Background Immunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus also suppresses T-cell function in the skin, contributing to a high incidence of skin cancer and associated mortality and morbidity in solid organ transplant recipients. Here, we aimed to identify a compound capable of re-establishing antitumor T-cell control in the skin despite the presence of tacrolimus.
Methods In this study, we performed time-resolved fluorescence resonance energy transfer to identify molecules capable of antagonizing the interaction between tacrolimus and FKBP12. The capacity of these molecules to rescue mouse and human T-cell function in the presence of tacrolimus was determined in vitro, and the antitumor effect of the lead compound, Q-2361, was assessed in “regressor” models of skin cancer in immunosuppressed mice. Systemic CD8 T-cell depletion and analyses of intratumoral T-cell activation markers and effector molecule production were performed to determine the mechanism of tumor rejection. Pharmacokinetic studies of topically applied Q-2361 were performed to assess skin and systemic drug exposure.
Results Q-2361 potently blocked the interaction between tacrolimus and FKBP12 and reversed the inhibition of the nuclear factor of activated T cells activation by tacrolimus following T-cell receptor engagement in human Jurkat cells. Q-2361 rescued T-cell function in the presence of tacrolimus, rapamycin, and everolimus. Intratumoral injection of Q-2361-induced tumor regression in mice systemically immune suppressed with tacrolimus. Mechanistically, Q-2361 treatment permitted T-cell activation, proliferation, and effector function within tumors. When CD8 T cells were depleted, Q-2361 could not induce tumor regression. A simple solution-based Q-2361 topical formulation achieved high and sustained residence in the skin with negligible drug in the blood.
Conclusions Our findings demonstrate that the local application of Q-2361 permits T-cells to become activated driving tumor rejection in the presence of tacrolimus. The data presented here suggests that topically applied Q-2361 has great potential for the reactivation of T-cells in the skin but not systemically, and therefore represents a promising strategy to prevent or treat skin malignancies in immunosuppressed organ transplant recipients.
- Skin Neoplasms
- CD8-Positive T-Lymphocytes
- CD4-Positive T-Lymphocytes
- Therapies, Investigational
- Transplantation Immunology
Data availability statement
No data are available.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.
- Skin Neoplasms
- CD8-Positive T-Lymphocytes
- CD4-Positive T-Lymphocytes
- Therapies, Investigational
- Transplantation Immunology
WHAT IS ALREADY KNOWN ON THIS TOPIC
Altering, reducing, or removing immunosuppressive drugs can lead to reduced tumor incidence or tumor rejection in solid organ transplant patients, but carries a serious risk of transplant rejection. The most commonly prescribed immunosuppressive drug in these patients is the calcineurin inhibitor, tacrolimus.
WHAT THIS STUDY ADDS
This study demonstrates a novel approach using the compound Q-2361, which permits T-cell function in the presence of tacrolimus. When applied locally, Q-2361 drives T-cell-mediated tumor regression in systemically immune-suppressed mice.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study shows that localized Q-2361 therapy has great potential to combat skin cancer in transplant patients without altering their immunosuppressive drug regimens, hence providing a new strategy to improve the efficacy of skin cancer treatment in immunosuppressed solid organ transplant recipients.
Solid organ transplant recipients require life-long immunosuppression in order to prevent the immune-mediated rejection of their transplanted organs. As a consequence of the systemic distribution of immunosuppressive drugs within the body however, these patients suffer a 65-fold to 250-fold increased risk of developing cutaneous malignancies including squamous cell carcinoma (SCC) and Kaposi’s sarcoma.1–3 The incidence of post-transplant skin cancer in transplant patients in the USA was recently reported to be 1651 per 100,000 person-years (post 2008), which is 3.7 times the rate of all cancers combined in the overall US population.4 The development of subsequent skin cancers is a particular hallmark of immunosuppression regimes, with approximately 50% of all patients who develop a post-transplant skin cancer developing a subsequent skin cancer within 2 years,5 increasing to approximately 70% within 5 years.6 7 The development of multiple primary lesions is also a serious concern, with some kidney transplant patients currently presenting to the Princess Alexandra Hospital, Brisbane, Australia requiring the removal of ~120 primary skin tumors each year. Patients with 10 or more cutaneous SCCs have markedly elevated risks of recurrence and metastasis.8 SCC is the most prevalent cancer in solid organ transplant recipients, with a more aggressive clinical course compared with the general population, and is a significant contributor to morbidity and mortality.9 10
The success of donor graft acceptance can be largely attributed to the inhibition of alloreactive T-cell activation by the calcineurin inhibitors ciclosporine and tacrolimus. Tacrolimus in particular has become the mainstay of combination immunosuppressant regimes due to its more favorable cardiovascular risk profile, its improved ability to prevent acute rejection and promote long-term graft survival, and its low rate of drug discontinuation.11 Consequently, in 2017, tacrolimus was used in 95% of heart transplant, 93% of kidney transplant, 90% of pancreas transplant, 83% of lung transplant, and 81% of liver transplant patients in the USA.12 Following oral administration, tacrolimus acts systemically to inhibit the signaling phosphatase calcineurin. By complexing calcineurin with the FK506-binding protein (FKBP12), tacrolimus prevents calcineurin from binding to activated calmodulin and enables the translocation of nuclear factor of activated T cells (NFAT) transcription factors from the cytoplasm to the nucleus.13 The lack of NFAT translocation prevents the transcription of NFAT-regulated proinflammatory cytokine genes in T-cells,14 subsequently blocking T-cell function. Interestingly, the use of calcineurin inhibitors does not appear to affect T-cell production, differentiation, or migration to patient skin, as T cells can be readily detected in the skin of kidney transplant recipients receiving ciclosporine and tacrolimus-based immunosuppression regimens.15
