Reduction of soluble PD-L1 by plasma exchange and radiation therapy in patients with refractory melanoma re-sensitizes to immunotherapy.

Reduction of soluble PD-L1 by plasma exchange and radiation therapy in patients with refractory melanoma re-sensitizes to immunotherapy. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reduction of soluble PD-L1 by plasma exchange and radiation therapy in patients with refractory melanoma re-sensitizes to immunotherapy. Jacob Orme, Henan Zhang, Prashanth Lingamaneni, Yohan Kim, Roxane Lavoie, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4542220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Introduction : Immune checkpoint inhibitors (ICI) are an essential systemic therapy for advanced melanoma. However, most melanomas develop resistance to ICI. Tumor-derived soluble PD-L1 (sPD-L1) and other soluble immunosuppressive factors drive checkpoint inhibitor resistance and correlate with inferior survival. We previously showed that therapeutic plasma exchange (TPE) removes sPD-L1 from circulation. Thus, we hypothesized that TPE-mediated removal of sPD-L1 and other immunosuppressive factors could overcome immunotherapy resistance in refractory melanoma. Methods: In this clinical trial (NCT04581382), we prospectively enrolled eighteen (18) patients with widely metastatic melanoma with progression despite anti-PD-1 ICI and elevated sPD-L1 by ELISA (≥1.7ng/mL). Each patient received radiotherapy to between one and three metastatic lesions (at least two unirradiated lesions) followed by three TPE sessions on consecutive days and re-challenge with checkpoint inhibitor. The primary safety and efficacy endpoints of the study were adverse events (AEs) and sPD-L1 reduction by TPE, respectively. Secondary endpoints included RECIST-based response in unirradiated lesions and overall survival. Correlative studies included kinetics of sPD-L1 and soluble immonsuppressive factors and dynamics of peripheral immune cell phenotypes. Results : Mean age was 62 (SD 13) and seven of eighteen (39%) were female. Mean baseline sPD-L1 was 26.31 ng/mL (40.01). The treatment was well-tolerated with one (6%) patient with grade 3 and 4 adverse events from a central line infection after TPE. Levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p<0.0001). Two patients (11.1%) experienced complete response (CR), one (5.6%) partial response, three (16.7%) stable disease, and 12 (66.7%) progressive disease. In one case, immunotherapy was discontinued after two years due to no detectable lesions. Changes in tumor-reactive (T TR ) GZMB + /CX3CR1 + /CD11a high and other peripheral immune cell populations predicted overall survival in this cohort. In addition to sPD-L1, other soluble mediators of ICI resistance were also reduced by TPE and predicted overall survival in this cohort. Summary/Conclusion : sPD-L1 and other soluble immunoregulatory signaling molecules are important mediators of ICI resistance. SBRT and TPE can resensitize ICI-refractory melanoma by removing these factors. Patients with persistently elevated or rapid rebound of sPD-L1 following TPE experienced poor response and overall survival. Serial monitoring of sPD-L1 may predict response to ICI and multiple courses of TPE may be necessary. Our findings may apply in other ICI-resistant cancers with elevated sPD-L1. ClinicalTrials.gov registration: NCT04581382, ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma 1). Biological sciences/Immunology/Immunotherapy Biological sciences/Cell biology/Cell death Immunotherapy resistance Therapeutic Plasma Exchange Programmed Cell Death 1 Receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 MAIN Programmed death-ligand 1 (PD-L1) on the surface of cancer cells engages receptor Programmed cell death protein 1 (PD-1) on the surface of immune cells to prevent anti-tumor immunity. 1 Immune checkpoint inhibitor therapies (ICI) blocking PD-1/PD-L1 and other immunosuppressive ligand-receptor interactions are an important tool in the fight against many cancers, including melanoma. 