FIELD

Described herein are antigen specific T regulatory cells (Tregs). Such cells can be used in therapy to minimize undesirable immune responses, such as those observed in autoimmunity and hemophilia as well as in response to protein therapy for genetic diseases. Methods for producing antigen specific Tregs also are described.

BACKGROUND

T regulatory cells suppress immune responses of other cells. They come in several forms with the most well-understood being those that express CD4, CD25, Foxp3, and Helios (CD4 ⁺ CD25 ⁺ Tregs). These cells are involved in shutting down immune responses after they have successfully eliminated invading organisms, and in preventing autoimmunity. Methods for expanding non-specific regulatory T cells have been described in the literature. However, because the T cells employed in those methods were non-specific, the activity of such cells could be immunosuppressive for responses to pathogenic infections and cancer, where an immune response would be desirable.

There currently exists a need for effective therapies to minimize or eliminate the undesirable immune response to self components that is the underlying cause of autoimmune disorders, including type 1 diabetes, uveitis and multiple sclerosis, and the unfavorable response to treatment of genetic diseases like hemophilia and

Pompe's with biotherapeutics.

SUMMARY

In accordance with some embodiments, there is provided a method for producing antigen specific T regulatory cells comprising transducing T cells with an expression vector comprising a nucleotide sequence encoding a T cell receptor that specifically recognizes the antigen, and expanding the transduced T cells ex vivo. In some embodiments, transducing the T cells comprises transducing HLA matched restricted natural T regulatory cells. In some embodiments, the transducing comprises transducing cells with an amphotrophic packaging retrovirus. In some embodiments, the T cells are obtained from a donor's buffy coat prior to being transduced.

In accordance with any of these embodiments, the antigen is selected from the group consisting of the C2 domain of human clotting factor VIII and antigens associated with multiple sclerosis, such as myelin basic protein or myelin

oligodendrocyte glycoprotein (MBP, MOG), diabetes (e.g., GAD65), or uveitis (S- antigen). In some embodiments, the antigen is a human clotting factor, such as factor VIII having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen is involved in a subject's reaction to biotherapeutics in genetic diseases like Pompe's and hemophilia.

In accordance with any of these embodiments, the cells may be subjected to a stimulation step prior to transduction, and/or an expansion step following

transduction.

Also provided is a method for expanding T regulatory cells and/or T effector cells transduced with a T cell receptor-encoding sequence, comprising culturing the cells in IL-2 containing RPMI1640 media with 10% fetal bovine serum. In some embodiments, the culturing is effected for up to 4 days.

Also provided is a method for expanding T regulatory cells transduced with a T cell receptor-encoding sequence, comprising comprising culturing the cells in the presence of the specific antigen recognized by the T cell receptor and

oligodeoxynucleotides, and optionally further in the presence of irradiated DRl HLA- typed peripheral blood mononuclear cells and IL2. In some embodiments, the culturing is effected for about 21 days.

Also provided are antigen specific T regulatory cells prepared by any method described herein.

Also provided are methods of reducing an immune response to an antigen, comprising administering to a subject in need thereof a composition comprising an antigen specific T regulatory cell as described herein that is specific to the antigen. In some embodiments, the subject is suffering from hemophelia, multiple sclerosis, diabetes or uveitis. In some embodiments, the subject is receiving biotherapeutics for a genetic disease such as Pompe's or hemophilia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Identification and retroviral expression of a FVIII-2191-2220- specific TCR in primary CD4 T cells

(A) Scheme of FVIII-2191-2220-specific TCR cloning from a hemophilia A subject's T-cell clone. The cloning procedure is described in Materials and Methods. Briefly, to amplify TCR cDNA from the clone, poly C oligonucleotide was linked onto the 3' terminus of total cDNAs by terminal deoxynucleotidyl transferase (TdT). The variable region was amplified using semi-nested PCR with a poly GI primer and two reverse primers (pCl and pC2) corresponding to two different 5' upstream coding regions of the a and β chain constant regions.

(B) Amplification of V regions of the 17195TCR using semi-nested PCR. Primary amplification and nested amplification were carried out with pCl and pC2 (figure

1 A). PGI was commonly used as the forward primer for the primary and nested PCR step. Bold arrows indicate the predicted sizes of the amplified Va and νβ PCR products.

(C) Retroviral expression construct of the FVIII-2191-2220-specific TCR. To build the FVIII-2191-2220-specific TCR, the variable regions (Va and νβ) from the FVIII- 2191-2220-specific T effector clone were combined with human constant regions (Ca and CP) extracted from the NCBI database. To produce the individual a and β TCR chains from a single transcript, a P2A cleavage peptide was inserted between the a and β chain sequences. Expression of the GFP reporter is controlled by IRES, which is located downstream of the TCR construct.

(D) Amino acid sequence of a FVIII C2-specific human T cell receptor (TCR17195).

