments were performed using anti-CD3c antibody in Jurkat cells with the same condition as in (A), followed by immunoblot with anti-CD45 antibody. (D) CD45-specific phosphatase activity measured in immunoprecipitate from samples in panel C using FDP (Fluorescein Diphosphate, Tetraammonium Salt) substrate as described in the methods. Data depicted as mean ± SD (N=3). One-way ANOVA with Tukey's multiple comparison test; p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0132] Figure 2A-2H (panels A-H) shows that CD45 phosphatase coimmunoprecipitated with BTN2A2. (A) Jurkat cells were immunoprecipitated with anti- BTN2A2 antibody or IgG and blotted with CD45 antibody at unstimulated state or after activation anti-CD3 (1 mg / ml) for 48 hours. Input is ~3% of cell lysate. One representative experiment is depicted. (B) Jurkat cells were treated with both plate-bound anti-CD3 antibodyAttorney Docket No. 2969027.002013( Ipg / m I ) for 48 hours in the presence of recombinant BTN2A2-Fc or Fc-tag protein. Cells were lysed in IP buffer and immunoprecipitated with protein G and immunoblotting performed with anti-CD45 antibody. Input is ~3% of cell lysate. One representative experiment is depicted. (C) Jurkat cells were treated with anti-CD3 antibody (lpg / ml) in the presence of recombinant BTN2A2-Fc or Fc-tag protein for 48 hours and crosslinked with BS3, lysed, immunoprecipitated with protein G and immunoblotted with anti-CD45 antibody. Input is ~3% of cell lysate. One representative blot is depicted. (D) Ribbon diagram representation of protein-protein interaction between BTN2A2 (pink) and CD45 (PTPRC)(white) showing potential amino acids (blue sticks) determining their interaction. Two residues N419 and N468, key glycosylation sites, located in the fibronectin domain of CD45 that is critical for the interaction is indicated in a spherical model. (E) Western blot demonstrating the expression of CD45 in wild type Jurkat cells (lane 1) and Jurkat cells with CD45 knock-down (CD45-KD) (lane 2); CD45 expression in CD45-KD cells in which wild type CD45 or mutant CD45 (double mutant - N419A and N468A) were expressed is depicted in lanes 3-4. GAPDH was used internal control. (F) Co-immunoprecipitation of CD45 with BTN2A2 as described in panel B in Jurkat cells lacking CD45 in which wild type CD45 or mutant CD45 (double mutant - N419A and N468) were expressed and activated (1 mg / ml of anti-CD3 antibody for 48hrs) in the presence of recombinant BTN2A2-Fc or Fc. (G) Western blot demonstrating expression of the CD45 isoforms at baseline and at 48 hours after stimulation with anti-CD3 antibody in Jurkat cells. (H) Western blot analysis of CD45 isoforms (CD45RO and RA) following immunoprecipitation with protein G agarose beads of lysates obtained from activated Jurkat cells (1 mg / ml of anti-CD3 antibody for 48hrs) in the presence of recombinant BTN2A2-Fc or Fc. One representative experiment is depicted.

[0133] Figure 3A-3D (panels A-D) shows that BTN2A2-Fc enhances Tregs and suppress Thl7 cells differentiation in in-vitro mixed lymphocytes reaction (MLR) (A) Flow cytometry analysis plot of primary CD4+ T cells (from spleen and lymph nodes of Foxp3-GFP transgenic mice) incubated with bound 1 g / ml anti-CD3 and in the presence or absence of 10 pg / ml BTN2A2-Fc fusion protein in MLR for 7 days as described in methods and analyzed for CD4+CD25+ Foxp3-GFP+ve cells expression. As positive control, effects of TGF-b (1 ng / ml) under the same conditions are also depicted. Right panel show summary plot depicted as mean±SD for 3 independent experiments. One-way ANOVA with Tukey's multiple comparisonAttorney Docket No. 2969027.002013 test; p<0.05 (*). (B) Flow cytometry analysis of primary CD4 +T cells (isolated from murine spleen and lymph nodes) incubated for 5 days with anti-CD3 (0.5 mg / ml) antibody alone or anti-CD3 +TGF-b (1.5 ng / ml) +IL6 (10 ng / ml) +IL-1(3 (10 ng / ml) and / or recombinant BTN2A2- Fc (10 g / ml) in MLR and analyzed for CD4+RORgt+ve cells. Right panel show summary plot depicted as mean±SD for 3 independent experiments. One-way ANOVA with Tukey's multiple comparison test; p<0.05 (*). (C) Flow cytometry analysis of Foxp3-GFP +ve cell (%) population in total CD4+ T cells co-cultured with dendritic cells (DC) at day 7. Purified T cells were incubated in RPMI ex-vivo for day7 with DC cells (1:10 ratio) isolated from wild-type or BTN2A2- / - mice. (D) Flow cytometry analysis of Foxp3-GFP -i-ve cell (%) population in total CD4+ cells co-cultured with B cells at day 7. Purified T-cells were incubated ex-vivo for day7 with B-cell (1:5 ratio) isolated from wild-type or BTN2A2- / - mice. Data for experiments in (C- D) are depicted as mean ± SD (n=3). Unpaired t-test; p<0.05 (*), p<0.001 (***).

[0134] Figure 4A-4C (panels A-C) shows that inhibition of CD45 phosphatase activity in primary immune cells blocks BTN2A2 mediated Treg differentiation and Thl7 suppression. (A) Immunoblot analysis of phosphorylated ZAP-70 (p-Zap70) and total Zap70 in Jurkat cells pretreated with CD45 phosphatase inhibitor for 1 hr and stimulated for 3 min with platebound anti-CD3 (10 | g / ml) antibody in the presence or absence of recombinant BTN2A2-Fc (10 |jg / ml). (B) Flow cytometry analysis plot of primary CD4+ T cells (from spleen and lymph nodes of Foxp3-GFP transgenic mice) incubated with bound 1 p.g / ml anti-CD3 and / or 10 |jg / ml BTN2A2-Fc fusion protein in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM) and analyzed for CD4+CD25+ Foxp3-GFP+ve cells expression. Right panel shows summary plot depicted as mean ± SD for 3 independent experiments. (C) Flow cytometry analysis of primary CD4 +T cells (isolated from murine spleen and lymph nodes) incubated for 5 days with anti-CD3 (0.5 mg / ml) antibody alone or anti-CD3 +TGF-b (1.5 ng / ml) +IL6 (10 ng / ml) +IL-1£ (10 ng / ml) and / or recombinant BTN2A2-Fc (10 pg / ml) in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM) and analyzed for CD4+RORgt+ve cells. Right panel shows summary plot depicted as mean ± SD. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**), p<0.001 (***).

[0135] Figure 5A-5I (panels A-l) shows that BTN2A2-Fc ameliorates crescentic glomerulonephritis in mice induced by nephrotoxic serum (NTS). (A) Schematic protocol of NTS-induced nephrotoxic glomerulonephritis model and histological changes. GBM =Attorney Docket No. 2969027.002013 glomerular basement membrane. (B) Quantification of mean ± SEM proteinuria (on Day 7) in mice with NTS induced glomerulonephritis treated with recombinant BTN2A2-Fc protein or vehicle control (control) (N=12 / group); Mann-Whitney p<0.001(***). (C-D) Representative images of glomerular injury by PAS staining. Crescent formation in mice with NTS induced glomerulonephritis treated with treated with recombinant BTN2A2-Fc protein or control (D). Scale bars are 25 mm. Summary panel (C) depicts quantification of % glomeruli with crescents (mean ± SEM, N=12 / group); Mann-Whitney p<0.001(***). (E-F) Relative Foxp3 (D) and RORgt (E) expression compared to GAPDH as internal control in CD4+ T cells purified from spleen and lymph nodes from mice with NTS induced glomerulonephritis treated with BTN2A2-Fc protein or control. Data depicted as mean ± SEM (N=8 / group). Mann-Whitney test; p<0.05 (*). (G) Quantitative PCR analysis show CD5 expression in CD4+T cells from mice with NTS induced glomerulonephritis treated with BTN2A2-Fc protein or control.. N=8 / group. (H-l) Immunoblot analysis of IL17A in kidney tissue lysate of mice with glomerulonephritis treated with recombinant BTN2A2-Fc or control (H). Panel I shows quantification of western blot. Data represented as mean ± SEM. N=4 per group. Unpaired t-test; p<0.05 (*).

[0136] Figure 6A-6H (panels A-H) depicts BTN2A2 (- / -) mice showing exacerbated crescentic glomerulonephritis. (A) Scatter plot shows quantification of mean ± SEM 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 injury by PAS staining. Wild-type mice show minimal glomerular injury with a low dose of nephrotoxic serum, while BTN2A2 (- / -) mice show severe glomerulonephritis (B). Scale bars are 25 mm. Summary data are depicted as mean ± SEM of %Glomerular crescents (C). N=15 per group for all experiments. Mann-Whitney test; p<0.001 (***). (D-E) Relative Foxp3 mRNA (D) and RORgt mRNA (E) compared to GAPDH as internal control in CD4+ve T cells purified from spleen and lymph nodes cells from wild-type and BTN2A2-knockout mice with nephrotoxic serum induced glomerulonephritis. (F) Quantitative PCR analysis show CD5 expression in CD4+T cells from wild-type and BTN2A2-knockout mice with nephrotoxic serum induced glomerulonephritis. N=7 / group. (G-H) Immunoblotting for IL17A expression in in kidney tissue lysate using a nti-l L-17A antibody (G) and quantitation (H) from wild-type and BTN2A2 null mice with nephrotoxic serum induced glomerulonephritis. n>4 per group. Unpaired t-test; p<0.01 (**), p<0.001 (***).Attorney Docket No. 2969027.002013

[0137] Figure 7A-7H (panels A-H) shows that BTN2A2-Fc rescues fetus resorption and increases litter size in CBA / J x DBA / 2 pregnancy model. (A-B). Pregnant CBA / J x DBA / 2 mice treated with BTN2A2 throughout pregnancy has improved litter size (A), reduced resorption (B) compared to untreated mice. As additional control, DBA / 2 x CBA / J mice has been included. Data (A, B) represented as mean ± SEM. N=ll per group for all experiments. Kruskal-Wallis test with Dunn's test for multiple comparison; p<0.05 (*), p<0.01 (**), p<0.001 (***). (C-D) Quantitative PCR (C) and flowcytometry (D) analysis show increase in Foxp3 expressing CD4+T cells in pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc as compared to control mice. N=8 / group (E) Flow cytometry analysis (left panel) show reduced number of CD4+RORgt +ve cells in pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc as compared to control mice. Right panel shows quantification of the summary data. N=8 / group. (F) Quantitative PCR analysis show CD5 expression in CD4+T cells in pregnant CBA / J x DBA / 2J mice and treated with recombinant BTN2A2-Fc as compared to control mice. N=8 / group. (G, H) Immunoblot analysis of IL17A in placental tissue lysate of pregnant CBA / J x DBA / 2J mice treated with recombinant BTN2A2-Fc or control (left panel). Right panel shows quantification of summary data. N=3 per group. One-way ANOVA test with Tukey's multiple comparison test for experiments in C-G; p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0138] Figure 8A-8D (panels A-D) shows that BTN2A2-Fc enhances Tregs and suppress Thl7 cells differentiation in in vitro human PBMC mixed lymphocyte reactions. (A-B). Flow cytometry plots showing percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) in human PBMC incubated with / without recombinant BTN2A2-Fc (10 mg / ml) in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM). Data depicted as mean + / -SD showing percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) among total CD4+ cells. N=4 per group for all experiments. Unpaired 2-tailed t test; p<0.001 (***). (C-D). Flow cytometry plots showing percentage of CD4+RORgt +ve (Thl7 cells) in human PBMC incubated for 3 days with anti-CD3 antibody (0.5 mg / ml) only or anti-CD3 antibody +TGF- b (1.5 ng / ml) +IL-6 (10 ng / ml) +IL-lb (10 ng / ml) in absence or presence of recombinant BTN2A2-Fc (10 mg / ml) in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM). Data represent mean ± standard deviation. N=3 per group for all experiments. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**).

[0139] Figure 9 shows a representative schematic of the model by which BTN2A2 as a co-inhibitor of TCR signaling via CD45 phosphatase.Attorney Docket No. 2969027.002013

[0140] Figure 10A-10D (panels A-D) shows that BTN2A2-Fc regulates T-cell receptor signaling. (A) Schematic diagram showing the recombinant human BTN2A2-Fc fusion. The recombinant fusion gene consisting ofthe two extracellulardomains (IgV and lgC2) of BTN2A2 molecule (green color) cloned along with the Fc region of a human IgGl antibody (orange color). The chimeric protein (BTN2A2-Fc) is separated by IEGRMD (SEQ ID NO:57) spacer polypeptide. (B) Coomassie blue staining of purified recombinant BTN2A2-Fc protein on reducing (SDS) gel with a protein ladder in the left column and a band corresponding to BTN2A2-Fc (~55 kDa) in the right two columns. Lower panel shows western blot of recombinant BTN2A2-Fc using specific anti-human BTN2A2 antibody. (C) IL-2 secretion from Jurkat cells incubated for 24hrs with plate-immobilized anti- CD3 antibody (lpg / ml) in the absence or presence of immobilized recombinant BTN2A2-Fc (lOpg / ml), 293-cell derived human BTN2A2-Fc (R&D systems) or mouse BTN2A2 (R&D systems), and immobilized immunoglobulin Fc domain as negative control is depicted (N=3 or 4). (D) Immunoblot analysis of phosphorylated proteins, p-Zap70 and p-CD3- and total Zap70 and CD3- in Jurkat cells stimulated for 3 min with immobilized anti-CD3 antibody (10 pg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 pg / ml) or immunoglobulin Fc domain (10 pg / ml). Right panel shows relative intensity plots as mean+SD (N=3). One-way ANOVA with Tukey's multiple comparison test; p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0141] Figure 11 depicts pervanadate studies. Jurkat cells treated with permanent phosphatase inhibitor (Pervanadate) plus minus BTN2A2-Fc or left untreated. After cell lysis, phosphorylated protein, p-Zap70 and p-CD3- were analyzed by Western blot. Total Zap70 and CD3- were controls.

[0142] Figure 12 depicts Jurkat cells stimulated with / without anti-CD3 antibody (lpg / ml) for 48 hrs and the lysates were immunoblotted with anti-CD45 or anti- BTN2A2 antibody. GAPDH expression was used as a loading control.

[0143] Figure 13A-13B (panels A-B) depicts CRISPR-Cas9 deletion of CD45 and site directed mutagenesis of CD45. (A) a representative schematic of CD45 sequences showing location of gRNA sequences complementarity. Lower panel show electropherogram of the sequence deleted upstream of exon-1 to downstream of exon-3, nucleotide sequence: SEQ ID NO:46; amino acid sequence: SEQ ID NO:47. (B) Electropherogram showing change of nucleotides, AAT to GCT at Asn419Ala and AAT to GCT at Asn468Ala in CD45 mutants gene. Top left: nucleotide sequence-SEQ ID NO:48, amino acid sequence-SEQ ID NO:49; Top right:Attorney Docket No. 2969027.002013 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.

[0144] Figure 14A-14C (panels A-C) shows that BTN2A2-Fc inhibits CD4+ve T-cells proliferation, apoptosis and IL2 secretion. (A) Flow cytometry plots of CellTrace violet stained purified mouse CD4+ve cells incubated with immobilized anti-CD3 and anti-CD28 antibody (0.5pg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 pg / ml) or immunoglobulin Fc domain (10 pg / ml) for Day 3. Right panels shows bar graph of unproliferated cells (GO) percentage. (B) Flow cytometry plots of Annexin-V stained purified mouse CD4+ve cells incubated with immobilized anti-CD3 and anti-CD28 antibody (0.5pg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 pg / ml) or immunoglobulin Fc domain (10 pg / ml) for Day 3. Right panels shows bar graph of apoptotic cells percentage. (C) Flow cytometry plots of IL2 positive purified mouse CD4+ve cells incubated with immobilized anti-CD3 and anti-CD28 antibody (0.5pg / ml) in the presence of immobilized recombinant BTN2A2-Fc (10 pg / ml) or immunoglobulin Fc domain (10 pg / ml) for Day 3. Right panels shows bar graph of IL2 positive cells percentage. Data are depicted as mean ± SD (n=4). Unpaired t- test; p<0.001 (***).

[0145] Figure 15A-15F (panels A-F) show qPCR studies for candidate molecules in T cell differentiation pathway. Quantitative PCR analysis for IL-2RB (A), Foxp3 (B), IL-21 (C), TBX21 (D), GATA3 (E) and Smad-3 (F) in primary CD4 cells incubated for 1 Day and 5 days in absence or presence of anti-CD3 antibody and / or recombinant BTN2A2-Fc. All data represented as mean ± standard deviation. n=3 per group for all experiments. One-way ANOVA with Tukey's multiple comparisons test; p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0146] Figure 16A-16B (panels A-B) shows BTN2A2 null mice generation / characterization. (A) Btn2a2- / - mouse m by 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 are identified, with the ATG start codon in exon-2 and the TAG stop codon in exon-8 (Transcript Btn2a2-203: ENSMUST00000110433). Exons 2~8 (covers 100.0% of the coding region) were selected as target site. Cas9 and gRNA co-injected into fertilized eggs for KO mouse production. The pups were genotyped by PCR followed by sequencing analysis. The size of effective KO region: "10500 bp. gRNA target sequences SEQ ID NOs: gRNAl: SEQ ID NO:39;Attorney Docket No. 2969027.002013 gRNA2: SEQ ID NO:40; gRNA3: SEQ ID N0:41; gRNA4: SEQ ID NO:42. Genotyping primer SEQ ID NOs: Fl: SEQ ID NO:43; F2: SEQ ID NO:44; Rl: SEQ ID NO:45. (B) Agarose gel electrophoresis of genotyping PCR. Homozygotes (Homo) mouse shows one band with 700 bp, heterozygotes (Het) mouse with two bands at 700 bp and 500 bp; wildtype mouse (WT) one band at 500 bp.

[0147] Figure 17A-17B (panels A-B) shows BTN2A2 expression in antigen presenting cells. qPCR analysis of BTN2A2 expression in B cells (A) and Dendritic cells (B) populations purified using magnetic beads from spleen and lymph nodes of wildtype and BTN2A2-KO mice. Left panels shows flow cytometry plots of purified cells, and right panels shows fold change in RNA expression of BTN2A2 transcript compared to GAPDH.

[0148] Figure 18 contains a schematic protocol of abortion prone model.

[0149] Figure 19 depicts a schematic showing binding of BTN2A2 with Myelin oligodendrocyte glycoprotein (MOG). The protein structure prediction software (Prime, www.schrodinger.com) was used to evaluate the binding of BTN2A2 to MOG, the antigen for multiple sclerosis and other MOG-related autoimmune diseases. Root mean square of atomic positions (RMSD) was <1A.

[0150] Figure 20A & 20B contain flow cytometry plots. Figure 20A shows the percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) in human PBMC incubated with / without recombinant BTN2A2-Fc (10 mg / ml) in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM). Figure 20B shows the percentage of CD4+RORgt +ve (Thl7 cells) in human PBMC incubated for 3 days with anti-CD3 antibody (0.5 mg / ml) only or anti-CD3 antibody +TGF- b (1.5 ng / ml) +IL-6 (10 ng / ml) +IL-lb (10 ng / ml) in absence or presence of recombinant BTN2A2-Fc (10 mg / ml) in MLR in absence or presence of CD45 phosphatase inhibitor (125nM). Data represent mean ± standard deviation. N=3 per group for all experiments. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**).

[0151] Figure 21 shows data from a nephrotoxic nephritis model of glomerulonephritis wherein animals were immunized with 0.5mg Rabbit IgG and then 20 ml iv nephrotoxic serum showing that BTN2A2-Fc treatment reduced the T-effector pathway Thl7 cells (RORyt+)while increasing Treg cell populations(FoxP3+)IL-17 levels were also decreased.

[0152] Figure 22 shows data in an NTSN model in BTN2A2 (- / -) animals showing that NTS induced extremely severe crescentic GN in BTN2A2 (- / -) animals compared to WT.Attorney Docket No. 2969027.002013

[0153] Figure 23 contains a schematic of a DNA construct, pFUSE-hlgGle2-Fcl, available from Invivogen which may be used to express an exemplary BTN2A2-variant IgGl Fc fusion protein according to the invention.

