WO2018237003A1

WO2018237003A1 - Methods for producing induced t regulatory cells

Info

Publication number: WO2018237003A1
Application number: PCT/US2018/038503
Authority: WO
Inventors: Alexei Savinov; Ilian RADICHEV; Christina AMATYA
Original assignee: Sanford Health
Priority date: 2017-06-23
Filing date: 2018-06-20
Publication date: 2018-12-27

Abstract

Disclosed herein are methods for producing induced T regulatory cells (iTregs), involving contacting naive CD4+CD25- T cells and/or naive CD8+CD25- T cells in vitro with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein (also known as VTCNl, B7x or B7S 1), wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) interleukin-2 (IL-2), wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4+CD25- T cells and/or naive CD8+CD25- T cells into iTregs.

Description

Methods for producing induced T regulatory cells Cross-Reference

This application claims priority to U.S. Provisional Patent Application Serial Number 62/523,904 filed June 23, 2017, incorporated by reference herein it its entirety.

Background

Regulatory T-cells, formerly known as suppressor T-cells, modulate or suppress immune responses, particularly to prevent autoimmunity and maintain tolerance to self- antigens. The potential for Tregs to actively regulate autoimmunity and induce long term tolerance has great potential application as a strategy for inducing long-lived tolerance. Taking advantage of Tregs has been complicated by an inability to expand and characterize this minor T cell subset, a population of cells reduced even further in autoimmune-prone animals and patients. Prior efforts to generate and expand Tregs in vitro or ex vivo have not been very successful or adequate for clinical use.

Summary

Disclosed herein in a first aspect are methods for producing induced T regulatory cells

(iTregs), comprising contacting naive CD4¹CD25^" T cells and/or naive CD8¹CD25^" T cells in vitro with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein (also known as VTCN1, B7x or B7S1), wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein lgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) interleukin-2 (IL-2), wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells into iTregs.

In one embodiment, the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

NO:2). In another embodiment, the IgC domain is the only portion of the B7-H4 protein present in the polypeptide. In a further embodiment, the polypeptide comprises a signal peptide targeting the polypeptide for secretion; in one such embodiment, the signal peptide is a heterologous signal peptide, such as an insulin signal peptide.

In another embodiment, the contacting comprises contacting cells with the polypeptide and the antibodies for at least 4 days. In a further embodiment, least one (or two, or all three) of the polypeptide, the anti-CD3 antibodies, and the anti-CD28 antibodies are present on a bead. In one embodiment, the naive T-cells are present at a concentration of at least 2x 10⁶ cells per mL. In another embodiment, the anti-CD3 antibodies and the anti-CD28 antibodies are attached to a surface, such as attached to beads. In a further embodiment, there is a ratio of about 1 : 1 of naive cells to antibody-bound beads.

Disclosed herein in a second embodiment are recombinant polypeptides, comprising an lg-C domain from a B7-H4 protein linked to a heterologous signal peptide, wherein the IgC domain is the only portion of the B7-H4 protein present in the polypeptide. In one embodiment, the recombinant polypeptide further comprises an amino acid linker between the Ig-C domain and the heterologous signal peptide. In another embodiment, the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

SMPEINVOYNASSESLRCEAPRWFPQPTVAWASQVIXKJANFSEVSNT^FELN SENVTTUKWSVLYN\TINNTYSCMIENDIAKATGDlK\n^{^}DSEVKRRSQLQLLNS (SEQ ID NO: l); and

SMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFEL NSENVTMKWSVLYNVTTNNTYSCMIENDIAKATCDIKVTCSEiKRRSHLQLLNS (SEQ ID NO:2). In a further embodiment, the heterologous signal peptide comprises or consists of the amino acid sequence malwmrllpllallalwgpdpaaa (SEQ ID NO:3). In further embodiments, the recombinant polypeptide comprises or consists of a polypeptide selected from the group consisting of:

VAWASQVD(X}ANFSEVSNT¾FELNSE>m^iKWSVLYNVTTONWSCMIENDIAKA TGDIKVTDSEVKRRSQLQLLNS (SEQ ID NO:4); and

VWASQVDQGANFSEVSNT^FELNSENVTTVIKVVSVLYNVTINNTYSCMIENDIAKA TGDIKVTESEiKRRSHLQLLNS (SEQ ID NO:5). In another embodiment, the recombinant polypeptide is present on a solid support, such as a bead. Disclosed herein in further aspects are isolated nucleic acids encoding the polypeptide of any embodiment or combination of embodiments of the disclosure; recombinant expression vectors comprising an isolated nucleic acid of the disclosure operatively linked to a promoter sequence, and recombinant host cells comprising recombinant expression vectors of the disclosure.