2–4 Unfortunately, the use of ICI in most metastatic cancers does not lead to a cure, as these cancers eventually develop resistance. Overcoming acquired ICI resistance is a major unmet need in melanoma and other cancers. One promising method to address ICI resistance has been the combined use of multiple classes of ICI. For example, an anti-PD-1 ICI such as nivolumab may achieve partial immune cell activation but fail to fully mobilize anti-tumor immunity. In some patients, adding an anti-Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) ICI such as ipilimumab may provide the necessary added activation to completely control a tumor. Indeed, the combination of nivolumab and ipilimumab leads to a robust immune response in multiple cancers, including melanoma, and represents an important clinical tool. 5,6 Unfortunately, cancers employ many defenses and, while the number of available ICI classes has grown, it may not be practical to combine more than a few such treatments in a clinical setting due to overlapping and expanding toxicities, including treatment-related death. 6,7 Furthermore, successive ICI regimens in later lines of therapy have shown mixed results in clinical trials. 6,8 This challenge is explained in part by the presence of soluble immunosuppressive factors that prevent the therapeutic effect of ICI through multiple mechanisms. The most well-studied of these factors is soluble PD-L1 (sPD-L1). Produced by proteolytic cleavage or by alternative splicing, sPD-L1 binds PD-1 directly on the surface of T cells to dampen anti-tumor immunity despite ICI treatment. 9–12 These experimental observations have been confirmed in clinical studies showing that high plasma sPD-L1 predicts ICI resistance in multiple cancers. 13–19 Unsurprisingly, sPD-L1 impairs circulating tumor-reactive (T TR ) CD8 + CD11a high T cells that are critical for ICI response. 20–22 Malignant cells also release exosomal PD-L1 (exPD-L1) to drive a similar immunosuppression. 23–26 While sPD-L1-mediated resistance is formidable on its own, evidence is emerging that similar properties are shared by other cancer-derived soluble factors. These include soluble forms of B7H3 (CD276), 27,28 CD27, 29 CD80 (B7.1), 29–31 CD137, 32,33 CTLA-4 (sCD152), 34–37 Lymphocyte-activation gene 3 (LAG3), 37–39 major histocompatibility complex I chain-related molecule (MIC), 40,41 PD-1, 34 PD-L2, 29 and T-cell immunoglobulin and mucin domain proteins (sTIM1, 3, and 4). 42 Although their mechanisms and contributions to tumor cell immune checkpoint evasion vary, each of these soluble factors has the potential to overwhelm ICI therapy and prevent anti-tumor immunity in cancer. There is a critical need to address this Gordian knot of soluble factors mediating immunosuppression and ICI resistance in cancer. Therapeutic plasma exchange (TPE) is a safe and widely available clinical procedure in which patient blood is filtered or centrifuged to remove deleterious substances. We previously demonstrated that TPE removes sPD-L1 and exosomes from circulation. 43 In our pilot study, TPE removed 70.8% of circulating sPD-L1 from the plasma. In other studies, we have demonstrated that stereotactic body radiotherapy (SBRT) to metastatic sites of disease can expand critical T cell populations that drive ICI response. 26,44,45 Based on these observations, we proposed clinical use of SBRT and TPE to re-sensitize ICI-refractory cancers. In the present study (NCT04581382), we explored sequential limited SBRT, TPE, and ICI re-challenge in the setting of ICI-refractory metastatic melanoma with high (≥1.7 ng/ml) sPD-L1 ( Supplemental Fig 1 ). Each patient received SBRT to three or fewer (and never all) sites of metastasis, three daily sessions of TPE, and finally ICI re-challenge. The primary safety endpoint of this study was adverse events (AE) per version 5.0 of the revised National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). The primary efficacy endpoint was sPD-L1 removal after TPE. Secondary efficacy endpoints were the number of patients achieving complete response (CR), partial response (PR), or stable disease (SD) based on unirradiated lesions and overall survival (OS). Correlative analysis included peripheral blood mononuclear cell (PBMC) subpopulations (including a prespecified change in tumor-reactive CD8+ [T TR ] and regulatory T cells), 44–46 extracellular vesicle (EV) kinetics, and exploratory plasma proteomic analysis. RESULTS Patients and baseline characteristics Patients with metastatic melanoma progressing on any PD-1 