[0154] Figure 24 contains the sequence of an exemplary BTN2A2 fusion protein (SEQ ID NO: 61) according to the invention. The first 237 amino acids at the N-terminus of the fusion protein comprise the BTN2A2 domain which is linked via a 2 amino acid linker, (RS) to a variant IgGl Fc protein comprising 5 mutations, M252Y, S254T, T256E, H433K and N434F, which enhance FcRn binding and thereby promote serum half-life of the BTN2A2 fusion protein. (See Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8).

[0155] Figure 25 contains the results of a mixed lymphocyte reaction (MLR) showing that BTN2A2-Fc fusion enhances Tregs and suppresses Thl7 differentiation in in vitro PGMC MLRs. In panel A the data is depicted as mean + / -SD showing percentage of Treg cells (CD4+CD25+Foxp3+ T cells) in human PBMCs (responder) incubated for 7 days with recombinant hBTN2A2-Fc (10 pg / ml) in one-way MLR. Data represent mean ± standard deviation. N=3 per group for all experiments. Unpaired 2-tailed t test; p<0.05 (*). In panel B the data is depicted as mean + / -SD showing percentage of CD4+RORyt +T cells (Thl7 cells) in human PBMCs (responder) incubated for 5 days with anti-CD3 antibody (0.5 pg / ml) only or anti-CD3 antibody +TGF- 3 (1.5 ng / ml) +IL-6 (10 ng / ml) +IL-1(3 (10 ng / ml) in absence or presence of recombinant hBTN2A2-Fc (10 pg / ml) in one-way MLR. Data represent mean ± standard deviation. N=3 per group for all experiments. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**), p<0.001 (***)

[0156] Figure 26 contains the results of experiments showing the inhibitory effect of hBTN2A2 on B cell Proliferation in a one-way MLR with two Human PBMCs (set 1) using a BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay. In this one-way MLR, responder and stimulator PBMCs are pre-incubated with or without hBTN2A2-Fc (10 pg / ml) for 10 minutes before being combined for culture. Cells are cultured with a medium containing BrdU, a thymidine analog. During DNA replication, BrdU is incorporated into the DNA in place of thymidine. After incubation (7 days and 14 days), the cells are fixed, and the DNA is denatured to make the incorporated BrdU accessible to detection antibodies. An anti-BrdU antibody is used to detect the BrdU incorporated into the DNA. The intensity of anti-BrdU antibody is proportional to the amount of BrdU incorporated, which reflects the level of cell proliferation.Attorney Docket No. 2969027.002013The results clearly show that recombinant hBTN2A2-Fc significantly inhibited CD19+ B cell proliferation in a one-way MLR at both 7 days (4.2 to 1.2%) and 14 days (8.5 to 3.5%).

[0157] Figure 27 contains the results of additional experiments showing the inhibitory effect of hBTN2A2 on B cell proliferation in a one-way MLR with two Human PBMCs (set 2). These experiments used the same one-way MLR conditions with two different human PBMCs (set 2) and the same BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay conditions as the experiments in Figure 27. The results similarly show that recombinant hBTN2A2-Fc significantly inhibited CD19+ B cell proliferation in this one-way MLR at both 7 days (5.0 to 3.1%) and at 14 days (7.0 to 3.8%).

[0158] Figure 28 contains the results of one-way MLR experiments showing the inhibitory effect of hBTN2A2-Fc on human IgG production. The figure shows total IgG production by human B cells stimulated in a one-way MLR. As is shown therein unstimulated cells from individuals A and B produced IgG after 14 days. Stimulating A cells with irradiated B cells, and vice versa, also resulted in IgG production. However, adding hBTN2A2-Fc to these stimulated cultures inhibited IgG production entirely. This suggests that hBTN2A2-Fc has an important (blocking) impact of B-cell proliferation after stimulation and on IgG production.

[0159] Figure 29A-E contains the results of experiments disclosed in Example 11 showing that BTN2A2 Inhibits R848 / IL-2 Induced Activation of B-Cells. In these experiments PBMCs were cultured for five days in the indicated conditions. Control: no drugs. BTN: 5 pg / mL BTN2A2. Stim: 10 ng / mL IL-2 and 1 pg / mL R848. Samples were then plated following a Fluorospot protocol. Representative images are shown for (A) Control, (B) BTN, (C) Stim, and (D) Stim / BTN. (E) Number of spots were calculated using ImageJ and normalized to cell density. Data presented as mean ± s.d. N = 6.

[0160] Figure 30A-G contains a composite of the images obtained in the imaging experiments of Figure 29 (which are disclosed in Example 11). The composite was created by an analysis of individual well photos. Rows D and H are irrelevant for this analysis, they were used to test out a new protocol for the reagents involved. The experiments were done in triplicate at two densities (2000 ce I Is / we 11, Rows A-C and 1000 cel Is / wel I, Rows E-G). Column 1 = Positive Control; Column 2 = Stim; Column 3 = BTN; Column 4 = Stim / BTN; Column 5 = Negative control (no cells).Attorney Docket No. 2969027.002013

[0161] Figure 31 contains results from experiments wherein cells from normal individuals were exposed to (BTN-2A2-FCLALAPG), CTLA4-lg and no additives.

[0162] Figure 32A-B contains results from experiments wherein cultures in the experiments of Figure 31 were harvested at day #6 and analyzed for proliferation of (CD4+ / CD25+ / CD127low,FoxP3+) Treg cells.

[0163] Figure 33 d iscloses and shows schematically the methods which were used to assess T-cell and NK cell reactivity to SARS-CoV2 peptide incubation.

[0164] Figure 34 contains the data of the experiments described in Figure 33.

[0165] Figure 35 contains the data of in vitro experiments comparing the effects of different BTN2A2 Fc fusions on IL-2 production by Jurkat cells activated with an anti-CD3 antibody.

[0166] Figure 36 contains a schematic protocol of the NTS-induced nephrotoxic glomerulonephritis model comparing the effects of different BTN2A2 Fc fusions on the reduction of nephrotoxicity.

[0167] Figure 37 and Figure 38 both contain renal histologic results which compared the effects of mice which were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch).

[0168] Figure 39 contains data comparing crescent scores and fibrinoid necrosis of the mice which were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). The data shows that there is a marked improvement in all the BTN- treated group compared to control Fc treated group with the most dramatic results in the mice treated with the BTN2A2-LALAPG variant.

[0169] Figure 40 contains data comparing body weights of the mice which were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch).

[0170] Figure 41 contains data comparing proteinuria of the mice which were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). The data show that while both hBTN2A2-Fc-WT and hBTN2A2-Fc-LALAPG reduced proteinuria, and inhibitedAttorney Docket No. 2969027.002013 renal histological changes, the mice treated with the LALAPG variant had more dramatic and also exhibited less toxicity.

[0171] Figure 42 contains data comparing Foxp3 expression in CD4+ T cells purified from spleen and lymph nodes of the mice which were treated with the Fc control, BTN2A2- hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2- hlgGlFc-LALAPG (CHO expressed batch). The data show that the mice treated with the BTN1A2FC fusions all had substantially higher Foxp3 expression levels compared to the Fc control group.

[0172] Figure 43 contains data showing the correlation between proteinuria and Foxp3 expression in CD4+ T cells purified from spleen and lymph nodes of the mice which were treated with the Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2- hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). The data show that increased Foxp3 expression correlates to a reduction in proteinuria.DESCRIPTION OF THE INVENTION

[0173] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0174] As used herein the term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. Forexample, the language "about 50%" covers the range of 45% to 55%. In various embodiments, the term "about" when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, / / specifically provided for in the claims.Attorney Docket No. 2969027.002013

[0175] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST- 2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0176] "Linked" as used herein in the context of linked peptides, polypeptides, or proteins refers to being "connected to" either directly or indirectly. Indirect linkage can be mediated by a polypeptide linker such as poly-glycine or a glycine-serine polypeptide, for example, (GGGGS)n (SEQ ID NO:6)n or (GGGGGS)n (SEQ ID NO:7)n, wherein n is an integer. In various embodiments, n is an integer from 1-10, 10-20, 20-30, 30-40, or 50-100. Other such linkers are known in the art and is considered to be encompassed by this term.

[0177] Described herein we demonstrate that BTN2A2 functions as a ligand for CD45, binding both to the TCR complex and CD45RO isoform on T cell surfaces, resulting in retention of CD45 phosphatase activity proximal to the TCR complex. Consequently, the enhanced phosphatase activity reduces downstream TCR signaling, which in turn promotes regulatory T cell (Treg) expansion, suppressing T effector cell differentiation. We further demonstrate the significant immunomodulatory effects of BTN2A2 in a mouse model of autoimmune glomerulonephritis, showing that BTN2A2 genetic deficiency exacerbated kidney injury, whereas administration of recombinant BTN2A2 limited disease severity. Furthermore, in a second mouse model of immune-mediated spontaneous abortion, treatment of pregnant dams with recombinant BTN2A2 helped animals tolerate their pregnancies, reducing fetal loss.

[0178] Data included herein provide multiple lines of evidence showing that BTN2A2 functions as a ligand for CD45, preferentially binding to CD45RO isoform, and that it functions to prevent segregation of CD45 from the TCR / CD3 complex, resulting in sustained CD45's phosphatase activity within the immune synapse which reduces downstream signaling cascade (See Figure 9) Consequently, BTN2A2 reduces proliferation of effector Thl7 cells while promoting differentiation of T regulatory cells, and reduces severity of NTS-inducedAttorney Docket No. 2969027.002013 glomerulonephritis and immune mediated pregnancy loss in mouse models of immunologic diseases. Our findings suggest that during cognate T cell / APC interactions, APC-produce BTN2A2 which ligates T cell-expressed CD45RO to the TCR, functioning as a molecular brake at the time of activation of the TCR complex by peptide / MHC complexes. Our data suggest that in CD45RO+ T cells, BTN2A2 binds to both CD45 and CD3e, likely maintaining them in proximity, enabling CD45 phosphatase activity to persist, enabling dephosphorylation of CDS ITAM domains and Zap70 and limiting downstream TCR signals required for full T cell activation.

[0179] Our data also indicate that BTN2A2 enhances CD45's phosphatase activity within the TCR complex which modulates the TCR signaling but does not fully prevent it, and consequently, permits expansion of Tregs while reducing effector T cell differentiation / expansion. In prior studies, transient targeting of CD45 induced potent antigen-specific regulatory T cells and induced transplant tolerance. Our data suggests that BTN2A2 expressed on APCs and DCs is likely the endogenous ligand for CD45 on T cells. CD45 is expressed as multiple spliced products and it was believed that CD45RA and CD45RB are replaced by CD45RO following T cell activation. These studies suggest that BTN2A2 predominantly binds to the CD45RO isoform that is expressed on activated T cells, signifying specificity for BTN2A2's actions. While multiple human glycoproteins, including galectin-1 and CD22, were shown by others to bind CD45, there is no clear evidence that these putative ligands modulate CD45 phosphatase activity. Other identified ligands forCD45 include pULll, a viral protein expressed by cytomegalovirus-infected cells, as well as E3 / 49K protein expressed by adenovirus-infected cells; both of these viral proteins may play roles in inhibiting anti-viral immunity by inhibiting TCR signaling, although precise mechanisms remain unclear.

[0180] We show that BTN2A2-Fc limits clinical manifestations of glomerular crescent formation and proteinuria in murine NTS glomerulonephritis and reduces miscarriages in a distinct orthogonal mouse model, both of which were associated with augmented Treg and reduced Thl7 cell populations. CD5, a T cell activation marker was attenuated by BTN2A2-Fc. Our data further indicate that BTN2A2-Fc is a potentially useful immune modulator with translational potential in a variety of immune-mediated diseases and transplantation. Our observations are an important step forward in understanding how naturally occurring proteins can modify inflammatory events by interacting with CD45 to sustain TCR complex phosphatase activity, increasing Treg and decreasing Thl7 cell populations.Attorney Docket No. 2969027.002013

[0181] Our findings suggest that BTN2A2 ligation of CD45 phosphatase to the TCR complex leads to dampened TCR signaling resulting in expansion of Tregs and suppression of Thl7 cells. While we demonstrate a beneficial effect of BTN2A2 in two orthogonal models of autoimmunity / immune tolerance, our data suggest that BTN2A2 / CD45 signaling pathway could be targeted in a wide variety of immune-mediated diseases such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, and transplant rejections.

[0182] Moreover, since CD45 is also expressed on B cells, we predicted that some of the beneficial effects of BTN2A2 in our animal studies may be derived from BTN2A2's actions on B cells. Indeed, we show herein in MLR experiments that BTN2A2 inhibits B cell activation and / or B cell proliferation and based thereon potentially may be used to elicit humoral suppression and / or B cell tolerance in a subject in need thereof, e.g., a subject with an autoimmune, inflammatory or allergic or proliferative condition (e.g., cancer) involving pathologic antibodies or pathologic B cell proliferation.

[0183] In particular, this will be accomplished by contacting immune cells of the subject or donor immune cells with a Butyrophilin, optionally Butyrophilin A2, or a conjugate or fusion protein comprising said Butyrophilin, optionally Butyrophilin A2, e.g., wherein said contacting occurs before, proximate to, or after the subject is to be or has been exposed to an antigen against which humoral suppression and / or B cell tolerance is to be elicited.

[0184] In exemplary embodiments this will induce antigen-specific humoral suppression and / or B cell tolerance, which may be prolonged or even reverse the disease (i.e., potentially "reset" or "reverse" B cell responses being elicited against the antigen).

[0185] Accordingly, various embodiments of the present invention are based, at least in part, on these finding.

[0186] Based on the phylogenetic tree, BTN1A1, B2N2A1, BTN2A2, BTN3A1, BTN3A2 and BTN3A3 have similar structure, particularly their extracellular domains, and are closely related. It is therefore expected that all of these isoforms, particularly, their extracellular domains, to have similar function to BTN2A2 in its ability to regulate the balance of Treg and Thl7 cells and to inhibit B cell activation and / or B cell proliferation and based thereon potentially may be used to elicit humoral suppression and / or B cell tolerance in a subject in need thereof, and be used in accordance with various embodiments of the invention.

[0187] In some embodiments, the subject is an organ, tissue or cell therapy transplant recipient. In various embodiments, the method reduces the likelihood of organ, tissue or cellAttorney Docket No. 2969027.002013 transplant rejection by inhibiting a humoral immune response against the transplanted organ, tissue or cell therapy. In various embodiments, the method reduces a need for an immunosuppressive therapy or reduces the amount of immunosuppressive therapy administered to the subject. In various embodiments, the method reduces the toxic effects of immunosuppressive therapy on the subject.

[0188] In various embodiments, the subject has an autoimmune, inflammatory or allergic disorder involving pathologic antibodies or pathologic B cell proliferation, e.g., a neurological disorder, SLE, multiple sclerosis, an autoimmune kidney disease (optionally IgA nephropathy, ANCA vasculitis, or membranous glomerulonephritis), Sjogren's Syndrome, B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, advanced stage follicular lymphoma, chronic lymphocytic leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, Pulmonary arterial hypertension (PAH), Systemic sclerosis, Refractory hypertension, Autoimmune hypoglycemia, and / or Anti-NMDA receptor encephalitis.

[0189] In some embodiments, the subject has received a transplanted cell, tissue or organ and shows signs and / or has a history of antibody mediated transplant rejection.

[0190] In some embodiments, the subject has or is to receive cell therapy, or a tissue or organ transplant, optionally one or more of kidney, heart, lung, pancreas, bone marrow, and liver.

[0191] In some embodiments, the subject comprises a cytopathy mediated by allo- & auto-antibodies and / or pathogenic B-cells.

[0192] In some embodiments, the subject comprises a condition selected from immune thrombocytopenic purpura (ITP), hemolysis, hemolytic anemia, and / or a condition characterized by antibodies to lymphocytes and / or antibodies against other lymphoid and myeloid cellular elements.

[0193] In some embodiments, the method is effected in vitro.

[0194] In some embodiments the method is effected in vivo.

[0195] Typically the method comprises contacting immune cells in vitro and / or in vivo with butyrophilin A2 (BTN2A2), fragment thereof, a BTN2A2-related isoform, a BTN2A2- related isoform fragment or a fusion polypeptide comprising any of the foregoing, and optionally an antigen to which antigen-specific humoral suppression and / or B cell tolerance is to be elicited in the subject.Attorney Docket No. 2969027.002013

[0196] In some instances, the subject comprises multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease and the method further comprises contacting said immune cells ex vivo or in vivo with myelin oligodendrocyte glycoprotein (MOG) or amyloid protein proximate to when the immune cells are contacted with Butyrophilin, optionally Butyrophilin A2, or a conjugate orfusion protein comprising said Butyrophilin, optionally Butyrophilin A2.

[0197] In some instances, the subject has a chronic inflammatory, autoimmune or allergic disorder involving pathologic antibodies or pathologic B cell expansion, e.g., one that remits and relapses, optionally multiple sclerosis, optionally Relapsing-Remitting Multiple Sclerosis (RRMS), vasculitis, optionally ANCA-Associated Vasculitis, rheumatoid arthritis, or lupus, optionally relapsing focal or diffuse lupus nephritis, and the Butyrophilin, optionally Butyrophilin A2, or a conjugate or fusion protein comprising said Butyrophilin, optionally Butyrophilin A2 is administered during active bouts of the disease.

[0198] In some instances, the autoimmune disorder or inflammatory disorder comprises allergy, asthma, scleroderma, glomerulonephritis, vasculitis, inflammatory bowel disease or Crohn's disease, rheumatoid arthritis, an autoimmune neurological disease, antibody mediated transplant rejection, infantile cholestasis, haemophagocytic lymphohistiocytosis, erythrocytic haemophagocytosis, malnutrition, systemic lupus erythematosus (lupus), psoriasis, sarcoidosis, myasthenia gravis or HIV. In various embodiments, the autoimmune neurological disease is multiple sclerosis. In various embodiments, the autoimmune neurological disease is myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease.

[0199] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is fused to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusion protein") and / or another half-life extending moiety such as PEG, albumin, lipidation, cholesterol, or glycosylation. In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is fused to the Fc domain or the fragment of the Fc domain ("BTN2A2-related isoform-Fc fusion protein"), typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.

[0200] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusionAttorney Docket No. 2969027.002013 protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function. In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain ("BTN2A2- related isoform-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc- associated effector function.

[0201] I n various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, or helical peptides.

[0202] In various embodiments, a spacer peptide is between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the Fc domain or the fragment of the Fc domain. That is, there is a spacer between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein comprises BTN2A2- spacer-Fc domain, or BTN2A2-spacer-fragment of the Fc domain, or BTN2A2 fragment- spacer-Fc domain, or BTN2A2 fragment-spacer-fragment of the Fc domain. These examples are not to limit the possible combinations, but rather to provide clarity.

[0203] Examples of spacers include but are not limited to IEGRMDDISSTMVRS (SEQ ID NO:56), IEGRMD (SEQ ID NO:57), EAEAEAK (SEQ ID NO:58), SIINFEKL (SEQ ID NO:59), and GGGS (SEQ ID NQ:60).

[0204] In various embodiments, the Fc domain comprises mutations selected from Met 208Leu, Asn214Ser or both, in reference to SEQ ID NO:4. The mutation is capable of extending the half-life of B2N2A2Fc. (See e.g., Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life", Front Immunol. 2019; 10: 1296)

[0205] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is fused to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusion protein") and / or another half-life extending moiety such as PEG, albumin, lipidation, cholesterol, or glycosylation. In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is fused to the Fc domain or the fragment of the Fc domain ("BTN2A2-related isoform-Fc fusion protein"), typically that of a human IgG such as humanAttorney Docket No. 2969027.002013IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.

[0206] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function. In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain ("BTN2A2- related isoform-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc- associated effector function.

[0207] I n various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, or helical peptides.

[0208] In various embodiments, a spacer peptide is between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the Fc domain or the fragment of the Fc domain. That is, there is a spacer between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein comprises BTN2A2- spacer-Fc domain, or BTN2A2-spacer-fragment of the Fc domain, or BTN2A2 fragment- spacer-Fc domain, or BTN2A2 fragment-spacer-fragment of the Fc domain. These examples are not to limit the possible combinations, but rather to provide clarity.

[0209] Examples of spacers include but are not limited to IEGRMDDISSTMVRS (SEQ ID NO:56), IEGRMD (SEQ ID NO:57), EAEAEAK (SEQ ID NO:58), SIINFEKL (SEQ ID NO:59), and GGGS (SEQ ID NQ:60).

