Methods for monitoring an allogenic transplant recipient for potential indications of rejection

Description

METHODS FOR MONITORING AN ALLOGENIC TRANSPLANT RECIPIENT FOR POTENTIAL INDICATIONS OF REJECTIONCross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 504,885, filed on May 30, 2023, the disclosure of which is incorporated by reference.Background

[0002] The need for prolonged immunosuppressive therapy has been the main drawback in considering allogeneic hair transplantation between two histocompatible persons. Two previous case reports of allogenic transplantation have exclusively involved bone marrow (BM) transplant recipients, whereby, apart from HLA compatibility, the chimeric immune tolerance inherent to the procedure obviates the need for immunosuppressive treatment.3,4 In the absence of chimeric immune tolerance, person-to-person hair transplantation is a viable option between two unrelated histocompatible individuals in selected patients that already require lifelong immunosuppressive therapy. Because more solid organ transplants are performed annually than bone marrow transplants, solid organ transplant recipients constitute a larger transplant recipient population that may benefit from person-to-person hair transplantation based on proven histocompatibility, successful solid organ transplantation, and a pre-existing condition of lifelong immunosuppressive treatment. Furthermore, the less invasive nature of the procedure used to harvest donor hair follicles, the follicular unit excision (FUE) technique, makes the donor harvesting procedure more acceptable to matched donors.Summary of the Invention

[0003] Applicant recognized that transplanted allogenic hair follicles could serve as proxies for the viability of allotransplants of solid organs, like kidneys. According to one aspect of the invention, methods monitor for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, such methods having steps of: (A) transplanting one or more allogenic follicular units from the same donor of the solid organ to one or more sites on the skin of the recipient; (B) monitoring the recipient for one or more indications of potential acute or chronic transplant rejection by monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of an immune response and potential rejection of the transplanted solid organ; and (C)if one or more indications of inflammation and an immune response against one or more of the transplanted follicular units are detected: (i) administer immunosuppressive therapy to the recipient or modify the recipient's existing immunosuppressive therapy to ameliorate or prevent the onset of acute or chronic organ rejection; and / or (ii) perform additional diagnostic procedures to confirm onset of acute or chronic organ rejection.

[0004] Follicular units can be placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions.

[0005] One or more indications of potential acute or chronic transplant rejection are the detection of one or more of the following events at the one or more follicular unit placement sites: an antibody- mediated response; unwanted alloreactivity; an acute or chronic host versus graft disease; de novo development of donor specific human leukocyte antigen (HLA) antibodies; an increase in the concentration of polycytotoxic T cells, an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates; and an imbalance of CD4:CD8 T-cell ratios.

[0006] Immunosuppressive therapy is administered to a recipient, or preexisting immunosuppressive therapy is modified for a recipient, wherein the therapy comprises the administration of one or more of: a corticosteroid; a cytostatic drug; an anti-TNFa antibody; an IL-2 receptor inhibitor, or other antibodybased immunosuppressive therapy.

[0007] A corticosteroid can be, for example, prednisone; a cytostatic drug can be, for example, tacrolimus, mycophenolate, mycophenolate mofetil, or ciclosporin; and an IL-2 receptor inhibitor can be basiliximab.

[0008] In another aspect of the invention, methods for select a candidate allogeneic solid organ donor for a recipient, including the steps of: (A) transplanting one or more follicular units excised from one or more candidate organ donors and placing the excised follicular units to one or more sites on the skin of the recipient; (B) monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of the potential rejection of a transplanted solid organ from the donor; (C) selecting one or more donor candidates for organ harvesting if no indications or therapeutically acceptable indications of inflammation and an immune response against one or more of the transplanted follicular units are detected; and (D) transplanting the donated organ to the recipient.

[0009] In yet another aspect of the invention, methods ameliorate the onset of an acute rejection of a transplanted allogeneic solid organ, like a kidney, in a recipient, the steps of such a method include (A)transplanting one or more allogenic follicular units from the donor of transplanted allogeneic solid organ to one or more sites on the skin of the recipient; ( B) monitoring the recipient for one or more indications of potential acute transplant rejection by monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of acute rejection of the transplanted solid organ; and (C) administering immunosuppressive therapy to the recipient or modify the recipient's existing immunosuppressive therapy to ameliorate acute rejection of the transplanted organ if one or more indications of acute rejection are detected.Brief Description of the Figures

[0010] Fig. 1A contains a "before photo" of a patient depicting a Norwood VI-VI I bald state. 119x137mm (300 x 300 DPI)

[0011] Fig. 1B contains a photo of the same patient in Fig. 1A immediately after grafting frontal scalp with 2350 allogenic follicular units. 119x137mm (300 x 300 DPI)

[0012] Fig. 1C contains a photo of the same patient in Figs. 1A-B at 6-9 Months post-surgery, the grafted area shows robust growth of grafted follicles. 119x137mm (300 x 300 DPI)

[0013] Fig. 2A shows that the histology of the transplanted recipient zone at 6 months post allogenic transplantation includes perifollicular infundibulo-isthmal lymphocytic infiltrates.