Disclosed herein in a further aspect are compositions, comprising:

(a) the polypeptide of any embodiment or combination of embodiments of the disclosure;

(b) anti-CD3 antibodies; and

(c) anti-CD28 antibodies.

In one embodiment, the composition is present on a solid support, such as a bead. Disclosed herein in a further aspect are iTreg cell populations generated by the method of any embodiment or combination of embodiments of the disclosure.

Disclosed herein in a still further aspect are methods for treating an autoimmune disorder and/or transplant rejection, comprising:

(a) in vitro contacting naive CD4XD25^" T cells and/or naive CD8XD25^" T cells with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein, wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) IL-2, wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4*CD25^" T cells and/or naive CD8*CD25^" T cells into iTregs; and

(b) administering to a subject with an autoimmune disorder or who has received or is receiving a transplant an amount effective to treat the autoimmune disorder and/or transplant rejection of the iTregs generated in step (a).

In one embodiment, the polypeptide comprises or consists of the polypeptide of any embodiment or combination of embodiments of the disclosure. In another embodiment, step (a) is carried out according to the method of any embodiment or combination of embodiments of the disclosure. In one embodiment, the naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells are obtained from the subject to be treated. In another embodiment, the naive

CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells are obtained from peripheral blood. In further embodiments, the autoimmune disorder is selected from the group consisting of type I diabetes, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis, or wherein the subject has graft versus host disease. Description of the Figures

Figure 1. (A) Schematic representation of generated soluble 6xHis-tagged VTCN1 (VI), VTCNl's IgV (IgV) and VTCNl's IgC (IgC) constructs. (B) Western blot analysis of VI , IgV and IgC proteins isolated from conditioned media of HEK cells stably expressing the indicated mutant construct. (C) Naive CD4⁺CD25^" and CD8⁺CD25^" T cells were isolated from mouse spleen, activated in the presence of indicated recombinant protein and then analyzed by FACS for the presence of CD25⁺FoxP3⁺ cells. Stronger increase of the double positive population (in square) representing Treg-like cells is observed in IgC and IgV+IgC treated naive T cells. (D) Quantification of Tregs and their proliferation. Data are represented as mean

Figure 2. (A) Outline of the suppression assay with classical Tregs. First, naive CD4^1" T cells were activated for 5 days in the presence of indicated recombinant protein. Next, insulin peptide-loaded macrophages were co-cultured for 6 days with l: l ratio of CFSE- labeled G9C8 CD8^* cells and the activated naive CD4⁺ cells. G9C8 CD8⁺ T cells originate from insulin-specific diabetogenic T cell clone isolated from the mouse model for T1D, NOD. These cells are robustly activated when a short insulin peptide (InsB^15"23) is presented to them by antigen presenting cells (in our case, macrophages). At the end, the co-cultures were gated for live CD8⁺ cells and their proliferation was analyzed by the dilution of the fluorescent dye, CFSE (only G9C8 cells are labeled with it). (B-C) Histograms - Representative FACS of G9C8 CD8⁺ T cell proliferation. It is noticeable that the most resilient decrease of T cell proliferation is achieved by IgC domain, not IgV. Right bar graph - quantitative analysis. Data are represented as mean ±

***/K0.001. (D) Representative FACS analysis of CD4⁺CD25⁺FoxP3⁺ and

CD8⁺CD25⁺FoxP3⁺ after activation of human CD4⁺ or CD8⁺ T cells in the presence of the indicated protein. (E) Quantification of Tregs numbers. Data are represented as mean ±SEM

Figure 3. Bar graph showing mean concentration (ng/ml) of TGFp ±SEM (n=6) in an example of the methods of the disclosure; * * *p<0.001. Detailed Description

As used herein and unless otherwise indicated, the terms "a" and "an" are taken to mean "one", "at least one" or "one or more". Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular. All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified.

As used herein, "about" means +/- 5% of the recited dimension or unit.

All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

In a first aspect, the disclosure provides methods for producing induced T regulatory cells (iTregs), comprising contacting naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells in vitro with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein (also known as VTCN1, B7x or B7S 1), wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) interleukin-2 (IL-2), wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4⁺CD25^" T cells and/or naive

CD8TD25^" T cells into iTregs.