[0210] In various embodiments, the Fc domain comprises mutations selected from Met 208Leu, Asn214Ser or both, in reference to SEQ ID NO:4. The mutation is capable of extending the half-life of B2N2A2Fc. (See e.g., Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life", Front Immunol. 2019; 10: 1296)

[0211] In some embodiments, the Fc domain comprises a modified Fc, e.g., IgGl Fc which comprises one, two, three, four or all five of the following mutations: M252Y, S254T, T256E, H433K and N434F.Attorney Docket No. 2969027.002013

[0212] In some embodiments, the Fc domain comprises a modified Fc, e.g., IgGl Fc which comprises the amino acid sequence in FIGURE 24 (SEQ ID NO: 61), and which may be expressed from the construct shown in in FIGURE 23. BTNs comprising this modified Ig Fc will possess longer serum half-life as these mutations increase IgG binding to the neonatal Fc receptor, which results in reduced FcRN-mediated clearance. The IgGl Fc may in addition or alternatively comprise mutations which impair FcR or complement binding, e.g., the LALAPG mutations identified in Table 1 infra.

[0213] In some embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO: 6.

[0214] In some embodiments, the BTN2A2 fragment comprises the amino acid sequence of SEQ ID NO:3.

[0215] In some embodiments, a fusion protein having SEQ ID NO:1 or SEQ ID NO:2 is administered in accordance with the embodiments described herein. In various embodiments, a fusion protein having SEQ ID NO:1 or SEQ ID NO:2 wherein the spacer IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced with IEGRMD (SEQ ID NO:57), is administered in accordance with the embodiments described herein.

[0216] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is modified by glycosylation or PEGylation. These modifications are capable of extending the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2- related isoform or the BTN2A2-related isoform fragment.

[0217] Modification by PEGylation can extend the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2- related isoform fragment. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies", Biodrugs 2008; 22 (5): 315-329, incorporated herein by reference as though fully set forth. Thus, for example PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include but are not limited 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, 60-75 kDa.

[0218] Another approach to improve the pharmacokinetic (PK) properties of BTN2A2 or a fragment thereof is its attachment to albumin, e.g., human serum albumin (HSA), whichAttorney Docket No. 2969027.002013 precludes proteolytic degradation and rapid renal filtration (due to the relatively large size of HSA of approximately 66 kDa). Still another approach to improve the pharmacokinetic (PK) properties of BTN2A2 ora fragment thereof is glycosylation. Yet another approach to improve the pharmacokinetic (PK) properties of BTN2A2 or a fragment thereof is its attachment to lipids or to cholesterol.

[0219] In various embodiments, the BTN2A2 fragment comprises an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise a transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise a cytoplasmic domain. In various embodiments the BTNA2 fragment binds to CD45, e.g., CD45RO.

[0220] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3 or a fragment or fusion polypeptide comprising any of the foregoing, optionally an Fc fusion polypeptide typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.

[0221] In various embodiments, the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both. In various embodiments the BTN2A2-related isoform fragment binds to CD45, e.g., CD45RO.

[0222] Various embodiments of the invention provide for a method of treating IBD, e.g., Crohn's or DC in a subject in need thereof, comprising: administering butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a fusion polypeptide comprising any of the foregoing, optionally an Fc fusion polypeptide, to the subject.

[0223] In some embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is fused to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function. . In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is fused to the Fc domain or the fragment of the Fc domain ("BTN2A2-related isoform-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.Attorney Docket No. 2969027.002013

[0224] In some embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain ("BTN2A2-Fc fusion protein") typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.

[0225] In various embodiments, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain ("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, leucine zipper, or aliphatic, or helical peptides.

[0226] In some embodiments, a spacer peptide, optionally from 2-50 amino acids, is between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the Fc domain or the fragment of the Fc domain, typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function. That is, there is a spacer between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein comprises BTN2A2-spacer-Fc domain, or BTN2A2-spacer-fragment of the Fc domain, or BTN2A2 fragment-spacer-Fc domain, or BTN2A2 fragment-spacer-fragment of the Fc domain. These examples are not to limit the possible combinations, but rather to provide clarity.

[0227] Examples of spacers include but are not limited to IEGRMDDISSTMVRS (SEQ ID NO:56), IEGRMD (SEQ ID NO:57), EAEAEAK (SEQ ID NO:58), SIINFEKL (SEQ ID NO:59), and GGGS (SEQ ID NQ:60).

[0228] In various embodiments, the Fc domain, e.g., an IgGl Fc domain, comprises mutations selected from Met 208Leu, Asn214Ser or both, in reference to SEQ ID NO:4. The mutation is capable of extending the half-life of B2N2A2Fc. (See e.g., Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life", Front Immunol. 2019; 10: 1296)

[0229] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO: 6.

[0230] In various embodiments, a fusion protein having SEQ ID NO:1 or SEQ ID NO:2 is administered in accordance with the embodiments described herein. In various embodiments, a fusion protein having SEQ ID NO:1 or SEQ ID NO:2 wherein the spacerAttorney Docket No. 2969027.002013IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced with IEGRMD (SEQ ID NO:57), is administered in accordance with the embodiments described herein.

[0231] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is modified by attachment to an albumin, e.g., human serum albumin (HSA).

[0232] In various embodiments, the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2-related isoform fragment is modified by glycosylation or PEGylation. These modifications are capable of extending the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2- related isoform or the BTN2A2-related isoform fragment.

[0233] Modification by PEGylation can extend the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2- related isoform fragment. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies", Biodrugs 2008; 22 (5): 315-329, incorporated herein by reference as though fully set forth. Thus, for example PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include but are not limited 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, 60-75 kDa.

[0234] In various embodiments, the BTN2A2 fragment comprises an extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise a transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise a cytoplasmic domain. In various embodiments the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0235] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0236] Various embodiments of the invention provide for a fusion protein, comprising: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment; and an Fc domain or a fragment of the Fc domain, typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function.Attorney Docket No. 2969027.002013

[0237] In various embodiments, the fusion protein is not a fusion protein having SEQ ID NO:1. In various embodiments, the fusion protein is not a fusion protein having SEQ ID NO:1, wherein the spacer IEGRMDDISSTMVRS (SEQ ID NO:56) is replaced with IEGRMD (SEQ ID NO:57).

[0238] In various embodiments, the Fc domain or the fragment of the Fc domain is from an IgGl antibody.

[0239] In various embodiments, the Fc domain or the fragment of the Fc domain, typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function and may comprise one or more mutations. In various embodiments, the mutation is Met208Leu, Asn214Ser, or both, in reference to SEQ ID NO:4. Mutations to the Fc domain provide for extended half-life of the fusion protein.

[0240] In various embodiments, the Fc domain has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5 or in SEQ ID NO: 6. Alternatively, the BTN2A2 may comprise an Fc variant selected from any of the following:

[0241] Alternative Fc Variants

[0242] As mentioned the subject BTN2A2 polypeptides and fragments may comprise an Fc region which optionally may be mutagenized to modulate an Fc-associated effector function. In some embodiments, one or more amino acid modifications are introduced into the Fc region, thereby generating an Fc region variant or Fc variant. An Fc region may comprise a C-terminal region of an immunoglobulin heavy chain that comprises a hinge region, CH2 domain, CH3 domain, or any combination thereof. As used herein, an Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., a substitution, addition, or deletion) at one or more amino acid positions.

[0243] In some embodiments, a variant Fc region comprises at least one amino acid modification in the Fc region. Combining amino acid modifications are also useful. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein, e.g. of the specific Fc region positions identified herein.Attorney Docket No. 2969027.002013

[0244] In some embodiments, the altered effector function is reduced effector function. In some embodiments, the altered effector function is increased effector function Effector functions generally refer to a biological event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Non-limiting effector functions include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation. In some cases, antibody -dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (e.g., natural killer cells, neutrophils, macrophages) recognize bound antibody on a target cell, subsequently causing lysis of the target cell. In some cases, complement dependent cytotoxicity (CDC) refers to lysing of a target cells in the presence of complement, where the complement action pathway is initiated by the binding of Clq to antibody bound with the target.

[0245] In certain cases, it is beneficial to reduce the effector function. In some instances, modifications in the Fc region generate an Fc variant with (a) decreased antibodydependent cell-mediated cytotoxicity ADCC), (b) decreased complement mediated cytotoxicity (CDC), and / or (c) decreased affinity for Cl q. In some embodiments, the Fc region is modified to decrease antibody dependent cellular cytotoxicity (ADCC), decreased antibodydependent cell-mediated phagocytosis (ADCP), decreased complement mediated cytotoxicity (CDC), and / or decreased affinity for Clq by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254,255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324,325, 326, 327, 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 numbering). In some embodiments, the variant Fc region is selected from TABLE 1.

[0246] TABLE 1Attorney Docket No. 2969027.002013Attorney Docket No. 2969027.002013Attorney Docket No. 2969027.002013

[0247] In vitro assays to assess ADCC activity of a molecule of interest are well known in the art. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 and 5,821,337. Useful effector cells for use in such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and Natural Killer (NK) cells.

[0248] In some embodiments, variant Fc regions exhibit reduced effector function as compared with wild-type human IgGl. Non-limiting examples of Fc mutations in IgGl that, in certain instances, reduce ADCC and / or CDC include substitutions at one or more of positions: 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 IgGl, where the numbering system of the constant region is that of the EU index as set forth by Kabat.

[0249] In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an N297A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an N297Q substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an N297D substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an D265 A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an S228P substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L235 A substitution, according to the EUAttorney Docket No. 2969027.002013 numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L237A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L234A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an E233P substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L234 V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an C236 deletion, according to the Kabat numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a P238 A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an A327Q substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a P329 A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an P329G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L235E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an P331S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an L234F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 235G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 235 Q substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 235R substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 235S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 236F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 236R substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 237E substitution, according to the EU numbering system. In some embodiments, the variant FcAttorney Docket No. 2969027.002013 region comprises an IgGl Fc region comprising an 237K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 237N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 237R substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 238 A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 2381 substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238W substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 238Y substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 248 A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254D substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 2541 substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254P substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc regionAttorney Docket No. 2969027.002013 comprising an 254Q substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254T substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 254V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 255N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 256H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 256K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 256R substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 256V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 264S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 265H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 265K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 265S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 265Y substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 267G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 267H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 2671 substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 267K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 268K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 269N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 269QAttorney Docket No. 2969027.002013 substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 270A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 270G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 270M substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 270N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 271T substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 272N substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 279F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 279K substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 279L substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 292E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 292F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 292G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 292L substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 293S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 301W substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 304E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 31IE substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 311 G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 311S substitution, according to the EUAttorney Docket No. 2969027.002013 numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 316F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 327T substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 328V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 329 Y substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 330R substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 339E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 339L substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 3431 substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 343 V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 373A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 373G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising a 373S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 376E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 376W substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 376Y substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 380D substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 382D substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 382P substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 385P substitution, according to the EU numbering system. In someAttorney Docket No. 2969027.002013 embodiments, the variant Fc region comprises an IgGl Fc region comprising an 424H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 424M substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 424V substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 434L substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 438G substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 439E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 439H substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 439Q substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440D substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440E substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440F substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440M substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440T Fc region substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising an 440V substitution, according to the EU numbering system.

[0250] In some embodiments, the variant Fc region comprises an IgGl Fc region L234A, L235E, G237A, A330S, and / or P331S by EU numbering. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising E233P, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising S228P and L235E. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L235E, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A and L235A, according toAttorney Docket No. 2969027.002013 the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A, L235A, and G237A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A, L235A, P329G, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234F, L235E, and P331S, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A, L235E, and G237A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A, L235E, G237A, and P331S, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234 A, L235A, G237A, P238S, H268A, A330S, and P331S (IgGl), according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising L234A, L235A, and P329A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising G236R and L328R, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising G237A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising F241A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising V264A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising D265A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising D265A and N297A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising D265A and N297G, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising D270A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising N297A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising N297G, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising N297D, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising N297Q, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising P329A, according to the EU numbering system. In some embodiments, the variant Fc regionAttorney Docket No. 2969027.002013 comprises an IgGl Fc region comprising P329G, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising P329R, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising A330L, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising P331 A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgGl Fc region comprising P331S, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2 Fc region. In some embodiments, the variant Fc region comprises an lgG4 Fc region. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising S228P, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising S228P, F234A, and L235A, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2-lgG4 cross-subclass (lgG2 / G4) Fc region. In some embodiments, the variant Fc region comprises an lgG2-lgG3 cross-subclass Fc region. In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising H268Q, V309L, A330S, and P33 IS, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising V234A, G237A, P238S, H268A, V309L, A330S, and P331S, according to the EU numbering system. In some embodiments, an antibody comprises a Fc region comprising high mannose glycosylation.

[0251] In some embodiments, the one or more mutations relative to a wildtype Fc region comprises or consists of L234A, L235A, and P329G by EU numbering. In some embodiments, the one or more mutations relative to a wildtype Fc region comprises or consists of L234 A, L235E, G237A, A330S, and P331S by EU numbering. In some embodiments, the one or more mutations relative to a wildtype Fc region is selected from the group consisting of:N297A / Q / G; L235A / G237A / E318A; L234A / L235A; G236R / L328R; S298G / T299A; L234F / L235E / P331 S; H268Q / V309L / A330S / P331S; L234A / L235A / P329G;V234A / G237A / P238S / H268A / V309L / A330S / P331 S; and L234F / L235E / D265A. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising a S228P substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising an A330S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG4 Fc region comprising a P331S substitution, according to the EU numbering system.Attorney Docket No. 2969027.002013

[0252] In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising an A330S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising an P331S substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising an 234A substitution, according to the EU numbering system. In some embodiments, the variant Fc region comprises an lgG2 Fc region comprising an 237A substitution, according to the EU numbering system.In some embodiments, the variant Fc region comprises IgGl Fc region, and wherein the one or more mutations comprises (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328 A, (i) 327Q or 327T, (j) 329 A, 329G, 329Y, or 329R (k) 331 S, (1) 236F or 236R, (m) 238 A, 238E, 238G, 238H, 238L, 238V, 238 W, or 238Y, (n) 248 A, (o) 254D, 254E, 254G, 254H, 2541, 254N, 254P, 254Q, 254T, or 254V, ( p) 255N, (q ) 256H, 256K, 256R, or 256V, (r) 2645, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267L, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292L, (aa) 2935, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 3115, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343L or 343V, (jj) 373 A, 373G, or 373 S, (kk) 376E, 376W, or 376Y, (II) 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 andL235A, (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 P331S (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, (III) A330L, (mmm) P331A orP331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S or (ppp) any combination of (a) - (uu), per EU numbering.

[0253] In various embodiments, the fusion protein further comprising a spacer peptide, optionally from 2-50 amino acids, between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the Fc domain or the fragment of the Fc domain. That is, there is a spacer between the BTN2A2 portion and the Fc domain portion. For example, the fusion protein comprises BTN2A2-spacer-Fc domain, or BTN2A2-spacer-fragment of the Fc domain, or BTN2A2 fragment-spacer-Fc domain, orAttorney Docket No. 2969027.002013BTN2A2 fragment-spacer-fragment of the Fc domain. Preferably the linker is not cleaved by serum proteases.

[0254] These examples are not to limit the possible combinations, but rather to provide clarity. Examples of spacers include but are not limited to IEGRMDDISSTMVRS (SEQ ID NO:56), IEGRMD (SEQ ID NO:57), EAEAEAK (SEQ ID NO:58), SIINFEKL (SEQ ID NO:59), and GGGS (SEQ ID NQ:60).

[0255] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain. In various embodiments, the BTN2A2 fragment does not comprise the cytoplasmic domain. In various embodiments the BTN2A2 fragment binds to CD45, e.g., CD45RO.

[0256] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0257] In various embodiments, the fusion protein has the sequence of SEQ ID NO:2.

[0258] Various embodiments provide for a modified polypeptide, comprising: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2- related isoform fragment or a combination thereof, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or the combination thereof is PEGylated, lipidated or glycosylated.

[0259] These modifications are capable of extending the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2- related isoform fragment.

[0260] Modification by PEGylation can extend the half-life of the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof, the BTN2A2-related isoform or the BTN2A2- related isoform fragment. Examples of PEGylation include Veronese and Mero, "The Impact of PEGylation on Biological Therapies", Biodrugs 2008; 22 (5): 315-329, incorporated herein by reference as though fully set forth. Thus, for example PEG or branched PEG (PEG2) can be used. Exemplary sizes of PEG or PEG2 include but are not limited 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, 60-75 kDa.Attorney Docket No. 2969027.002013

[0261] In various embodiments, the BTN2A2 fragment comprises the extracellular domain of BTN2A2. In various embodiments, the BTN2A2 fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both. In various embodiments the BTN2A2 fragment binds to CD45, e.g. CD45RO,

[0262] In various embodiments, the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3. In various embodiments, the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

[0263] Various embodiments provide for an immunoassay to detect butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof.

[0264] In various embodiments, the immunoassay is an ELISA.

[0265] In various embodiments, the immunoassay comprises an anti-butyrophilin A2(BTN2A2) antibody, an anti-BTN2A2 fragment antibody, an anti-BTN2A2-related isoform antibody, or an anti-BTN2A2-related isoform fragment antibody, or a combination thereof immobilized on a solid support; and the solid support.

[0266] In various embodiments, the immunoassay further comprises a biological sample obtained from a subject. The subject may be one who desires information regarding levels butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment.

[0267] Various embodiments, provide for a method of using an immunoassay of the present invention for patient monitoring, the method comprises: contacting a biological sample obtained from a patient to an immunoassay of the present invention; and detecting the level of butyrophilin A2 (BTN2A2), BTN2A2 fragments, BTN2A2-related isoforms, or BTN2A2-related isoform fragments, or a combination thereof.

[0268] In various embodiments, the method further comprises selecting or administering a treatment of the present invention as described herein. In various embodiments, the method further comprises selecting or administering one of the following treatments(i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment;(ii) a fusion polypeptide, comprising:Attorney Docket No. 2969027.002013 a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof; and an albumin, e.g., HSA, or an Fc domain or a fragment of an Fc domain, e.g., that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function;(iii) a modified polypeptide, comprising: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or the combination thereof is PEGylated, lipidated or glycosylated; or(iv) a non-BTN2A2 related treatment.

[0269] Various embodiments provide for a method of using an immunoassay of the present invention for patient stratification, the method comprising: contacting a biological sample obtained from a patient to an immunoassay of the present invention; detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; and stratifying the patient for treatment with a treatment of the present invention as described herein.

[0270] Various embodiments provide for a method of using an immunoassay of the present invention for patient stratification, the method comprising: contacting a biological sample obtained from a patient to an immunoassay of the present invention; detecting the level of butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, or a combination thereof; and stratifying the patient for treatment with one of the following treatments:(i) butyrophilin A2 (BTN2A2), a BTN2A2 fragment thereof, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment;(ii) a fusion polypeptide, comprising: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof; and an Fc domain or a fragment of the Fc domain, typically that of a human IgG such as human IgGl, lgG2, lgG3 or lgG4, which optionally may be modified to increase or decrease a particular Fc-associated effector function;Attorney Docket No. 2969027.002013(iii) a modified polypeptide, comprising: a butyroph ili n A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or the combination thereof is PEGylated, lipidated or glycosylated; or(iv) a non-BTN2A2 related treatment.Wild type Amino acid sequence (SEQ ID NO:1)(237aa BTN2A2 EC domain (bold and italicized) + 15aa Xa and vector cloning seq + 227aa Fc region (bold))MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTWGPANPILAMVGENTTLRCHLSPEKNAEDMEVR WFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSY DEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFM VTTAVIIRDKYVRNVSCSVNNTLLGQEKETV\EGRMDD\SSTM\ / RSDKTH7CPPCPAPE.U.GGPS\ / FLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KMutant Amino acid sequence with long-acting protein (SEQ ID NO:2)(237aa BTN2A2 EC domain (bold and italicized)+ 2 aa vector cloning seq + 227aa Fc region(bold), underline shows the aa substitutions)MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTWGPANPILAMVGENTTLRCHLSPEKNAEDMEVR WFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSY DEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFM VTTAVlIRDKYVRNVSCSVNNTLLGQEKETVRSDKlHTCPPCPAPELLGGPSX / FLFPPKPKDTLMiSRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGKSEQ ID NO: 3 (BTN2A2 EC domain)Attorney Docket No. 2969027.002013MEPAAALH FSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCH LSPEKNAEDMEVRWF RSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIH NVTAQENGIYRCYFQEGRSYDEAIL RLWAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVII RDKYVRNVSCSVNNTLLGQEKETVSEQ ID N0:4 (Fc region (wt))DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO:5 (Fc region (mutant))DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLH EALHSHYTQKSLSLSPGK

[0271] SEQ ID NO: 61 BTN2A2 domain 237aa (1-237) XX linker 2aa Fc (e2) tag 227aa(221-447)

[0272] MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCH LSPEK NAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLWAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYIT REPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALKFHYTQKSLSLSPGK*

[0273] SEQ ID NO: 6 above comprises a variant IgGl Fc region comprising the following mutations: M252Y, S254T, T256E, and / or N433K and N434F which enhance FcRn binding and thereby increase serum half-life of the BTN2A2-fusion protein.Attorney Docket No. 2969027.002013

[0274] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, the fusion protein, or the modified polypeptide described herein. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0275] In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts, esters, amides, and prodrugs" as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0276] The term "pharmaceutically acceptable esters" refers to the relatively nontoxic, esterified 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 separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in theAttorney Docket No. 2969027.002013 presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.