Cancer is a serious and potentially fatal complication for patients with otherwise successful transplants, hence several pharmacological approaches to skin cancer risk reduction have been examined. However, no strategies have proven effective without unacceptable impacts on transplanted organs and/or the patients themselves. For example, reducing the dose of immunosuppressive medications can decrease malignancy rates, however, such approaches have been linked to an increased risk of graft failure16 and an increased frequency of transplant rejection.17 Alternatively, switching from a calcineurin inhibitor-based treatment regime to a mammalian target of rapamycin (mTOR) inhibitor-based treatment regimen can reduce de novo SCC formation18–21 and induce the regression of pre-existing premalignant lesions.20 However, conversion from calcineurin inhibitors to mTOR inhibitors is associated with a high degree of mTOR inhibitor discontinuation due to a significant increase in adverse events, including edema, diarrhea, headache, mouth ulceration, pneumonitis, and impaired wound healing.18–24
There are no US Food and Drug Administration-approved drugs for cutaneous SCC treatment in organ transplant recipients.25 Current consensus-based treatment recommendations are lacking for first invasive low-risk SCCs; however, for field cancerization (areas with multiple keratotic lesions that may include subclinical lesions), the consensus is for lesion-directed therapy (eg, surgery, cryotherapy) followed by some form of field therapy.26 One of the most common field therapies is the topical application of 5-fluorouracil (Efudex), although other therapies such as photodynamic therapy and imiquimod (Aldara) are also used.27 5-Fluorouracil is an antimetabolite chemotherapy which causes blistering, pain, swelling, and scarring, and its side effects are exacerbated by exposure to ultraviolet (UV) wavelengths present in sunlight. Consequently, its use is associated with tolerance issues and adherence concerns.26 Importantly, a recent meta-analysis encompassing 92 clinical trials concluded that there is limited evidence for the efficacy of current field therapies to prevent the emergence of skin cancer in solid organ transplant recipients.28 Thus, the current standard of care for prevention and management of cutaneous malignancies in the organ transplant population is far from optimal and does not protect patients from the adverse health outcomes associated with skin cancer.
Based on the forecasted continued dominance of tacrolimus in immunosuppressant regimes, there will be an ongoing and significant need for improved treatment options for premalignant and malignant lesions in the skin of organ transplant patients being treated with tacrolimus. We hypothesize that an antagonist of tacrolimus could be employed topically, in organ transplant recipients being maintained on tacrolimus, to reduce the immunosuppressive effects of tacrolimus locally and safely in the skin. Distinct from other field therapies, the mechanism of action is the reactivation of T-cells which are vital for tumor immunosurveillance.29 Here, we describe the identification and characterization of Q-2361, a reversible antagonist of the tacrolimus–FKBP12 binding interaction. Q-2361 was efficacious in rescuing T-cell function and promoting tumor regression in the presence of tacrolimus and displayed sustained residence in the skin following topical application.
The details about peptidyl-prolyl isomerase (PPIase), mouse T-cell proliferation, human T-cell proliferation, and NFAT activation assays are provided in online supplemental methods.
GPI-1046 was purchased from Key Organics. Q-2361, VX-710, and V10-367 were synthesized by a contract research organization (O2h discovery) and liquid chromatography-mass spectrometry (LCMS) and proton nuclear magnetic resonance data were consistent with structure.30–33 Ciclosporin A and everolimus were purchased from Merck, tacrolimus was purchased from MedChemExpress, and rapamycin was purchased from LC Laboratories.
Time-resolved fluorescence resonance energy transfer (TR-FRET) assay
TR-FRET was conducted by Eurofins Selcia to determine the capacity of tacrolimus antagonists to compete for tacrolimus binding to the FKBP12 enzyme. The FKBP12 enzyme used in the assay is tagged with a polyhistidine sequence. An anti-6xHis antibody, labeled with a fluorescent donor, F(d), binds the tagged enzyme. The enzyme ligand, tacrolimus, is tagged with a fluorescent acceptor, F(a), and binds the enzyme. When the components of the antibody/enzyme/ligand complex are in close proximity, excitation of the F(d) labeled antibody at a particular wavelength A results in F(a) emission at wavelength B due to non-radiative energy transfer. In the presence of a test inhibitor which competes for tacrolimus, the complex is disrupted and is no longer in close proximity, resulting in emission at wavelength A due to the F(d). An 8-point dilution series was performed for tacrolimus antagonists and unlabeled tacrolimus (used as a control) over a concentration range of 0.001–10 µM. The inhibitors were added to the master mix in the assay plate containing the enzyme/antibody/ligand complex, with a final detergent concentration of 0.005%. The reaction was incubated for 30 min at room temperature and then read on a SpectraMax M5 (Molecular Devices) at wavelengths A (615 nm) and B (665 nm). The ratio between light emission (wavelength B/A) was calculated and the blank subtracted values were plotted against the inhibitor concentration in Log10 molar and fitted using one site Ki non-linear regression to determine the Kd of the bound test inhibitor.
