Methods for monitoring potential rejection indications of allograft recipients

Abstract

The present invention relates to methods for monitoring potential indications of allogeneic organ rejection by monitoring transplanted hair follicles from the same donor of a transplanted organ, and for performing or modifying immunosuppressive therapy to ameliorate or prevent the occurrence of acute or chronic organ rejection. The methods of the invention may also be used to select candidate allogeneic solid organ donors for recipients, as well as to improve the occurrence of acute rejection of transplanted allogeneic solid organs, such as kidneys.

Description

Cross-references to related applications

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

[0002] The need for long-term immunosuppressive therapy has been a major obstacle to considering allogeneic hair transplantation between two histocompatible individuals. The two previous case reports of allogeneic transplantation involved only bone marrow (BM) transplant recipients; therefore, in addition to HLA compatibility, the inherent chimeric immune tolerance of this procedure eliminated the need for immunosuppressive therapy.3,4 In the absence of chimeric immune tolerance, human-to-human hair transplantation between two unrelated but histocompatible individuals is a viable option for selected patients who already require lifelong immunosuppressive therapy. Since solid organ transplants outnumber bone marrow transplants annually, solid organ transplant recipients constitute a larger transplant recipient population, which may benefit from human-to-human hair transplantation based on proven histocompatibility, successful solid organ transplantation, and pre-existing lifelong immunosuppressive therapy. Furthermore, the less invasive nature of the procedure for harvesting donor hair follicles (follicular unit excision, FUE) makes donor harvesting procedures more readily accepted by matched donors. Summary of the Invention

[0003] The applicant recognizes that transplanted allogeneic hair follicles can serve as a proxy for the feasibility of allogeneic transplantation of solid organs (such as kidneys). According to one aspect of the invention, a method for monitoring potential indications of organ rejection in recipients who have previously received allogeneic solid organ transplants comprises the following steps: (A) transplanting one or more allogeneic hair follicle units from the same donor as the solid organ to one or more sites on the recipient's skin; (B) monitoring one or more potential acute or chronic transplant rejection indications in the recipient by monitoring indications of inflammation and immune response at the one or more hair follicle unit placement sites in response to the one or more transplanted hair follicle units, wherein the detection of indications of inflammation and immune response in response to the one or more transplanted hair follicle units indicates an immune response and potential rejection of the transplanted solid organ; and (C) if one or more indications of inflammation and immune response in response to the one or more transplanted hair follicle units are detected: (i) administering immunosuppressive therapy to the recipient or modifying the recipient's existing immunosuppressive therapy to improve or prevent the occurrence of acute or chronic organ rejection; and / or (ii) performing additional diagnostic procedures to confirm the occurrence of acute or chronic organ rejection.

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

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

[0006] Immunosuppressive therapy is administered to the recipient, or a pre-existing immunosuppressive therapy is modified for the recipient, wherein the therapy includes administration of one or more of the following: corticosteroids; cell-suppressing drugs; anti-TNFα antibodies; IL-2 receptor inhibitors; or other antibody-based immunosuppressive therapies.

[0007] Corticosteroids can be, for example, prednisone; cell inhibitors can be, for example, tacrolimus, mycophenolate, mycophenolate mofetil, or cyclosporine; IL-2 receptor inhibitors can be balithiabumab.

[0008] In another aspect of the invention, a method for selecting candidate allogeneic solid organ donors for a recipient includes the following steps: (A) transplanting one or more hair follicle units excised from one or more candidate organ donors and placing the excised hair follicle units on one or more sites on the recipient's skin; (B) monitoring for indications of inflammatory and immune responses at the one or more hair follicle unit placement sites in response to the one or more transplanted hair follicle units, wherein the detection of indications of inflammatory and immune responses in response to the one or more transplanted hair follicle units indicates potential rejection of the transplanted solid organ from the donor; (C) if no indications of inflammatory and immune responses in response to the one or more transplanted hair follicle units are detected, or if therapeutically acceptable indications are detected, selecting one or more donor candidates for organ harvesting; and (D) transplanting the donated organ to the recipient.