[0014] Fig. 2B shows that the transplanted recipient zone at 6 months post allogenic transplantation also contains CD8 lymphocytes.

[0015] Fig. 2C shows that the transplanted recipient zone at 6 months post allogenic transplantation also contains CD4 lymphocytes, and that the distribution of CD8:CD4 lymphocytes is 2:1.Detailed Description

[0016] This disclosure describes compositions and methods that utilize transplanted allogenic follicular units as proxies for detecting, monitoring, or detecting and monitoring rejection of transplanted allogenic solid organs or organ tissue harvested from the same donor and transplanted into the same host. In other words, the disclosure describes inventions that utilize transplanted allogenic hair follicles as "witness follicles" or "witness follicular units" to track the viability of transplanted organs harvested from the same allogenic donor of the hair follicles. Thus, it follows that compositions and methods of the invention can be used clinically in therapies to ameliorate rejection of transplanted allogenic organs and preserve the viability of the transplanted organs by administeringtherapies to prevent further tissue destruction. Typically, such therapies are immunosuppressive therapies administered to downregulate the organ recipient's immune response to the transplanted, allogenic solid organ or organ tissue.

[0017] Some methods of the invention monitor an allogenic transplant recipient for potential indications of solid organ rejection by transplanting one or more follicular units harvested from the same donor of the allogenic solid organ that was previously transplanted in the recipient. Accordingly, the same individual is the donor of the allogenic follicular units and the allogenic solid organ. In the context of this disclosure, a "solid organ" is typically, but not limited to, a kidney. Other examples of solid organs include liver and one or more lobes of a lung. Indeed, any solid organ or functional portion thereof, that can be harvested from a living donor that survives the harvesting procedure can be considered a solid organ that is transplanted to an allogenic recipient in a method of the invention. A transplanted organ or follicular unit that is "allogenic" means that the donor of an organ or follicular unit recipient is genetically non-identical to the recipient of the transplanted tissue, which may also be referred to as an allogeneic transplant.

[0018] In methods of the invention, follicular units are typically harvested by a technique called follicular unit excision (FUE), which is a process by which individual follicular units are harvested for implantation at an incision site on the scalp. The harvested follicular units can be placed at any site on the skin of the recipient. For example, in a method of the invention, follicular units are placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions.

[0019] In methods of the invention, the step of monitoring an allogenic transplant recipient for potential indications of organ rejection is carried out by monitoring the skin site, where donor follicular units were placed, for indications of inflammation and an immune response against one or more of the transplanted follicular units. More specifically, some methods of the invention detect an immune response at or near the allogenic follicular unit placement sites. In a method of the invention, for example, detecting an immune response against follicular units includes the detection of an antibody- mediated response, while in another method of the invention, the method detects unwanted al loreactivity. Other examples of detectable immune responses at one or more allogenic follicular unit placement sites include: the detection of an acute or chronic host versus graft disease, the detection of the de novo development of donor specific human leukocyte antigen (HLA) antibodies, the detection of an increase in the concentration of polycytotoxic T cells, the detection of an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates, and the detection of imbalances of CD4:CD8 T-cell ratios.The detection of one or more immune responses at one or more allogenic follicular unit placement sites is indicative of an immune response against the transplanted solid organ.

[0020] Accordingly, in a method of the invention, follicular units are placed at a site on the skin of a subject that was previously the recipient of an allogenic kidney transplant from the same donor of the follicular units, and the follicular unit placement sites are monitored for one or more of: the detection of an acute or chronic host versus graft disease, the detection of the de novo development of donor specific human leukocyte antigen (HLA) antibodies, the detection of an increase in the concentration of polycytotoxic T cells, the detection of an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates, and the detection of imbalances of CD4:CD8 T-cell ratios. In such a method in which donor HLA-specific antibodies are detected, the detected antibodies can be IgA, IgM, IgD, IgE, or IgG antibodies. In such a method in which the CD4:CD8 T-cell ratio is determined to be imbalanced, the imbalance is relative to a CD4:CD8 ratio typically found in healthy tissue and blood levels. In some methods of the invention the CD4:CD8 ratio in healthy tissue or blood levels is 1:2 and an imbalanced CD4:CD8 ratio is: 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10, or more.