Figure 2. Flow chart depicting a protocol for producing FVIII C2-specific human regulatory T cells and effector T cells. To produce FVIII C2-specific T effector cells, na ^' ive human T cells (CD4 ⁺ CD25 ^" CD127 ⁺ CD45RA ⁺ ) were isolated by FACS sorting (FACSAria, BIC, USUHS) from healthy donor's buffy coat. The cells were pre-stimulated with plate-coated anti-CD3e antibody and plate-coated anti-CD28 antibody for 48 hrs. Then, the cells were transduced with amphotropic TCR-17195- IRES-GFP-containing retroviral culture supernatant in retronectin-coated plates (to express the TCR from a patient clone reactive to FVIII). The transduced cells were expanded in IL2-containing RPMI 1640/10% FBS for 4 days. To confirm activation of T cells by FVIII C2, the expanded effector T cells in which TCR17195 was expressed (Teff-17195) were cultured for 4 days with gamma-irradiated DR1-HLA typed PBMCs (6000 rad by Co60) in the presence of pOVA (negative control), pFVIII C2, or soluble anti-CD3e antibody. To produce TCR17195-expressing T regulatory cell (Treg-17195), human Tregs (CD4 ⁺ CD25 ^hi CD127 ^l0 ) were isolated from healthy donor's buffy coat. Pre-stimulation, retroviral transduction, and expansion of Treg-17195 was done as described for T effector cells. To confirm C2-specific Treg activation through TCR17195, expanded Treg-17195 cells were co-cultured with gamma-irradiated DR1-HLA typed PBMCs (6000 rad by Co60) for 24 hrs in the presence of pOVA, pFVIII C2, or soluble anti-CD3e antibody.

Figure 3. Activation of 17195 T effectors by FVIII-2191-2220

(A) Proliferation of 17195TCR-transduced CD4 T cells with FVIII-2191-2220. Pre- stimulated primary CD4 T cells were transduced with 17195TCR or with a mock vector and maintained for 10 days in culture media supplemented with IL-2. For proliferation assays, cells were labeled with cell proliferation dye (eFluor 450) and then re-activated with irradiated DR1 -PBMCs plus peptide FVIII-2191-2220 (0.5 μg /ml), pOVA (0.5 μg /ml), or anti-CD38 antibody (0.5 μg /ml) for 4 days. Cell proliferation was measured by flow cytometry using a standard dye dilution assay. Representative results are shown for CD4 cells from two different donors that were transduced with 17195TCR.

(B) Staining of transduced, expanded GFP ⁺ 17195 T effectors shown in (A) using PE- labeled DR1 tetramers loaded with FVIII-2191-2220.

(C) Proliferation of GFP ^" and GFP ⁺ T cell effectors stimulated with OVA peptide, FVIII-2191-2220 or anti-CD38 antibody. Transduced T effectors (2X10 ⁶ /well) were stimulated using irradiated DRl-APCs (Responder:Stimulator=l :5) as in (A). After culturing for 10 days, The cells were harvested and, viable GFP ^" and GFP ⁺ CD4 cells were counted using flow cytometry.

(D) Retroviral expression of 17195 TCR in CD4 T naive and CD4 Treg cells. Naive T cells were pre-stimulated in plate-bound anti-CD3 antibody and anti-CD28 antibody for 48 hrs. Then, these cells were transduced in virus-coated plate for 24hrs.

Expression of GFP and staining with FVIII peptide/DRl tetramer was performed by FACS analysis at 3 days after transduction of 17195TCR.

(E) Cell surface expression of 17195 TCR in transduced T cells (10 day-cultured). Expression of retroviral 17195 TCR was measured by flow cytometry with anti-Vp2 versus GFP.

(F) FVIII peptide-specific induction of Treg-specific surface markers in transduced Tregs. Two week-expanded mock and 17195 Tregs (transduction followed as in Figure 2A and then expansion with irradiated DR1 PBMCs in the presence of FVIII peptide) were re-activated for 48 hrs with irradiated APCs and FVIII peptide (0.5 g/ml) in the presence of IL-2 (200 U/ml). Histograms show the relative expression of LAP, GARP, and GITR in GFP-positive transduced population.

Figure 4. Data showing that TCR17195 activates transduced human Tregs in an FVIII C2-specific manner. To confirm C2-specific Treg activation through TCR17195, expanded Treg-17195 cells were co-cultured with gamma-irradiated DR1-HLA typed PBMCs (6000 rad by Co60) for 24 hrs in the presence of pOVA, pFVIII C2, or soluble anti-CD3e antibody. Foxp3 and GARP (glycoprotein A repetitions predominant, LRRC32) staining was then done on fixed, co-cultured cells. Foxp3 and GARP are transiently induced at the early stage (24-48 hrs) of activated Tregs. In GFP negative population, the induction of Foxp3 and Helios was shown only in the anti-CD3e Ab stimulation group and pC2-mediated stimulation did not give any induction of Foxp3 and Helios (dot plots and histograms in left box). In GFP positive cells (expressing the TCR), stimulation by C2 induces expression of Foxp3 and Helios in a portion of cells as well as stimulation by CD3e. Figure 5. Optimized ex vivo expansion of 17195TCR-Tregs with FVIII- 2191-2220 plus oligodeoxynucleotides

(A) FVIII-2191-2220-specific enrichment of transduced Foxp3 Helios ⁺ Tregs via long-term expansion. After a 2 ^nd round of expansion (described in Figure 10), the CD4 T cells were stained using DRl tetramers loaded with FVIII -2191-2220 (top dot plots). Expression of Foxp3 and Helios in gated CD4 ⁺ GFP ⁺ population were also evaluated with intracellular staining (bottom contour plots).

(B) Comparison of FVIII-2191-2220-specificity and Fox3 Helios ⁺ phenotypes for short-term and long-term expanded 17195TCR-Tregs. First-round expanded (Exp) cells (1 ^st Exp) were harvested 8 days after viral transduction and second-round expanded cells (2 ^nd Exp) were harvested 16 days after transduction. Tetramer staining (to determine FVIII-2191-2220 specificity) and intracellular FACS staining (to quantify Foxp3 and Helios expression) were performed as in Figure 5A.