[0277] As used herein, "pharmaceutically acceptable salts or prodrugs" are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0278] The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the functionally active one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. A prodrug of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be designed to alter the metabolic stability or the transport characteristics of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to mask side effects or toxicity, to improve the flavor of a compound or to alter other characteristics or properties of a compound. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, once a pharmaceutically active form of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, those of skill in the pharmaceutical art generally can design prodrugs of the compound. Suitable examples of prodrugs include methyl, ethyl and glycerol esters of the corresponding acid.

[0279] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Transdermal" administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilizedAttorney Docket No. 2969027.002013 powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. When administered via the ocular route, they may be in the form of eye drops.

[0280] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0281] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.Attorney Docket No. 2969027.002013

[0282] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0283] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20thedition, Williams & Wilkins PA, USA) (2000).KITS

[0284] The present invention is also directed to a kit. Thus, in some embodiments the kit contains a composition including: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment, the fusion protein, or the modified polypeptide as described herein.

[0285] The exact nature of the components configured in the inventive kit depends on its intended purpose. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals. In another embodiments, the kit is configured for detectingAttorney Docket No. 2969027.002013 a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2- related isoform fragment.

[0286] Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0287] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. 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, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing: a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2- related isoform fragment, the fusion protein, or the modified polypeptide as described herein. In another example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing: an anti-butyrophilin A2 (BTN2A2) antibody, an anti- BTN2A2 fragment antibody, an anti-BTN2A2-related isoform antibody, or an antiBTN2A2- related isoform fragment antibody described herein. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.EXAMPLES

[0288] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limitAttorney Docket No. 2969027.002013 the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1 Materials and MethodsBTN2A2-Fc construct generation and purification of recombinant protein

[0289] Soluble recombinant BTN2A2-Fc protein was generated using baculovirus infected insect cell system, a method that is both scalable and has been successfully used for generation of recombinant proteins in the immune system. An upstream 711 bp region of human BTN2A2 gene (NP_008926.2) containing signal peptide and two extracellular domains (IgV and lgC2) was amplified using specific primers (See TABLE 2) from cDNA from 293T cells. Amplified region was first cloned into pFUSE-hlgGl-Fcl vector (InvivoGen Catalog # pfuse- hglfcl) using Agel and EcoRV restriction sites. Subsequently, BTN2A2 region along with Fc region was amplified using specific primers (TABLE 2) and cloned into pBacPakS vector using Xbal and Sacl restriction sites. Paired-end sequencing of the clone was performed by sanger sequencing to confirm the sequence. Primers were synthesized from Integrated DNA Technologies, Inc (IDT) and Phusion® High-Fidelity PCR Master Mix with HF Buffer was used in all PCR amplification reaction (New England Biolabs). Baculovirus were generated by cotransfecting pBacPak8-BTN2A2-Fc clone with linearized baculovirus DNA (TakaraBio Catalog# 631401) into Sf9 insect cells according to manufacturer protocol. Preparation of baculovirus passage (P0, Pl, P2) was done as described in manufactures protocol (TakaraBio, Cat # 631402). P3 baculovirus stock was used for large scale protein purification using Hi5 cells grown in serum free media (Express Five™ SFM, ThermoFisher, Cat# 10486025). Pierce™ Protein G Agarose columns (ThermoFisher Scientific, Cat# 20398) was used for purification of secreted soluble-BTN2A2-Fc protein from the supernatant. Purified protein was finally suspended in lx phosphate buffer saline (lxPBS). Protein purity was checked by SDS-PAGE followed by Coomassie blue staining. Specificity of the purified protein was confirmed by western blot analysis using specific BTN2A2 antibody. Commercially available recombinant 293 cells derived human BTN2A2-FC (Cat# 8918-BT; R&D systems) and mouse BTN2A2 (Cat# 8997-BT-050; R&D systems) was used for comparison studies with baculoviral-generated recombinant B2N2A2-Fc.TABLE 2. Primer SequenceAttorney Docket No. 2969027.002013T-cells activation and IL-2 secretion assay

[0290] In vitro T cell activation was performed. Briefly, 96-well plates were coated with l .g / ml concentrations of anti-CD3 mAb (OKT3, clone x) in PBS at 4°C overnight in absence or presence of recombinant BTN2A2-Fc (10 |ig / ml) or Fc tag protein (Cat#10702- HNAH, Sino Biological Inc) or 293 cells derived human BTN2A2-Fc or mouse BTN2A2. A totalAttorney Docket No. 2969027.002013 of 2.5x10sJurkat cells / well were added to precoated flat-bottom 96-well plates. Cells were incubated for 24 hrs in 37°C incubator with 5% CO2. Total 100 pl of medial was used to measure IL-2 production using an IL-2 ELISA kit from Millipore-Sigma (Cat# RAB0286-1KT) according to the manufacturer' protocol.In vitro TCR stimulation and signal transduction analysis

[0291] For short-term activation of the Jurkat cells, a 48-well tissue culture plate was coated with anti-CD3 mAb (OKT3, lOpg / mL) and BTN2A2-FC (lOpg / mL) or Fc-Tag (lOpg / mL). Plate was incubated overnight at 4°C and each well was washed twice with PBS. A total of 2xl06cells in 75pl lxPBS were then added to each well for 3 min at 37°C. The reaction was then stopped with ice cold PBS. Pervanadate was prepared by incubating vanadate (200mM) and H2O2 (200mM) in 1:2 ratio for 15 min at room temperature. Jurkat cells in lxPBS were treated with pervanadate at final concentration of O.lmM for 5 min in 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 reaction or immunoblotting.Immunoprecipitation and Immunoblotting

[0292] J urkat cells were activated with plate-bound anti-CD3 BTN2A2-Fc or Fc-tag protein as control or left un-activated. Cells were collected then washed in PBS and resuspended in IP lysis buffer (lOmM HEPES pH 7.5, 0.5mM EDTA, 0.5% NP-40, 250mM NaCI, lx phosSTOP and protease inhibitors), incubated on ice for 30 min with vortexing, and cleared by centrifugation at 15,000g for 10 min. An aliquot of protein lysate was used for western blotting, while the remainder were incubated for 4 hrs at 4°C with specific antibodies or IgG control. Protein A / G Agarose (ThermoFisher Scientific) was added to lysate and incubated for additional 2 hrs. Beads were washed once in IP-lysis buffer with 0.5% NP-40 and twice in IP- lysis buffer with 0.2% NP-40. Proteins were eluted with SDS sample buffer supplemented with DTT by heating at 70°C for 5-10 min. For Western blot analysis, 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-tag control, and their respective input controls were blotted as described below. Approximately 12xl06Jurkat cells were used for each condition and lysed in 300 ml IP-lysis buffer. For immunoprecipitation, lysate was incubated with lmg of antibody to specific protein or respective IgG control. Protein A / G Agarose beads were used to pulldown the complex and eluted in lOOul sample buffer. Western blots were probed with anti-phospho-Zap70 anti-phospho-CD3z antibody and also antibody against totalAttorney Docket No. 2969027.002013Zap70 and CD3z protein (See TABLE 3). For phosphatase activity, immunoprecipitated material was directly mixed in Fluorescein Diphosphate, Tetraammonium Salt substrate (FDP, Catalog # F2999, Thermofisher Scientific) and activity was measured using Molecular Devices SpectraMax® M2 plate readers as recommended by protocol included by manufacturer.TABLE 3. Antibody ListAttorney Docket No. 2969027.002013

[0293] Extracellular crosslinking with BS3 (bis[sulfosuccinimidyl] suberate) was performed according to user manual (Thermo Scientific MAN0011240). Jurkat cells were washed with PBS and then incubated on ice with 10 pg / mL BTN2A2-Fc and Fc-tag protein for 1 hr. To induce crosslinking, BS3 (ThermoFisher) was added at a final concentration of 5 mM and incubated for 30 min at room temperature. The reaction was stopped using Tris HCI, pH 7.5, at a final concentration of 20 mM for 15 minutes at room temperature. The cells were then washed extensively in PBS and resuspended in IP-lysis buffer and cleared by centrifugation at 14,000g for 10 min. Protein G Agarose beads were used to pulldown BTN2A2-Fc and Fc-tagged protein. Beads were washed and eluted with SDS sample buffer supplemented with dithiothreitol. Eluted samples were analyzed by western blot probed with anti-CD45 antibody.

[0294] For Western blot analysis, Jurkat cells were collected and centrifuged. Cell pellets were resuspended in 2.5x volume of RIPA buffer (20mM Tris pH 7.5, 150 mM NaCI, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.5% SDS containing protease and phosphatase inhibitors). Total protein concentrations in the lysates were determined using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) by measuring absorption at 595 nm on the microplate reader (Bio-Rad). Total of 40-80 pg of each protein sample was used for western blot analysis. Protein lysate prepared after Jurkat cell treatment and / or immunoprecipitation was separated in 4-12% Bis-Tris gel (ThermoFisher Scientific) and transferred to nitrocellulose membrane. Membrane was blocked for 1 h in blocking solution (5% BSA in PBS with 0.1% Tween [PBST]). The membrane was incubated with primary antibodies overnight at 4°C, washed with PBST three times (10 minutes each), incubated with secondary antibodies for 1 h at room temperature, and finally washed with PBST three timesAttorney Docket No. 2969027.002013(10 minutes each). The blots were visualized using an ECL assay (ThermoFisher Scientific). Antibodies used for immunoblotting analysis are listed in TABLE 3.Molecular modeling of BTN2A2 and PTPRC

[0295] A molecular model of BTN2A2 was generated using homology modeling. A search for the homologous structure of the extracellular domain of BTN2A2 revealed that BTN3A2 shares about 47% of sequence homology with BTN2A2. Using the crystal structure of BTN3A2 as a template, the three-dimensional model of BTN2A2 was generated using SWISS- MODEL workspace. Subsequently, the molecular structure of BTN2A2 was subjected to a short 1.2ns molecular dynamics simulation using Desmond (Schrodinger, Inc., San Diego, CA). The three-dimensional structure of PTPRC was retrieved from the protein data bank (PDB code: 5FN7). To determine the interaction between BTN2A2 and PTPRC proteins, RosettaDock 4.0 was used. The top 10 predicted models were then subjected to 1.2ns molecular simulation followed by minimization using Desmond. Then, the most energetically stable docking model was used to identify potential amino acids for mutation.CRISPR-Cas9 deletion of CD45 gene and site directed mutagenesis of CD45

[0296] CD45 knockdown cell lines were generated using the CRISPR-Cas9 system. Two different guide RNAs were designed complementary to upstream of CD45 exon-1 and downstream to exon-3 based on its protospacer adjacent motif (PAM) sequence (Table 2). These guide RNA sequences were cloned into lenti-Crispr v2 blast vector containing the Cas9 coding gene and a blasticidine resistant gene. Lentivirus were generated using envelop and packaging plasmid. Ju rkat cells were then transduced with lentivirus expressing guideRNA and Cas9 gene and selected for blasticidine resistant cells. Single-cell cloning was performed to isolate independent clones. Deletion of Exon-1 to Exon-3 was confirmed by direct Sanger sequencing (Figure 13A).

[0297] CD45 (PTPRC) (NM_002838) Human Tagged ORF Clone was purchased from origene technologies. Inc. Site-directed mutagenesis were performed using the QuickChange mutagenesis kit (Invitrogen), to introduce the N419A and N468A mutations. Introduction of the mutation was confirmed by direct sequencing (Figure 13B). Lentivirus were generated using envelop and packaging plasmid. Jurkat cells deleted for CD45 gene were then transduced with lentivirus expressing mutant CD45 gene.In vitro Treg and Thl7 Differentiation by mixed lymphocyte reaction (MLR)Attorney Docket No. 2969027.002013

[0298] For Treg differentiation, fresh spleen, and lymph nodes (inguinal, brachial and axillary) were harvested from mice and washed with lx RPMI media. Spleen was excised into small pieces and single cells were prepared by mashing the tissue with the plunger end of syringe through a 70 micron strainer and cells were suspended in 5 ml of RPMI media. Cells were stimulated with plate bound anti-CD3 (l|ig / ml) with or without BTN2A2-Fc fusion protein (10 .g / ml) and co-cultured with CD1 mice irradiated splenocytes for 7 days. Cells were labeled with immunofluorescent antibodies and analyzed for CD4+CD25+Foxp3-GFP+ve coexpression using flow cytometry. CD4+ve cells incubated with anti-CD3 antibody in absence or presence of recombinant BTN2A2-Fc for day-1 and day-5 were used to extract RNA for qPCR analysis. CD4+ve T cells were isolated using EasySep™ Mouse CD4+ T Cell Isolation Kit (Stem Cell Tech); B cells using EasySep™ Mouse B Cell Isolation Kit (Stem Cell Tech); Pan-DC cells using EasySep™ Mouse Pan-DC Cell Enrichment Kit II (Stem Cell Tech); T cells using EasySep™ Mouse T Cell Isolation Kit (Stem Cell Tech) per protocols recommended by manufacturer.

[0299] To explore effects of BTN2A2 on Thl7 differentiation, lxlO6spleen cells were stimulated with soluble anti-CD3 antibody (0.5 mg / ml) and CD1 mice irradiated spleen cells (1:1 ratio) and Thl7 differentiation cytokines cocktail (TGF-bl 1.5ng / ml, IL-6 lOng / ml, IL-lb lOng / ml) with or without recombinant BTN2A2-Fc (10|ig / ml). Cells were co-cultured for 5 days and subjected to intracellular immunofluorescence staining of RORgt (Thl7 cell marker). Cells were analyzed for CD4+RORgt +ve co-expression using flow cytometry. CD45 phosphatase inhibitor (Compound 211, Catalog # 530197; Millipore Sigma) that has previously been characterized as irreversible, and selective blocker of the allosteric pocket at the D1-D2 domains interface away from the substrate-binding / catalytic site, was used at 125nM concentration during the course of T cell differentiation studies.Flow Cytometry Analysis

[0300] Immune cells were freshly harvested from spleen and lymph nodes (inguinal, brachial and axillary) of mice. Cells were counted using a Hemavet 950FS hematology analyzer (Drew Scientific, Miami Lake, FL). Immunostaining and flow cytometry analysis of cells were performed as described previously (Li et al., 2018). Briefly, cells isolated from mice or treated in vitro cultures were harvested and stained with Ghost Dye™ Red 780 Viability Dye (Cell Signaling). Cells were washed extensively with 0.5% BSA in lxPBS and resuspended in staining buffer (FBS); and further stained with fluorochrome conjugated antibodies for cells surfaceAttorney Docket No. 2969027.002013 marker anti-CD3, anti-CD4 and anti-CD25. Cells were washed with staining buffer to remove excess antibodies and then fixed / permeabilized using mouse Foxp3 Buffer Set (BD Biosciences, San Jose, CA) according to manufacturer protocol. Fixed / permeabilized cells were incubated in rat serum for 15 minutes and stained with intracellular markers (Foxp3 and / or RORgt). Cells harvested from Foxp3-GFP transgenic mice were stained with anti-CD3, anti-CD4 and anti-CD25 antibodies, washed and suspended buffer containing DAPI. Cells were directly analyzed for CD4+CD25+Foxp3-GFP or CD4+RORgt expression. The stained cells were analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA). Details of the commercial antibodies used in this study are provided in TABLE 3.Cells proliferation and apoptosis assay

[0301] CD4+ cells were isolated from freshly harvested spleen of wild type B6 mice as described above. Cells were stained with CellTrace™ Violet dye at 5mM dye concentration in 105cells / ml dilution according to manufacturer protocol (Invitrogen). 2.5x106 CellTrace™ Violet stained cells were incubated on bound anti-CD3 (0.5 g / ml) plus anti-CD28 (0.5 / ml) with BTN2A2-Fc fusion protein (10 .g / ml) or Fc control (10 .g / ml). Cells were culture for 3 days at 37°C and 5% CO2 in RPMI medium supplemented with 10%FBS and 1% Pen-Strp. Unstimulated parent generation (GO) indicated as the brightest peak on the far-right side of the histogram (Fig. 14). Cells were stained with Annexin V-FITC conjugate for apoptotic cells assay. For intracellular staining of IL2, cells were fixed, permeabilized and stained with IL2 antibody and analyzed using a BD LSR Fortessa (BD Biosciences, San Jose, CA).Immunofluorescence Studies

[0302] I mmunofluorescence staining for CD45 and CD3^ was performed. Anti-CD3 antibody (lOmg / ml) and recombinant BTN2A2-Fc (lOmg / ml) were coated on glass poly-L- lysine cover slips in PBS overnight at 4°C. lxlO6Jurkat cells in 75ul of IxPBS were exposed to coated coverslip for 3 min at 37°C and fixed using Cytofix / Cytoperm- Fixation / Permeabilization Kit (BD biosciences). Coverslips were then gently washed two times with lx PBS. Cells were blocked with blocking buffer (5% goat / mouse serum in 0.3% triton- xlOO in IxPBS) and stained with mouse anti-human CD45 (abeam cat# ab8216) and rabbit anti-human CD3 (abeam cat# Ab226263). Coverslips were then gently washed 3 time with lx PBS for 10 minutes each and stained with Donkey anti-Mouse IgG Antibody, Alexa Fluor™ 555 and Goat anti-Rabbit IgG Antibody, Alexa Fluor™ 488 (ThermoFisher Scientific). CoverslipsAttorney Docket No. 2969027.002013 were then gently washed 3 time with lx PBS for 10 minutes each, incubated in DAPI for 5 minutes and mounted on slides using Prolong™ Diamond Antifade Mountant (ThermoFisher Scientific). Glass slides were then imaged with a Zeiss LSM 780 confocal microscope through a 63x resolution with oil immersion. The image was analysed as an 8-bit image and intensity of red and green channel was measured from 0-255 grayscale fluorescent units. Colocalization analysis was performed on Image-J plugin "Colocalization Finder". At least seven fields were analysed per experimental condition, in which an average of 50 T cells were analysed to yield the average amount of co-localization.Quantitative Polymerase Chain Reaction (QPCR)

[0303] For gene expression analysis, the total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific). The cDNA was synthesized from 500 ng of total RNA using SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific). Quantitative real time PCR was performed with Powerllp™ SYBR™ Green Master Mix (Thermo Fisher Scientific) on a QuantStudio Real-Time PCR system (Thermo Fisher Scientific). GAPDH was used as internal controls. Gene expression was compared using the AACt method. Primers details are provided in (Table 2).Animal Models

[0304] All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and all the protocols used in this study were approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee.

[0305] Foxp3EGFP (C.CR-Foxp3tm2Tch / i, Strain # 006769) mice: Foxp3EGF mice that coexpress EGFP and the regulatory T cell-specific transcription factor Foxp3 under the control of the endogenous promoter was acquired from the Jackson Laboratory.