Mice and cell lines
All animal procedures were approved by the University of Queensland Animal Ethics Committee; Approval Number UQDI/512/17. C57BL/6 and Balb/c mice were purchased from the Animal Resources Facility (Perth, Australia). HPV38E6E7-FVB mice34 were bred and maintained locally at the Translational Research Institute Biological Research Facility (Brisbane, Australia). All mice used were females aged 8–14 weeks and were housed under specific pathogen-free conditions. The UV-induced SCC cell line (HPV38 SCC), described previously,29 was created in-house. HPV38 SCC cells were maintained in modified Ham’s F12 media: 25% Dulbecco’s Modified Eagle’s Medium/high glucose (Thermo Fisher Scientific), 5% fetal bovine serum (FBS, Thermo Fisher Scientific), 5 µg/mL insulin (Merck), 0.4 µg/mL hydrocortisone (Merck), 10 ng/mL human recombinant epidermal growth factor (Invitrogen), 8.4 ng/mL Cholera toxin from Vibrio cholera (Merck), 24 µg/mL adenine (Merck) and 1X penicillin/streptomycin/glutamine (Life Technologies). The UV-induced 5117-RE cell line was a kind gift from Professor Hans Schreiber, The University of Chicago, Chicago, USA.35 5117-RE cells were maintained in Roswell Park Memorial Institute (RPMI) medium (Life Technologies), 10% FBS (Life Technologies), and 1X penicillin/streptomycin/glutamine (Life Technologies).
In vivo tumor studies
HPV38 SCC and 5117-RE are regressor cell lines which are unable to form tumors in immune-competent animals. However, these cell lines readily form tumors when injected into mice immunosuppressed with tacrolimus. Tacrolimus-infused diet (150 parts per million, provided ad libitum) was manufactured by Specialty Feeds (Perth, Western Australia, Australia) as described previously.36 HPV38E6E7-FVB (for HPV38 SCC) and Balb/c (for 5117-RE) mice>20 g in body weight were placed on tacrolimus-infused diet for 7 days prior to subcutaneous tumor cell challenge on the lower back (1×106 cells/mouse). Mice were maintained on tacrolimus-infused diet throughout experiments (unless indicated in the figure legend). Tumor growth was recorded every 1–3 days by measuring the major dimension (D) and minor dimension (d) of the tumor via a digital caliper. Measurements were transformed into tumor volume using the formula: tumor volume (cm3)=D×d2/2. Once tumors reached approximately 0.05–0.1 cm3 mice were regrouped to ensure an even distribution based on tumor size. The mice were then treated two times per day with intratumoral injections of Q-2361 (40 µL of 2 mg/mL stock formulated in 4% ethanol/0.2% Tween-80/phosphate-buffered saline) or vehicle control. In experiments involving CD8 T-cell depletion, mice were injected with 250 µg of anti-CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 µg of antibody on day 22 post tumor challenge. Mice were bled on day 10 post tumor challenge to check depletion efficiency via flow cytometry. Euthanasia was performed when tumors reached a maximum volume of 1 cm3. Immunohistochemistry on formalin-fixed tumor fragments was performed by the QIMR Berghofer Histology Facility (Brisbane, Australia) using anti-pan-cytokeratin (clone AE1/AE3, Dako), anti-vimentin (clone RV202, Santa Cruz), anti-E-cadherin (clone NCH-38, Dako), and anti-CD34 (clone EP373Y, Abcam) primary antibodies.
Analysis of T-cell function in tumors
To release cells from tumors, harvested tissue was cut into small fragments and digested for 60 min at 37°C in RPMI media containing 2% FBS, 3 mg/mL collagenase D and 5 µg/mL DNase I. Tissues were then gently pressed through a 70 µm cell strainer to create a single-cell suspension. Cells from each tumor were resuspended in 300 µl of RPMI media containing 10% FBS, 1X penicillin–streptomycin–glutamine, and 100 µM 2-mercaptoethanol (Merck), before staining with appropriate antibodies to assess phenotypic markers and effector molecules as follows.