Representative data from one of two experiments are shown.

(C) Plasticity of long-term expanded 17195TCR-Tregs and Treg phenotype stability induced by antigen. Second-round expanded 17195TCR-transduced Tregs were rested for 3 -days of culture without IL-2 and then re-stimulated for 4 hrs with PMA and ionomycin in the presence of Golgi-block reagent. Intracellular IFNy (top contour plots and graph) and IL-2 (bottom contour plots and graph) production were measured by FACs analysis.

(D) DNA methylation of TSDR in long-term expanded 17195TCR Tregs with FVIII- 2191-2220 plus ODN. To analyze DNA methylation in the TSDR, mock-transduced and 17195TCR-transduced Tregs were expanded with anti- CD3s antibody or FVIII- 2191-2220 in the presence of ODN, as in figure 4A. TSDR is an unmethylated CpGs- enriched region within Foxp3 genome of natural Treg. Heat map analysis shows methylation status of specific nine out of total eleven CpGs. The histogram

summarized the mean percent methylation of the 9 TSDR CpGs in 17195TCR- transduced Tregs with FVIII 2191-2220/ODN stimulation vs. anti- CD3s antibody stimulation condition. Figure 6. Data illustrating C2-specific immunosuppression by Tregs expressing TCR17195 in vitro. For the immunosuppression assay, naive T effector cells and Tregs were prepared as in Figures 2-4. T na ^' ive cells were expanded with C2 and irradiated antigen-presenting cells (APC) after viral transduction of TCR17195 for 14 days. The expanded T effector- 17195 population was used as responder cells. To monitor cell division of GFP positive cells, responders were labeled with the Cell Proliferation Dye-eFluor450 (CPD450). Tregs cells were transduced with mock virus or TCR17195, then were expanded for 14 days with anti-CD3s antibody or pC2 peptide. In the co-culture, the ratio of Responders to Stimulators was fixed at 1 : 1 and the amount of Tregs was varied as indicated. Cell mixtures were cultured with anti- CD3e antibody or pC2 for 2 days (A) or 4 days (B) without IL-2 addition. A. C2- specific inhibition of division of the proliferation dye CPD450 labeled GFP ⁺ responders by Treg-17195. Left histograms show cell division of labeled responder co-cultured with Treg-GFP or Treg-17195 in the presence of anti-CD3e antibody and irradiated APCs. Co-culture in the presence of pC2 is shown in right histograms. B. C2-specific immunosuppression by Treg-17195. To confirm and measure cell growth in mixed culture, [ ³ H] -thymidine was added at 18 hrs before harvest, with similar results.

(C) Suppression of FVIII-specific cytokine secretion by 17195TCR-Tregs. Four week-expanded mock-transduced Tregs or 17195TCR-Tregs were mixed with 17195TCR-T effectors (responders) at the indicated ratios with γ-irradiated DR1- PBMCs and rFVIII (0.2 μg/ml) and cultured for 36 hrs without IL-2. Cytokines in culture media were measured using a human Thl, Th2, Thl7 CBA kit (BD

Bioscience). The quality of the mock-transduced and 17195TCR-transduced Tregs was evaluated by measuring their GFP expression (left panels) and the expression of Foxp3 and Helios in the GFP ⁺ cells (right panels). Raw MFI data from the CBA assays were converted to cytokine concentrations according to standard curves generated for each experiment. Data are presented as mean ± SD. Figure 7. In vitro suppression of anti-FVIII antibody production by engineered FVIII-specific human Tregs

Pooled splenocytes from two immunized DR1/E16-FVIII-KO mice were used as T- responders. In a T25 flask, 1 x 10 ⁷ splenocytes were co-cultured with 17195TCR- transduced or mock-transduced Tregs at different ratios in the absence/presence of ^g/ml rFVIII. After 6 days in culture, FVIII-specific antibody secreting cells (ASC) were detected using an ELISPOT assay as described in Methods.

(A) FVIII-specific ASC detected in splenocyte cultures that contained no TCR- or mock-transduced Tregs.

(B) FVIII-specific ASCs detected in splenocytes co-cultured with various ratios of 17195TCR-transduced or mock-transduced Tregs. Co-culture with 17195TCR-Tregs profoundly inhibited anti-FVIII ASC formation, even with responder to suppressor ratios as low as 1 : 0.0625 (1/16). The histograms in (C) and (D) summarize the ELISPOT data in (A) and (B), respectively. Data are presented as mean ± SEM. (E) The quality of the Tregs used in these experiments was evaluated by measuring their GFP expression (left panels) and the expression of Foxp3 and Helios in the GFP ⁺ cells (right panels).

Figure 8. Identification and retroviral expression of a Ob2.F3 -specific TCR in primary CD4 T cells. Retroviral expression construct of the Ob2.F3- specific TCR.

Figure 9. Expression of Ob.2F3 TCR in polyclonal human T cells.

Specific proliferation vs. MBP peptide is shown in the third vertical column; TCR νβ expression is in the bottom row.

Figure 10. A diagram of three ex vivo culture conditions for long-term expansion of functionally stable specific Tregs.