[0009] In another aspect of the invention, a method for improving the occurrence of acute rejection of a transplanted allogeneic solid organ (such as a kidney) in a recipient, the steps of such a method comprising: (A) transplanting one or more allogeneic hair follicle units from a donor of the transplanted allogeneic solid organ to one or more sites on the recipient's skin; (B) monitoring one or more potential indications of acute transplant rejection in the recipient by monitoring indications of inflammation and immune response in response to the one or more transplanted hair follicle units at the placement site, wherein the detection of indications of inflammation and immune response in response to the one or more transplanted hair follicle units indicates acute rejection of the transplanted solid organ; and (C) if one or more indications of acute rejection are detected, administering immunosuppressive therapy to the recipient or modifying the recipient's existing immunosuppressive therapy to improve acute rejection of the transplanted organ. Attached Figure Description

[0010] Figure 1A This is a "previous photo" of the patient, showing Norwood stage VI-VII hair loss. 119 x 137 mm (300 x 300 DPI).

[0011] Figure 1B for Figure 1A Photograph taken immediately after a 2350 allogeneic hair follicle units were transplanted onto the forehead scalp of the same patient. 119 x 137 mm (300 x 300 DPI).

[0012] Figure 1C for Figure 1A-Figure 1B Photos of the same patient 6-9 months post-surgery show robust growth of transplanted hair follicles in the transplanted area. 119x137mm (300 x 300 DPI).

[0013] Figure 2A The histology of the recipient zone 6 months after allogeneic transplantation is shown, including perifollicular infundibulum-isthmus lymphocyte infiltration.

[0014] Figure 2B The results showed that the recipient area still contained CD8 lymphocytes 6 months after allogeneic transplantation.

[0015] Figure 2C The results showed that the recipient site of the allogeneic transplant still contained CD4 lymphocytes 6 months after transplantation, and the CD8:CD4 lymphocyte ratio was 2:1. Detailed Implementation

[0016] This disclosure describes compositions and methods that utilize transplanted allogeneic hair follicle units as proxies for detecting, monitoring, or detecting and monitoring rejection of transplanted allogeneic solid organs or organ tissues collected from the same donor and transplanted into the same host. In other words, the invention described herein utilizes transplanted allogeneic hair follicles as “witness follicles” or “witness follicle units” to track the viability of transplanted organs collected from the same allogeneic donor of the hair follicles. Therefore, the compositions and methods of this invention can be used clinically for treatment to improve rejection of transplanted allogeneic organs and maintain the viability of transplanted organs by preventing further tissue damage. Typically, such treatments are immunosuppressive therapies used to downregulate the organ recipient's immune response to transplanted allogeneic solid organs or organ tissues.

[0017] Some methods of the present invention monitor potential indications of solid organ rejection in allogeneic transplant recipients by transplanting one or more hair follicle units harvested from the same donor of an allogeneic solid organ previously transplanted into the recipient. Thus, the donor of the allogeneic hair follicle unit and the allogeneic solid organ are the same individual. In the context of this disclosure, "solid organ" is generally, but not limited to, a kidney. Other examples of solid organs include the liver and one or more lobes of the lung. In fact, any solid organ or functional portion thereof harvested from a living donor who survives a harvesting procedure can be considered a solid organ transplanted into an allogeneic recipient in the methods of the present invention. The transplanted organ or hair follicle unit is "allogeneic" because the donor of the organ or the recipient of the hair follicle unit is genetically different from the recipient of the transplanted tissue; this can also be referred to as allogeneic transplantation.