As shown in the examples that follow, the inventors have demonstrated that the methods of the invention can be used to cause conversion of nai ve CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells into induced T regulatory cells (iTregs). Regulatory T-cells, formerly known as suppressor T-cells, modulate or suppress immune responses, particularly to prevent autoimmunity and maintain tolerance to self-antigens. The potential for Tregs to actively regulate autoimmunity and induce long term tolerance has great potential application as a strategy for inducing long-lived tolerance. Taking advantage of Tregs has been complicated by an inability to expand and characterize this minor T cell subset, a population of cells reduced even further in autoimmune-prone animals and patients. Prior efforts to generate and expand Tregs in vitro or ex vivo have not been very successful or adequate for clinical use. The methods of the present invention provide a significant improvement in the ability to generate and expand iTregs for clinical use.

While not being bound by any specific hypothesis, the inventors believe that the methods trigger Treg-inducing signals and stimulate TGF-beta secretion, a key cytokine which promotes iTreg differentiation from naive T-cells.

As used herein, "naive CD4⁺CD25^" T cells" are mature helper T cells that have not been contacted with their cognate antigen, which express the CD4 marker and negative for the CD25 marker.

As used herein, "naive CD8⁺CD25^" T cells" are mature cytotoxic T cells that have not been contacted with their cognate antigen, which express the CD8 marker and negative for the CD25 marker. As used herein, "iTregs" are induced T regulatory cells that express the either the CD4 or the CD8 proteins together with the CD25 marker, and the FOXP3 marker.

As will be understood by those of skill in the art, the naive CD4*CD25^" T cells and/or naive CD8⁺CD25^" T cells may be present in a population of T-cells (i.e: the methods do not require a pure population of naive CD4⁺CD25^" T cells and/or naive CD8XD25^" T cells). The T cell population may be obtained from any suitable source. In various embodiments in which the methods arc for clinical use, the T cell population is harvested from peripheral blood obtained from a subject to be treated or from a donor distinct from said subject. The T cell population may be derived from any source in which naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells exist, such as peripheral blood, the thymus, lymph nodes, spleen, and bone marrow.

In particular embodiments, the naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells are enriched from a T cell population of cells prior to the contacting step. Naive CD4*CD25^" T cells and/or naive CD8*CD25^" T cells can be enriched using standard techniques, such as by targeting for selection of CD4⁺ or CD8⁺ cell surface markers, and separating using automated cell sorting such as fluorescence-activated cell sorting (FACS), solid-phase magnetic beads, etc. To enhance enrichment, positive selection may be combined with negative selection against CD25^"cells, using standard techniques. In various embodiments, the T cell population may enriched to at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater of naive CD4⁺CD25^" T cells or naive CD8*CD25^" T cells prior to the contacting step. Any suitable polypeptide comprising an Ig-C domain from a B7-H4 protein, wherein the polypeptide is not the full B7- H4 protein and does not include the B7-H4 protein IgV domain can be used in the methods disclosed herein. In one embodiment, the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

In one embodiment, the IgC domain is the only portion of the B7-H4 protein present in the polypeptide. In another embodiment, the polypeptide comprises a signal peptide targeting the polypeptide for secretion. Any suitable signal peptide may be used for secretion of the polypeptide; it is well within the level of those of skill in the ait to determine an appropriate signal peptide for an intended use. In one embodiment, the signal peptide is a heterologous signal peptide, such as an insulin signal peptide. In one non-limiting embodiment, the signal peptide comprises or consists of the amino acid sequence malwmrllpllallalwgpdpaaa (SEQ ID NO:3). As used herein, a heterologous signal peptide is any signal peptide that is not the B7-H4 signal peptide.

In a further embodiment, the recombinant polypeptide comprises or consists of the amino acid sequence selected from the group consisting of:

TGDIKVTESEIKRRSHLQLLNS (human) (SEQ ID NO:5).

Any suitable anti-CD3 antibodies, anti-CD28 antibodies, and interleukin-2 (IL-2) concentration may be used in the methods disclosed herein, based on the teachings of the disclosure. Anti-CD3 antibodies, anti-CD28 antibodies, and IL-2 are all commercially available.