Pre-stimulated Treg cells (CD4+CD25hiCD1271o) were transduced with retroviral 17195 TCR and then 1st round expanded for 8-10 days with three different stimuli: anti-CD3e antibody, FVIII peptide, or FVIII peptide plus oligonucleotide (FVIII peptide/ODN) in the presence of irradiated DR1 PBMCs and IL-2 (200 U/ml). For 2nd round expansion culture, the cells were rested in the media without IL-2 for 48-72 hrs. The stimulating and culturing condition for 2nd expansion was identical with 1st expansion. After 2nd expansion, the expanded cells were rested for 48 hrs in the absence of IL-2 for further experiments. To monitor the quality of each grouped 17195 Tregs, FVIII peptide/DRl tetramer staining and intracellular staining of Foxp3 and Helios were performed every 5 days during the expansion culture.

Figure 11. FVIII pep tide-specific enrichment of transduced

Foxp3+Helios+ Tregs in short-term expansion culture (1st round expansion).

DETAILED DESCRIPTION

Described herein are methods of producing specific human T regulatory cells (Tregs), useful, for example, to reduce undesirable immune responses in conditions such as autoimmunity, including hemophilia, type 1 diabetes, uveitis and multiple sclerosis, as well as in the response to protein therapy for genetic diseases. As used herein the term "specific" Tregs designates Tregs that respond to a specific antigen and inhibit effector T cells that are reactive to a specific antigen.

Specific embodiments of the methods are illustrated as follows: T cell receptors (TCR) from a patient with an autoimmune disease (such as, for example, a hemophilia A patient) are cloned. TCRs can be cloned according to procedures known in the art, including those described in Nature Medicine, 19(11) 1534-41 (2013) and Nature Medicine, 19(11) 1542-46 (2013). Stimulated patient's polyclonal T cells are transduced with the TCRs, thus rendering the expanded cells antigen specific. When the polyclonal Tregs, isolated from peripheral blood, are stimulated under conditions that lead to non-specific Tregs, the cells can then be transduced to become antigen-specific Tregs, capable of specifically suppressing the undesirable immune response to the antigen.

T cells previously have been rendered specific for cancer antigens (CAR's) to specifically destroy cancer cells (turn on immunity). However, the methods described herein produce specific Tregs to modulate (turn off) undesirable immune responses, e.g., to reduce an immune response. The present inventors have now obtained definitive evidence that specific Tregs can be produced using the methods described herein, and that the resulting Tregs are functionally active to inhibit effector T cells that are reactive to a specific antigen (e.g., an FVIII peptide). By expressing a specific TCR directed to a specific antigen, (e.g., human clotting factor VIII, MBP, MOG, GAD65, S-Antigen, or other target), a patient's Tregs can be made specific and used for immunotherapy of the associated disease.

In addition, the methods described herein provide for the first time a protocol for preferential expansion of functionally stable, specific Tregs using specific antigen and oligonucleotide components. The methods therefore are useful for producing Tregsin a cellular therapy to treat, for example, hemophilia patients who are prone to making antibodies against FVIII. Further, the methods can be applied to other diseases associated with undesirable immune responses, such as type I diabetes, multiple sclerosis, uveitis, and other conditions of autoimmunity, or undesireable immune response to protein therapy. .

Thus, described herein are methods for developing antigen-specific T regulatory cells (Tregs), which can be used to treat or minimize unwanted immune responses, such as those that occur in autoimmune diseases and in protein therapy for genetic diseases. Conditions for preferential expansion of functionally stable, specific Tregs using specific antigen and oligonucleotide components also are described.

In accordance with one aspect, there is provided a method for producing antigen specific T regulatory cells comprising transducing T cells with an expression vector comprising a nucleotide sequence encoding a T cell receptor that specifically recognizes the antigen, and expanding the transduced T cells ex vivo. In some embodiments, the T cells are expanded ex vivo to obtain a greater number of functional Tregs and without experiencing functional loss.

In some embodiments, the T cells are obtained from a donor's buffy coat by,for example, FACS sorting (FACSAria, BIC, USUHS) CD4+CD25-CD45RA+ for T naive cells and CD4+CD25 ^hl CD127 ^l0 for Treg cells, prior to being transduced. In some embodiments a pre-stimulation step precedes the transduction step. For example, cells may be pre-stimulated with plate-coated anti-CD38 antibody and plate- coated anti-CD28 antibody for 36 to 48 hours.

In accordance with any of the foregoing embodiments, transducing the T cells may comprise transducing HLA DR1 restricted natural T regulatory cells and/or effector T cells. In some embodiments, the transducing comprises transducing pre- stimulated cells with an amphotrophic packaging retrovirus containing a sequencing encoding a T cell receptor. In specific embodiments, the T cell receptor is specific for clotting factor FVIII. In further specific embodiments, the cells are transduced with an amphotrophic TCR-17195 packaging retrovirus designated TCR-17195-IRES-GFP (i.e., a retroviral construct comprising a sequence encoding the TCR-17195 linked with green fluorescent protein (GFP) expression via intra ribosome entry site (IRES)).

In accordance with any of the foregoing embodiments, the transduction protocol may involve transduction with retroviral culture supernatant in retronectin- coated plates.

In some embodiments, after transduction by viral particles, expressed TCR can be detected flow cytometry based on GFP expression.

In specific embodiments, expansion of transduced Treg or T effector cells may be carried out in IL-2 containing RPMI1640 media with 10% fetal bovine serum (FBS) for up to 4 days.

In specific embodiments, the expansion step of specific Tregs is conducted in accordance with oligonucleotide technology as disclosed in Kim et al,

"Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion," Blood 2012 Mar 22; 119(12):2810-8. doi: 10.1182/blood-

2011-09-377895. Epub 2012 Jan 31, incorporated herein by reference in its entirety.