Phenotypic and activation analysis
Isolated cells were incubated with Fc-block (Purified Rat Anti-Mouse CD16/CD32: isotype Rat IgG2a, clone: 93, Biolegend) for 20 min on ice to block non-specific antibody staining. Monoclonal antibodies for surface staining (CD45.1-PE-Dazzle (clone A20), TCRβ-FITC (clone H57-597), CD8α-PE-Cy7 (clone 53–6.7), CD4-AF700 (clone RM4-5), CD69-APC (clone H1.2F3); all Biolegend) were subsequently added and incubated on ice for 30 min in concert with Live/Dead Aqua Stain (Biolegend) to elucidate live cell populations. Cells were then resuspended in fixation buffer (eBioscience) and incubated in the dark at room temperature for 20 min. Cells were then washed and resuspended in 1X permeabilisation buffer (eBioscience) and incubated with anti-Ki67-BV605 (clone 16A8; Biolegend) antibody in the dark at room temperature for 20 min. Counting beads were then added to each sample before analysis using an LSR Fortessa X20 (BD Biosciences) flow cytometer with FACSDiva software (Becton Dickinson). Data were exported and analyzed using FlowJo software (Treestar).
Cytokine/effector molecule analysis
Isolated cells were stimulated ex vivo for 30 min in 96-well plates with plate-bound anti-CD3 (clone 145-2 C11; Biolegend) and soluble anti-CD28 (2.5 µg/mL—clone 37.51, Biolegend) at 37°C with 5% CO2. A total of 5 µg/mL brefeldin A was added to all wells, and cells were incubated at 37°C for a further 3.5 hours. Cells were then harvested and incubated with Fc-block for 20 min on ice. Monoclonal antibodies for surface staining (CD45.1-PE-Dazzle, TCRβ-FITC, CD8α-PE-Cy7, CD4-AF700) were subsequently added and incubated on ice for 30 min in concert with Live/Dead Aqua Stain (Biolegend). Cells were then resuspended in fixation buffer (eBioscience) and incubated in the dark at room temperature for 20 min. Cells were then washed and resuspended in 1X permeabilisation buffer (eBioscience) and incubated with intracellular antibodies including anti-IFN-γ-APC (clone XMG1.2; eBioscience), anti-TNFα-BV785 (clone MP6-XT22; Biolegend), anti-IL-2-BV605 (clone JES6-5H4; Biolegend), anti-granzyme B-BV421 (clone QA18A28; Biolegend), anti-perforin-PE (clone S16009A; Biolegend) in the dark at room temperature for 20 min. The addition of counting beads and flow cytometric analysis was as described above.
Topical pharmacokinetic study
The single dose pharmacokinetics of Q-2361 was assessed by topical administration to C57BL/6 mouse ears. Q-2361 (10 µL of 3% w/v (37.1 mM) solution in propylene glycol) was administered topically to one ear only (per mouse) using a silicone brush. At 1, 6, and 24 hours blood was harvested via cardiac bleed into cryovials containing 10 µL 0.5 M ethylenediaminetetraacetic acid, snap frozen on dry ice, and stored at −80°C. Ears were cleaned with distilled water and dried with cotton balls/swab before performing two tape strips (one piece of tape per strip). Ears were then removed and weights recorded. Ears were then placed into cryovials, snap frozen on dry ice, and stored at −80°C. The quantitation of Q-2361 using LC/MS/MS was performed by TheraIndx Lifesciences. Briefly, calibration standards and quality control samples were prepared by adding 2.5 µL of stock solutions of test compound of different concentrations into 25 µL of naïve mouse blood or ear homogenates. Control samples were prepared by spiking 2.5 µL of water or acetonitrile into 25 µL of naïve mouse blood or ear homogenates. The blood or ear samples were transferred into polypropylene Eppendorf tubes. 100 µL of 0.1 M zinc sulfate was added into the tubes, vortexed for 10 s, and 250 µL of HPLC-grade acetonitrile containing internal standard (pimecrolimus) was added, vortexed for 2 min, and centrifuged for 3 min at 800×g. A total of 20–40 µL of the supernatant was analyzed by LCMS/MS. Instrument: Acquity UPLC, Waters. Column: Acquity BEH C18 100×2.1 mm, 1.7µm; Mobile phase A: methanol; Mobile phase B: 5 mM ammonium acetate with 0.1% formic acid; Mobile phase gradient details: T=0 min (10% A, 90% B); T=0.01 min (10% A, 90% B); gradient to T=1.5 min (95% A, 5% B); T=3.2 min (95% A, 5% B). Flow rate: 0.3 mL/min, run time: 4.5 min; Ionization mode: Electrospray ionization (positive).
All statistical analyses were carried out using GraphPad Prism V.9.0 (GraphPad Software, San Diego, California, USA). Statistical tests are as indicated in figure legends. A p value of <0.05 (*) was considered significant. P<0.01 (**), p<0.001 (***), and p<0.0001 (****) are indicated.
