CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International Patent Application No. PCT/EP2022/074646, filed Sep. 5, 2022, which claims the benefit of priority to U.S. Ser. No. 63/241,358, filed Sep. 7, 2021, the disclosure of each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a computer readable Sequence Listing which has been submitted in XML file format via Patent Center, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted via Patent Center is entitled “14526-027-999_SEQLISTING.xml”, was created on Jan. 26, 2024 and is 36,002 bytes in size.

1. INTRODUCTION

Provided herein are methods for inducing tolerance to an organ or tissue transplant in a patient. Compositions for use with these methods and kits are also disclosed.

2. BACKGROUND

Long-term results of organ transplantation remain unsatisfactory. Chronic rejection of the donor graft and health issues associated with long-term immunosuppressant use continue to be serious complications of the procedure. These complications, however, could be overcome by inducing tolerance to the donor graft by the recipient's immune system. Tolerance avoids a destructive immune response following transplantation without the need for systemic immunosuppression.

Combined kidney and bone marrow transplantation has been described. See, e.g., Kawai et al. 2014, American Journal of Transplantation 14:1599-1611. This study showed long-term stable kidney allograft survival without maintenance immunosuppression. However, revisions to the conditioning regimen are needed for optimized clinical outcome. The methods disclosed herein describes the refined treatment regimens to provide consistent tolerance to organ or tissue transplantation.

Siplizumab is a humanized IgG1κ monoclonal antibody directed against the human CD2 glycoprotein, a receptor expressed on virtually all mature human T cells and on the vast majority of thymocytes (Bierer B E, Burakoff S J., Immunol Rev. 1989; 111:267-294). CD2 promotes the adhesion of T cells to antigen-presenting cells through its interaction with the ligand LFA-3 (CD58) (van der Merwe P A, Barclay A N, Mason D W, et al. Biochemistry. 1994; 33(33):10149-10160). Binding of CD2 to LFA-3 also leads to a cascade of intracellular signals necessary for T-cell activation, conferring an important costimulatory function to this molecule (Bockenstedt L K. et al., J Immunol. 1988; 141(6):1904-1911; Binder, Christian, et al., (2020). CD2 Immunobiology. Front. Immunol. 11:1090. doi: 10.3389/fimmu.2020.01090).

Ligation of either siplizumab or its parent antibody (BTI-322/Lo-CD2a) to human CD2 promotes T-cell depletion through antibody-dependent cell-mediated cytotoxicity (ADCC), an effect that is notably more pronounced on activated T cells (Branco L. et al., Transplantation. 1999; 68(10):1588-1596; Nizet Y, et al. Transplantation. 2000; 69(7):1420-1428). In addition, these drugs efficiently inhibit the in vitro allogeneic mixed-lymphocyte reaction (MLR), with subsequent hyporesponsiveness upon allogeneic restimulation, while preserving reactivity to non-specific stimulation. This effect was found to be mediated, at least in part, by specific activation-associated T-cell depletion (Latinne D. et al., Int Immunol. 1996; 8(7):1113-1119; Xu Y. et al., Clin Exp Immunol. 2004; 138(3):476-483; Podestà MA, Binder C, Sellberg F, et al. Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro. Am J Transplant. 2019; 20:88-100. https://doi.org/10.1111/ajt.15533; Binder Christian, et al. (2020) Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit AntiThymocyte Globulin In Vitro. Front. Immunol. 11:592553. doi: 10.3389/fimmu.2020.592553).

Siplizumab has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney-bone marrow transplantation (Dey, Bimalangshu, et al. Anti-tumour response despite loss of donor chimaerism in patients treated with non-myeloablative conditioning and allogeneic stem cell transplantation. British Journal of Haematology, 2005, 128, 351-359, doi:10.1111/j.1365-2141.2004.05328.x; Shaffer, Juanita, et al. Regulatory T-cell recovery in recipients of haploidentical nonmyeloablative hematopoietic cell transplantation with a humanized anti-CD2 mAb, MEDI-507, with or without fludarabine. Exp Hematol. 2007 July;35(7):1140-52. doi: 10.1016/j.exphem.2007.03.018).