[0306] BTN2A2 Knock-out mice (- / -): BTN2A2(- / -) mice (strain: C57BL / 6J- Btn2a2emlcyagen) were generated by deleting 10460 base pair region of btn2a2 gene comprising exon2-8 using CRISPR-Cas9 technique at commercial facility (Cyagen Biosciences) (Figure 16). Three PCR primers set (Table 1) were used for genotyping mice using polymerase chain reaction method. Wildtype mice show single band of 500bp, heterozygous shows two bands of 700bp and 500bp, while homozygous mice show 500bp single band on agarose gel electrophoresis (Figure 16).Attorney Docket No. 2969027.002013

[0307] Glomerulonephritis Model: The detailed protocol to induce crescentic glomerulonephritis (GN) has been described previously. Nephrotoxic sera were raised in rabbits by repeated immunization with the purified glomeruli in complete and incomplete Freund's adjuvant. The mice were preimmunized with normal rabbit IgG and complete Freund's adjuvant five days prior to administration of nephrotoxic serum. Nephrotoxic serum nephritis was induced by the injection of 20 ml or 10 ml nephrotoxic serum intravenously at day 0. Four doses of 25 mg BTN2A2-Fc fusion protein or vehicle (control) were injected i.p. at day 0, 2, 4, 6 of nephrotoxic serum injection. Mice were scarified at day 7 to collect tissues, urine, blood cells and plasma. Immune cells were harvested from fresh whole spleen and lymph-nodes (Inguinal, Brachial and Axillary) and CD4+ve T cells were isolated using EasySep™ Mouse CD4+T Cell Isolation Kit (STEM CELLTECH). Protein lysate of snap-freeze tissue in liquid nitrogen was prepared by homogenizing 5mg tissue in 500ul ice-cold RIPA lysis buffer. Lysate was agitated for 2 hrs at 4°C, centrifuged at 16000g for 20min at 4°C and supernatant was collected. About 80 mg of tissue lysate was used to detect IL17A expression using western blot.

[0308] To obtain the protein-to-creatinine ratio (mg / mg) in urine for evaluation of proteinuria, urine protein was measured with a protein assay dye (No. 500-0006, Bio-Rad, Hercules, CA), and urinary creatinine was measured using a Creatinine Assay Kit (No. DICT- 500, BioAssay Systems, Hayward, CA). Fibrinoid necrosis (a precursor lesion for crescents, defined by finding GBM rupture, fibrin deposition, and karyorrhexis), and crescents were assessed in all glomeruli on one paraffin section for each mouse using periodic acid- methenamine silver and PAS stains, respectively. Representative images were captured using a light microscope (Nikon Eclipse 50i, Nikon, Tokyo, Japan). Histopathologic diagnosis of GN was evaluated by a board-certified renal pathologist (M.Y.)

[0309] Miscarriage Model: DBA / 2 and CBA / J mice were purchased from Jackson Laboratory. We crossed male DBA / 2 with female CBA / J to document immunologically mediated pregnancy loss as described elsewhere (Zenclussen et al., 2005). As controls, we crossed female DBA / 2 with male CBA / J strain and we evaluated litter size, pup weight and litter resorption rates. To study the therapeutic effects of BTN2A2, four doses of 25mg BTN2A2-Fc fusion protein were injected intraperitoneally at gestational days 6, 9. 12 and 15 in CBA / J female mice that were crossed with male DBA / 2 mice. Animals were sacrificed at gestational day 18 and blood / tissues harvested for molecular analysis.Attorney Docket No. 2969027.002013Statistics

[0310] Data were graphed and statistics performed using GraphPad Prism version 9.2. Data are presented mean ± standard deviation (SD) as indicated. Shapiro-Wilk test was used to test the normality of the data. Unpaired t-test was used to compare the data of two groups that passed normality test otherwise compared with Mann- Whitney test. For data with three or more groups, one-way ANOVA test with Tukey's test for multiple comparison was used for data that passed normality test otherwise compared with Kruskal-Wallis test followed by Dunn's test for multiple comparison. Statistically significant differences were defined as p<0.05 (*), p<0.01 (**), p<0.001 (***). The number of experiments per experiment is indicated in the legends to figures.Example 2 Human Studies

[0311] This study was approved by the Institutional Review Board at the Cedars-Sinai Medical Center.

[0312] Treg Assays: Whole blood was drawn from healthy unrelated individuals to prepare peripheral blood mononuclear cells (PBMC) using the Ficoll-Hypaque gradient centrifugation method. After the stimulator PBMC was irradiated, it was exposed to the responder PBMC at 1:1 (1 x 106 / ml of each PBMCs) in the absence or presence of BTN2A2-Fc at 0, 10 mg / ml, and then incubated for 7 days for measurement of Tregs. MLR mixture were first stained with antibodies to CD45, CD3, CD4, CD25, and CD127. After permeabilization, the cells were stained with antibody to Foxp3. After acquiring cells by flow cytometry, lymphocytes separated from CD45+ leukocytes were plotted against CD4. CD4+ cells were plotted as CD25 versus CD127 and then CD25+CD127low / “ cells against Foxp3. CD4+ / CD25+ / CD127low / “ / Foxp3+ cells were designated as Treg cells. Treg cell levels were expressed as Treg cel 1% in CD4+ T cells.

[0313] Thl7 assays: Human PBMCs (responder cells) isolated from Ficoll-Hypaque gradient centrifugation method were cultured in irradiated stimulator cells at 1:1 (1 x 106 / ml of each PBMCs) along with Thl7 differentiation cytokines cocktail (anti-CD3 antibody 0.5ug / ml, TGFb 1.5ng / ml, IL-6 lOng / ml, IL-lb lOng / ml) with or without recombinant BTN2A2- Fc (lOmg / ml) for 5 days. Cells were stained with surface marker CD3 and CD4. After permeabilization, the cells were stained anti-RORgt antibody and analyzed for CD4+RORgtAttorney Docket No. 2969027.002013+ve co-expression using flow cytometry. Thl7 cells were expressed as RORgt +ve cells % in CD4 T cells. CD45 phosphatase inhibitor was used at 125nM concentration during the course of T cell differentiation studies.Example 3BTN2A2 blocks CD3-dependent signaling in Jurkat Cells

[0314] To elucidate the mechanisms underlying BTN2A2's ability to regulate T cell immunity (Ammann et al., 2013; Sarter et al., 2016), we generated a soluble recombinant human BTN2A2-Fc fusion protein using baculoviral expression systems. Consistent with prior reports performed in murine systems (Ammann et al., 2013), recombinant human BTN2A2- Fc, but not recombinant Fc control protein, blocked anti-CD3-induced IL-2 production by Jurkat cells (Figure 10A-10C).

[0315] When we next evaluated signaling components downstream of the TCR, we observed that BTN2A2-Fc reduced anti-CD3 induced phosphorylation of Zap70, and CD3z in Jurkat cells (Figure 10D). Addition of the phosphatase inhibitor pervanadate resulted in hyperphosphorylation of Zap70 and CD3z at baseline (unstimulated), and in contrast to the findings in the absence of pervanadate, addition of BTN2A2-Fc to the culture had no effect on the hyperphosphorylated proteins (Figure 11). Together these data suggested that BTN2A2- Fc activates a phosphatase that downregulates TCR signaling.BTN2A2 blocks TCR signaling by binding to and enhancing CD45 phosphatase

[0316] Based on the known molecular mechanisms linking CD45's phosphatase activity to TCR signaling (Jung et al., 2021; Leupin et al., 2000), we tested the hypothesis BTN2A2's inhibitory effect on TCR activation is mediated through interaction with CD45. Western blotting of CD45-associated proteins after co-immunoprecipitation (co-IP) of CD45 in unstimulated Jurkat cells demonstrated that in the absence of TCR activation, CD45 was normally associated with several components of the TCR complex (Zap70 and CD3z). However, activation of the TCR complex with anti-CD3 antibodies resulted in CD45 dissociating from Zap 70 and CD3z. In contrast, TCR activation in the presence of BTNT2A2 caused CD45 to remain associated with the TCR complex (Figure 1A), I im iti ng T-cel I activation. Immunocytochemistry confirmed colocalization of CD45 and CD3z in untreated cells at baseline, segregation of CD45 following anti-CD3-induced TCR activation, and maintenance of CD45 and CD3z colocalization when cells were stimulated in the presence of BTN2A2,Attorney Docket No. 2969027.002013 suggesting that BTN2A2 prevents the exclusion of CD45 from the immune synapse during the early phases of T cell activation (Figure IB).

[0317] Co-IP experiments additionally demonstrated that compared to unstimulated cells, anti-CD3 activation reduced the association of CD3e with CD45, whereas TCR activation in the presence of BTN2A2 augmented the interaction between CD45 and CD3e (Figure 1C). Functional experiments looking at TCR-associated CD45 phosphatase activity showed that when TCR complexes were isolated by co-IP with anti-CD3e antibodies, CD45 phosphatase activity was detectable at baseline, decreased following TCR activation, and maintained when cells were activated in the presence of BTN2A2, suggesting segregation of CD45 from the TCR complex after activation and retention in the presence of BTN2A2 (Jung et al., 2021; Leupin et al., 2000) (Figure ID). Together with prior studies that activating CD45 phosphatase on lipid microdomains on T cell surfaces results in decreased sensitivity of TCR-mediated signaling (He et al., 2002), our new data indicate BTN2A2 dampens TCR signaling by activating CD45 phosphatase activity in the TCR complex which in turn blocks critical phosphorylation events downstream of the TCR.BTN2A2 interacts with CD45 phosphatase

[0318] We next employed a co-IP experimental strategy using Jurkat cells to test for direct interactions between CD45 and endogenously expressed BTN2A2 (Figure 2A). These assays showed CD45 co-immunoprecipitated with endogenous BTN2A2 in unstimulated Jurkat cells, and binding was significantly enhanced in activated cells. Anti-CD3 activation of Jurkat cells did not change endogenous CD45 and BTN2A2 expression levels (Figure 12). Complementary experiments of cells treated with exogenous BTN2A2-Fc protein or Fc protein control confirmed that exogenous BTN2A2-Fc but not control Fc-tag protein binds to CD45 in activated T cells (Figure 2B-C). We then performed molecular modeling to identify regions in the extracellular region of CD45 that might interact with BTN2A2. Protein homology modeling showed that BTN2A2 interacted with the extracellular fibronectin domain of CD45 protein and in particular amino acids - Asn-419 and Asn-468 (putative N-glycosylation sites based on their location within N-X-S / T consensus motifs) were critical for this interaction (Figure 2D). We then performed site-directed mutagenesis of asparagine to alanine and confirmed that mutant CD45 interaction with BTN2A2 is significantly attenuated (Figure 13 and Figure 2E-F).

[0319] Based on our observations CD45 binding to endogenous BTN2A2 after activation, we suspected that BTN2A2 was preferentially binding with the CD45RO isoform,Attorney Docket No. 2969027.002013 because CD45RO is predominantly expressed in activated T cells (Hermiston et al., 2003) and is upregulated after Jurkat cell activation (Figure 2G). Considering this, we repeated the pulldown assay using isoform specific antibodies (Figure 2H). These experiments confirmed that BTN2A2 preferentially binds CD45RO isoform, the predominant isoform in activated T cells.BTN2A2 enhances regulatory T cell expansion and suppresses Thl7 cell populations in primary mouse T cells

[0320] Activated CD4+ T cells differentiate into several effector cell subsets based on activation events and the cytokine milieu present during activation. Since it was previously reported that BTN2A2 induced Foxp3 expression in CD4+ T cells (Ammann et al., 2013) in short-term culture studies we tested whether T cells activated in the presence of BTN2A2 increased Treg populations in splenocytes from Foxp3-EGFP mice that co-express EGFP when Foxp3 is expressed. We cultured murine T cells (Foxp3+ reporter mice -BALB / cJ strain) in the presence of irradiated, allogeneic mouse splenocytes (CD1 strain) with or without recombinant BTN2A2-Fc. These assays showed a 50% increase in CD4+CD25+Foxp3+ T cells, in BTN2A2-Fc treated cells compared to controls, a similar magnitude to that observed in positive control cultures using exogenous TGF-P (Figure 3A). Interestingly, short-term exposure of BTN2A2 to activated CD4+T cells led to less proliferation and enhanced survival, phenotypes noted with a pro-differentiation state and similar to what has been reported with TGF-b (McKarns and Schwartz, 2005) (Figure 14). Parallel assays showed that recombinant BTN2A2 blocked IL-6 / TGF-p / IL-ip-induced production of RORgt, the signature transcription factor of Thl7 cells (Figure 3B).

[0321] To further explore the short-term effects of BTN2A2 on T cell subset expression patterns in mixed lymphocyte reaction (MLR) culture conditions, we assessed gene expression patterns for molecules related to Thl, Th2, Treg and Thl7 cells by RT-PCR. BTN2A2 upregulated IL2Rbeta and Foxp3 and downregulated IL-21 consistent with our in vitro findings of expansion ofTregs and suppression of Thl7 cells (Figure 15A-C). The assays further showed downregulation of transcription factors regulating Thl (TBX21) and Th2 (GATA3) differentiation pathway at day 5 (Figure 15D-F).

[0322] To explore the role of endogenous BTN2A2, we genetically knocked out murine BTN2A2 using CRISPR-Cas9 strategy (Figure 16) and confirmed that expression of BTN2A2 was absent in professional APCs (CDllc+ dendritic cells and CD19+ B cells) in knock-out miceAttorney Docket No. 2969027.002013 compared to wild-type littermates (Figure 17). Interestingly, when isolated APCs from BTN2A2- / - animals were co-cultured with murine T cells from Foxp3+ reporter mice, absence of BTN2A2 from both isolated dendritic cells and isolated B cells from BTN2A2- / - animals impaired mouse T cell differentiation to Foxp3+ Tregs when compared to dendritic cells and B cells from wildtype littermates (Figure 3C-D)BTN2A2-induced enhancement of Treg / Thl7 balance is dependent on CD45 phosphatase activity.

[0323] To test whether CD45 phosphatase activity was essential for BTN2A2's actions on TCR signaling and Treg expansion, we used a previously validated CD45-specific phosphatase inhibitor (Perron et al., 2014). Addition of BTN2A2-Fc to activated Jurkat T cells decreased phosphorylation of ZAP70 kinase, while inhibition of CD45 phosphatase activity with this small molecule inhibitor (Perron et al., 2014) abrogated the effect (Figure 4A). When we inhibited CD45 phosphatase activity during BTN2A2-Fc treatment we also observed complete abrogation of the effects of BTN2A2-Fc on increased Treg and decreased Thl7 populations (Figure 4B-C). Taken together with other experiments described above, these studies link BTN2A2 effects on CD45 phosphatase activity within the TCR complex and immune synapse and Treg and Thl7 cellular balance.BTN2A2-Fc therapy exhibits immunoregulatory function in vivo

[0324] To test whether and how the in vitro observed immunoregulatory effects of BTN2A2 correlate to findings in vivo, we employed two distinct murine 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 significant proteinuria (Figure 5A-D). Administration of BTN2A2-Fc during the days following NTS administration reduced proteinuria and glomerular crescent formation. Administration of BTN2A2-Fc also increased CD4+ Foxp3 gene expression (Figure 5E), decreased CD4+ RORgt gene expression (Figure 5F), attenuated T cell activation marker CD5 levels (Figure 5G) in CD4+ T cells purified from spleen and lymph nodes. Further, BTN2A2-Fc lowered IL17A protein expression in the kidneys compared to controls (Figure 5G-I).

[0325] We next administered a lower dose of NTS (50% less than the amount used in experiments above) to BTN2A2- / - mice (Figure 16) and wildtype littermate controls (Figure 6). These experiments demonstrated that lower doses of NTS induced relatively mild crescentic glomerulonephritis and proteinuria among wild-type control animals, however, inAttorney Docket No. 2969027.002013BTN2A2- / - animals we saw an exacerbation of crescentic glomerulonephritis and severe proteinuria (Figure 6A-C). We also observed reduced Foxp3 and enhanced RORgt gene expression in splenic / lymph node CD4+ T cells from the BTN2A2- / - mice vs. controls (Figure 6D-E). CD5 was enhanced in CD4+ T cells in BTN2A2- / - mice compared to wild type mice (Figure 6F). Analysis of kidney tissue one week after administration of nephrotoxic serum also showed increased IL17A protein expression in the kidneys of BTN2A2- / - vs controls animals (Figure 6G-H).

[0326] To evaluate the generalizability of the role of BTN2A2 in immunoregulation, we studied the effects of recombinant BTN2A2-Fc in DBA / 2 mice mated with CBA / J mice, a model of immunologically mediated abortions (Clark et al., 2008). We confirmed that DBA / 2 male x CBA / J female had reduced litter sizes and higher spontaneous adsorption rates compared to CBA / J male x DBA / 2 females. Administration of BTN2A2-Fc improved litter size and rescued the excess abortion rates that were noted in the DBA / 2 male x CBA / J female (Figure 7A-B and Figure 18). The beneficial effects of BTN2A2-Fc were associated with increased frequencies of splenic / lymph node Foxp3+ Tregs, reduced frequencies of splenic / lymph nodeThl7 cells and relative attenuation of CDS, consistent with our findings in the autoimmune GN model (Figure 7D-F). In addition, BTN2A2-Fc protein also reduced placental IL-17 protein expression that correlated with improved litter sizes (Figure 7G-H).BTN2A2 enhances Treg cell expansion and suppresses Thl7 cell populations in human PBMCs

[0327] To evaluate whether the effects of recombinant BTN2A2 apply to human T cells, we analyzed human PBMCs activated by mixed lymphocyte reactions (MLR, using allogeneic stimulator cells) with and without recombinant BTN2A2-Fc, and quantified CD4+CD25+CD127low / _Foxp3+ Treg cell numbers 7 days later. These co-cultures showed that the BTN2A2-Fc induced 2-fold expansion of Tregs under MLR conditions compared with controls; however BTN2A2-Fc was unable to induce Tregs in the presence of CD45 inhibitor (Figure 8A-B). Parallel experiments revealed that recombinant BTN2A2-Fc robustly blocked the TG Fb-I L6-I Lib induced increase in Thl7 cells (Figure 8C-D). Interestingly, BTN2A2-Fc was unable to block cytokines induced Thl7 cells in the presence of CD45 inhibitor. Taken together, these observations suggest that CD45 phosphatase activity is necessary for BTN2A2's actions in humans as well.Example 4Attorney Docket No. 2969027.002013Effect of BTN2A2-Fc Fusion Protein on the Percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) in human PBMCs

[0328] This example relates to the experiments in Figure 20A & B. Figure 20A contains flow cytometry plots showing percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) in human PBMC incubated with / without recombinant BTN2A2-Fc (10 mg / ml) in MLR for 7 days in absence or presence of CD45 phosphatase inhibitor (125nM). Data depicted as mean + / -SD showing percentage of Treg cells (CD4+CD25+Foxp3 +ve cells) among total CD4+ cells. N=4 per group for all experiments. Unpaired 2-tailed t test; p<0.001 (***).

[0329] Figure 20B contains flow cytometry plots showing percentage of CD4+RORgt +ve (Thl7 cells) in human PBMC incubated for 3 days with anti-CD3 antibody (0.5 mg / ml) only or anti-CD3 antibody +TGF- b (1.5 ng / ml) +IL-6 (10 ng / ml) +IL-lb (10 ng / ml) in absence or presence of recombinant BTN2A2-Fc (10 mg / ml) in MLR in absence or presence of CD45 phosphatase inhibitor (125nM). Data represent mean ± standard deviation. N=3 per group for all experiments. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**).

[0330] The results show the potentiating effects of the recombinant BTN2A2-Fc on the numbers of Tregs and the effects of CD45 inhibition on the numbers of Tregs.Example 5BTN2A2-Fc Fusion Protein Ameliorates Crescenta Glomerulonephritis Elicited by Nephrotoxic Serum

[0331] This example relates to the experiments in Figure 21. This figure contains data in a nephrotoxic serum nephritis (NTSN) model of glomerulonephritis. Animals were immunized with 0.5mg Rabbit IgG and then 20 pl iv nephrotoxic serum. Animals were sacrificed at day 7. As can be seen, NTS induced severe crescentic GN in control animals that was inhibited by BTN2A2-Fc. Similar reductions in proteinuria were also seen. Importantly, BTN2A2-Fc treatment reduced the T-effector pathway Thl7 cells (RORyt+)while increasing Treg cell populations (FoxP3+)IL-17 levels were also decreased by BTN2A2-Fc. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**).

[0332] These results show that BTN2A2-Fc treatment may be used to treat conditions wherein reduced T-effector pathway Thl7 cells (RORyt+) and / or increased numbers of Tregs is therapeutically desirable.Attorney Docket No. 2969027.002013Example 6BTN2A2(- / -) Mice Show an Exacerbated Crescenta Glomerulonephritis in NTSN Model

[0333] This example relates to the experiments in Figure 22. As shown therein, the inventors examined the NTSN model in BTN2A2 (- / -) animals. Animals were sacrificed at day 7 as above. As can be seen, NTS induced extremely severe crescentic GN in BTN2A2 (- / -) animals compared to WT. As can be seen significantly higher elevations in (RORgt+) and suppression of FoxP3 cells were seen in the BTN2A2 (- / -) compared to WT. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**).