ICI were eligible for this study. Potentially eligible patients provided informed consent prior to a baseline screening blood draw. Patients were screened from December 2020 to February 2023 with an accrual goal of between 17 and 20 evaluable patients. Thirty-four (34) patients were screened for eligibility (see CONSORT diagram in Supplemental Fig 2 ). Sixteen (16) patients were excluded due to low sPD-L1 (n=8), poor venous access (n=2), unwillingness to undergo treatment or follow-up (n=2), contraindications to ICI (n=2), or death before sPD-L1 level could be assessed (n=2). Eighteen (18) patients were eligible ( Table 1 ). Mean age of patients in the study was 63 years (SD 13), and 7 (39%) were female. Mean baseline sPD-L1 measured by ELISA was 26.31 ng/mL. All patients had previously received at least one PD-1 inhibitor that had failed, and fifteen (83%) had previously received at least one ICI doublet ( e.g. , ipilimumab and nivolumab) that had failed. Half of patients had received two prior lines of therapy, and an additional one third of patients had received four or more lines of therapy before enrollment in the study. All eligible patients (n=18) underwent treatment ( Supplemental Fig 1 and 2 ). Treatment comprised 1-5 days of stereotactic body radiotherapy (SBRT) to at least one—but not all—sites of disease seen on imaging. This was followed by three consecutive days of one session each of TPE, followed by re-initiation of ICI therapy. ICI therapy was the same as previously received in 16 patients (89%). One patient was unable to complete the third TPE treatments due to a line infection. All patients were included in the intent to treat analysis regardless of treatment completion. SBRT, TPE, and ICI re-challenge is safe and well-tolerated. The primary safety endpoint of this trial was to assess the adverse events (AE) observed during SBRT, TPE, and ICI re-challenge in patients with melanoma receiving PD-1 immunotherapy ( Table 2 ). AEs were collected and reported according to CTCAE V.5.0. In a pre-planned stopping rule, if 3 of the first 10 patients were to experience grade 4 or greater adverse events attributable to the intervention, the study would be halted. In total, one patient experienced AE grade 3 and 4 thromboembolic event and sepsis, respectively, secondary to an infection of a vascular access device. This patient required hospitalization with line removal and intravenous antibiotics. Other AEs were grade 1-2 and included fatigue and arthralgias/myalgias. No deaths were attributable to therapy. Therapeutic plasma exchange removes soluble PD-L1 with varying reconstitution rates. The primary efficacy endpoint of the study was the reduction of sPD-L1 levels by TPE ( Fig 1A, Supplemental Figure 3A ). Relative and absolute levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p=6.1*10 -5 ). Mean recovery rate of sPD-L1 at the second cycle of ICI re-challenge was 21.4% of pre-TPE levels. SBRT, TPE, and ICI re-challenge resensitized melanomas to immunotherapy. A secondary efficacy endpoint of the study was the proportion of patients achieving CR (defined as absence of radiologically apparent disease), PR (defined as greater than 30% radiographic reduction), or SD (defined as less than 30% radiographic reduction) as a best response per Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 in unirradiated lesions assessed radiographically. 47 Response was assessed by a trained radiologist. Two patients (11.1%) experienced CR, one (5.6%) PR, three (16.7%) SD, and 12 (66.7%) progressive disease (PD) ( Table 3 ). Notably, there was a deeper absolute sPD-L1 reduction with TPE in patients who experienced CR or PR (p=0.02, Supplemental Fig 3B ). A further secondary endpoint of the study was overall survival (OS). Median overall survival for the cohort by Kaplan-Meier estimate was 18.9 months (95% CI 15.1-NR) ( Supplemental Fig 4 ). CR, PR, or SD response to treatment predicted superior overall survival (OS) (p=0.002) ( Fig 1B, Supplemental Fig 5 ). Median OS for patients experiencing PD was 15 months. Median OS for responders was not reached. Representative irradiated and unirradiated lesions were selected and measured by a blinded radiologist across time in approximately 3-month intervals and assessed for standardized uptake value (SUV) and tumor size. The majority of measured irradiated and unirradiated lesions showed a greater than 50% reduction in SUV ( Fig 1C ). Approximately one third of measured irradiated and unirradiated lesions showed a greater than 50% reduction in size ( Fig 1D) . An example FDG PET scan image of irradiated and non-irradiated lesions in a patient experiencing CR is shown in Fig 1E . A swimmer plot of all patients is shown in Fig 1F . Tumor-reactive effector T cell changes predict outcomes to immunotherapy re-challenge after radiotherapy and TPE. Peripheral blood mononuclear cell (PBMC) subpopulations act as liquid biomarkers and as critical substrates of ICI immunotherapy. Changes in these subpopulations reflect the effects of therapy on anti-tumor immunity. We isolated PBMCs and measured subpopulations by flow cytometry according to a broad panel ( Supplemental Tables 1-2, Supplemental Fig 6-7 ). We compared subpopulations from pre-treatment to the beginning of the second cycle of ICI re-challenge. A heatmap of changes is shown ( Fig 2A). We further evaluated how these changes correlated with overall survival in our cohort by Cox proportional hazards regression ( Fig 2B, Supplemental Tables 3-4 ). Our group previously identified tumor-reactive CD8+ T cells (T TR ) with high expression of CD11a, Granzyme B, and CX3CR1 (GZMB + /CX3CR1 + /CD11a high or T TR ). 44–46 In a prespecified analysis, peripheral blood T TR changes from baseline to the next cycle of ICI re-challenge predicted overall survival (OS) with a Cox proportional hazard ratio (HR) of 0.16 (95% confidence interval [CI] 95% CI 0.03-0.8, p=0.026). We plotted survival by T TR change using Kaplan-Meier estimates ( Fig 2C , log rank p=0.01). When T TR levels were analyzed dichotomously as increasing versus decreasing (cutoff ratio=1), the results were identical. Regulatory T cells marked by CD4, CD25, and FOXP3 expression (T reg ) are well-established predictors of poor response to immunotherapy. In a prespecified analysis, peripheral blood T reg changes from baseline to the next cycle of ICI re-challenge predicted OS (HR 7.87 [95% CI 1.6-38.7], p=0.01). We plotted survival by T reg change using Kaplan-Meier estimates ( Fig 2D , log rank p=0.004). When T reg levels were analyzed dichotomously as increasing versus decreasing, these cells did not predict a significant difference in OS (HR 2.89 [95% CI 0.74-11.3], p=0.13). We previously showed that NKG7 marks resilient T cells that are critical to immunotherapy response. 44,48 In an exploratory analysis, changes in peripheral blood CD11a high CD8+ T cells with NKG7 expression from baseline to the next cycle of ICI re-challenge predicted superior OS (HR 0.19 [95% CI 0.04-0.88], p=0.03). We plotted survival by NKG7-positive T cell change using Kaplan-Meier estimates ( Fig 2E , log rank p=0.02). In addition, we previously reported that Bim (BCL-2-interacting mediator of cell death) is upregulated in T cells by PD-L1/PD-1 engagement and that T cell Bim expression drives poor anti-tumor immunity. 44,49 Persistent CD8+ T cell Bim expression despite ICI predicts poor responses to immunotherapy. 50 In an exploratory analysis, changes in peripheral blood CD11a high CD8+ T cells with Bim expression from baseline to the next cycle of ICI re-challenge predicted inferior OS (HR 6.8 [95% CI 1.58-29.32], p=0.01). We plotted survival by Bim + T cell change using Kaplan-Meier estimates ( Fig 2F , log rank p=0.003). Soluble mediators of immunosuppression are elevated in patients with ICI-refractory melanoma, are reduced by TPE, and rapidly reaccumulate. As noted above, soluble factors beyond sPD-L1 contribute to ICI resistance. 