Q-2361 binds FKBP12 with high affinity
Tacrolimus acts as a “molecular glue” to complex FKBP12 with calcineurin. This prevents calcineurin from binding to activated calmodulin to enable the translocation of NFAT to the nucleus, which in turn prevents the transcription of NFAT-regulated proinflammatory cytokine genes in T cells (figure 1A, left panel). We sought to enable NFAT gene transcription in the presence of tacrolimus by competitively outcompeting the binding of tacrolimus to FKBP12 with ligands that do not bind to calcineurin (figure 1A, right panel). A selection of compounds reported as potent non-immunosuppressive ligands of FKBP12 (figure 1B) were assessed for their capacity to compete with tacrolimus in binding to the FKBP12 enzyme in a TR-FRET assay (figure 1C and table 1). Surprisingly, with the exception of Q-2361 (L-685,818),30 these compounds displayed significantly weaker binding affinity for FKBP12 in the TR-FRET assay than previously reported, though discrepancies in the FKBP12 binding affinity and inhibition of FKBP12 PPIase activity of GPI-1046 have been observed by others.32 37 Q-2361 bound potently to FKBP12 (Kd=2.0 nM) and was selected for further study. Because high lipophilicity may have led to adherence to plastic surfaces in the TR-FRET assay, GPI-1046,33 VX-710,32 and V10-36731 were additionally assessed in a functional FKBP12 PPIase in glass cuvettes. Once again, the compounds were found to have reduced inhibitory activity relative to reported values (online supplemental table S1).
Q-2361 rescues mouse CD8+ T-cell function
To determine the capacity of FKBP12-ligands to rescue T-cell function in the presence of tacrolimus, naïve mouse T cells were labeled with an intracellular dye and stimulated with anti-CD3/CD28 beads in the presence of tacrolimus. After 1 hour, increasing concentrations of FKBP12-ligands were added to the culture. CD8+ T-cell proliferation and viability was assessed by flow cytometry 3 days later. Based on poor binding affinity to FKBP12 (table 1), GPI-1046 was not included in functional testing. V10-367 was found to be incapable of rescuing CD8+ T-cell proliferation in the presence of tacrolimus (0.3 ng/mL), and also to be toxic to T cells at 10 µM (online supplemental figure 1). VX-710 at 3 µM partially rescued CD8+ T-cell proliferation in the presence of tacrolimus (0.6 ng/mL), but responses were not improved with 6 µM VX-710 (figure 2A, left panel), possibly due to inherent immunosuppressive qualities of VX-710 at this dose (figure 2A, central panel). By comparison, Q-2361 dose-dependently and significantly rescued CD8+ T-cell proliferation in the presence of tacrolimus at all concentrations tested (figure 2B, left panel). Full rescue was achieved at 1 µM Q-2361. In the absence of tacrolimus, Q-2361 had no significant effect on CD8+ T-cell proliferation (figure 2B, central panel) or viability (figure 2B, right panel) at any of the tested concentrations compared with the no drug control. In summary, Q-2361 was the only compound that rescued mouse CD8+ T-cell function in the presence of tacrolimus in vitro, was not inherently immunosuppressive, and did not impact on T-cell viability.
Mechanisms of Q-2361 rescue
The immunosuppressive drugs tacrolimus and ciclosporin A are both calcineurin inhibitors; however, while tacrolimus complexes calcineurin with FKBP12, ciclosporin A complexes calcineurin with cyclophilin (figure 3A). Therefore, the FKBP12-binding compound Q-2361 should rescue NFAT activation in the presence of tacrolimus, but not in the presence of ciclosporin A. To test these hypotheses, we used human Jurkat-Lucia NFAT reporter cells to study NFAT activation in treated cells. Cells were initially treated with tacrolimus, ciclosporin A, rapamycin, or everolimus for 1 hour before the addition of anti-CD3 antibody and the indicated concentrations of Q-2361 or control media. After 24 hours, NFAT activity was assessed by luminescence following the addition of a luciferin substrate and expressed as relative light units. As shown in figure 3B, Q-2361 enabled NFAT activity in the presence of tacrolimus but not in the presence of ciclosporin A, as expected. Rapamycin and its analog everolimus had no impact on NFAT activation in line with their separate mechanism of action as mTOR inhibitors (figure 3B). However, because rapamycin and everolimus bind to the same FKPB12-binding site as tacrolimus, it was expected that Q-2361 would rescue T-cell function in the presence of these drugs by outcompeting their binding to FKBP12. To test this, we collected peripheral blood mononuclear cells from healthy human volunteers and set up T-cell rescue experiments similar to those described in figure 2. As shown in figure 3C, Q-2361 was confirmed to rescue T-cell function in the presence of rapamycin and everolimus, but not ciclosporin A, confirming that Q-2361 is also a rapamycin and everolimus antagonist. Together, these results confirm the expected mechanism of action of Q-2361 as a competitive FKBP12-binding moiety.