[0021] Methods of the invention for monitoring an allogenic transplant recipient for potential indications of organ rejection can also include one or more steps to administer therapy to the recipient to prevent or ameliorate organ rejection if one or more indications of an immune response at a recipient's allogenic follicular unit placement site. Typically, such therapy is an immunosuppressive therapy. For example, an immunosuppressive therapy of the invention administers a corticosteroid (e.g., prednisone), cytostatic drug (e.g., tacrolimus, mycophenolate, mycophenolate mofetil, ciclosporin), an anti-TNFa antibody, IL-2 receptor inhibitors such as basiliximab, or other antibody-based immunosuppressive therapy. Because transplant recipients are typically administered lifetime regimens of immunosuppressive drugs or cocktails of immunosuppressive drugs, if a method of the invention detects one or more indications of an immune response at a recipient's allogenic follicular unit placement site, and the recipient had received immunosuppressive therapy prior to and concurrently with an allogenic follicular unit transplant procedure, the immunosuppressive therapy administered according to the invention will modify or replace the immunosuppressive therapy that the recipient had been receiving prior to the detection of one or more indications of an immune response at a recipient's allogenic follicular unit placement site. In some methods of the invention, the recipient undergoes diagnostic procedures to confirm the onset of acute or chronic organ rejection.

[0022] As stated above, the invention also includes methods for selecting a candidate allogeneic solid organ donor for transplant recipients. In such a methods, one or more follicular units are excised fromone or more candidate organ donors and transplanted to one or more sites on the skin of the recipient, and then monitored for indications of inflammation and an immune response against one or more of the transplanted follicular units. The detection of indications of inflammation and / or an immune response against one or more of the transplanted follicular units is indicative of the potential rejection of a transplanted solid organ from the donor. Accordingly, one or more organ donor candidates are selected for harvesting of an organ for transplantation if there are no indications or therapeutically acceptable indications of inflammation and an immune response against one or more of the transplanted follicular detected. In some methods of the invention for selecting a candidate allogeneic solid organ donor for transplant recipients, the organ is a kidney.

[0023] Harvested follicular units can be placed at any site on the skin of the recipient. For example, in a method of the invention, follicular units are placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions. In methods of the invention for selecting a candidate allogeneic solid organ donor for a recipient, one or more indications of potential acute or chronic transplant rejection can include the detection of one or more of the following events at the one or more follicular unit placement sites: an antibody-mediated response; unwanted alloreactivity; an acute or chronic host versus graft disease; de novo development of donor specific human leukocyte antigen (HLA) antibodies; an increase in the concentration of polycytotoxic T cells, an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates; and an imbalance of CD4:CD8 T-cell ratios.

[0024] As stated above, the invention also includes methods for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ in a recipient. In some such methods, the onset of acute rejection of a transplanted kidney is ameliorated.

[0025] In methods of the invention for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, like a kidney, one or more allogenic follicular units harvested from the donor of transplanted allogeneic solid organ are transplanted to one or more sites on the skin of the recipient, and then monitored for one or more indications of potential acute transplant rejection at one or more of the transplanted follicular unit sites. Immunosuppressive therapy is administered to the recipient or the recipient's existing immunosuppressive therapy is modified to ameliorate acute rejection of the transplanted organ if one or more indications of acute rejection are detected.

[0026] Methods of the invention for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, like a kidney, can also include one or more steps to administer therapy to the recipient to ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ if oneor more indications of an immune response are detected at a recipient's allogenic follicular unit placement site. Typically, such therapy is an immunosuppressive therapy. For example, an immunosuppressive therapy of the invention administers a corticosteroid (e.g., prednisone), cytostatic drug (e.g., tacrolimus, mycophenolate, mycophenolate mofetil, ciclosporin), an anti-TNFa antibody, IL-2 receptor inhibitors such as basiliximab, or other antibody-based immunosuppressive therapy. Because transplant recipients are typically administered lifetime regimens of immunosuppressive drugs or cocktails of immunosuppressive drugs, if a method of the invention detects one or more indications of an immune response at a recipient's allogenic follicular unit placement site, and the recipient had received immunosuppressive therapy prior to and concurrently with an allogenic follicular unit transplant procedure, the immunosuppressive therapy administered according to the invention will modify or replace the immunosuppressive therapy that the recipient had been receiving prior to the detection of one or more indications of an immune response at a recipient's allogenic follicular unit placement site. In some methods of the invention, the recipient undergoes diagnostic procedures to confirm the onset of acute or chronic organ rejection.Examples