In one embodiment the contacting comprises contacting cells with the polypeptide and the antibodies for at least 4 days (i.e.: 4 days, 5 days, 6 days, 7 days, or more). The contacting is carried out under conditions for any suitable time sufficient to cause conversion of nai ve CD4⁺CD25^" T cells or nai ve CD8⁺CD25^" T cells into iTregs. In various

embodiments, the contacting is carried out for at least 4 days, at least 5 days, or more. Any suitable culture conditions can be used, the determination of which are well within the level of those of skill in the art. Any suitable culture media (including but not limited to Roswell Park Memorial Institute (RPMI) medium), and supplements (including but not limited to fetal bovine serum, antibiotics, etc.) can be used, and any suitable culture environmental conditions can be used (including but not limited to incubation at 37°C at 5% C0₂).

The polypeptide and/or antibodies may be bound or attached to a carrier or substrate, e.g., microtiter plates (ex: for ELISA), membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface of such supports may be solid or porous and of any convenient shape. In one embodiment, at least one (or 2, 3, or all 4) of the polypeptide, the anti-CD3 antibodies, the anti-CD28 antibodies, and the IL-2 are present on a bead. Any suitable concentration of Ig-C domain, CD3 antibodies, CD28 antibodies, and IL-2 may be used to carry out the methods of the invention. It is well within the level of those of skill in the art to determine suitable concentrations depending on the intended use, based on the present disclosure. In various embodiments, the Ig-C domain is present in a concentration of at least SO ng/ml, 100 ng/ml, 250 ng/ml, 500 ng/ml, 1 ug/ml, 2.5 ug/ml, 5 ug/ml, 7 ug/ml, or more. In various further embodiments, 11-2 is present at a concentration of at least 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 2 ng/ml, or more.

The naive T-cells may be present at any concentration suitable to generate a desired amount of iTregs. In one embodiment, the naive T-cells are present at a concentration of at least 2x 10⁶ cells per niL. In another embodiment, the anti-CD3 antibodies and the anti-CD28 antibodies are attached to a surface, such as attached to beads. In a further embodiment, there is a ratio of about 1: 1 of naive cells to antibody-bound beads.

The contacting may require other components as deemed appropriate for an intended use. In certain embodiments, the contacting may comprise contacting with one or more additional agents, e.g., a costimulatory agent, a second regulatory T cell stimulatory agent, or agents that generally promote the survival and/or growth of T cells.

Also provided herein are iTreg cell populations generated by the method of any embodiment or combination of embodiments of the disclosure. The iTreg cell population may comprise at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater of iTregs.

In another aspect, the disclosure provides recombinant polypeptides, comprising an Ig-C domain from a B7-H4 protein linked to a heterologous signal peptide, wherein the IgC domain is the only portion of the B7-H4 protein present in the polypeptide. In one embodiment, the recombinant polypeptide further comprises an amino acid linker between the Ig-C domain and the heterologous signal peptide. In another embodiment, the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

In another embodiment, the heterologous signal peptide comprises or consists of the amino acid sequence malwmrllpllallalwgpdpaaa (SEQ ID NO:3). In a further embodiment, the recombinant polypeptide comprises or consists of a polypeptide selected from the group consisting of:

TGDIKVTESEIKRRSHLQLLNS (human) (SEQ ID NO:5).

The polypeptides of the invention may be bound or attached to a carrier or substrate, e.g., microtiter plates (ex: for ELISA), membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface of such supports may be solid or porous and of any convenient shape.

In another aspect, the present invention provides isolated nucleic acids encoding a polypeptide of the present disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, "isolated nucleic acids" are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to poly A sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, , and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the polypeptides of the invention.

In a further aspect, the present disclosure provides recombinant expression vectors comprising the isolated nucleic acid of any aspect of the invention operatively linked to a suitable control sequence. "Recombinant expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. "Control sequences" operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting all types of cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N J.), and the Ambion 1998 Catalog (Ambion, Austin, TX). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid or a viral vector.

In another aspect, the present disclosure provides host cells that have been transfected with the recombinant expression vectors disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, T^d Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY). A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide.

In another aspect, the disclosure provides compositions and/or kits, comprising:

(b) anti-CD3 antibodies; and (c) anti-CD28 antibodies.