Regression of established SCC tumors
UV radiation-induced tumors are known to carry a high mutational load and to be inherently immunogenic.38 39 Previously, we developed a transplantable SCC tumor cell line derived from SCC tumors that arose as a result of the treatment of HPV38E6E7-FVB mice with chronic UV (figure 4A). Transplanted SCC cells are unable to form tumors when injected subcutaneously into immune-competent mice, but readily form SCC tumors (as confirmed histopathologically through positive pan-cytokeratin staining; Online supplemental figure 2) when injected into mice fed tacrolimus in their diet. Tumors grew rapidly beneath the skin reminiscent of invasive SCC (online supplemental figure 3). Removing tacrolimus from the diet of mice bearing established tumors results in systemic immune-reactivation and SCC regression29 (figure 4B). To explore whether the local administration of Q-2361 would enable the regression of established tumors in mice maintained on a tacrolimus diet (figure 4C), we allowed tumors to become established beneath the skin and then administered Q-2361 or vehicle intratumorally two times per day (40 µL; 2 mg/mL: 2.48 mM) to ensure Q-2361 reached the tumor (figure 4D). We further subdivided Q-2361 and vehicle groups by receipt of either anti-CD8b depleting antibody or isotype control antibody, to explore a role for CD8 T cells in responses to Q-2361. As shown in figure 4E, the treatment with Q-2361 significantly reduced tacrolimus-dependent SCC tumor growth. Evidence of tumor regression was apparent in 100% of treated mice, with complete regression occurring in 4 of 12 animals (not shown). The ability of Q-2361 to mediate SCC regression was completely lost in animals depleted of CD8 T cells, both ruling out a direct role for Q-2361 as an antitumor agent and confirming a key role for CD8 T-cell function in Q-2361-mediated SCC regression. Intratumoral treatment with Q-2361 had no apparent impact on animal body weight (figure 4F). In summary, Q-2361 induces SCC tumor regression in tacrolimus-suppressed mice in vivo through a CD8 T cell-dependent mechanism.
Reactivation of T cells within regressing SCC tumors
To further explore the mechanism of action of Q-2361 in driving tumor regression, we compared the tumor-derived T-cell phenotype during SCC tumor regression after Q-2361 treatment or tacrolimus withdrawal. In the first model, tumors were initially established in tacrolimus-fed mice and then tacrolimus was withdrawn from the diet (figure 5A). This resulted in SCC regression as a consequence of systemic immune reactivation. In the second model, mice were maintained on a tacrolimus diet throughout the experiment and established tumors were treated with Q-2361 (two times a day, 40 µL; 2 mg/mL: 2.48 mM) intratumorally to reactivate the immune system locally (as in figure 4E,F and figure 5B). In both models, we harvested tumors soon after the induction of tumor regression (12 days after tacrolimus withdrawal in figure 5A, and after 11 days of Q-2361 treatment in figure 5B) and analyzed them for evidence of T-cell activation and effector function by flow cytometry (figure 5C,D; representative flow plots and gating strategy shown in online supplemental figure 4). Systemic immune reactivation following tacrolimus withdrawal and local immune reactivation following Q-2361 treatment resulted in a strikingly similar pattern of tumor regression (figure 5A,B) and CD8/CD4 T-cell activation and effector molecule production within regressing tumors (figure 5C,D and online supplemental figure 5A). Large increases in the absolute number of CD8 T cells, the numbers of CD8 T cells expressing the CD69 activation marker, and the percentage of CD8 T cells producing the effector cytokines IFN-γ and TNF-α were clearly evident in regressing tumors in both models (figure 5C,D). The production of the cytotoxic effector molecules granzyme B and perforin was also increased and showed statistical significance in a repeat Q-2361 treatment experiment (online supplemental figure 5B) and at an earlier timepoint in the tacrolimus withdrawal experiment (online supplemental figure 5C). Q-2361 also significantly increased the percentage of CD4 and CD8 T cells positive for the proliferation marker Ki67 (online supplemental figure 5B). In summary, the intratumoral injection of Q-2361 enables the local activation and effector function of CD8 and CD4 T cells resulting in SCC tumor regression in immune-suppressed mice.
Reduced tumor growth in a model of spindle cell sarcoma
To evaluate the efficacy of Q-2361 in a different tumor model and in a different inbred mouse strain, we examined the 5117-RE tumor model in TH2-prone Balb/c mice. Similar to our SCC model, 5117-RE tumors originated following the chronic UV treatment of mice,35 and do not form tumors when injected into immune-competent mice (not shown). However, 5117-RE cells readily formed tumors when injected into mice fed a tacrolimus diet (online supplemental figure 2). CD34 staining revealed 5117-RE tumors to be highly vascularized, but negative for pan-cytokeratin, vimentin, or E-cadherin staining (online supplemental figure 2). In the absence of defining marker expression, they can be described as “UV-induced undifferentiated spindle cell sarcoma”. Morphologically, they appear very similar to leiomyosarcoma and fibrosarcoma. Like the model described in figure 5B, we established tumors in mice maintained on a tacrolimus diet and treated them with Q-2361 intratumorally (two times per day, 40 µL; 2 mg/mL: 2.48 mM; figure 6A). These dense tumors had a tendency to split rather than absorb injected volumes; however, we saw evidence of regression in ~41% of Q-2361-treated mice, with complete regression occurring in 2 of 24 animals (not shown). We harvested 10 tumors at random from each group on day 20 following 12 days of intratumoral injection, and analyzed them for evidence of T-cell activation and effector function by flow cytometry (figure 6B). Q-2361 treatment was associated with increased total T-cell infiltration, increased CD4 and CD8 T-cell activation as determined by CD69 staining, and increased production of IFN-γ and granzyme B in both CD4 and CD8 T cells. A large increase in the production of TNF-α was also detected in CD8 T cells but not in CD4 T cells. Overall, these results demonstrate the potential for Q-2361 to elicit both CD8 and CD4 T-cell activation and antitumor immunity in the presence of tacrolimus-induced immunosuppression in vivo.