For example, a 25mer DNA oligonucleotide of random composition may be added during the expansion of Tregs in vitro to prolong stabilization of the Foxp3 Helios ⁺ subpopulation and to yield a population particularly suitable for use in cellular biotherapy. In the oligonucleotide treatment protocol in the long term expansion of antigen-specific Tregs, expanded Tregs express almost the same level of Foxp3 and

Helios as freshly isolated Tregs. Moreover, oligonucleotide treatment maintains demethylation status of Treg-specific demethylation region (TSDR) in the expanded Tregs.

In some embodiments the T cells are subjected to a stimulation step prior to being transduced, and then subjected to an expansion step after transduction.

In some embodiments, the antigen for which the antigen-specific T regulatory cells and/or T effector cells are specific is selected from the group consisting of the C2 domain of human clotting factor VIII (FVIII) or an antigen associated with multiple sclerosis, type I diabetes, uveitis, or other diseases.

An exemplary sequence of a FVIII C2-specific human T cell receptor (TCR17195) is the 620 amino acid sequence of SEQ ID NO:l below:

RQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQ

QFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVG

DAPNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ

TNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP

EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLR

LWSSRAKRGSGATNFSLLKQAGDVEENPGPMLLLLLLLGPGSGLGAVVSQHP

SRVICKSGTSVKIECRSLDFQATTMFWYRQFPKKSLMLMATSNEGSKATYEQ

GVEKDKFLINHASLTLSTLTVTSAQPEDSSFYICSAHTRANYGYTFGSGTRLTV

VEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKE

VHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL

SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGK

ATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 1)

In another embodiment, the antigen for which the antigen-specific T regulatory cells and/or T effector cells are specific is selected is an antigen associated with multiple sclerosis. An exemplary sequence of a Ob.2F3 -specific human T cell receptor is the amino acid sequence of SEQ ID NO:2 below:

ETLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQ WYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSK SSSLLITASRAADTAS YFCATDTTSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTD FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN

NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLL

MTLRLWSSRAKRGSGATNFSLLKQAGDVEENPGPMLLLLLLLGPGISLLLPGS

LAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLML

MATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDL

TSGSLNEQFFGPGTRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATG

FFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFW

QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY

QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Also described herein is a protocol for preferential expansion of functionally stable, specific Tregs using specific antigen and, optionally, oligonucleotide components as described above. This protocol comprises gamma-irradiated DR1- HLA typed PBMCs as antigen presenting cells (6000 rad by Co60) and recombinant IL2 as a growth factor in the media.

EXAMPLES

Identification of FVIII-specific TCR

The cloning strategy for the FVIII-specific TCR from a hemophilia A T effector clone is shown in Fig 1A \ Briefly, cDNA from clone 17A-19wk-5 ² ' ³ (abbreviated

17195TCR) was tagged with a poly-C tail using a terminal transferase reaction. The V regions were then amplified using a common poly-GI forward primer (pGI: 5'- CACCGGGIIGGGIIGGGII-3') and two different sets of human constant region- specific reverse primers (pCl and pC2: pCal, 5'- AGTCAGATTTGTTGCTCC AGGCC-3 ' ; pCbl, 5'- TTCACCCACCAGCTCAGCTCC-3'; pCa2, 5'- ATACGCGTTCTCTC AGCTGGTACACGG-3 ' ; pCb2, 5'-

ATACGCGTAGATCTCTGCTTCTGATGGC-3'). After a second round of PCR with these primers, amplified V regions (500-600 base pairs) were cloned into a TA cloning vector (Invitrogen) (Fig IB). Insertion of PCR product was confirmed by restriction enzyme digestion of plasmid DNA. Extracted individual sequences were nBlast-matched with reference database sequences from the International

ImMunoGeneTics (IMGT) and NCBI. Once the identified a and β chain V regions were identified, the TCR of clone 17195TCR was constructed utilizing the human TCR constant region C region reference sequence. To incorporate the a and β sequences, the a and β chain regions were connected by a P2A peptide linker ⁴ to create a single construct (Fig 1C), and cDNA for green fluorescent protein (GFP) was inserted downstream of the TCR coding region separated by an internal ribosomal entry site (IRES). The re-designed retroviral 17195TCR construct was synthesized by GenScript USA, Inc. (Piscataway, NJ). Retrovirus was produced using a Phoenix- Ampho packaging system.

Isolation, transduction, and expansion of TCR-transduced T effectors and Tregs

Buffy-coat fractions from healthy 20-70-year-old males were provided by the Department of Transfusion Medicine at the National Institutes of Health (NIH) or purchased from the American Red Cross. All procedures were approved by the USUHS Institutional Review Board, and all blood donors provided written informed consent in accordance with the Declaration of Helsinki. To obtain CD4 T-nai ^' ve and Treg cells, peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by ficoll separation (Ficoll-Paque™ Plus, GE Healthcare). CD4 T cells were enriched by positive selection with MACS (Miltenyi Biotech, Auburn, CA). Naive CD4 T cells (CD4 ⁺ CD25 ^~ CD127 ⁺ CD45RA ⁺ ) and Tregs (CD4 ⁺ CD25 ^hi CD127 ^l0 ) were then sorted on a FACSAria (BD Biosciences).