[0027] Example 1. - Allogeneic Hair Transplant in a Kidney Transplant Recipient

[0028] The first allogeneic hair transplant based solely on HLA compatibility was performed using a follicular unit excision (FUE) method to harvest follicular units from an individual that had previously donated a kidney and transplant the follicular units to a severely bald recipient that had also been the recipient of the follicular unit donor's kidney. The recipient of the follicular units was a 60-year-old male, who received a matched kidney allograft from an unrelated friend - the follicular units donor - two decades earlier. The recipient presented with Norwood VI-VI I level androgenic alopecia Fig. 1. The recipient's alopecia had been unsuccessfully treated over the course of the preceding three years by twice daily topical minoxidil 5%, one year of oral finasteride 1 mg daily, and several monthly sessions of platelet-rich plasma. The patient had also received a well-tolerated two-decade-long immunosuppressant regimen that included the administration of tacrolimus 2.5 mg / kg / day, mycophenolate mofetil 500 mg / day, and prednisone 0.5 mg / kg / day for his renal allotransplant.

[0029] The follicular units recipient requested the use of hair from his willing kidney donor because the recipient an insufficient amount of hair for autotransplantation because of his extensive baldnessand to and his preference to minimize the amount of surgery. The recipient was also already on lifelong immunosuppressive therapy to mitigate kidney allograft rejection.

[0030] The FUE method of hair transplantation was used because it is minimally invasive and avoids the linear scars of other techniques. After obtaining informed consent from the recipient and the donor, 2350 follicular units were harvested from the safe donor scalp zones of the kidney donor by FUE and grafted into the bald frontal scalp of the kidney recipient (Fig, 2). The procedure was well tolerated, and the patient was discharged to continue his usual immunosuppressive treatment.

[0031] The recovery period was uneventful, with an 85% yield manifesting at 9 months (Fig. 3) based on hair density measurements. At 6 month follow-up, blood work and trichoscopy of the recipient area were unremarkable, while histologic analysis revealed moderate perifollicular infundibulo-isthmic lymphocytic infiltration with no fibrosis or remarkable epithelial changes in the grafted zones (Fig. 4). The non-grafted zone showed rare interfol licular dermal lymphocytes with no fibrosis or epithelial changes. Immunohistochemical staining for CD4 and CD, 8 of the grafted vs. non-grafted zones, showed 30-45% CD4; 45-60% CD8 - Ratio 1:2 in both zones. Additional hair transplants are planned to address remaining bald areas using an expanded donor supply by combining autologous and allogenic grafts.

[0032] The finding of moderate perifollicular infundibulo-isthmic lymphocytic infiltration in the recipient area is consistent with results reported in successful autologous hair transplants. It is encouraging that the CD4:CD8 ratio mirrors the finding in the non-transplanted area, suggesting no indication of impending graft rejection. This is the first report of a successful allogeneic hair transplant from a solid organ donor to its recipient.

Claims

What is claimed:

1. A method of monitoring for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, the method comprising:(A) transplanting one or more allogenic follicular units from the same donor of the solid organ to one or more sites on the skin of the recipient;IB) monitoring the recipient for one or more Indications of potential acute or chronic transplant rejection by monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of an immune response and potential rejection of the transplanted solid organ; and(C) if one or more indications of inflammation and an immune response against one or more of the transplanted follicular units are detected:(I) administer immunosuppressive therapy to the recipient or modify the recipients existing immunosuppressive therapy to ameliorate or prevent the onset of acute or chronic organ rejection; and / or(ii) perform additional diagnostic procedures to confirm onset of acute or chronic organ rejection.

2. The method of claim 1 for monitoring for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, wherein the follicular units are placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions.