[0334] These results show that the expression of BTN2A2 plays an important role in controlling (reducing) the numbers of RORgt-i- cells and increasing the number of FoxP3+ cells.Example 7BTN2A2-Fc Enhances Tregs and Suppresses Thl7 Cell Differentiation in One-way Mixed Lymphocyte Reaction using Human PBMCs

[0335] This example relates to the Mixed Lymphocyte Reaction experiments the results of which are in Figure 25.

[0336] One-Way MLR with hBTN2A2-Fc

[0337] Mixed Lymphocyte Reaction (MLR) is an in vitro assay used to measure the response of lymphocytes to foreign cells. Peripheral Blood Mononuclear Cells (PBMCs) are a type of blood cell with a single round nucleus. PBMCs include lymphocytes (T cells, B cells, and natural killer cells) and monocytes. In MLR, PBMCs are isolated from two genetically distinct individuals and then are mixed in a culture.

[0338] The experiments were based on a one-way MLR with hBTN2A2-Fc wherein only one lymphocyte population (Responder) can respond or proliferate. The other lymphocyte population (Stimulator) is prevented from replicating by irradiation. In these experiments responder and stimulator PBMCs are pre-incubated with or without hBTN2A2- Fc for 10 minutes before being combined for culture.

[0339] hBTN2A2-Fc construct generation and purification of recombinant protein

[0340] Soluble recombinant hBTN2A2-Fc protein was generated using baculovirus infected insect cell system, a method that is both scalable and has been successfully used for generation of recombinant proteins in the immune system. An upstream 711 bp region of human BTN2A2 gene (NP_008926.2) containing signal peptide and two extracellular domainsAttorney Docket No. 2969027.002013(IgV and lgC2) was amplified using specific primers* from cDNA from 293T cells. Amplified region was first cloned into pFUSE-hlgGl-Fcl vector (InvivoGen Catalog # pfuse-hglfcl) using Agel and EcoRV restriction sites. Subsequently, hBTN2A2 region along with Fc region was amplified using specific primers* and cloned into pBacPak8 vector using Xbal and Sacl restriction sites. Paired-end sequencing of the clone was performed by sanger sequencing to confirm the sequence. Primers were synthesized from Integrated DNA Technologies, Inc (IDT) and Phusion® High-Fidelity PCR Master Mix with HF Buffer was used in all PCR amplification reaction (New England Biolabs). Baculovirus were generated by co-transfecting pBacPak8- hBTN2A2-Fc clone with linearized baculovirus DNA (TakaraBio Catalog# 631401) into Sf9 insect cells according to manufacturer protocol. Preparation of baculovirus passage (P0, Pl, P2) was done as described in manufactures protocol (TakaraBio, Cat # 631402). P3 baculovirus stock was used for large scale protein purification using Hi5 cells grown in serum free media (Express Five™ SFM, ThermoFisher, Cat# 10486025). Pierce™ Protein G Agarose columns (ThermoFisher Scientific, Cat# 20398) was used for purification of secreted soluble-hBTN2A2- Fc protein from the supernatant. Purified protein was finally suspended in lx phosphate buffer saline (lxPBS). Protein purity was checked by SDS-PAGE followed by Coomassie blue staining. Specificity of the purified protein was confirmed by western blot analysis using specific hBTN2A2 antibody. Commercially available recombinant 293 cells derived human BTN2A2-FC (Cat# 8918-BT; R&D systems) and mouse BTN2A2 (Cat# 8997-BT-050; R&D systems) was used for comparison studies with baculoviral-generated recombinant hB2N2A2- Fc.

[0341] Experimental Conditions of One-Way MLR with hBTN2A2-Fc

[0342] In the experiments whole blood was drawn from healthy unrelated individuals to prepare peripheral blood mononuclear cells (PBMC) using the Ficoll-Hypaque gradient centrifugation. Only one lymphocyte population (Responder) can respond or proliferate. The other population (Stimulator) is prevented from replicating by irradiation. Responder and stimulator PBMCs are pre-incubated with or without hBTN2A2-Fc (lOmg / ml) for 10 minutes before being combined for culture.

[0343] The irradiated stimulator PBMCs was irradiated was exposed to the responder PBMCs at 1:1 (1 x 106 / ml of each PBMCs), and then incubated for measurements of Tregs (7 days), Thl7 cell (5 days), B cell proliferation (7 days and 14 days) and IgG production.

[0344] Treg AssayAttorney Docket No. 2969027.002013

[0345] MLR mixture were first stained with antibodies to CD45, CDS, CD4, CD25, and CD127 (BD Bioscience). After permeabilization, the cells were stained with antibody to Foxp3. After acquiring cells by flow cytometry, lymphocytes separated from CD45+ leukocytes were plotted against CD4. CD4+ cells were plotted as CD25 versus CD127 and then CD25+CD127 low / - cells against Foxp3. CD4+ / CD25+ / CD127low / - / Foxp3+ cells were designated as Treg cells. Treg cell levels were expressed as Treg ce 11% in CD4+ T cells.

[0346] Thl7 Assay

[0347] H uman PBMCs (responder cells) isolated from Ficoll-Hypaque gradient centrifugation were cultured in irradiated stimulator cells at 1:1 (1 x 106 / m I of each PBMCs) along with Thl7 differentiation cytokines cocktail (anti-CD3 antibody 0.5ug / ml, TGF£ 1.5ng / ml, IL-6 lOng / ml, IL-1[3 lOng / ml) with or without recombinant

[0348] hBTN2A2-Fc (lOpg / ml) for 5 days. Cells were stained with surface marker CD3 and CD4 (BD bioscience). After permeabilization, the cells were stained anti-RORyt antibody and analyzed for CD4+RORyt + co-expression using flow cytometry. Thl7 cells were expressed as RORyt + cells % in CD4 T cells.

[0349] RESULTS

[0350] The results of these experiments are in Figure 25. Panel A of Figure 25 contains data depicted as mean + / -SD showing percentage of Treg cells (CD4+CD25+Foxp3+ T cells) in human PBMCs (responder) incubated for 7 days with recombinant hBTN2A2-Fc (10 pg / ml) in one-way MLR. The data represent mean ± standard deviation. N=3 per group for all experiments. Unpaired 2-tailed t test; p<0.05 (*). Panel B of Figure 25 contains data depicted as mean + / -SD showing percentage of CD4+RORyt +T cells (Thl7 cells) in human PBMCs (responder) incubated for 5 days with anti-CD3 antibody (0.5 pg / ml) only or anti-CD3 antibody +TGF- P (1.5 ng / ml) +IL-6 (10 ng / ml) +1 L-10 (10 ng / ml) in absence or presence of recombinant hBTN2A2-Fc (10 pg / ml) in one-way MLR. Data represent mean ± standard deviation. N=3 per group for all experiments. One-way ANOVA test with Tukey's multiple comparison test; p<0.01 (**), p<0.001 (***). These results show that hBTN2A2-Fc Enhances Tregs and Suppresses Thl7 Cell differentiation in this one-way MLR using Human PBMCs.Example 8Inhibitory Effect of BTN2A2-Fc on B Cell Proliferation in One-way Mixed Lymphocyte Reaction using Human PBMCs (Set 1) at Day 7 and Day 14Attorney Docket No. 2969027.002013

[0351] This example relates to the one-way MLR experiments the results of which are contained in Figure 26.

[0352] One-Way MLR with hBTN2A2-Fc

[0353] The one-way mixed lymphocyte reaction experiments were effected using recombinant hBTN2A2-Fc protein as in the previous example.

[0354] hBTN2A2-Fc construct generation and purification of recombinant protein

[0355] Soluble recombinant hBTN2A2-Fc protein used in these experiments was generated as described in the previous example.

[0356] Experimental Conditions of One-Way MLR with hBTN2A2-Fc

[0357] The experimental conditions for the one-way mixed lymphocyte reaction was effected as in the prior example except that the effects of recombinant hBTN2A2-Fc protein on B cell proliferation was assayed using the BrdU cell proliferation assay as described below.

[0358] BrdU Cell Proliferation Assay Conditions

[0359] The BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay is a method used to measure cell proliferation by detecting the incorporation of BrdU into newly synthesized DNA of actively dividing cells. The Assay followed the manufacture's manual for Phase-Flow™ BrdU Cell Proliferation Kit for Flow Cytometry (BioLegend). Cells are cultured with a medium containing BrdU, a thymidine analog. During DNA replication, BrdU is incorporated into the DNA in place of thymidine. After incubation (7 days and 14 days), CD3-CD19+ B cells are fixed, and the DNA is denatured to make the incorporated BrdU accessible to detection antibodies. An anti-BrdU antibody is used to detect the BrdU incorporated into the DNA. The intensity of anti-BrdU antibody is proportional to the amount of BrdU incorporated, which reflects the level of cell proliferation.

[0360] RESULTS

[0361] The effects of recombinant hBTN2A2-Fc protein on B cell proliferation in this one-way MLR using the BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay is contained in Figure 26. As is noted above, this method measures cell proliferation by detecting the incorporation of BrdU into newly synthesized DNA of actively dividing cells and is widely used in studying cell growth and the effects of drugs on cell proliferation. In the one-way MLR, responder and stimulator PBMCs are pre-incubated with or without hBTN2A2-Fc (10 pg / ml) for 10 minutes before being combined for culture. Cells are cultured with a medium containing BrdU, a thymidine analog. During DNA replication, BrdU is incorporated into theAttorney Docket No. 2969027.002013DNA in place of thymidine. After incubation (7 days and 14 days), the cells are fixed, and the DNA is denatured to make the incorporated Brdll accessible to detection antibodies. An anti- Brdll antibody is used to detect the BrdU incorporated into the DNA. The intensity of anti- Brdll antibody is proportional to the amount of BrdU incorporated, which reflects the level of cell proliferation.

[0362] As can be seen from the data in Figure 26, recombinant hBTN2A2-Fc significantly inhibited CD19+ B cell proliferation in this one-way MLR at both 7 days (4.2 to 1.2%) and 14 days (8.5 to 3.5%).Example 9Inhibitory Effect of BTN2A2-Fc on B Cell Proliferation in One-way Mixed Lymphocyte Reaction using Human PBMCs (Set 2) at Day 7 and Day 14

[0363] This example relates to the one-way MLR experiments the results of which are in Figure 27. These experiments were effected using the same methods as in the previous example except the experiments were conducted with a different set (Set 2) of human PBMCs.

[0364] RESULTS

[0365] As is noted above, the one-way MLR experiments described in the previous example, which assessed the effects of hBTN2A2-Fc on B cell proliferation, were repeated using a different batch of human PBMCs. The results of these experiments are contained in Figure 27.

[0366] These results again clearly show that recombinant hBTN2A2-Fc significantly inhibited CD19+ B cell proliferation in this one-way MLR at both day 7 (5.0 to 3.1%) and day 14 (7.0 to 3.8%).Example 10Inhibitory Effect of BTN2A2-Fc on Human IgG Production in One-way Mixed Lymphocyte Reaction at Day 14 Analyzed using ELISA

[0367] This example relates to the one-way MLR experiments the results of which are in Figure 28.

[0368] One-Way MLR with hBTN2A2-Fc

[0369] The one-way mixed lymphocyte reaction experiments were effected using recombinant hBTN2A2-Fc protein as in the previous example.

[0370] hBTN2A2-Fc construct generation and purification of recombinant proteinAttorney Docket No. 2969027.002013

[0371] Soluble recombinant hBTN2A2-Fc protein used in these experiments was generated as described in the previous example.

[0372] Experimental Conditions of One-Way MLR with hBTN2A2-Fc

[0373] The experimental conditions for the one-way mixed lymphocyte reaction were as in the prior example except that the effects of recombinant hBTN2A2-Fc protein on human IgG production was assayed using a human IgG ELISA as described below.

[0374] Human IgG ELISA

[0375] To detect IgG production in MLR, residual IgG was removed by washing PBMCs with lxPBS before combining them. They were then cultured in serum-free RPMI 1640 medium (Thermo Fisher Scientific) without any supplements or media changes during the culture period. After 14 days of culture, media from unstimulated cells from individuals A, B, stimulated A cells with irradiated B cells, and vice versa were collected. The Assay followed the manufacture's manual for Human IgG ELISA kit (Raybiotech). This assay employs an antibody specific for human IgG coated on a 96-well plate. Standards and undiluted samples are pipetted into the wells and IgG present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human IgG antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IgG bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

[0376] RESULTS

[0377] The data in Figure 28 shows total IgG production by human B cells stimulated in the afore-described one-way mixed lymphocyte reaction (MLR). As can be seen from the data therein, in the experiments unstimulated cells from individuals A and B produced IgG after 14 days. Stimulating A cells with irradiated B cells, and vice versa, also resulted in IgG production. By contrast, as can be seen from the results in Figure 28 the addition of hBTN2A2- Fc to these stimulated cultures inhibited IgG production entirely.

[0378] This result indicates that hBTN2A2-Fc has an important (blocking) effect on B- cell proliferation after stimulation and on antibody production, e.g., IgG production which indicates that hBTN2A2-Fc potentially may be used in the treatment of human autoimmune, allergic, inflammatory and alloimmune diseases wherein the suppression of pathologic B cells and antibody production, e.g., pathologic, autoimmune or interfering antibodies, e.g., IgGs,Attorney Docket No. 2969027.002013IgAs, IgMs, IgEs, or IgDs associated with disease pathology and / or IgGs, IgAs, IgMs, IgEs, or IgDs which suppress innate host immune reactions, e.g., against an infectious agent or antibodies which suppress the efficacy of a drug or which suppress innate host immune reactions elicited by an immune modulator, e.g., a cytokine, hormone, growth factor is therapeutically desirable.Example 11BTN2A2 Inhibits R848 / IL-2 Induced Activation of B-Cells

[0379] I n this example experiments were conducted which further demonstrated that BTN2A2 inhibits the activation of B-cells.EXPERIMENTAL PROTOCOLS

[0380] The experimental protocols used in these experiments are disclosed below.CELL CULTURE

[0381] PBMCs were seeded in 12-well plates at 1x106 cells / well in RPMI supplemented with Penicillin-Streptomycin and L-glutamine. Media was further supplemented depending on the treatment group: Control (no additional supplementation), BTN (5 pg / mL BTN2A2), Stim (10 ng / mL IL-2 and 1 pg / mL R848), or Stim / BTN (combination of Stim and BTN). Samples were treated for five days in a 379C / 5% CO2, high relative humidity incubator.FLUOROSPOT ASSAY

[0382] Post-treatment, samples were collected and assayed for B-cell activation. Briefly, samples were seeded onto a 96-well PVDF plate, that was previously coated with anti- hlgG antibody (Mabtech cat #3850-3-250), at two densities (1000 and 2000 cells / well). Cells were incubated and allowed to bind for approximately 18 hours in 379C / 5% CO2, high relative humidity incubator. Wells were then washed three times with PBST. Then, a biotinylated detection anti-hlgG antibody (Mabtech cat#3850-6-250) was incubated for 2 h at room temperature, followed by three washes with PBST. Lastly, streptavidin-conjugated CTL-Red was incubated in the wells for 1 h at room temperature in the dark (Cellular Technology Ltd. cat #hT36). Wells were washed three times with DI-H2O, allowed to dry overnight in the dark, and fluorescent images were taken.Attorney Docket No. 2969027.002013

[0383] Figure 29A-E : contains the results of these experiments which show that BTN2A2 Inhibits R848 / IL-2 Induced Activation of B-Cells wherein the PBMCs were cultured for five days in the indicated conditions. In the figure Control refers to no drugs; BTN refers to 5 pg / mL BTN2A2. Stim refers to 10 ng / mL IL-2 and 1 pg / mL R848. Samples were then plated following the Fluorospot protocol. Representative images are shown for (A) Control, (B) BTN, (C) Stim, and (D) Stim / BTN. In panel (E) of Figure 29 the number of spots were calculated using ImageJ and normalized to cell density. Data presented as mean ± s.d. N = 6.SPOT COUNTING AND STATISTICS

[0384] Images were analyzed via ImageJ to obtain number of spots per well. Spots were then normalized to cell number and plotted. Statistics were done via student's T-test, P < 0.05 (*), 0.01 (**), 0.001 (***), 0.0001 (****).

[0385] IMAGES

[0386] Figure 30A-G contains a composite of all images obtained. These experiments were done in triplicate at two densities (2000 cells / well, Rows A-C and 1000 cells / well, Rows E-G). Column 1 = control; Column 2 = Stim; Column 3 = BTN; Column 4 = Stim / BTN).EXPERIMENTAL RESULTS

[0387] Addition of BTN2A2 significantly reduced R848 / IL-2 induced activation of B- cells, reducing number of spots detected by approximately 75% (Figure 29A-E). By contrast, BTN2A2 alone did not induce any off-target activation (Figure 29A-B). Of note, all samples showed similar cell health after the five-day treatment. When collected and counted in preparation for seeding on the fluorescent plate, all groups showed similar cell densities (0.6- 0.75 x 106cells) and health (~80% viability) via a live / dead stain.CONCLUSIONS

[0388] As is noted above, the addition of BTN2A2 significantly reduced R848 / IL-2 induced activation of B-cells, reducing the number of spots detected by approximately 75% (Figure 29A-E); whereas BTN2A2 alone did not induce any off-target activation (Figure 29B).

[0389] Based on these results and the results of the previous Example, it is anticipated that Butyrophilins and conjugates thereof, especially BTN2A2 and BTN2A2 conjugates, e.g., BTN2A2 Fc conjugates or BTN2A2 albumin conjugates may be used to treat or preventAttorney Docket No. 2969027.002013 conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production and / or for eliciting prolonged humoral suppression or humoral tolerance in subjects with conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production, e.g., autoimmune, allergic and inflammatory conditions involving aberrant or pathologic B cell activation and / or aberrant or pathologic antibody production.

[0390] This will be confirmed in further experiments using BTN2A2 Fc conjugates and Fc controls (to corroborate that the inhibition of B cell activation is specific to the BTN moiety). Additionally such experiments may in addition elucidate the mechanism(s) by which BTN2A2 reduces the activation of B-cells.Example 12Evaluation of Functional Properties of BTN2A2 Fc Construct Comprising LALAPG Mutations

[0391] As traditional Fc fusion constructs may elicit off-target effects such as complement activation and activation of Fc receptor bearing cells such as NK cells, macrophages and B-cells, we created a new fusion protein (BTN-2A2-FCLALAPG). The LALAPG mutation, a triple substitution (L234A, L235A, P329G) in the Fc region of an antibody, significantly reduces or eliminates Fc-mediated effector functions like complement activation (CDC) and antibody-dependent cellular phagocytosis (ADCP), while maintaining the antibody's antigen-binding activity and stability. This "effector-silencing" property makes LALAPG-mutated antibodies useful in applications requiring strong antigen binding but minimal immune activation, aimed at reducing off-target liver accumulation. The sequence of the hBTN2A2- FcLALAPGfusion protein we constructed is below. In the construct the hBTN2A2 and FCI AI APGsequences are intervened by a linker (LI).

[0392] hBTN2A2-Ll-Fc-LALAPG protein sequence

[0393] QFTVVGPAN PILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQE DGSIWLECISGGWYPEPLTVWRDPYGEWPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLG QEKETVIEGRMDDISSTMVRSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAttorney Docket No. 2969027.002013AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG sequence (SEQ ID NO: 68)

[0394] In EXPERIMENT 1 (described below) we evaluated the effects of BTN2A2- FCLALAPGon (CD4+ / CD25+ / CD127low,FoxP3+) Treg cells in unstimulated PMBCs.

[0395] In EXPERIMENT 2 (also described below) we evaluated the effect of BTN2A2- FCLALAPGO NCD8+ Teffector / T Memory cell & NK cell responses in SARS CoV2 peptide-stimulated PMBCs.

[0396] EXPERIMENT 1: Effect of BTN2A2-FCLALAPGon(CD4+ / CD25+ / CD127low,FoxP3+) Treg cells in unstimulated PMBCs

[0397] Cells from normal individuals were exposed to (BTN-2A2-FcLALAPG), CTLA4-lg and no additives using the methods described in Figure 31 and below:

[0398] METHODS

[0399] Step 1: Cryopreserved PMBCs from a single subject were stained with CellTrace Violet and cultured in complete media in the presence of BTN-2A2-FcLALAPG (100 mg / ml), CTLA4-lg (100 mg / ml), anti-CD3 / anti-CD28 ) (100 mg / ml), or no treatment.