The compositions and/or kits may be used, for example, in the methods of the disclosure. In another embodiment, the compositions and/or kits further comprise IL-2. The compositions and/or kits may be bound or attached to a solid support, e.g., microliter plates (ex: for ELISA), membranes and beads, etc. Solid supports may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface of such supports may be solid or porous and of any convenient shape. In one embodiment, the solid support comprises a bead. In another aspect the disclosure provides methods for treating an autoimmune disorder and/or transplant rejection, comprising:

(a) in vitro contacting naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells with (i) a polypeptide comprising an lg-C domain from a B7-H4 protein, wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) IL-2, wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells into iTregs; and

The methods of this aspect of the disclosure can be used to treat any T cell-mediated autoimmunity, or to treat and/or transplant rejection. In non-limiting embodiments, the autoimmune disorder is selected from the group consisting of type I diabetes, rheumatoid arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, and graft versus host disease.

In one embodiment, the polypeptide comprises or consists of the polypeptide of any embodiment or combination of embodiments disclosed herein. In another embodiment, step (a) is carried out according to the method of any embodiment or combination of embodiments disclosed herein

As will be understood by those of skill in the art, the naive CD4XD25^" T cells or naive CD8⁺CD25^" T cells may be present in a population of T-cells (i.e: the methods do not require a pure population of naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells). The T cell population for clinical use may be obtained from a subject to be treated and/or obtained from a donor distinct from said subject. The T cell population may be derived from any source in which naive CD4XD25^" T cells or naive CD8⁺CD25^" T cells exist, such as peripheral blood, the thymus, lymph nodes, spleen, and bone marrow.

In particular embodiments, the naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells may be enriched from a T cell population of cells prior to the contacting step. Naive

CD4XD25^" T cells or naive CD8⁺CD25^" T cells can be enriched using standard negative selection techniques.

The subject can be any mammal in which modulation of an autoimmune reaction is desired. Mammals of interest include, but are not limited to: rodents, e.g. mice, rats;

livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats; and primates, e.g. humans. In one embodiment, the subject is a human.

In a preferred embodiment, the naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells are obtained from the subject to be treated. These methods generally involve reintroducing expanded iTreg cells extracted from the same patient. In an alternate embodiment, the T cell population of cells is obtained from a donor distinct from the subject. The donor is preferably syngeneic, but can also be allogeneic, or even xenogeneic provided the cells obtained are subject-compatible in that they can be introduced into the subject, optionally in conjunction with an immunosuppressive therapy, without resulting in extensive chronic graft versus host disease (GvHD). Allogeneic donor cells are preferably human-leukocyte-antigen (HLA)- compatible, and are typically administered in conjunction with immunosuppressive therapy. To be rendered subject-compatible, xenogenic cells may be subject to gamma irradiation or PEN110 treatment.

As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorders) being treated; (c) inhibiting worsening of symptoms characteristic of the disorders) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

Examples:

Non-limiting example of generation of .Trees from naive T cells according to the methode of the disclosure.

1.. Isolate naive CD4⁺ or CD8⁺ T cells from mouse spleen or human peripheral blood mononuclear cells using standard protocols. Naive T cell isolation kits were obtained from Miltenyi Biotec (including their CD4⁺CD25⁺ Mouse Regulatory T Cell Isolation Kit; Naive CD8a+ Mouse T Cell Isolation Kit; Naive CD4+ Human T Cell Isolation Kit Π; and CD4⁺CD25⁺ Naive Human CD8+ T Cell Isolation Kit.)

2. Count the cells.

3. Resuspend cells at concentration 2 ^χ 10⁶ cells per mL in RPMI supplemented with 10% FBS and antibiotics (penicillin and streptomycin) - medium and additives can be obtained from different suppliers.

4. Add 25 μΐ per 1 x 10⁶ cells anti-CD3/anti-CD28 T cell activation beads + 7 Mg/ml IgC domain of VTCN1 (B7H4) + 1L-2 (50 ng/ml when activating mouse T cells and 2 ng/ml when activating human T cells).

5. Incubate 5 days at 37°C in a humidified incubator with 5% C0₂.

6. Analyze percentage of Treg cells by staining with variety of antibodies (anti- CD4/CD8, anti-CD25, anti-FoxP3).

Activation beads were obtained from ThermoFisher Scientific (Dynabeads® Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation; Dynabeads® Human T- Activator CD3/CD28 for T Cell Expansion and Activation

Mouse IL-2 was obtained from R&D Systems. Human IL-2 can be obtained from, for example, Cell Signaling Technology).