27–42 We next sought to determine the effect of TPE on these soluble factors in systemic circulation. We measured a large library of soluble factors in sufficient plasma samples from patients on the study and compared these to matched healthy controls using a high-throughput O-link multiplex assay. A variety of immunosuppressive soluble factors were elevated at baseline in the blood of patients with ICI-refractory melanoma versus healthy controls ( Fig 3A, Data File 1 ), including sCTLA-4, sLAG3, sPD-1, and sTIM-3. We next measured reduction and rebound of these factors in the plasma of patients from SBRT to TPE, pre- to post-TPE, and at the second cycle of ICI re-challenge (see Data File 1 ). TPE significantly reduced exemplary immunosuppressive factors sPD-1, sLAG3, and sTIM3 (HAVCR2) ( Fig 3B-D ). Notably, sCTLA4, which was only moderately elevated in this cohort versus healthy controls, was unchanged ( Fig 3E ). Conversely, Prostaglandin E synthase 2 (PTGES2), a marker of systemic inflammatory response, increased significantly after TPE (p=0.0004) and recovered by cycle 2 of ICI re-challenge (p=0.016) ( Fig 3F ). Given the direct connection between soluble immunosuppressive factors and ICI resistance, we sought to determine whether the level of these factors at the time of ICI re-challenge ( i.e. , post-TPE) could predict overall survival. Level of sPD-L1 (log rank p=0.003) and sTIM3 (log rank p=0.007) after TPE predicted OS ( Fig 4A-B ). Conversely, OS was not predicted by sPD-1, sLAG3, or sCTLA-4 in this cohort. As outlined above, we observed rapid reconstitution of most factors prior to second cycle ICI re-challenge. We next sought to determine whether levels of these factors at the second cycle of ICI re-challenge could predict outcomes. These factors did not predict OS in our cohort ( Fig 4C-D ). Exosomal PD-L1 was not significantly changed on treatment. We and others have previously showed that malignant cells release exosomes or extracellular vesicles bearing PD-L1 that can drive immunosuppression and can release sPD-L1 by proteolytic cleavage. 23–26 Radiation can cause tumor exosome shedding by multiple mechanisms, including cell death. 26 However, exosomal PD-L1 (exPD-L1) was previously reduced by TPE in a pilot study. 43 Thus, it is unclear whether exPD-L1 would be expected to be reduced or increased after both SBRT and TPE. We measured exPD-L1 by flow cytometry before and after TPE in the present cohort ( Supplemental Fig 8A ). Circulating exPD-L1 was not significantly changed. However, change in exPD-L1 after TPE predicted improved OS (HR 0.28 [95% CI 0.08-0.98], p=0.047) in the cohort ( Supplemental Fig 8B ). DISCUSSION Soluble immunosuppressive factors present a significant challenge to ICI therapy. While PD-1 inhibitors and sPD-L1 are the best studied classes of ICI and resistance-inducing soluble immunosuppressive factors, respectively, they are archetypal of other ICI treatments and the multi-layered evasions employed by cancer cells. Current ICI therapies can be overwhelmed by the volume and variety of these factors in the blood of patients with ICI-resistant malignancies. This study provides the first clinical evidence that a combination of limited SBRT, TPE, and ICI re-challenge can restore responsiveness to immune checkpoint inhibitors (ICIs) in patients with metastatic melanoma who had previously progressed on the same ICI regimen. We observed a disease control rate (DCR) of 33% among these patients, meeting our prespecified secondary endpoint. This DCR is remarkable in such a heavily pre-treated population. 89% of patients had previously received multiple lines of therapy that had failed. 83% had previously received at least one ICI doublet that had failed in a prior line of therapy. 89% of patients on the study received the same PD-1 ICI that had failed in a prior line of therapy. This study met its primary safety and efficacy endpoints. AEs were commensurate with other studies of immunotherapy. TPE effectively reduced plasma sPD-L1, although the level of sPD-L1 and other soluble factors known to limit anti-tumor immunity rebounded rapidly by the second cycle of ICI in this study. The study also provides provocative correlative markers that may help inform future approaches. First, treatment induced significant changes in peripheral circulating immune cells. Some of these changes, including tumor reactive CD8+ T cells that express CD11, Granzyme B, and CX3CR1 and classical regulatory T cells, predicted clinical outcomes despite the limited number of patients in the study. A favorable shift in the balance of anti-tumor immunity and tolerance provides evidence that ICI-resistant tumors remain potentially sensitive to immunotherapy. Furthermore, these changes reflect the accepted mechanism of treatment response and may help guide future studies as biomarkers. Second, TPE significantly reduced soluble mediators of immunosuppression. The