[0400] Step 2: Additional IL-2 (50 lU / ml) was added after 24 hours.

[0401] Step 3 : Cultures are harvested on Day 6.

[0402] Step 4. Cells are stained and analyzed by use of flow cytometry.

[0403] The stained cell cultures harvested at Day 6 were then analyzed for the proliferation of (CD4+ / CD25+ / CD127low,FoxP3+) Treg cells. The experimental results are shown in Fig 32A & 32B.

[0404] RESULTS

[0405] The results of EXPERIMENT 1 are shown in Figure 32A and 32B. As can be seen, unstimulated PBMCs incubated with BTN-2A2-FCLALAPGshow a significant increase in CD4+ / CD25+ / CD127low,FoxP3+) Treg cells compared to PBMCs + CTLA4-lg and PBMCs alone. The numerical data of Figure 32A is shown in Figure 32B:Attorney Docket No. 2969027.002013

[0406] These results demonstrate the induction of CD4+ / CD25+ / CD127low,FoxP3+Treg cells in resting PBMCs by BTN-2A2-FCLALAPGis a novel observation. In a 6-day culture of PBMCs under non-MLR conditions, BTN-LALAPG treatment showed an overall trend toward increased Treg proliferation, as assessed by CellTrace Violet dye dilution. In contrast, CTLA4- Ig showed a trend toward decreased Treg proliferation, compared to untreated controls. These results suggest that BTN-2A2-FCLALAPGtreatment alone can modulate immune cell proliferation and composition even in the absence of allogeneic stimulation.

[0407] EXPERIMENT 2: Effect of BTN2A2-FcLALAPGon CD8+Teffector / Memory cell & NK cell responses in SARS CoV2 peptide-stimulated PMBCs

[0408] METHODS

[0409] The methods used in EXPERIMENT 2 to assess T-cell and NK cell reactivity to SARS-CoV2 peptide incubation are described and shown schematically in Figure 33 and below:

[0410] Step 1: Unstimulated PMBCs were pretreated with or without anti-CD45RO antibody or BTN-2A2-FCLALAPG

[0411] Step 2: The pretreated PMBCs were incubated overnight with cov-2 spike peptides or POHA in the presence of costimulatory molecules (CD28 / CD49d) and Golgi complex inhibitor (BFA).

[0412] Step 3: After incubation the cell surface antigens of the PMBCs were stained (CD45, CD3, CD4, CD56) and the intracellular cytokine components of permeabilized PMBCs were stained (IL-2, TNF-a, I FN-¥).

[0413] Step 4: After staining the stained PMBCs were recovered using flow cytometry.

[0414] Step 5 : The recovered stained PMBCs were then analyzed using forward light scatter (FSC) and side light scatter (SSC) analysis. (Forward scatter (FSC) and Side light scatter (SSC) are measurements in flow cytometry used to analyze cell populations by detecting lightAttorney Docket No. 2969027.002013 scattered from a laser beam interacting with individual cells. FSC correlates with cell size, while SSC indicates internal complexity or granularity).

[0415] RESULTS

[0416] Using the above-described methods in EXPERIMENT 2 the effects of BTN-2A2- FCLAL PGon$ARS-CoV2 peptide stimulation of PBMCs was evaluated. In these experiments PMBCs were obtained from 4 highly-HLA sensitized patients awaiting kidney transplantation. PBMCs were incubated with SARS-CoV peptides and PHA as + control as shown.

[0417] Figure 34 shows I FN-¥ responses in the 4 patients wherein the PBMCs thereof were incubated with SARS-CoV2 peptides and the response was evaluated with and without anti-CD45RO and BTN-2A2-FCLALAPG. These data indicate that BTN-2A2-FCLALAPGinhibits IFN-¥ responses to SARS-CoV2 peptides elicited by NK cells and by CD8+ T-effector / memory cells.

[0418] The results of EXPERIMENT 2 show that BTN-2A2-FCLALAPGpotently modulates T-cell and NK cell IFN-¥ responses to SARS-CoV2 peptides. In addition, the results of EXPERIMENT 1 show that BTN-2A2-FCLALAPGstimulates T-reg cells in resting human PMBCs.

[0419] Therefore, has been demonstrated to BTN-2A2-FCLALAPGpotentially be useful as a therapeutic, e.g., for promoting Treg activity and for modulating (suppressing) proinflammatory cytokine responses, e.g., I FN-¥ responses to antigens elicited by T-cell and NK cells. Based thereon BTN2A2-FCLALAPGpotentially may be used in the treatment of allergic, inflammatory and autoimmune and in the treatment of conditions or treatment methods which are often associated with autoimmune, allergic or inflammatory responses such as chemotherapy, infection, and transplant indications.Example 14Comparison of Different BTN2A2 Constructs Including BTN2A2-FCLALAPGin Jurkat IL2 Assays

[0420] In order to further assess the impact of the LALAPG mutations on the desired activities different BTN2A2 Fc fusion proteins were compared in a Jurkat IL-2 assay and in an NTS-induced nephrotoxic glomerulonephritis model.

[0421] The specific BTN2A2 Fc fusion proteins, the effects of which were compared are:

[0422] hBTN2A2-hlgGl_(Original)

[0423] BTN2A2-FC (WT original protein)Attorney Docket No. 2969027.002013

[0424] QFTVVGPAN PILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLWAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEWPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIEGRMDDISSTMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHN HYTQKSLSLSPG (SEQ ID NO: 69)

[0425] hBTN2A2-hlgGlLALAPG

[0426] QFTVVGPAN PILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLWAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEWPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIEGRMDDISSTMVRSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG sequence (SEQ ID NO: 70)

[0427] hBTN2a-ECD_Ser249Fc (short)

[0428] QFTVVGPAN PILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEWPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFM PSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG (SEQ ID NO: 71)

[0429] hBTN2a_lgV-hlgGl

[0430] QFTVVGPAN PILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG (SEQ ID NO: 72)

[0431] In vitro Jurkat IL-2 ExperimentsAttorney Docket No. 2969027.002013

[0432] BTN2A2 Fc fusion proteins having the sequences identified above were compared in an in vitro Jurkat IL-2 assay along with appropriate controls (PBS and anti-CD3 antibody treatment).

[0433] Jurkat Protocol

[0434] The Jurkat assay used comprises in vitro T cell activation of Jurkat cells with an anti-CD3 antibody. Briefly, 96-well plates (Corning cat#2580) were coated with 2 .g / ml concentrations of anti-CD3 mAb (clone OKT3, BD Bioscience cat# 566685) in PBS at 4°C overnight in absence or presence of variable concentration of recombinant human BTN2A2 variants (50, 25, 10, 5, 1, 0.5 and 0.1 .g / ml). A total of 10x105 Jurkat cells / well / 200ul RPMI complete media were added to precoated flat-bottom 96-well plates. Cells were incubated for 48 hrs in 37°C incubator with 5% CO2. Total 100 pl of medium was used to measure IL-2 production using an IL-2 ELISA kit from R and D systems (Cat# D2050) according to the manufacturer' protocol.

[0435] RESULTS

[0436] The results of the Jurkat IL-2 assay is shown in Figure 35. The results revealed that all of the comparator BTN2A2-Fc constructs performed better (more potently reduced IL-2 levels) than the parental hBTN2A2-hlgGl_ construct (i.e., comprising entire BTN2A2 extracellular region and hlgGl with no Fc mutations) in vitro experiments comparing the effects of different BTN2A2 Fc fusions on IL-2 production byJurkat cells activated with an anti- CD3 antibody.

[0437] 2. In vivo NTS-lnduced Nephrotoxic Glomerulonephritis ModelExperiments

[0438] Specific BTN2A2 Fc fusion protein identified above were also compared in vivo in the NTS-lnduced Nephrotoxic Glomerulonephritis Model. Essentially, mice are primed intraperitoneally (IP) with normal rabbit IgGs and Complete Freund's Adjuvant (CFA) and are injected intravenously with nephrotoxic serum to induce glomerulonephritis.

[0439] Figure 36 contains a schematic protocol of the nephrotoxic serum (NTS)- induced nephrotoxic glomerulonephritis model which was used to compare the in vivo effects of different BTN2A2 Fc fusions. Particularly the inventors compared the in vivo effects of different BTN2A2 Fc fusions on the reduction of nephrotoxicity by evaluating crescent formation and glomerular basement membrane (GBM) rupture in mice groups treated with different BTN2A2 Fc fusions.Attorney Docket No. 2969027.002013

[0440] RESULTS

[0441] Figure 37 and Figure 38 contain renal histologic results showing crescent formation and glomerular basement membrane (GBM) rupture in the NTS-induced nephrotoxic glomerulonephritis model in mice groups which respectively were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch).

[0442] Figure 39 contains data comparing the crescent scores and fibrinoid necrosis in the mice which respectively were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). It can be seen from the data that there is a marked improvement in all the BTN2A2-Fc treated group compared to control Fc treated group with the most dramatic results in the mice treated with the BTN2A2-LALAPG variant.

[0443] Figure 40 contains data comparing body weights of the mice which were treated with the Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). The mice groups treated with the BTN2A2-Fc proteins all exhibited higher body weights compared to the mice treated with the Fc control.

[0444] Figure 41 contains data comparing proteinuria of the mice which were treated with an Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). It can be seen from these results that while both hBTN2A2-Fc-WT and hBTN2A2-Fc-LALAPG appreciably reduced proteinuria, and inhibited renal histological changes, the mice treated with the LALAPG variant had more dramatic effects on the reduction of proteinuria, and inhibition of renal histological changes, and moreover exhibited no detectable toxicity.

[0445] Figure 42 contains data of experiments comparing Foxp3 expression by CD4+ T cells purified from spleen and lymph nodes of the mice which respectively were treated with the Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). It can be seen from these results that the mice treated with the BTN1A2FC fusions all had substantially higher Foxp3 expression levels compared to the Fc control group treated mice.

[0446] Figure 43 contains data showing the correlation between proteinuria and Foxp3 expression by CD4+ T cells purified from spleen and lymph nodes of the mice whichAttorney Docket No. 2969027.002013 were respectively treated with the Fc control, BTN2A2-hlgGlFc WT (CHO expressed batch), BTN2A2-hlgGl-WT (sf9 expressed batch) or BTN2A2-hlgGlFc-LALAPG (CHO expressed batch). The data show that increased Foxp3 expression correlates to a reduction in proteinuria.

[0447] In the present invention "pathologic antibodies" include antibodies which are involved in disease pathology, e.g., an autoimmune, allergic or inflammatory condition; antibodies which preclude or inhibit innate host immunity, e.g., "interfering antibodies" which block or inhibit an effective (neutralizing) immune reaction against an infectious agent, e.g., a virus, bacterium, fungus, yeast, protozoan, or parasite; "anti-drug antibodies" which preclude or inhibit the efficacy of a drug, e.g., a small molecule or biologic, and autoimmune antibodies which suppress the efficacy of a naturally occurring protein or polypeptide, e.g., an immune modulator such as a cytokine, a hormone, a neuromodulator, a growth factor, a blood factor, and the like.

[0448] CONCLUSIONS

[0449] These results indicate that Butyrophilin polypeptides and conjugates, e.g., BTN2A2-Fc fusion polypeptides may be used alone or in association with a desired antigen to treat conditions wherein inhibition of [antigen-specific] B cell activation, B cell proliferation, antibody production, e.g., human IgGs, IgAs, IgEs, IgDs, IgMs, and / or eliciting prolonged or acute humoral suppression and / or B cell tolerance in a subject is warranted, e.g., wherein the subject comprises a B cell mediated inflammatory, autoimmune or allergic disorder and / or a condition involving pathologic antibodies or pathologic B cell activation and / or B cell expansion, optionally wherein the subject comprises one or more of the following: a neurological disorder, optionally anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, neuromyelitis optica spectrum disorder (NMOSD), anti-myelin oligodendrocyte glycoprotein (MOG) antibody encephalomyelitis, SLE, multiple sclerosis, rheumatoid arthritis, an autoimmune kidney disease (optionally IgA nephropathy or membranous glomerulonephritis), lupus, Addison's disease, Ankylosing spondylitis, Behcet's syndrome, Congenital adrenal hyperplasia, dermatitis herpetiformis, Goodpasture syndrome, Graves' disease, Hereditary hemochromatosis, Insulin-dependent diabetes mellitus, Idiopathic membranous glomerulonephritis, Myasthenia gravis, Narcolepsy, a skin disease associatedAttorney Docket No. 2969027.002013 with pathologic antibodies, optionally Psoriasis vulgaris, Pemphigus vulgaris, Sarcoidosis, scleroderma, ANCA vasculitis, celiac disease, Sjogren's Syndrome, B cell lymphoma, nonHodgkin's lymphoma, Hodgkin's lymphoma, advanced stage follicular lymphoma, chronic lymphocytic leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, Pulmonary arterial hypertension (PAH), Systemic sclerosis, Refractory hypertension, Autoimmune hypoglycemia, Hashimoto's thyroiditis, the subject expresses anti-drug antibodies, or the subject comprises an infectious condition, optionally a viral, bacterial or parasitic infectious condition associated with pathologic, autoimmune or interfering antibodies, optionally Mycobacterial disease, Mucocutaneous candidiasis, Schistosoma infection, Leishmania infection, Trypanosoma infection, Toxoplasma, Plasmodium infection, coronavirus infection, Staphylococcal infection, Disseminated M. bovis-BCG disease, Disseminated environmental mycobacteria disease, Herpes virus encephalitis, Severe influenza, Yellow fever, or COVID-19 pneumonia.

[0450] In specific embodiments optionally wherein the subject comprises one or more of the following: a neurological disorder involving pathologic antibodies or pathologic B cell expansion, SLE, multiple sclerosis, an autoimmune kidney disease (optionally IgA nephropathy, ANCA vasculitis, or membranous glomerulonephritis), Sjogren's Syndrome, B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, advanced stage follicular lymphoma, chronic lymphocytic leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, Pulmonary arterial hypertension (PAH), Systemic sclerosis, Refractory hypertension, Autoimmune hypoglycemia, and / or Anti-NMDA receptor encephalitis; the subject has received a transplanted cell, tissue or organ and shows signs and / or has a history of antibody mediated transplant rejection; the subject has or is to receive cell therapy, or a tissue or organ transplant, optionally one or more of kidney, heart, lung, pancreas, bone marrow, and liver; the subject comprises a cytopathy mediated by allo- & auto-antibodies and / or pathogenic B-cells; and / or the subject comprises a condition selected from immune thrombocytopenic purpura (ITP), hemolysis, hemolytic anemia, and / or a condition characterized by antibodies to lymphocytes and / or antibodies against other lymphoid and myeloid cellular elements.

[0451] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to theAttorney Docket No. 2969027.002013 specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0452] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve 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 as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0453] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Although the open-ended term "comprising," as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0454] Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in theAttorney Docket No. 2969027.002013 context of claims) may be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0455] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0456] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Claims

Attorney Docket No. 2969027.002013WHAT IS CLAIMED IS:

1. A method of inhibiting B cell activation, B cell proliferation, antibody production, and / or for eliciting humoral suppression and / or B cell tolerance optionally against a target antigen in a subject in need thereof by contacting immune cells of the subject or donor immune cells with a Butyrophilin or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising any of the foregoing, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising said Butyrophilin or fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof.

2. The method of claim 1, wherein(i) said Butyrophilin, optionally Butyrophilin A2 ora fragment, variant or modified form thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to at least one half-life extending moiety, optionally an albumin, an Fc protein, polyethylene glycol, a lipid, PASylation (attachment to poly-aspartate and serine) polymers, glycosylation, albumin binding moieties, or a combination of any of the foregoing.(ii) said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which optionally comprises one or more mutations which impair or promote an Fc effector function;(iii) said Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment thereof, is directly or indirectly conjugated or linked, optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which optionally comprises one or more mutations which impair or promote an Fc effector function which (i) enhances FcRN binding, (ii) impairs or eliminate binding to one or more FcRs and / or (iii) impairs or eliminates binding to complement proteins such as Clq, further optionally wherein the Fc comprises one or more of the mutations identified in Table 1;(iv) said Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment thereof, is directly or indirectly conjugated or linked,Attorney Docket No. 2969027.002013 optionally via a peptide linker, to an Fc protein, preferably a human IgGl, lgG2, lgG3 or lgG4, which Fc comprises the following mutations: L234A, L235A, and P329G (LALAPG);(v) said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof, possesses at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to the corresponding native Butyrophilin, optionally human Butyrophilin A2 or the corresponding fragment thereof, e.g., IgV and / or IgC domain or fragment thereof, wherein said fragment optionally comprises the extracellular domain of the Butyrophilin, optionally human Butyrophilin A2 and / or the Butyrophilin fragment, optionally Butyrophilin A2 fragment does not comprise the transmembrane domain, cytoplasmic domain, or both of the corresponding native Butyrophilin, optionally Butyrophilin A2;(vi) the Butyrophilin or a fragment, variant or modified form thereof comprises BTN1A1, B2N2A1, B2N2A2, BTN3A1, BTN3A2 or BTN3A3 or a fragment of any of the foregoing and is preferably human BTN1A1, B2N2A1, B2N2A2, BTN3A1, BTN3A2 or BTN3A3 or a fragment thereof;(vii) said contacting occurs before, proximate to, or after the subject is to be or has been exposed to an antigen against which humoral suppression and / or B cell tolerance is to be elicited;(viii) the method induces antigen-specific humoral suppression and / or B cell tolerance;(ix) the method induces prolonged antigen-specific humoral suppression and / or B cell tolerance;(x) the method induces acute antigen-specific humoral suppression and / or B cell tolerance;(xi) said contacting is effected ex vivo;(xii) said contacting is effected in vivo;(xiii) said contacting is effected ex vivo and in vivo;(xiv) said immune cells comprise bone marrow, peripheral blood cells, B cells, B cell progenitors, stem cells, Tregs, Bregs, NK cells, hematopoietic stem cells or any combination of the foregoing;Attorney Docket No. 2969027.002013(xv) the method further com rises contacting said immune cells with a desired antigen to which humoral suppression and / or B cell tolerance is to be induced;(xvi) the method further comprises contacting said immune cells with a soluble antigen to which humoral suppression and / or B cell tolerance is to be induced;(xvii) the method further comprises contacting said immune cells with an antigen expressed on a cell which humoral suppression and / or B cell tolerance is to be induced;(xviii) the method further comprises contacting said immune cells with a nucleic acid, optionally an mRNA, further optionally comprised in a lipid nanoparticle, viral vector or plasmid which encodes an antigen to which humoral suppression and / or B cell tolerance is to be induced;(xix) the method further comprises contacting said immune cells with an antigen wherein said antigen is selected from an autoantigen, allergen, therapeutic agent, parasitic antigen, or an antigen expressed by donor cells;(xx) the method further comprises contacting said immune cells with an antigen wherein said antigen is a food allergen, an aeroallergen, an animal product, a plant derived allergen, a microbial allergen, optionally a mite, fungus, yeast, etc., a drug, a hormone, a therapeutic enzyme, a therapeutic antibody, a blood or clotting factor, insect venom, or latex;(xxi) the method further comprises contacting said immune cells with an antigen wherein said antigen is a food allergen selected from a peanut allergen, a tree nut allergen, a dairy allergen, a wheat allergen, a soy allergen, an egg allergen, a shellfish allergen, a meat allergen, a sesame allergen, and a corn allergen;(xxii) said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or fragment thereof, optionally Butyrophilin A2 or fragment thereof, and optionally said antigen are administered in the same or different compositions, and further optionally are administered by one or more of injection, e.g., intravenous, intramuscular, subcutaneous, intrathecal, or injection into the bone marrow; mucosal administration, intranasal administration, oral administration, topical administration, or any combination of the foregoing;Attorney Docket No. 2969027.002013(xxiii) the immune cells or a graft, tissue or organ or composition comprising immune cells of a subject or the donor are contacted ex vivo with Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2, optionally human BTN2A2, or a fragment, variant or modified form thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment, variant or modified form thereof, optionally Butyrophilin A2 or a fragment, variant or modified form thereof and an antigen, the immune cells are optionally expanded in culture, and after contacting and optionally expansion are infused into the subject;(xxiv) the method comprises administering butyrophilin A2 (BTN2A2), fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment or a fusion polypeptide comprising any of the foregoing, and an antigen to which antigen-specific humoral suppression and / or B cell tolerance is to be elicited in the subject;(xxv) said Butyrophilin or fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment or conjugate comprising alleviates or reduces the expression pathologic antibodies or pathologic B cell expansion and / or results in longer periods or disease remission;(xxvi) the Butyrophilin or a fragment thereof, optionally Butyrophilin A2 (BTN2A2) or a BTN2A2 fragment is linked to an albumin, optionally human serum albumin;(xxvii) the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked or fused to a Fc domain or an Fc fragment ("BTN2A2-Fc fusion protein") or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2-related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally any of the mutations shown in TABLE 1;(xxvii) the BTN2A2 or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, and the linker is G, polyserine, polyglycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, or helical peptides;Attorney Docket No. 2969027.002013(xxviii) the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoform fragment is modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol;(xxix) the BTN2A2 fragment comprises an extracellular domain of BTN2A2 or fragment thereof;(xxx) the BTN2A2 fragment does not comprise a transmembrane domain, a cytoplasmic domain, or both;(xxxi) the BTN2A2 conjugate comprises an Fc, optionally human IgGl, lgG2, lgG3 or lgG4, optionally comprising a hinge region, further optionally wherein one or more cysteines of the hinge region are substituted with another amino acid, optionally serine, further optionally wherein the Fc comprises one or mutations which inhibit FcR binding, further optionally LAI.APG;(xxxii) the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3;(xxxiv) the BTN2A2 or BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both;(xxxv) a Butyrophilin A2 (BTN2A2)-Fc fusion protein is administered wherein (i) the BTN2A2 moiety of the fusion protein comprises at least one BTN2A2 polypeptide possessing at least 90% sequence identity to the IgV domain of human or murine BTN2A2 and which BTN2A2 polypeptide lacks or only comprises a fragment of the BTN2A2 IgC domain; (ii) an Fc region, and optionally (iii) a peptide linker which intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1- 50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG;(xxxvi) the BTN2A2-Fc fusion protein comprises or consists of a polypeptide possessing at least 95-99% sequence identity to the IgV domain of BTN2A2, optionally human BTN2A2 IgV domain, optionally wherein the BTN2A2-Fc fusion protein comprises two of said BTN2A2 polypeptides, wherein optionally a peptide linker intervenes said BTN2A2 polypeptides, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues;Attorney Docket No. 2969027.002013(xxxvii) the BTN2A2-Fc fusion protein comprises a human Fc region, optionally a human IgGl, lgG2, lgG3 or lgG4 Fc region;(xxxix) the BTN2A2-Fc fusion protein comprises a human IgGl or lgG2 Fc region, optionally one comprising one or more mutations which impair or enhance one or more effector functions, e.g., FcR binding, complement binding, FcRN binding, further optionally wherein said one or more mutations comprise the Fc mutations identified in Table 1;(xl) the BTN2A2-Fc fusion protein comprises a mutated IgGl Fc region wherein the mutations comprise or consist of (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G substitution mutation and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1- 50, 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues;(xli) the BTN2A2-Fc fusion protein comprises a BTN2A2 polypeptide or CD45 binding fragment thereof and a mutated IgGl Fc region, wherein the mutated IgGl Fc region comprises (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G mutation, and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, further optionally a 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues, and still further wherein the linker comprises or consists of GGSGG or a multimer thereof;Attorney Docket No. 2969027.002013(xlii) the BTN2A2-Fc fusion protein comprises at its N-terminus a signal peptide, further optionally the endogenous BTN2A2 signal peptide or an immunoglobulin signal peptide;(xliii) the BTN2A2-Fc fusion protein comprises a flexible linker intervening the BTN2A2 and Fc moiety;(xliv) the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked or fused to a Fc domain or an Fc fragment ("BTN2A2-Fc fusion protein") or the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2-related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally wherein the Fc, optionally an IgGl Fc comprises one or more of the mutations shown in TABLE 1 and / or comprises any one, two, three, four or all five of M252Y, S254T, T256E, and / or H433K and N434F, and / or is the variant IgGl Fc contained in SEQ ID NO: 6, further optionally wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment comprises the BTN2A2 polypeptide in SEQ ID NO: 6 or a variant thereof wherein the RS linker peptide intervening the BTNA2 polypeptide and the IgGl Fc variant optionally is replaced by another peptide linker, preferably a non- cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker;(xliii) the BTN2A2 or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, preferably a non- cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, furtherAttorney Docket No. 2969027.002013 optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker;(xliv) the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoform fragment is modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol; or(xlv) any combination of the foregoing.