Antibodies were obtained as noted below:

Antibody (Clone #) Origin Specificity Company

CD4-FITC (Clone # RM4-5) Rat Mouse eBioscience

CD4-FITC (Clone # RPA-T4) Mouse Human eBioscience

CD8-FITC (Clone # 53-6.7) Rat Mouse eBioscience

CD8-APC (Clone # 53-6.7) Rat Mouse eBioscience

CD8-FITC (Clone # SKI) Mouse Human eBioscience

CD25-APC (Clone # PC61.5) Rat Mouse eBioscience

CD25-APC (Clone # BC96) Mouse Human eBioscience

FOXP3-PE (Clone # 150D/E4) Mouse Mouse eBioscience

FOXP3-PE (Clone # PCH101) Rat Human eBioscience

Results

Individual VTCNl's Ig-like domains stimulate better than whole VTCN1 the conversion of CD4⁺ and CD8⁺ naive T cells into Treg-like cells (See figure 1). Figure 1(A) provides a schematic representation of generated soluble 6*His-tagged VTCN1 (VI), VTCNl's IgV (IgV) and VTCNl's IgC (IgC) constructs. Figure IB shows Western blot analysis of VI, IgV and IgC proteins isolated from conditioned media of HEK cells stably expressing the indicated mutant construct. Naive CD4⁺CD25^" and CD8⁺CD25^" T cells were isolated from mouse spleen, activated in the presence of indicated recombinant protein and then analyzed by FACS for the presence of CD25⁺FoxP3^1' cells. Stronger increase of the double positive population (in square) representing Treg-like cells is observed in IgC and IgV+IgC treated naive T cells (Figure lC). Tregs and their proliferation were quantified as shown in Figure ID; data are represented as mean ±SEM (n=6). *p≤0.05; **/K0.0l;

***/K0.00l.

The studies showed that VTCNl IgC-like domain stimulates the conversion of CD4⁺ naive T cells into functionally active Tregs (Figure 2). Figure 2A outlines of the suppression assay with classical Tregs. First, naive CD4⁺ T cells were activated for 5 days in the presence of indicated recombinant protein. Next, insulin peptide-loaded macrophages were co-cultured for 6 days with 1:1 ratio of CFSE-labeled G9C8 CD8⁺ cells and the activated naive CD4⁺ cells. G9C8 CD8⁺ T cells originate from insulin-specific diabetogenic T cell clone isolated from the mouse model for T1D, NOD. These cells are robustly activated when a short insulin peptide (InsB^15"23) is presented to them by antigen presenting cells (in our case,

macrophages). At the end, the co-cultures were gated for live CD8⁺ cells and their proliferation was analyzed by the dilution of the fluorescent dye, CFSE (only G9C8 cells are labeled with it).

Representative FACS of G9C8 CD8⁺ T cell proliferation are shown in Figure 2B, and a quantitative analysis is shown in Figure 2C. It is noticeable that the most resilient decrease of T cell proliferation is achieved by IgC domain, not IgV. Data are represented as mean

Representative FACS analysis of CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺FoxP3⁺ after activation of human CD4⁺ or CD8⁺ T cells in the presence of the indicated protein is shown in Figure 2D; quantification of Tregs numbers is shown in Figure 2E. Data are represented as mean

Naive CD4⁺CD25^" T cells from NOD mice were activated with anti-CD3/CD28- coated beads for 5 days in the presence of the indicated recombinant protein (VI is soluble VTCNl without its transmembrane portion). At the end of the activation, conditioned media was collected and analyzed by ELISA for the presence of TGFp (Figure 3). Data are represented as mean concentration (ng/ml) of TGFp ±SEM (n=6). ***p<0.001.

Claims

We claim:

1. A method for producing induced T regulatory cells (iTregs), comprising contacting naive CD4⁺CD25^' T cells and/or naive CD8⁺CD25^' T cells in vitro with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein (also known as VTCN1, B7x or B7S1), wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) interieukin-2 (IL- 2), wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells into iTregs.

2. The method of claim 1, wherein the naive T-cells are naive CD4⁺CD25^' T cells.

3. The method of claim 1, wherein the naive T-cells are naive CD8⁺CD25^" T cells.

4. The method of any one of claims 1-3, wherein the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

5. The method of any one of claims 1 -4, wherein the IgC domain is the only portion of the B7-H4 protein present in the polypeptide.

6. The method of any one of claims 1-5, wherein the polypeptide comprises a signal peptide targeting the polypeptide for secretion.