3. The method of claims 1 or 2 for monitoring for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, wherein the one or more indications of potential acute or chronic transplant rejection are the detection of one or more of the following events at the one or more follicular unit placement sites: an antibody-mediated response; unwanted ailoreactivity; an acute or chronic host versus graft disease; de novo development of donor specific human leukocyte antigen (HLA) antibodies; an increase in the concentration of polycytotoxic T cells, an increase in perifollicular infundibulo-isthrnal lymphocytic infiltrates; and an imbalance of CD4;CD8 T-cell ratios.

4. The method of any one of claims 1-3 for monitoring for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, wherein immunosuppressive therapy is administered to the recipient, or preexisting immunosuppressive therapy is modified, wherein the therapy comprises the administration of one or more of: a corticosteroid; a cytostatic drug; an anti-TNFct antibody; an IL-2 receptor inhibitor, or other antibody-based immunosuppressive therapy.

5. The method of claim 4, wherein: the corticosteroid is prednisone; the cytostatic drug is tacrolimus, mycophenolate, mycophenolate mofetii, or ciclosporin; and the IL-2 receptor inhibitor is basiliximab'6. The method of any one of claims 1-5 for monitoring for potential indications of organ rejection in a recipient of a previously transplanted allogenic solid organ, 'wherein the donated organ is a kidney or viable fragment thereof.

7. A method for selecting a candidate allogeneic solid organ donor for a recipient, the method comprising:(A) transplanting one or more follicular units excised from one or more candidate organ donors and placing the excised follicular units to one or more sites on the skin of the recipient;( B) monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of the potential rejection of a transplanted solid organ from the donor;(C) selecting one or more donor candidates for organ harvesting if no indications or therapeutically acceptable indications of inflammation and an immune response against one or more of the transplanted follicular units are detected; and(D) transplanting the donated organ to the recipient.

8. The method of claim 7 for selecting a candidate allogeneic solid organ donor for a recipient, wherein the follicular units are placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions.

9. The method of claim 7 or 8 for selecting a candidate allogeneic solid organ donor for a recipient, wherein the one or more indications of potential acute or chronic transplant rejection are the detection of one or more of the following events at the one or more follicular unit placement srtes: an antibody- mediated response; unwanted alloreactivity; an acute or chronic host versus graft disease; de novo development of donor specific human leukocyte antigen (HLA) antibodies; an increase in the concentration of polycytotoxic T ceils, an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates; and an imbalance of CD4:CD8 T-cell ratios.

10. The method of any one of claims 7-9 for selecting a candidate allogeneic solid organ donor for a recipient, wherein the donated solid organ is a kidney11. A method for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ in a recipient comprising:(A) transplanting one or more allogenic follicular units from the donor of transplanted allogeneic solid organ to one or more sites on the skin of the recipient;(B) monitoring the recipient for one or more indications of potential acute transplant rejection by monitoring the one or more follicular unit placement sites for indications of inflammation and an immune response against one or more of the transplanted follicular units, wherein the detection of indications of inflammation and an immune response against one or more of the transplanted follicular units is indicative of acute rejection of the transplanted solid organ; and(C) administer immunosuppressive therapy to the recipient or modify the recipient's existing immunosuppressive therapy to ameliorate acute rejection of the transplanted organ if one or more indications of acute rejection are detected.

12. The method of claim 11 for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, the follicular units are placed at one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back regions.

13. The method of claim 11 or 12 for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, wherein the one or more indications of potential acute or chronic transplant rejection are the detection of one or more of the following events at the one or more follicular unit placement sites: an antibody-mediated response: unwanted alloreactivity; an acute or chronic host versus graft disease; de novo development of donor specific human leukocyte antigen (HLA) antibodies;an increase in the concentration of polycytotoxic T ceils, an increase in perifollicular infundibulo-isthmal lymphocytic infiltrates; and an imbalance of CD4:CD8 T-cell ratios.

14. The method of any one of claims 11-13 for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, wherein immunosuppressive therapy is administered to the recipient, or preexisting immunosuppressive therapy is modified, wherein the therapy comprises the administration of one or more of: a corticosteroid; a cytostatic drug; an anti-TNFa antibody; an IL-2 receptor inhibitor, or other antibody-based immunosuppressive therapy.

15. The method of claim 14, wherein: the corticosteroid is prednisone; the cytostatic drug is tacrolimus, mycophenolate, mycophenolate mofetil, or ciclosporin; and the IL-2 receptor inhibitor is basiliximab.

16. The method of any one of claims 11-15 for ameliorating the onset of an acute rejection of a transplanted allogeneic solid organ, wherein the donated organ is a kidney.

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