Siplizumab depletes T cells globally while enriching regulatory T cells (Tregs) early following transplantation. In recipients of hematopoietic cell transplantation to treat malignancies (Shaffer J. et al., Exp Hematol. 2007; 35(7):1140-1152) and CKBMT recipients (Andreola G. et al., Am J Transplant. 2011; 11(6):1236-1247), a marked early enrichment in regulatory T cells (Tregs) was observed during the T-cell reconstitution phase (Savage, Thomas, et al., Early expansion of donor-specific Tregs in tolerant kidney transplant recipients. JCI Insight. 2018 Nov. 15; 3(22): e124086. doi: 10.1172/jci.insight.124086; Sprangers, Ben, et al. Origin of Enriched regulatory t cells in patients receiving combined kidney/bone marrow transplantation to induce transplantation tolerance. Am J Transplant. 2017 Aug; 17(8): 2020-2032. doi: 10.1111/ajt.14251; LoCascio, Samuel, et al. Mixed Chimerism, Lymphocyte Recovery and Evidence for Early Donor-Specific Unresponsiveness in Patients Receiving CKBMT to Induce Tolerance. Transplantation. 2010 Dec. 27; 90(12): 1607-1615. doi:10.1097/TP.0b013e318lffbaff).

Previous studies have shown that anti-IL6R treatment restores the physiological Treg/Th17 balance which may be important for the clinical response observed in patients with rheumatoid arthritis when administered humanized IL6R antibodies (Schinnerling, et al., Clin Exp Immunol. 2017 189(1):12-20). Anti-IL6R treatment has been shown to promote the expansion of protective Treg cells; Tada, BMC Musculoskelet Disord. 2016 17:290). Additionally, the use of an anti-IL6R blockade is efficient in treating cytokine release syndrome (CRS) (Brudno J N. and Kochenderfer J N. Blood. 2016 127(26):3321-30; Hay, K. Br J Haematol. 183(3):364-374. 2018). The use of an anti-IL6R antibody to block IL6 signaling is FDA approved as a therapeutic for CRS in CAR-T cell patients, (Le, et al., Oncologist. 2018 (8):943-947. 2018; Highlights of Prescribing Information; ACTEMRA® (tocilizumab) injection, for intravenous or subcutaneous use. Genentech, Inc. 2017).

3. SUMMARY

Provided herein are methods for inducing transient mixed chimerism in a transplant-eligible patient, wherein the methods comprise administering an anti-CD2 antibody or antigen binding fragment thereof to the patient, subjecting the patient to an organ or tissue transplant, infusing bone marrow cells from a donor to the patient; and administering one or more than one agent(s) to the subject prior to and/or after transplant. In certain embodiments, the chimerism can persist for several weeks to months after the transplant surgery. In certain embodiments, the methods include a conditioning regimen and a postoperative regimen. In certain embodiments, the methods induce tolerance in the recipient towards the transplanted organ or tissue. This tolerance can be long-lasting or permanent and minimized risk of Graft versus Host Disease.

Without being bound by theory, hematopoeitic cells, obtained from the bone marrow of the donor, can be infused into the recipient to create a state in which donor and recipient hematopoeitic cells coexist. In combination with non-myeloablative conditioning, bone marrow transplant results in chimerism of the recipient, leading to the induction of donor-specific tolerance. Advantages of this approach include the avoidance of ablating the recipient's immune system and inhibiting the development of Graft versus Host diseases without the need for long-term systemic immunosuppressive use.

In one aspect provided herein is a method of transplanting an organ or a tissue from a donor to a subject, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) administering fludarabine to the subject; d) transplanting the organ or the tissue into the subject; and e) infusing bone marrow cells from the donor to the subject.

In one aspect provided herein is a method of transplanting an organ or a tissue from a donor to a subject, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein the B-cell depleting antibody is administered to the subject more than 7 days prior to the transplanting.

In one aspect provided herein is a method of transplanting an organ or a tissue from a donor to a subject, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein a first dose of the anti-CD2 antibody or antigen binding fragment thereof is 1 day prior to the transplanting.

In one aspect provided herein is a method of transplanting an organ or a tissue from a donor to a subject, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject more than 2 days prior to the transplanting.

In one aspect provided herein is a method of minimizing chimeric transition syndrome in a subject in need of an organ or a tissue transplant, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) administering fludarabine to the subject; d) transplanting the organ or the tissue into the subject; and e) infusing bone marrow cells from the donor to the subject.