For transduction of 17195TCR, T-nai ^' ve cells or Tregs (2X10 ⁵ /ml) were stimulated with plate-coated anti-CD38 (5 μg/ml) and anti-CD28 antibodies (2 μg/ml) for 48-72 hrs plus recombinant human IL-2 (200 U/ml). Stimulated cells were transferred into 17195TCR viral particle-coated, retronectin (10 μg/ml)-pretreated plates and incubated for 24 hrs. After transduction, the cells were expanded for 3-5 weeks with 6000r gamma-irradiated HLA-DRB 1 *01 :01 (abbreviated hereafter as DR1) PBMCs and peptide FVIII-2191-2220 (0.2-0.5 μg/ml) plus IL-2 (200 U/ml). Irradiated PBMCs (PBMC : T cells = 2 : 1) and FVIII-2191-2220 (0.5 μg/ml) were added to the culture every 2 weeks. Expanded 17195TCR-effectors and 17195TCR- Tregs were rested in the media (RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1% human AB serum, IX Glutamax, and antibiotics) for 48-72 hrs.

Intracellular staining for Foxp3, Helios, and cytokines

Rested cells were re-stimulated with phorbol myristate acetate (PMA; 50 ng/ml) and Ionomycin (1 μg/ml) for 4 hours in the presence of Golgi Stop (0.75 μΐ/ml). Cells were then fixed with 4% paraformaldehyde solution and permeabilized in BSA-containing 0.1% Triton X-100/PBS. Permeabilized cells were stained for Foxp3-APC, Helios-PE, IL-2-PE and IFNy-PECy7 (BD Bioscience).

Cell proliferation and suppression assays

For standard suppression assays, fresh isolated or expanded T effectors were washed 2X in PBS and labeled with 10 μΜ eFluor-450 for 20 min. Labeled cells were washed in FBS-supplemented medium and 2-5 x lO ⁴ cells co-cultured with γ-irradiated DR1-PBMC as APCs in the presence of specific FVIII-2191-2220 or a non-specific peptide (OVA) for 4-5 days. Proliferation was measured by a flow cytometric dye- dilution assay.

An in vitro Treg suppression assay was also performed as previously described ⁵ ' ⁶ . Briefly, 3-4 week-expanded 17195TCR-effectors (4 x 10 ⁴ ) and γ- irradiated DRl-PBMCs were mixed at a ratio of 1 :2 in the absence of IL-2.

17195TCR-Tregs were then added at various ratios and stimulated with soluble anti- CD3 antibody (0.5 μ^ιηΐ), FVIII-2191-2220 (0.5 μ^ιηΐ), or rFVIII (0.2 μ^ιηΐ) for 4- 6 days and cell proliferation was assayed by dye dilution or by thymidine

incorporation following incubation with [ ³ H]-thymidine (1 μΟΛνεΙΙ) during the final 16-18 hours of culture. To measure suppression of cytokine production by Tregs, T effectors were incubated with Tregs (mock or 17195TCR) for 24 hours in the presence of FVIII-2191 -2220 with irradiated DR1 -PBMC and without IL-2. The supernatants were harvested and assayed for cytokine expression using a Thl, Th2 and Thl 7 human CBA kit (BD Bioscience).

DNA methylation analysis of human Treg-specific de-methylation regions (TSDR) Three-week-expanded 17195TCR-Tregs were harvested, washed and genomic DNA extracted using a Wizard Genomic DNA Purification Kit (Promega). Epitect Bisulfite kits (Qiagen) were used to convert unmethylated Cytosines in the genomic DNAs to Uracils. A Promark Q24 instrument was used to detect and quantify methylated TSDR CpGs in the TSDR, using human TSDR-specific primers. Nine CpGs were determined to be methylation-sensitive TSDR CpGs within the Foxp3 genome (-2376 to -2263 from translation starting site).

ELISPOT assay and in vitro suppression of antibody production by FVIII-specific B cells from HLA-transgenic hemophilia A mice

To quantify the in vitro anti-FVIII suppression, "humanized" hemophilia A mice were created by crossing El 6- FVIII knockout (KO) mice ⁷ with DR1- transgenic mice (Dr. Chella David, Mayo Clinic). These mice were immunized subcutaneously with 2μg rFVIII in incomplete Freund's adjuvant and boosted twice with 2μg rFVIII in PBS intraperitoneally 3 and 5 weeks later. Pooled splenocytes from two immunized mice having established titers against FVIII were used as T- responders. Next, lxl 0 ⁷ HLA-DR1-FVIII-KO splenocytes were co-cultured with 17195TCR-Tregs or mock transduced Tregs at various ratios in T25 flasks for 6 days in RPMI 1640 medium supplemented with 10% FBS, 2mM L-glutamine, 50μΜ 2- mercaptoethanol, and 1 μg/ml rFVIII. The effect of these human Tregs on FVIII- specific antibody secreting cell (ASC) formation was measured using an enzyme- linked immunospot (ELISPOT) assay with rFVIII-coated plates ⁸ ' ⁹ . Cells were washed, added to ELISPOT wells in triplicate and cultured overnight. The captured anti-FVIII antibodies were detected by HRP-conjugated anti-mouse IgG (H+L) (Invitrogen), and anti-FVIII ASC revealed with AEC substrate (BD Biosciences). Plates were read on a CTL ELISPOT plate reader (Cellular Technology Limited, Shaker Heights, OH).