3. The method of claim 1 or 2, wherein(i) the subject comprises a B cell mediated inflammatory, autoimmune or allergic disorder and / or a condition;(ii) the subject comprises a neurological disorder, SLE, multiple sclerosis, an autoimmune kidney disease (optionally IgA nephropathy, ANCA vasculitis, or membranous glomerulonephritis), Sjogren's Syndrome, B cell lymphoma, nonHodgkin's lymphoma, Hodgkin's lymphoma, advanced stage follicular lymphoma, chronic lymphocytic leukemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, Pulmonary arterial hypertension (PAH), Systemic sclerosis, Refractory hypertension, Autoimmune hypoglycemia, and / or Anti-NMDA receptor encephalitis;(iii) the subject has received a transplanted cell, tissue or organ and shows signs and / or has a history of antibody mediated transplant rejection;(iv) the subject has or is to receive cell therapy, or a tissue or organ transplant, optionally one or more of kidney, heart, lung, pancreas, bone marrow, and liver;(v) the subject comprises a cytopathy mediated by allo- & auto-antibodies and / or pathogenic B-cells;(vi) the subject comprises a condition selected from immune thrombocytopenic purpura (ITP), hemolysis, hemolytic anemia, and / or a condition characterized byAttorney Docket No. 2969027.002013 antibodies to lymphocytes and / or antibodies against other lymphoid and myeloid cellular elements;(vii) the subject comprises multiple sclerosis, myelin oligodendrocyte glycoprotein (MOG) antibody disease or Alzheimer's disease and the method further comprises contacting said immune cells ex vivo or in vivo with myelin oligodendrocyte glycoprotein (MOG) or amyloid protein proximate to when the immune cells are contacted with Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof;(viii) the subject has a chronic inflammatory, autoimmu ne or allergic disorder involving pathologic antibodies or pathologic B cell expansion, e.g., one that remits and relapses, optionally multiple sclerosis, optionally Relapsing-Remitting Multiple Sclerosis (RRMS), vasculitis, optionally ANCA-Associated Vasculitis, rheumatoid arthritis, or lupus, optionally relapsing focal or diffuse lupus nephritis, and the Butyrophilin or a fragment thereof, optionally Butyrophilin A2 or a fragment thereof, or a conjugate or fusion protein comprising said Butyrophilin, optionally Butyrophilin A2 is administered during active bouts of the disease;(ix) the method further comprises the use or inclusion of one or more of other actives which comprise one or more of a steroid, optionally a glucocorticoid, further optionally prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone; methotrexate, azathioprine mycophenolate, cyclophosphamide, a TNF inhibitor, optionally HUMIRA, etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab, an IL-6 antagonist, optionally an antagonist anti-IL-6 antibody, an anti-IL-17 antagonist, optionally Secukinumab, Ixekizumab, or Brodalumab, or another B cell suppressant or B cell depleting agent, optionally a CD40 antagonist, further optionally an anti-CD40 antagonist antibody, a CD154 antagonist, further optionally an anti-CD154 antagonist antibody, a B cell depleting antibody, optionally an anti-CD20 antibody, further optionally rituximab, ibritumomab, tiuxetan, or tositumomab; an anti-CD22 antibody, an anti-CD19 antibody, an anti-CD38 antibody,Attorney Docket No. 2969027.002013 an anti-BAFF receptor antibody, folic acid, or any other small molecule B cell suppressants;(x) the method further comprises the administration of one or more other active or actives, e.g., wherein the one or more actives are used in the treatment of autoimmunity, allergy or inflammatory conditions, optionally wherein the other active or actives include one or more of steroids, optionally a glucocorticoid, further optionally prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone; methotrexate, azathioprine mycophenolate, cyclophosphamide, a TNF inhibitor, optionally HUMIRA, etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab, an IL-6 antagonist, optionally an antagonist anti-IL-6 antibody, an anti-IL-17 antagonist, optionally Secukinumab, Ixekizumab, or Brodalumab, or another B cell suppressant or B cell depleting agent, optionally a CD40 antagonist, further optionally an anti-CD40 antagonist antibody, a CD154 antagonist, further optionally an anti-CD154 antagonist antibody, a B cell depleting antibody, optionally an anti-CD20 antibody, further optionally rituximab, ibritumomab, tiuxetan, or tositumomab; an anti-CD22 antibody, an anti-CD19 antibody, an anti-CD38 antibody, an anti-BAFF receptor antibody, folic acid, or any other small molecule B cell suppressants, and combinations of any of the foregoing;(xi) it is effected before, at the same time, or after administration of butyrophilin A2 (BTN2A2), a fragment thereof, a BTN2A2-related isoform, a BTN2A2-related isoform fragment or a fusion polypeptide comprising any of the foregoing;(xii) the method results in acute or prolonged suppression of B cell activation and / or proliferation;(xiii) the method results in acute or prolonged antigen specific B cell tolerance.(xiv) the method is used to desensitize a subject who is to / or has received HLA incompatible cells, tissue and / or organ;(xv) the method includes detecting overall antibody levels and / or antibodies specific to one or more target antigens in one or more samples obtained from the subject after and / or during treatment to assess the efficacy of the treatment method; orAttorney Docket No. 2969027.002013(xv) any combination of the foregoing.

4. A composition comprising(i) a butyrophilin fusion polypeptide or modified butyrophilin polypeptide, comprising(a) a butyrophilin A2 (BTN2A2), a BTN2A2 fragment, a BTN2A2-related isoform, or a BTN2A2-related isoform fragment or a combination thereof; and(b) optionally an albumin or Fc domain or Fc fragment, optionally an IgGl, lgG2, lgG3 or lgG4 Fc or Fc fragment, preferably an IgGl, which optionally may be mutated, which further is optionally mutated in order to enhance or reduce one or more Fc-associated effector functions, further optionally any of the mutations shown in Table 1, and / or is modified to enhance half-life such as by glycosylation or PEGylation or lipidation or by attachment to cholesterol, and wherein (a) and (b) are directly or indirectly linked, optionally by a linker peptide; and(ii) an antigen against which humoral suppression and / or B cell tolerance is to be elicited, or a nucleic acid encoding said antigen, optionally comprised in a viral vector, plasmid or lipid nanoparticle.

5. The composition of claim 4, wherein the butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises an Fc domain, optionally an IgGl Fc domain, which Fc comprises one or more mutations, optionally wherein the mutations comprise Met208Leu, Asn214Ser, or both, in reference to wild type human IgGl or SEQ ID NO:4.

6. The composition of claim 4, wherein the butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises a spacer peptide between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, and the albumin or Fc domain or the Fc fragment, optionally an IgGl Fc.

7. The composition of any one of the previous claims, wherein the butyrophilin fusion polypeptide or modified butyrophilin polypeptide comprises a spacer peptide between the BTN2A2, BTN2A2 fragment, the BTN2A2-related isoform, orthe BTN2A2- related isoform fragment, and the albumin or Fc domain or the Fc fragment, optionallyAttorney Docket No. 2969027.002013 an IgGl Fc selected from the group consisting of IEGRMDDISSTMVRS (SEQ ID NO:56), IEGRMD (SEQ ID NO:57), EAEAEAK (SEQ ID NO:58), Sil N FE KL(SEQ ID NO:59), and GGGS (SEQ ID NQ:60).

8. The composition of any one of the previous claims, wherein the BTN2A2 fragment comprises the extracellular domain of BTN2A2.

9. The composition of any one of the previous claims, wherein the BTN2A2 fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

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

11. The composition of any one of the previous claims, wherein the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

12. The composition of any one of the previous claims, wherein the butyrophilin A2 (BTN2A2), the BTN2A2 fragment, the BTN2A2-related isoform, or the BTN2A2-related isoform fragment, or the combination thereof is PEGylated or glycosylated.

13. The composition of any one of the previous claims, wherein the BTN2A2 fragment binds to CD45.

14. The composition of any one of the previous claims, which further comprises immune cells of the subject who is to be treated with the composition, and / or the composition comprises donor antigens and / or donor cells, of a living or non-living donor from which the subject is to be administered or transplanted cells, a tissue and / or an organ therefrom.

15. The composition of any one of the previous claims, wherein(i) said antigen is selected from an autoantigen, allergen, therapeutic agent, parasitic antigen, or an antigen expressed by donor cells;(ii) said antigen is a food allergen, an aeroallergen, an animal product, a plant derived allergen, a microbial allergen, optionally a mite, fungus, yeast, etc., a drug, a hormone, a therapeutic enzyme, a therapeutic antibody, a blood or clotting factor, insect venom, or latex.Attorney Docket No. 2969027.002013 said antigen is a food allergen selected from a peanut allergen, a tree nut allergen, a dairy allergen, a wheat allergen, a soy allergen, an egg allergen, a shellfish allergen, a meat allergen, a sesame allergen, and a corn allergen;(iii) said antigen is an allergen;(iv) said antigen is an autoantigen;(v) said antigen is an inflammatory agent;(vi) said antigen is that of a transplanted cell, tissue or organ; or(vii) any combination of the foregoing.

16. The composition of any one of the previous claims, which further comprises one or more active agents, of optionally selected from steroids, optionally a glucocorticoid, further optionally prednisolone, cortisone, hydrocortisone, methylprednisolone, dexamethasone; methotrexate, azathioprine mycophenolate, cyclophosphamide, a TNF inhibitor, optionally HUMIRA, etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab, an IL-6 antagonist, optionally an antagonist anti-IL-6 antibody, an anti-IL-17 antagonist, optionally Secukinumab, Ixekizumab, or Brodalumab, or another B cell suppressant or B cell depleting agent, optionally a CD40 antagonist, further optionally an anti-CD40 antagonist antibody, a CD154 antagonist, further optionally an anti-CD154 antagonist antibody, a B cell depleting antibody, optionally an anti-CD20 antibody, further optionally rituximab, ibritumomab, tiuxetan, or tositumomab; an anti-CD22 antibody, an anti-CD19 antibody, an anti-CD38 antibody, an anti-BAFF receptor antibody, folic acid, or any other small molecule B cell suppressants.

17. The composition of any one of the previous claims, which when administered to a subject in need thereof:(i) results in acute or prolonged suppression of B cell activation and / or proliferation;(ii) results in, or provides for, antigen specific B cell tolerance;(iii) when administered to a subject who is to / or has received HLA incompatible cells, tissue and / or organ desensitizes the subject, i.e., the subject does not elicit or elicits a reduced humoral response against the HLA incompatible cells, tissue and / or organ; or(iv) any combination of the foregoing.Attorney Docket No. 2969027.00201318. The composition of any one of the previous claims, wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment thereof is linked or fused to a Fc domain or an Fc fragment ("BTN2A2-Fc fusion protein") or the BTN2A2-related isoform orthe BTN2A2- related isoform fragment is linked or fused to an Fc domain or Fc fragment ("BTN2A2- related isoform-Fc fusion protein"), optionally that of human IgGl, lgG2, lgG3 or lgG4 Fc, which optionally may be mutated, further optionally mutated in order to enhance or reduce an Fc-associated effector function, further optionally wherein the Fc, optionally an IgGl Fc comprises one or more of the mutations shown in TABLE 1 and / or comprises any one, two, three, four or all five of M252Y, S254T, T256E, and / or H433K and N434F, and / or is the variant IgGl Fc contained in SEQ ID NO: 6, further optionally wherein the butyrophilin A2 (BTN2A2) or the BTN2A2 fragment comprises the BTN2A2 polypeptide in SEQ ID NO: 6 or a variant thereof wherein the RS linker peptide intervening the BTNA2 polypeptide and the IgGl Fc variant optionally is replaced by another peptide linker, preferably a non-cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

19. The composition of any one of the previous claims, wherein the BTN2A2 or the BTN2A2 fragment thereof is linked to a Fc domain or a fragment of the Fc domain by a linker, optionally a peptide of 2-50 amino acids, or wherein the BTN2A2-related isoform or the BTN2A2-related isoform fragment is linked to the Fc domain or the fragment of the Fc domain by a linker, preferably a non-cleavable peptide linker, optionally one 1-40, 2-25, 1-20, 2-10, 2-5, 4, 3, 2 or 1 amino acids in length, optionally any of the peptide linkers disclosed herein, further optionally polyserine, polyglycine, serine-serine, glycine-glycine, glycine-serine, GGGGSn (SEQ ID NO:6)n, GGGGGSn (SEQ ID NO:7)n, leucine zipper, or aliphatic, a helical peptide, or any other non-cleavable peptide linker.

20. The composition of any one of the previous claims, wherein the butyrophilin A2 (BTN2A2) or BTN2A2 fragment, BTN2A2-related isoform or BTN2A2-related isoformAttorney Docket No. 2969027.002013 fragment is modified by glycosylation or PEGylation or lipidation or by attachment to cholesterol.

21. The composition of any one of the previous claims, wherein the BTN2A2 fragment comprises an extracellular domain of BTN2A2.

22. The composition of any one of the previous claims, wherein the BTN2A2 fragment does not comprise a transmembrane domain, a cytoplasmic domain, or both.

23. The composition of any one of the previous claims, wherein the BTN2A2-related isoform is BTN1A1, B2N2A1, BTN3A1, BTN3A2 or BTN3A3.

24. The method of any one of the previous claims, wherein the BTN2A2-related isoform fragment does not comprise the transmembrane domain, the cytoplasmic domain, or both.

25. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein wherein (i) the BTN2A2 moiety of the fusion protein comprises at least one BTN2A2 polypeptide possessing at least 90% sequence identity to the IgV domain of human or murine BTN2A2 and which BTN2A2 polypeptide lacks or only comprises a fragment of the BTN2A2 IgC domain; (ii) an Fc region, and optionally (iii) a peptide linker which intervenes the BTN2A2 and Fc region of the BTN2A2-FC fusion protein, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues, further optionally wherein the linker comprises or consists of GGSGG.

26. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein wherein the BTNA2 moiety in the BTN2A2-Fc fusion protein comprises or consists of a polypeptide possessing at least 95-99% sequence identity to the IgV domain of BTN2A2, optionally human BTN2A2 IgV domain.

27. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises two or more of said BTN2A2 polypeptides or fragments, optionally a IgV domain, wherein optionally a peptide linker intervenes said BTN2A2 polypeptides, optionally a 1-50, 2-25, 2-15, 2-10 or 2-5 peptide linker, further optionally a flexible peptide linker, still further optionally a flexible peptide linker entirely or predominantly of glycine and serine residues.Attorney Docket No. 2969027.00201328. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises a human Fc region, optionally a human IgGl, lgG2, lgG3 or lgG4 Fc region.

29. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises a human IgGl or lgG2 Fc region, optionally one comprising one or more mutations which impair or enhance one or more effector functions, e.g., FcR binding, complement binding, FcRN binding, further optionally wherein said one or more mutations comprise the Fc mutations identified in Table 1.

30. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises a mutated IgGl Fc region wherein the mutations comprise or consist of (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G substitution mutation and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, optionally a 1- 50, 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues.

31. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises a BTN2A2 polypeptide or CD45 binding fragment thereof and a mutated IgGl Fc region, wherein the mutated IgGl Fc region comprises (i) M252Y, S254T, T256E, H433K, and N434F mutations which enhance serum half-life, (ii) at least one mutation which inhibits or prevents CDC and / or ADCC mediated cytotoxicity, optionally a N297G mutation, and / or L234A, L235A, and P329G substitution mutations, and (iii) optionally lacks the C-terminal lysine residue, and wherein a flexible peptide linker optionally intervenes the BTN2A2 and Fc region of the BTN2A2-Fc fusion protein, further optionally a 2-25, 2-15, 2-10 or 2-5 peptide linker, optionally entirely or predominantly comprised of glycine and serine residues.

32. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises at its N-terminus a signal peptide,Attorney Docket No. 2969027.002013 further optionally the endogenous BTN2A2 signal peptide or an immunoglobulin signal peptide.

33. The composition of any one of the previous claims, which comprises a Butyrophilin A2 (BTN2A2)-Fc fusion protein which comprises a flexible linker intervening the BTN2A2 and Fc moiety and / or optionally comprises a hinge region optionally wherein one or more of the cysteine residues are substituted with another amino acid, optionally serine.

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