In one aspect provided herein is a method of minimizing chimeric transition syndrome in a subject in need of an organ or a tissue transplant, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein the B-cell depleting antibody is administered to the subject more than 7 days prior to the transplanting.

In one aspect provided herein is a method of minimizing chimeric transition syndrome in a subject in need of an organ or a tissue transplant, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein a first dose of the anti-CD2 antibody or antigen binding fragment thereof is 1 day prior to the transplanting.

In one aspect provided herein is a method of minimizing chimeric transition syndrome in a subject in need of an organ or a tissue transplant, wherein the method comprises: a) administering a B-cell depleting antibody to the subject; b) administering an anti-CD2 antibody or antigen binding fragment thereof to the subject; c) transplanting the organ or the tissue into the subject; and d) infusing bone marrow cells from the donor to the subject; wherein the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject more than 2 days prior to the transplanting.

In some aspects, the B-cell depleting antibody is administered to the subject 9 days prior to the transplanting. In some aspects, the B-cell depleting antibody is administered to the subject 9 days prior to and 2 days prior to the transplanting. In some aspects, the B-cell depleting antibody is administered to the subject 9 days prior to and 2 days prior to the transplanting and 5 days post and 12 days post the transplanting. In some aspects, the B-cell depleting antibody is rituximab. In some aspects, the B-cell depleting antibody is administered to the subject at a dose of about 375 mg/m². In some aspects, the B-cell depleting antibody is not administered 7 days prior to the transplanting. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject 1 day prior to the transplanting, on the day of the transplanting, and 1 day post the transplanting. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject 6 days prior to the transplanting, 1 day prior to the transplanting, on the day of the transplanting, and 1 day post the transplanting. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject 6 days prior to the transplanting, 1 day prior to the transplanting, on the day of the transplanting, 1 day post the transplanting, and 6 days post the transplanting. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is siplizumab. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject at a dose of about 0.6 mg/kg. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof comprises: a) a heavy chain variable region CDR 1 of SEQ ID NO:3; and b) a heavy chain variable region CDR 2 of SEQ ID NO:4; and c) a heavy chain variable region CDR 3 of SEQ ID NO:5; and d) a light chain variable region CDR 1 of SEQ ID NO:6; and e) a light chain variable region CDR 2 of SEQ ID NO:7; and f) a light chain variable region CDR 3 of SEQ ID NO:8. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is a humanized antibody. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject at a dose of more than about 0.1 mg/kg. In some aspects, the anti-CD2 antibody or antigen binding fragment thereof is administered to the subject at a dose of about 0.6 mg/kg. In some aspects, each dose of the anti-CD2 antibody or antigen binding fragment thereof that is administered to the subject comprises the same dose.

In some aspects, the method provided herein further comprises administering a non-myeloablative conditioning agent to the subject. In some aspects, the non-myeloablative conditioning agent comprises an immunosuppressive agent. In some aspects, the immunosuppressive agent is cyclophosphamide. In some aspects, the immunosuppressive agent is administered to the subject 5 days prior to and 4 days prior to the transplanting. In some aspects, the immunosuppressive agent is administered to the subject at a dose of about 60 mg/kg. In some aspects, the immunosuppressive agent is administered to the subject at a dose of about 22.5 mg/kg. In some aspects, the non-myeloablative conditioning agent comprises an anti-neoplastic agent. In some aspects, the anti-neoplastic agent is a chemotherapy drug. In some aspects, the anti-neoplastic agent is fludarabine. In some aspects, the anti-neoplastic agent is administered to the subject 6 days prior to, 5 days prior to, 4 days prior to, and 3 days prior to the transplanting. In some aspects, fludarabine is administered to the subject 6 days prior to, 5 days prior to, 4 days prior to, and 3 days prior to the transplanting. In some aspects, the anti-neoplastic agent is administered to the subject at a dose of about 10 mg/m². In some aspects, the fludarabine is administered to the subject at a dose of about 10 mg/m².

In some aspects, the infusing bone marrow cells is on the same day as the transplanting. In some aspects, the method further comprises providing thymus irradiation to the subject. In some aspects, the thymus irradiation is provided to the subject 1 day prior to the transplanting. In some aspects, the thymus irradiation is provided to the subject at about 7 Gy.