Topical delivery of Q-2361
The efficacy studies involved intratumoral administration of Q-2361 because of the subcutaneous nature of the tumors in the models. Separately, we wished to gather data on the pharmacokinetics of topically applied Q-2361 to assess skin and systemic drug exposure in order to reflect the intended clinical usage. The skin and blood concentrations of Q-2361 were measured following single and repeat topical application of Q-2361. After a single topical dose of Q-2361 to the ear (10 µL of 3% w/v (37.1 mM) solution in propylene glycol), mean concentrations of Q-2361 in mouse ear were between 22.8 and 30.7 µg/g over 24 hours (table 2). Blood concentrations of Q-2361 were below the limit of quantitation (BLQ; <3.5 ng/mL) for all timepoints. Mouse ear drug levels were increased following 4 days of everyday dosing (10 µL of 3% solution in propylene glycol) with measured concentrations of Q-2361 in mouse ear between 37.3 and 52.3 µg/g over 6 hours (table 3). Relative to tacrolimus levels in mouse skin from our previous study36 showing 20 ng/g in the skin when clinical blood concentrations were attained, this exposure represents >2000-fold level of Q-2361 over tacrolimus. Blood concentrations of Q-2361 were BLQ for all but one sample (table 3). There was no evidence of Q-2361-associated erythema or skin irritation following 4 days of topical application.
The global burden of cutaneous keratinocyte cancers in solid organ transplant recipients is on the rise as a consequence of the increased long-term survival of this patient cohort. Successful organ acceptance and function requires life-long immunosuppressive drug treatment to dampen down T-cell immunity. However, T-cell immunity also plays a critical role in the recognition and destruction of malignant cells, and due to the systemic distribution of orally administered drugs, immunosuppression that extends to the skin increases the risk of skin cancer.40 41 Tacrolimus, a calcineurin inhibitor, is the mainstay of immunosuppressant regimens due to its high rate of transplant viability and low rate of drug discontinuation.42
In an effort to locally and safely counteract the systemic side effects of tacrolimus in the skin, we screened compounds described in the literature to be potent non-immunosuppressive ligands of FKBP12. Initial TR-FRET and PPIase analyses of GPI-1046,33 VX-710,32 and V10-36731 revealed significantly lower affinity for and inhibitory activity against FKPB12 than previously reported. GPI-1046 showed minimal binding to FKBP12 and was not studied further. V10-367 did not rescue CD8 T-cell function in the presence of tacrolimus, and while VX-710 could partially rescue CD8 T-cell function in the presence of tacrolimus at one concentration, it appeared to be immunosuppressive. These compounds did not meet the necessary pharmacological criteria for the proposed therapy; potency on FKBP12 comparable to tacrolimus (KD<5 nM), complete and dose-responsive T-cell rescue from tacrolimus, and no effect of compound alone on T-cells, and accordingly were not pursued further for development. By comparison, Q-2361 displayed similar FKBP12-binding affinity to tacrolimus, dose-dependently and significantly rescued mouse CD8+ T-cell proliferation in the presence of tacrolimus at all concentrations tested and had no significant effect on CD8+ T-cell proliferation or viability in the absence of tacrolimus. Importantly, Q-2361 was similarly effective at rescuing human T-cell function in the presence of tacrolimus. Although an FKBP12-binding compound, Q-2361 did not inhibit T-cell proliferation and was able to rescue NFAT activation in the presence of tacrolimus. Q-2361 was also shown to be a rapamycin and everolimus antagonist. When administered to mice immunosuppressed with tacrolimus, Q-2361 was well tolerated, resulted in T-cell-mediated antitumor immune responses and tumor control/regression in two different tumor models, was shown to work through CD8 T-cell activation, and displayed favorable pharmacokinetic properties. Together, these findings demonstrate the potential of Q-2361 as a preclinical candidate.
Q-2361 (L-685,818) was first described by Dumont et al as a potent antagonist of the immunosuppressive activity of both tacrolimus and rapamycin.30 43 Unlike tacrolimus, Q-2361 did not affect calcineurin phosphatase activity and did not induce nephrotoxicity in mice (two daily intravenous injections of 100 mg/kg/day did not produce any increase in blood urea nitrogen). Further, the authors reported that when administered via oral gavage at 25 mg/kg/day to either rats or dogs for 14 days, Q-2361 did not cause nephrotoxicity, behavioral changes, or gastrointestinal pathology known to be associated with tacrolimus.30
Our discussions with US and Australian organ transplant clinicians confirm the clinical trigger for standard of care field therapies, such as 5-fluorouracil, photodynamic therapy, and imiquimod is a history of multiple actinic keratoses. The most common field therapy, 5-fluorouracil, has no SCC prevention data in organ transplant recipients44 and there are associated tolerance issues. 5-Fluorouracil is an antimetabolite chemotherapy that causes blistering, pain, and swelling, which is exacerbated by UV exposure.45 Consequently, treatment is limited to once yearly. Recently, 5-fluorouracil was determined to be mutagenic in its own right and may drive tumor evolution and increase the risk of secondary malignancies.46 A recent meta-analysis by Chung et al28 concluded that the two less common field therapies, photodynamic therapy and imiquimod, had no clinical evidence for SCC prevention in organ transplant recipients. Furthermore, they have limited efficacy for actinic keratosis clearance in organ transplant recipients, the typical registration endpoint. Currently, there is no sign that clinical treatment options will improve the high incidence rate of SCCs in organ transplant recipients.