Retroviral expression of an engineered FVIII-specific TCR and FVIII-mediated proliferation of polyclonal CD4 T effectors

To prepare T effectors for retroviral transduction of 17195TCR, PBMCs from normal healthy donors were sorted to obtain a naive CD4 ⁺ T population (CD4 ⁺ CD25 ^~ CD 127 CD45RA ⁺ ); natural T-regulatory cells (nTreg, CD4 ⁺ CD25 ^iu CD127 ^l0W ) were also sorted and transduced as described below, stimulated with plate-coated anti- CD3s antibody and anti-CD28 antibody, and transduced with retroviral particles encoding the 17195TCR. Transduced cells were maintained and rested in the presence of IL-2 for 8 days without additional stimuli. Twenty-four hours after transduction, initial transfection efficiency was determined by measuring the GFP expression. GFP ⁺ cells comprised 6-25% of the virus-treated CD4 cells, and this proportion did not change during the maintenance period (2-4 days) (Fig SI A and data not shown). Transduction yields were comparable for mock-transduced cells and 17195TCR- transduced T effectors. The transduced GFP ⁺ CD4 cells were next stained with an anti-Vp2 antibody (recognizing the 17195TCR). The GFP ⁺ cells were predominantly νβ2+ (67% of the GFP ⁺ cells) whereas 10% of the non-transduced (GFP ) cells were νβ2+, indicating endogenous TCR expression levels (Fig 3E). The transduced cells showed GFP intensity that was proportional to the 17195TCR expression level.

To validate the antigen specificity of the 17195TCR, the transduced T effector cells were stained with DR1 tetramers loaded with FVIII-2191-2220. Despite the strong expression of this TCR on GFP ⁺ cells, only a small percentage of expanded naive T cells (less than 5% of the transduced and 0.2% of the total CD4 population) showed a tetramer ⁺ phenotype (Fig 3D), presumably due to pairing of the a and β chains with endogenous TCR chains. In spite of the low percentage of tetramer ⁺ 17195TCR-transduced cells, their tetramer ⁺ staining intensity was more than ten- fold higher and distinguishable from the non-specific tetramer staining (0.41% of the transduced and 0.01% of the total naive CD4 ⁺ population) or of mock-transduced cells (no tetramer ⁺ cells). These results indicated that a subset of the primary CD4 ⁺ cells transduced with the 17195TCR expressed a TCR that recognized FVIII-2191-2220 presented on HLA-DR1.

The functional activity of these 17195TCR-transduced T effectors was tested directly as follows. Ten day-rested TCR-transduced CD4 cells were labeled with a cell proliferation dye, and a proliferation assay was performed with DR1-PBMC presenting FVIII-2191 -2220 or an OVA peptide (pOVA). When 17195TCR- effectors were cultured with FVIII-2191-2220 , almost 100% of the GFP cells

proliferated (Fig 3 A) and the proportion of GFP ⁺ cells increased to 61 - 81%, more than the non-proliferating cells. Expanded GFP ⁺ 17195TCR T effectors bound

specifically to DRl-tetramers loaded with FVIII-2191-2220 : 24% of 17195TCR T effectors were tetramer ⁺ (Fig 3B). These specifically stimulated 17195TCR T

effector GFP ⁺ cells expanded 50X more than those cultured with pOVA (Fig 3C).

Moreover, the increase in absolute cell count suggests that an initial stimulation with FVIII-2191-2220 efficiently enriched the specifically transduced T effector

population.

Production of antigen specific Treg cells and induction ofTreg-specific factors by antigenic stimulation

To produce TCR17195 expressing T regulatory cell (Treg-17195), human

Tregs (CD4 ⁺ CD25 ^hi CD127 ^l0 ) were isolated from healthy donor's buffy coat. Pre- stimulation, retroviral transduction, and expansion of Treg-17195 was done as

described above for effector T cells and as shown in Fig.2. To address C2-specific

Treg activation through TCR17195, expanded Treg-17195 cells were co-cultured with gamma-irradiated DR1-HLA typed PBMCs (6000 rad by Co60) for 24 hrs in the presence of pOVA, pFVIII C2, or soluble anti-CD3e antibody. Foxp3 and GARP

(glycoprotein A repetitions predominant, LRRC32) staining was then done on fixed, co-cultured cells. Foxp3 and GARP are transiently induced at the early stage (24-48 hrs) of activated Tregs. In GFP negative population, the induction of Foxp3 and

Helios was shown only in the anti-CD3e Ab stimulation group and pC2-mediated stimulation did not give any induction of Foxp3 and Helios (Fig. 4 dot plots and

histograms in left box). In GFP positive cells (expressing the TCR), stimulation by C2 induced expression of Foxp3 and Helios in a portion of cells as well as stimulation by CD3e. See Fig. 4. The data clearly show C2-mediated TCR stimulation triggers activation of transduced Treg cells (dot plots and histograms in right box of Figure 4).

Functional stability of transduced Tregs after long-term ex vivo expansion using specific FVIII-2191-2220 and ODN The function of expanded human Tregs in vitro can be stabilized using random oligodeoxynucleotides (ODN) ^10-13 . Thus, we hypothesized that long-term expansion in the presence of FVIII-2191-2220 and ODN would optimize the enrichment of antigen-specific, transduced Treg expressing high levels of Foxp3 and Helios.

Transduced Tregs were cultured with anti-CD3s antibody, FVIII-2191-2220, or FVIII-2191-2220 and ODN (FVIII-2191-2220/ODN), in the presence of DRl-PBMCs plus recombinant IL-2 (Fig 10). After one round of expansion (8 days), the rate of cellular division slowed. The cell numbers and GFP fluorescence intensity after this expansion step were comparable in all groups (data not shown). Foxp3 and Helios in the GFP ⁺ cells were well maintained at levels similar to those of freshly isolated Tregs (Fig 11).