In some aspects, the method provided herein further comprises administering an anti-IL6R antibody to the subject. In some aspects, the anti-IL6R antibody is tocilizumab. In some aspects, the anti-IL6R antibody is administered to the subject at a dose of about 8 mg/kg. In some aspects, the anti-IL6R antibody is administered to the subject 7 days and 14 days post the transplanting. In some aspects, the anti-IL6R antibody is administered to the subject after the transplanting. In some aspects, the anti-IL6R antibody is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the anti-IL6R antibody is administered to the subject after about 3-4 months after the transplanting.

In some aspects, the method provided herein further comprises administering a steroid to the subject. In some aspects, the steroid is administered to the subject on the day of the transplanting. In some aspects, the steroid is administered to the subject for about 20 days after the transplanting. In some aspects, the steroid is administered to the subject for about 6 months after the transplanting. In some aspects, the steroid is corticosteroid. In some aspects, the steroid is administered to the subject after the transplanting. In some aspects, the corticosteroid is administered to the subject after the transplanting. In some aspects, the steroid is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the corticosteroid is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the steroid is administered to the subject after about 3-4 months after the transplanting. In some aspects, the corticosteroid is administered to the subject after about 3-4 months after the transplanting.

In some aspects, the method provided herein further comprises administering tacrolimus to the subject. In some aspects, the tacrolimus is administered to the subject at a dose of about 4-11 ng/mL. In some aspects, the tacrolimus is administered to the subject at a dose of about 8-10 ng/mL. In some aspects, the tacrolimus is administered to the subject on the day of the transplanting. In some aspects, the tacrolimus is administered to the subject for about 9-12 months post the transplanting. In some aspects, the tacrolimus is administered to the subject for about 1 month post the transplanting. In some aspects, the tacrolimus is administered to the subject after the transplanting. In some aspects, the tacrolimus is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the tacrolimus is administered to the subject after about 3-4 months after the transplanting.

In some aspects, the method provided herein further comprises administering mycophenolate mofetil to the subject. In some aspects, the mycophenolate mofetil is administered to the subject at a dose of about 2 g/day. In some aspects, the mycophenolate mofetil is administered to the subject on the day of the transplanting. In some aspects, the mycophenolate mofetil is administered to the subject for about 2 months post the transplanting. In some aspects, the mycophenolate mofetil is administered to the subject after the transplanting. In some aspects, the mycophenolate mofetil is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the mycophenolate mofetil is administered to the subject after about 3-4 months after the transplanting.

In some aspects, the method provided herein further comprises administering sirolimus to the subject. In some aspects, the sirolimus is administered to the subject at a dose of about 5-8 ng/mL. In some aspects, the sirolimus is administered to the subject at about or after about 1 month post the transplanting. In some aspects, the sirolimus is administered to the subject for up to about 12 months after the transplanting. In some aspects, the sirolimus is administered to the subject after the transplanting. In some aspects, the sirolimus is administered to the subject after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplanting. In some aspects, the sirolimus is administered to the subject after about 3-4 months after the transplanting.

In some aspects, the method provided herein induces mixed chimerism in the subject. In some aspects, the mixed chimerism is characterized by a percentage of donor cells in the lymphohematopoietic system of the subject of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or at least about 90%. In some aspects, the mixed chimerism persists for about or at most about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or at most 12 months. In some aspects, the ratio of FoxP3+ T cells to CD4+ T cells is increased in the subject relative to the ratio of FoxP3+ T cells to CD4+ T cells in the absence of the anti-CD2 antibody or antigen binding fragment thereof. In some aspects, the FoxP3+ expression persists in actively proliferating T cells after the last administration of the anti-CD2 antibody or antigen binding fragment thereof.

In some aspects, the method provided herein reduces the risk of Graft versus Host Disease in the subject. In some aspects, the organ or the tissue is kidney. In some aspects, the subject is a human. In some aspects, the donor is a human. In some aspects, the method provided herein induces tolerance in the subject to the transplanted organ or tissue.

4. BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

FIG. 1. Graph showing tolerance overview.

FIG. 2. Table showing schedule of assessment.

FIG. 3. Graph showing tolerance study overview.