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

[0019] In the method of this invention, the step of monitoring potential indicators of organ rejection in an allogeneic transplant recipient is performed by monitoring indicators of inflammatory and immune responses at the skin site where donor hair follicle units have been placed in response to one or more transplanted hair follicle units. More specifically, some methods of this invention detect immune responses at or near the allogeneic hair follicle unit placement site. In the method of this invention, for example, detecting an immune response against the hair follicle unit includes detecting an antibody-mediated response; while in another method of this invention, the method detects undesirable allogeneic reactions. Other examples of immune responses detectable at one or more allogeneic hair follicle unit placement sites include: detecting acute or chronic host-resistant graft disease, detecting the reformation of donor-specific human leukocyte antigen (HLA) antibodies, detecting an increase in multicytotoxic T cell concentrations, detecting an increase in perifollicular infundibulum-isthmus lymphocyte infiltration, and detecting an imbalance in the CD4:CD8 T cell ratio. The detection of one or more immune responses at one or more allogeneic hair follicle unit placement sites indicates an immune response against the transplanted solid organ.

[0020] Therefore, in the method of the present invention, a hair follicle unit is placed at a site on the skin of a subject who has previously received an allogeneic kidney transplant from the same donor from whom the hair follicle unit was obtained, and one or more of the following are monitored at the hair follicle unit placement site: detection of acute or chronic host-versus-graft disease, detection of the reformation of donor-specific human leukocyte antigen (HLA) antibodies, detection of increased concentrations of multicytotoxic T cells, detection of increased perifollicular infundibulum-isthmus lymphocyte infiltration, and detection of an imbalance in the CD4:CD8 T cell ratio. In such methods of detecting donor HLA-specific antibodies, the antibodies detected may be IgA, IgM, IgD, IgE, or IgG antibodies. In such methods of determining an imbalance in the CD4:CD8 T cell ratio, the imbalance is related to the CD4:CD8 ratio normally found in healthy tissue and blood levels. In some methods of the present invention, the CD4:CD8 ratio in healthy tissue or blood is 1:2, while the imbalanced CD4:CD8 ratio is 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10 or higher.

[0021] The method of the present invention for monitoring potential indications of organ rejection in allogeneic transplant recipients may further include one or more steps: if one or more indications of an immune response are detected at the placement site of the allogeneic hair follicle unit in the recipient, the recipient is treated to prevent or improve organ rejection. Typically, this treatment is immunosuppressive therapy. For example, the immunosuppressive therapy of the present invention includes administration of corticosteroids (e.g., prednisone), cell-suppressing drugs (e.g., tacrolimus, mycophenolate mofetil, mycophenolate mofetil, cyclosporine), anti-TNFα antibodies, IL-2 receptor inhibitors (e.g., balithimab), or other antibody-based immunosuppressive therapies. Since transplant recipients typically receive a lifelong regimen of immunosuppressive drugs or cocktails, if the method of the present invention detects one or more indications of an immune response at the allogeneic hair follicle unit placement site in the recipient, and the recipient received immunosuppressive therapy prior to and concurrently with the allogeneic hair follicle unit transplantation procedure, then the immunosuppressive therapy performed according to the present invention will modify or replace the immunosuppressive therapy that was received prior to the detection of one or more indications of an immune response at the allogeneic hair follicle unit placement site in the recipient. In some methods of the present invention, the recipient undergoes diagnostic procedures to confirm the occurrence of acute or chronic organ rejection.

[0022] As described above, the present invention also includes a method for selecting candidate allogeneic solid organ donors for transplant recipients. In such methods, one or more hair follicle units are harvested from one or more candidate organ donors and transplanted to one or more sites on the recipient's skin, and then indications of inflammatory and immune responses to the one or more transplanted hair follicle units are monitored. The detection of indications of inflammatory and / or immune responses to the one or more transplanted hair follicle units indicates potential rejection of the transplanted solid organ from the donor. Therefore, if no indications of inflammatory and immune responses to the one or more transplanted hair follicles are detected, or if therapeutically acceptable indications are detected, one or more organ donor candidates are selected to harvest an organ for transplantation. In some methods of selecting candidate allogeneic solid organ donors for transplant recipients according to the present invention, the organ is a kidney.