7. The method of claim 6, wherein the signal peptide is a heterologous signal peptide, such as an insulin signal peptide.

8. The method of any one of claims 1-7, wherein the contacting comprises contacting cells with the polypeptide and the antibodies for at least 4 days.

9. The method of any one of claims 1 -8, wherein at least one (or two, or all three) of the polypeptide, the anti-CD3 antibodies, and the anti-CD28 antibodies are present on a bead.

10. The method of any one of claims 1-9, wherein the naive T-cells are present at a concentration of at least 2* 10⁶ cells per mL.

11. The method of any one of claims 1-10, wherein the anti-CD3 antibodies and the anti- CD28 antibodies are attached to a surface, such as attached to beads.

12. The method of claim 11, wherein there is a ratio of about 1: 1 of naive cells to antibody-bound beads.

13. A recombinant polypeptide, comprising an Ig-C domain from a B7-H4 protein linked to a heterologous signal peptide, wherein the IgC domain is the only portion of the B7-H4 protein present in the polypeptide.

14. The recombinant polypeptide of claim 13, further comprising an amino acid linker between the Ig-C domain and the heterologous signal peptide.

15. The recombinant polypeptide of claim 13 or 14, wherein the Ig-C domain comprises or consists of the amino acid sequence selected from the group consisting of

SMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVT MKWSVLYNVTTONWSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNS (SEQ ID NO: 1); and

SMPEVNVDYNASSETI.RCEAPRWFPQPWVWASQVDQGANFSEVSNTSFELNSENV TMKWSVLYNVTTNNTYSCMIENDIAKATGDIKVTESEIKRRSliLQLLNS (SEQ ID NO:2).

16. The recombinant polypeptide of any one of claims 13-15, wherein the heterologous signal peptide comprises or consists of the amino acid sequence malwmrllpUallalwgpdpaaa (SEQ ID NO:3).

17. The recombinant polypeptide of any one of claims 13-16, comprising or consisting of a polypeptide selected from the group consisting of:

VAWASQVD(X}ANFSEVSNT¾FELNSE>m^iKWSVLYNVTTONWSCMIENDIAKA TGDIKVTDSEVKRRSQLQLLNS (SEQ ID NO:4); and VVWASQVDQGANFSEVSNTSFFXNSENVTMKW TGDIKVTESEiKRRSHLQLLNS (SEQ ID NO:5).

18. The recombinant polypeptide of any one of claims 13-17, wherein the recombinant polypeptide is present on a solid support, such as a bead.

19. An isolated nucleic acid encoding the polypeptide of any one of claims 13-17.

20. A recombinant expression vector comprising the isolated nucleic acid of claim 19 operatively linked to a promoter sequence.

21. A recombinant host cell comprising the recombinant expression vector of claim 20.

22. A composition, comprising:

(a) the polypeptide of any one of claims 13-18; (b) anti-CD3 antibodies; and

(c) anti-CD28 antibodies.

23. The composition of claim 22, wherein the composition is present on a solid support, such as a bead.

24. An iTreg cell population generated by the method of any one of claims 1-12.

The iTreg cell population may comprise at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater of iTregs.

25. A method for treating an autoimmune disorder and/or transplant rejection, comprising:

(a) in vitro contacting naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells with (i) a polypeptide comprising an Ig-C domain from a B7-H4 protein, wherein the polypeptide is not the full B7-H4 protein and does not include the B7-H4 protein IgV domain, (ii) anti-CD3 antibodies, (iii) anti-CD28 antibodies, and (iv) IL-2, wherein the contacting occurs for a time and under conditions sufficient to cause conversion of naive CD4⁺CD25^" T cells and/or naive CD8⁺CD25^" T cells into iTregs; and

26. The method of claim 25, wherein the polypeptide comprises or consists of the polypeptide of any one of claims 13-18.

27. The method of any one of claims 25-26, wherein step (a) is carried out according to the method of any one of claims 1-12.

28. The method of any one of claims 25-27, wherein the naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells are obtained from the subject to be treated.

29. The method of any one of claims 25-28, wherein the naive CD4⁺CD25^" T cells or naive CD8⁺CD25^" T cells are obtained from peripheral blood.

30. The method of any one of claims 25-29, wherein the autoimmune disorder is selected from the group consisting of type I diabetes, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and multiple sclerosis, or wherein the subject has graft versus host disease.