After a second round of expansion, GFP ⁺ cells were dramatically enriched only in the FVIII-2191-2220 and FVIII-2191-2220/ODN culture conditions (72% and 74%), compared to anti-CD38 stimulation (top dot plots in Fig 5A). Moreover, the percentage of tetramer ⁺ cells in the GFP ⁺ cell population was increased in both FVIII- 2191-2220 and FVIII-2191-2220/ODN conditions. Foxp3 and Helios expression after FVIII-2191-2220 stimulation showed a significantly different phenotype than of anti- CD3s. Specifically expanded GFP ⁺ cells maintained Foxp3 and Helios expression, but cells cultured with anti-CD38 did not (bottom dot plots in Fig 5 A). The data in Fig 5B suggest that the FVIII-2191-2220 signal given to 17195TCR Treg during expansion contributed to the enrichment of these FVIII-specific Tregs.

Analysis of the cytokine expression profile (Fig 5C) showed that cells undergoing two rounds of expansion with FVIII-2191-2220/ODN had the lowest percentage of IFNy (20%>) versus anti-CD3 conditioned cells (80%>) and FVIII- 2191-2220 cells (40%) (top dot plots and graph in Fig 5C). The FVIII-2191-2220 stimulated groups had few IL-2 ⁺ cells (bottom dot plots and graph in Fig 5C). These results demonstrate that the FVIII-2191-2220/ODN co-treatment protocol stabilizes Foxp3 and Helios expression and prevents the conversion of Tregs to IFNy-secreting T effectors. The status of Treg-specific demethylated region (TSDR) in ex vivo expanded Foxp3 Helios ⁺ human and murine nTregs is an important marker for stabilization of Tregs ^13~17 . GFP ⁺ cells were FACS-sorted from expanded 17195TCR Tregs and the methylation status of their TSDR CpGs analyzed (Fig 5D). FVIII-2191-2220/ODN- conditioned Tregs showed well-preserved demethylation for each of the TSDR CpGs compared to anti-CD38 conditioned GFP ⁺ cells (Fig 5D).

Evaluation of suppressive function

For the immunosuppression assay, na ^' ive T effector cells and Tregs were prepared as in Figures 2-4. T na ^' ive cells were expanded with C2 and irradiated antigen presenting cells (APC) after viral transduction of TCR17195 for 14 days and the expanded T effector-17195 population was used as responder cells. To monitor cell division of GFP positive cells, responders were labeled with the Cell Proliferation Dye-eFluor450 (CPD450). Tregs cells were transduced with mock virus or

TCR17195, then were expanded for 14 days with anti-CD3e antibody or pC2 peptide. In the co-culture, the ratio of Responders to Stimulators was fixed at 1 : 1 and the amount of Tregs was varied as indicated in Fig. 6.

Cell mixtures were cultured with anti-CD3e antibody or pC2 for 2 days (Fig. 6 A) or 4 days (Fig. 6 B) without IL-2 addition. Treg-17195 produced C2-specific inhibition of division of the proliferation dye CPD450 labeled GFP ⁺ responders (see Fig. 6 A). Treg-17195 produced C2-specific immunosuppression (see Fig. 6 B). To confirm and measure cell growth in mixed culture, [ ³ H] -thymidine was added at 18 hrs before harvest, with similar results.

Since Tregs are known to inhibit the production of inflammatory cytokines ^{18~ 22} , we next determined the levels of IL-2, IFNy, IL-4, and IL-17 produced by T effectors in the presence of Tregs after 24h. At a ratio of 1 :8 (Tregs :T effectors), FVIII-specific Tregs significantly suppressed dominant Thl cytokine (IFNy and TNF) production by T effectors compared to mock Tregs (Fig. 6C). Interestingly, FVIII- specific IL-10 production was also reduced in a FVIII-specific Tregs-dependent manner. Effective suppression of FVIII-specific ASC formation by 17195TCR Tregs in vitro

To determine whether human FVIII-specific Tregs could suppress the humoral anti-FVIII response as well, splenocytes from FVIII -primed HLA-DR1-FVIII-KO mice were co-cultured with rFVIII plus 17195TCR-Tregs or mock-transduced Tregs at different ratios (Fig 7). Specific 17195TCR-Tregs dramatically inhibited anti- FVIII ASC formation at all ratios of Tregs: total splenocytes. Mock-transduced Tregs did not suppress the anti-FVIII antibody response, but at high Tregs: total splenocytes ratios more ASC were detected, presumably due to cytokines produced from xenogeneic recognition. The almost complete suppression of ASC formation by 17195TCR-Tregs suggests involvement of both FVIII-specific and non-specific xenogeneic responses (Fig. 7). In summary, T cell receptor from a patient clone specific for human FVIII was expressed in expanded human T effector and natural T regulatory cells. These cells responded specifically to the FVIII peptide recognized by the original T cell clone and increased in both the number of tetramer binding cells and Treg markers. Tregs also specifically suppressed responder effector cells recognizing FVIII. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production by FVIII-specific B cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, thus validating their translational potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients.

The methods described herein therefore are useful for producing Tregs useful in a cellular therapy to treat hemophilia patients who are prone to making antibodies against FVIII that inhibit its clotting function, as well as in patients with type I diabetes, uveitis, and multiple sclerosis. Further, the methods can be applied to other diseases associated with undesirable immune responses. References

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