[0023] The collected hair follicle units can be placed at any site on the recipient's skin. For example, in the method of the present invention, hair follicle 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. In the method of the present invention for selecting candidate allogeneic solid organ donors for a recipient, one or more potential acute or chronic transplant rejection indications may include the detection of one or more of the following events at one or more hair follicle unit placement sites: antibody-mediated reaction; undesirable allogeneic reaction; acute or chronic host-resistant graft disease; reformation of donor-specific human leukocyte antigen (HLA) antibodies; increased concentration of multicytotoxic T cells; increased perifollicular infundibulo-isthmic lymphocyte infiltration; and CD4:CD8 T cell imbalance.

[0024] As described above, the present invention also includes methods for improving the occurrence of acute rejection of transplanted allogeneic solid organs in recipients. In some such methods, the occurrence of acute rejection of transplanted kidneys is improved.

[0025] In the method of the present invention for improving the occurrence of acute rejection of transplanted allogeneic solid organs (such as kidneys), one or more allogeneic hair follicle units harvested from the donor of the transplanted allogeneic solid organ are transplanted to one or more sites on the recipient's skin, and then one or more potential signs of acute transplant rejection are monitored at the one or more transplanted hair follicle unit sites. If one or more signs of acute rejection are detected, the recipient is given immunosuppressive therapy or the recipient's existing immunosuppressive therapy is modified to improve the acute rejection of the transplanted organ.

[0026] The method of the present invention for improving the occurrence of acute rejection of transplanted allogeneic solid organs (such as kidneys) may further include one or more steps: if one or more indicators of an immune response are detected at the placement site of the allogeneic hair follicle unit in the recipient, the recipient is treated to improve the occurrence of acute rejection of the transplanted allogeneic solid organ. Typically, this treatment is immunosuppressive therapy. For example, the immunosuppressive therapy administration of the present invention includes corticosteroids (e.g., prednisone), cell-suppressing drugs (e.g., tacrolimus, mycophenolate mofetil, mycophenolate mofetil, cyclosporine), anti-TNFα antibodies, IL-2 receptor inhibitors (e.g., balithimab), or other antibody-based immunosuppressive therapies. Since transplant recipients typically receive a lifelong regimen of immunosuppressive drugs or mixtures of immunosuppressive drugs, if the method of the present invention detects one or more indications of an immune response at the allogeneic hair follicle unit placement site in the recipient, and the recipient received immunosuppressive therapy prior to and concurrently with the allogeneic hair follicle unit transplantation procedure, then the immunosuppressive therapy performed according to the present invention will modify or replace the immunosuppressive therapy that was received prior to the detection of one or more indications of an immune response at the allogeneic hair follicle unit placement site in the recipient. In some methods of the present invention, the recipient undergoes a diagnostic procedure to confirm the occurrence of acute or chronic organ rejection.

[0027] Example Example 1—Allogeneic hair transplantation in kidney transplant recipients The first fully HLA-compatible allogeneic hair transplant was performed using the follicular unit extraction (FUE) method. Follicular units were harvested from an individual who had previously donated a kidney and transplanted into a recipient with severe hair loss who was also the kidney recipient of the donor follicular unit. The recipient was a 60-year-old male who had received a matched kidney allogeneic transplant from an unrelated friend (the follicular unit donor) 20 years prior. The recipient exhibited androgenetic alopecia at Norwood VI-VII levels (Figure 1). Over the past three years, the recipient had unsuccessfully treated his hair loss with twice-daily topical 5% minoxidil, one year of oral 1 mg finasteride, and multiple monthly platelet-rich plasma (PRP) therapies. The patient also received a well-tolerated immunosuppressive therapy regimen for up to 20 years due to his kidney allogeneic transplant, which included administration of tacrolimus 2.5 mg / kg / day, mycophenolate mofetil 500 mg / day, and prednisone 0.5 mg / kg / day.

[0028] The recipient of the follicular unit requested the use of hair from his voluntary kidney donor because he did not have enough hair for autologous transplantation due to extensive hair loss and preferred to minimize the amount of surgery. The recipient is also undergoing lifelong immunosuppressive therapy to mitigate rejection from the kidney allogeneic transplant.