The key differentiation for Q-2361 from competitor field therapies is the potential for SCC prevention based on its unique local activation of CD8 T-cells in immunosuppressed patients. Furthermore, because Q-2361 works specifically through reactivating T-cells, tolerability may be improved relative to competitors that cause blistering and pain through cytotoxicity and general immune activation. If good tolerability is demonstrated clinically, Q-2361 could be administered earlier and regularly, aligned with quarterly organ transplant recipient dermatologist visits. Distinct from other field therapies, the potential for Q-2361 to reactivate CD4+ and CD8+ T-cells in the skin of organ transplant recipients regardless of a calcineurin inhibitor-based treatment regime or a mTOR inhibitor-based treatment regimen also presents the opportunity to trial it for the treatment of other high morbidity skin conditions that affect this patient cohort, including as a field treatment for multiple actinic keratoses,47 48 viral warts,49 50 superficial fungal infections,49 51 and Kaposi sarcoma.52–54
A limitation of our study is that, clinically, we propose to treat patients topically at the first stages of disease, ideally when malignant cells are restricted to the epidermis and upper dermis; however, our mouse tumor models are more representative of invasive disease where malignant cells are growing beneath the dermis. Nonetheless, it is highly encouraging that we demonstrate antitumor immunity in subcutaneous tumors experiencing an exponential growth phase. Additionally, Q-2361 achieves mouse skin concentrations 2000-fold greater than the skin levels of tacrolimus, shown in our previous study. A similar ratio of Q-2361 over tacrolimus achieves full tacrolimus rescue in mouse and human T-cells. A key preclinical objective is to demonstrate that topically delivered Q-2361 has minimal systemic exposure to avoid organ rejection. We have demonstrated in the topical mouse pharmacokinetic study (tables 2 and 3) that sustained, high levels of drug in the skin can be achieved with minimal flux from skin to plasma. Encouragingly, we see no evidence of skin irritation following the topical application of Q-2361 to mouse skin, and Dumont et al report that Q-2361 is non-toxic to mice, rats, and dogs when administered intravenously/orally.30 Towards a first-in-human study, it will be necessary to select a clinic-ready formulation supporting our desired pharmacokinetic profile. In vitro permeation testing and in vivo pharmacokinetic studies will enable formulation selection and confirm that systemic exposure is minimal to ensure a maximal safety window. Testing Q-2361 in human clinical trials will ultimately provide validation for this study.
In summary, we propose to use Q-2361 topically, in conjunction with systemic tacrolimus in organ transplant recipients, at sites of field cancerization or early clinical disease. This intervention promises to reduce the suppressive effects of tacrolimus on cytotoxic CD8 T-cells locally at these skin sites, without altering current patient immunosuppressive drug regimens. In so doing, we hope to achieve the best possible outcome for the patient; no organ rejection, and no cutaneous tumor development. Such an outcome has the potential to be transformative for organ transplant patients trapped between the need for immunosuppression and the lethality of associated malignant skin disorders.
Data availability statement
No data are available.
Patient consent for publication
This study involves human participants. Blood was obtained from healthy human volunteers. Metro South Human Research Ethics Committee—HREC/17/QPAH/778. Participants gave informed consent to participate in the study before taking part.
We thank the Translational Research Institute’s Biological Research Facility and Flow Cytometry Facility for excellent animal care and technical support. We also acknowledge the support provided by The University of Queensland and UniQuest through their drug discovery initiative, QEDDI. Graphics were created with BioRender.com.
Contributors Conceptualization: JWW. Methodology: MV, KB, RP, T-AC, and JWW. Investigation: MV, KB, RP, HYC, T-AC, and JWW. Visualization: MV and JWW. Funding acquisition: IHF, HPS, SC, and JWW. Project administration: BWD, AH, T-AC, and JWW. Supervision: BWD, AH, T-AC, and JWW. Writing—original draft: KB, RP, BWD, AH, and JWW. Writing—review and editing: MV, KB, RP, HYC, IHF, HPS, SC, BWD, AH, T-AC, and JWW. BWD, AH, and JWW are the guarantors of this work.
Funding National Health and Medical Research Council Development Grant APP2000135 (JWW, IHF, SC, and HPS) Merchant Foundation (JWW, IHF, and HPS) National Health and Medical Research Council MRFF Next Generation Clinical Researchers Program Practitioner Fellowship APP1137127 (HPS).
Competing interests The use of Q-2361 as disclosed in this study has been patented (WO2021248189), with JWW, BWD, AH, T-AC, RP, and KB as coinventors. All other authors declare that they have no competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.
Author note This article is dedicated to the memory of Matty Hempstalk, Transplant Australia.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.