FIG. 4. Table showing schedule of assessment.

FIG. 5. Graph showing IL-6 mAb (e.g., tocilizumab) treatment for chimeric transition syndrome (CTS). Graph shows administration of anti-CD2 antibody or antigen binding fragment thereof (e.g., siplizumab) on days −6, −1, 0, and 1 (where day 0 is day of transplant) and administration of IL-6 mAb (e.g., tocilizumab) only if chimeric transition syndrome (CTS) occurs.

FIG. 6. Graph showing prophylactic IL-6 mAb (e.g., tocilizumab) treatment on days 7 and 14 and administration of anti-CD2 antibody or antigen binding fragment thereof (e.g., siplizumab) on days−6,−1, 0, and 1 (where day 0 is day of transplant).

FIG. 7. Graph showing anti-CD2 antibody or antigen binding fragment thereof (e.g., siplizumab) administered 6 days prior to transplant, 1 day prior to transplant, on the same day as transplant, 1 day post transplant, and 6 days post transplant, and prophylactic anti-IL-6R mAb (e.g., tocilizumab) treatment on days 7 and 14.

5. DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods for the induction of tolerance of an organ or tissue transplant recipient's immune system towards the transplanted organ or tissue without the need for ongoing immunosuppressive therapy. Provided herein are methods for the combined organ/tissue and bone marrow transplantation preceded by specific conditioning treatment regimen and followed by a specific post-operative treatment regimen. The regimen can be a non-myeloablative procedure including administration of antibodies, immunosuppressors, and radiation that depletes the immune cells of the recipient. In certain embodiments, the components of the regimen can be modified as described herein to achieve mixed chimerism in the recipient. In certain embodiments, the components of the regimen can be modified as described herein to achieve induction of tolerance of an organ/tissue transplant recipient's immune system towards the transplanted organ/tissue without the need for ongoing immunosuppressive therapy.

Organ or tissue transplants and infusion of bone marrow for use with the methods provided herein are described in Section 5.2. Initial conditioning regimen and/or post operative treatment for use with the current methods are described in Section 5.3. Transplantation and bone marrow infusion are described in Section 5.4 and Section 5.5. Methods to assess clinical outcomes are described in Section 5.6. Pharmaceutical compositions are described in Section 5.7. Kits are described in Section 5.8. Examples of the methods provided herein are described in Section 6.

In a specific embodiment, an individual who has been selected to receive or has received an organ or tissue transplant may be treated using the methods described herein. Without being bound by theory, following the treatment regimen induces a state of transient, mixed chimerism. In a specific embodiment, a treatment regimen comprises:

- a. Administering rituximab (375 mg/m²) to the recipient at 9 days prior to, 2 days prior to, 5 days post, and 12 days post transplant surgery;
- b. Administering anti-CD2 antibody or antigen binding fragment thereof (e.g., siplizumab at about 0.6 mg/kg) 6 days prior to, 1 day prior to, on the same day, and 1 day post transplant surgery, OR administering anti-CD2 antibody or antigen binding fragment thereof (e.g., siplizumab at about 0.6 mg/kg) 6 days prior to, 1 day prior to, on the same day, 1 day post, and 6 days post transplant surgery;
- c. Administering cyclophosphamide (60 mg/kg) at 5 days prior and 4 days prior to the transplant surgery;
- d. Administering thymus irradiation (e.g., 7 Gy) 1 day prior to transplant surgery; and
- e. Administering tocilizumab (e.g., 8 mg/kg) if chimeric transition syndrome (CTS) occurs or on days 7 and 14 post transplant surgery.

In some embodiments, a treatment regimen further comprises administering tacrolimus (e.g., after the transplant (e.g., at a dose of about 4-11 ng/mL), after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplant, and/or 4-11 ng/mL on the day of transplant and for 9-12 months post transplant). In some embodiments, a treatment regimen further comprises administering mycophenolate mofetil (MMF) (e.g., after the transplant (e.g., at a dose of about 2 g/day), after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplant, and/or 2 g/day on the day of transplant and for 2 months post transplant). In some embodiments, a treatment regimen further comprises administering steroid or corticosteroids (e.g., after the transplant, after about or after at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months after the transplant, and/or on the day of transplant and for 20 days post transplant).