[0029] FUE was used for hair transplantation because it is minimally invasive and avoids the linear scarring seen in other techniques. After obtaining informed consent from both the recipient and donor, 2350 follicular units were harvested via FUE from a safe donor scalp area and transplanted into the balding forehead scalp of the kidney recipient (Figure 2). The procedure was well-tolerated, and the patient continued their usual immunosuppressive therapy after discharge.

[0030] The recovery period was stable, with a yield of 85% at 9 months based on hair density measurements (Figure 3). During the 6-month follow-up, blood and hair examinations of the recipient area were normal, while histological analysis showed moderate perifollicular infundibulocytic infiltration in the transplanted area, without fibrosis or significant epithelial changes (Figure 4). The non-transplanted area showed rare interfollicular dermal lymphocytes, without fibrosis or epithelial changes. Immunohistochemical staining of CD4 and CD8 in the transplanted and non-transplanted areas showed 30-45% CD4 and 45-60% CD8 in both areas, a ratio of 1:2. The plan is to expand donor supply by combining autologous and allogeneic grafts, followed by additional hair transplants to address remaining bald areas.

[0031] The moderate perifollicular infundibulocytic infiltration observed in the recipient area is consistent with results reported in successful autologous hair transplants. Encouragingly, the CD4:CD8 ratio was consistent with findings in non-transplant areas, suggesting no indication of impending graft rejection. This is the first reported case of successful allogeneic hair transplantation from a solid organ donor to its recipient.

Claims

1. A method for monitoring potential indications of organ rejection in recipients who have previously received allogeneic solid organs, the method comprising: (A) Transplanting one or more allogeneic hair follicle units from the same donor of the solid organ to one or more sites on the skin of the recipient; (B) Monitoring one or more potential acute or chronic transplant rejection indicators in the recipient by monitoring indicators of inflammatory and immune responses to one or more transplanted hair follicle units at the placement site of the one or more hair follicle units, wherein the detection of indicators of inflammatory and immune responses to one or more transplanted hair follicle units indicates an immune response and potential rejection of the transplanted solid organ; and (C) If one or more indicators of inflammation and immune response against one or more transplanted hair follicle units are detected: (i) administering immunosuppressive therapy to the recipient or modifying the recipient's existing immunosuppressive therapy to improve or prevent the occurrence of acute or chronic organ rejection; and / or (ii) Perform additional diagnostic procedures to confirm the occurrence of acute or chronic organ rejection.

2. The method for monitoring potential indicators of organ rejection in recipients who have previously received allogeneic solid organs, as described in claim 1, wherein, The hair follicle units are placed on one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back.

3. The method for monitoring potential indicators of organ rejection in recipients who have previously received allogeneic solid organs, as described in claim 1 or 2, wherein, The one or more potential indications for 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: antibody-mediated reaction; unwanted allogeneic reaction; acute or chronic host-resistant graft disease; reformation of donor-specific human leukocyte antigen (HLA) antibodies; increased concentration of multicytotoxic T cells; increased perifollicular infundibulum-isthmus lymphocyte infiltration; and CD4:CD8 T cell imbalance.

4. The method for monitoring potential indications of organ rejection in recipients who have previously received allogeneic solid organs, according to any one of claims 1-3, wherein, The recipient is given immunosuppressive therapy, or a pre-existing immunosuppressive therapy is modified, wherein the therapy includes administration of one or more of the following: corticosteroids, cell-suppressing drugs, anti-TNFα antibodies, IL-2 receptor inhibitors, or other antibody-based immunosuppressive therapies.

5. The method according to claim 4, wherein: The corticosteroid is prednisone; the cell-inhibiting drug is tacrolimus, mycophenolic acid, mycophenolate mofetil, or cyclosporine; and the IL-2 receptor inhibitor is balithiab.

6. The method for monitoring potential indications of organ rejection in recipients who have previously received allogeneic solid organs, according to any one of claims 1-5, wherein, The donated organ is a kidney or a viable part thereof.

7. A method for selecting candidate allogeneic solid organ donors for a recipient, the method comprising: (A) Transplanting one or more hair follicle units excised from one or more candidate organ donors and placing the excised hair follicle units on one or more sites on the recipient’s skin; (B) Monitor for signs of inflammation and immune response at the one or more hair follicle unit placement sites in response to one or more transplanted hair follicle units, wherein the detection of signs of inflammation and immune response in response to one or more transplanted hair follicle units indicates potential rejection of the transplanted solid organ from the donor. (C) If no indication of inflammatory and immune response to one or more transplanted hair follicle units is detected, or if a therapeutically acceptable indication is detected, one or more donor candidates are selected for organ harvesting. and (D) Transplanting the donated organs to the recipient.

8. The method for selecting candidate allogeneic solid organ donors for a recipient according to claim 7, wherein, The hair follicle units are placed on one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back.

9. The method for selecting candidate allogeneic solid organ donors for a recipient according to claim 7 or 8, wherein, One or more potential acute or chronic transplant rejection indications are the detection of one or more of the following events at the one or more hair follicle unit placement sites: antibody-mediated reaction; unwanted allogeneic reaction; acute or chronic host-resistant graft disease; The reformation of donor-specific human leukocyte antigen (HLA) antibodies; increased concentration of multicytotoxic T cells; increased infiltration of lymphocytes in the perifollicular infundibulum-isthmus; and an imbalance in the CD4:CD8 T cell ratio.

10. The method for selecting candidate allogeneic solid organ donors for a recipient according to any one of claims 7-9, wherein, The donated organ was a kidney.

11. A method for improving the incidence of acute rejection of transplanted allogeneic solid organs in recipients, the method comprising: (A) Transplanting one or more allogeneic hair follicle units from a donor of a transplanted allogeneic solid organ to one or more sites on the skin of the recipient; (B) Monitor one or more potential signs of acute transplant rejection in the recipient by monitoring signs of inflammation and immune response to one or more transplanted hair follicle units at the placement site of the one or more hair follicle units, wherein the detection of signs of inflammation and immune response to one or more transplanted hair follicle units indicates acute rejection of the transplanted solid organ. and (C) If one or more indications of acute rejection are detected, the recipient is given immunosuppressive therapy or the recipient’s existing immunosuppressive therapy is modified to improve acute rejection of the transplanted organ.

12. The method for improving the occurrence of acute rejection of transplanted allogeneic solid organs according to claim 11, wherein, The hair follicle units are placed on one or more sites on the scalp, forearm, upper arm, calf, lower thigh, upper thigh, abdomen, upper back, or lower back.

13. The method for improving the occurrence of acute rejection of transplanted allogeneic solid organs according to claim 11 or 12, wherein, One or more potential indications for acute or chronic transplant rejection are the detection of one or more of the following events at the site of placement of the one or more hair follicle units: antibody-mediated reaction; unwanted allogeneic reaction; acute or chronic host-resistant graft disease; reformation of donor-specific human leukocyte antigen (HLA) antibodies; increased concentration of multicytotoxic T cells; increased perifollicular infundibulum-isthmus lymphocyte infiltration; and CD4:CD8 T cell imbalance.

14. The method for improving the occurrence of acute rejection of transplanted allogeneic solid organs according to any one of claims 11-13, wherein, The recipient is given immunosuppressive therapy or a modified pre-existing immunosuppressive therapy, wherein the therapy includes administration of one or more of the following: corticosteroids, cell-suppressing drugs, anti-TNFα antibodies, IL-2 receptor inhibitors, or other antibody-based immunosuppressive therapies.

15. The method of claim 14, wherein: The corticosteroid is prednisone; the cell-inhibiting drug is tacrolimus, mycophenolic acid, mycophenolate mofetil, or cyclosporine; and the IL-2 receptor inhibitor is balithiab.

16. The method for improving the occurrence of acute rejection of transplanted allogeneic solid organs according to any one of claims 11-15, wherein, The donated organ was a kidney.

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