reduction of several of these factors also predicted clinical outcomes in this cohort. While soluble mediators of ICI resistance will vary widely from patient to patient and from cancer to cancer, TPE offers a broad way to address many such mediators. In all, this study suggests that SBRT and TPE may “reset” systemic immunity and restore immunotherapy sensitivity in ICI-refractory cancers. The recent SWOG study S1616 provides important context to our findings and an alternative approach to ICI resistance. In this study, patients with anti-PD-1 ICI-refractory melanoma were randomized to anti-CTLA-4 ipilimumab monotherapy or the ipilimumab and nivolumab doublet in the second line (NCT03033576). 6 Anti-CTLA-4 ICI monotherapy achieved a 9% response rate in this study as compared with 28% in the doublet arm, showing that anti-PD-(L)1 ICI resistance can be addressed clinically. A small minority of these patients had previously received an ICI doublet. It is unknown whether a combination of multiple ICI modalities and TPE could further improve response. Several limitations to this study should be acknowledged. First, this study combined three sequential treatments—SBRT, TPE, and PD-(L)1 ICI re-challenge—in an open label, single arm trial. While biomarkers were measured at multiple timepoints in the study, the individual contribution of SBRT and TPE to overcoming ICI resistance is uncertain. Although SBRT was highly effective against individual metastases in this study, most lesions were not irradiated. Of these unirradiated lesions, most showed a reduction in both size and FDG avidity. However, our observations must not be taken as evidence for a purported “abscopal” effect—meaning involution of unirradiated tumors after radiation of distant metastases—because this trial included neither an SBRT-free control nor a TPE-free comparator arm from which to draw such conclusions. A potential synergy of SBRT and TPE is that SBRT may reduce the de novo production of sPD-L1 from tumor sites and TPE may reduce the products (including sPD-L1) directly from circulation. While the clinical activity observed in this study—particularly against individual melanoma lesions—is favorable and warrants further study, most patients experienced disease progression and death. Even in patients with a favorable response, there was rapid re-accumulation of many immunosuppressive factors in the bloodstream. It is unclear whether a more sustained suppression of these factors would improve the robustness of ICI re-challenge response. Our approach was limited to a single three-day course of TPE. It is possible that serial TPE prior to each ICI cycle may be more effective, but this cannot be determined from the present study. Similarly, it is unclear to what extent SBRT affects these changes. This study did not include novel ICI classes. Thus, additional clinical trials are needed to determine how best to leverage SBRT and TPE in ICI resistance. As a related limitation, SBRT and TPE are medical procedures with attributes that may limit their use. First, while the current cost of each TPE session is approximately one sixth of the cost of each immunotherapy injection, the costs of immunotherapy are expected to decrease in the coming years through a combination of formulation changes and alternatives. 51 Conversely, the costs of SBRT and TPE are inelastic. This may limit the number of patients who are able to obtain the procedures. Further, while SBRT is widely available, TPE requires specialized equipment and expertise that are currently available only in limited quantity in major medical centers. Lastly, TPE requires wide-bore peripheral venous access. This increases risks of infection, hypotension, and hematoma formation. Taken together, this study suggests that SBRT, TPE, and ICI re-challenge provide a promising method to overcome ICI resistance in melanoma. Future studies are warranted to validate and optimize this approach as a tool to overcome ICI resistance in melanoma and other malignancies. These studies may include multiple cycles of TPE to achieve effective suppression of immunosuppressive factors. METHODS Study design and participants We conducted the ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma) study under a protocol approved by the local institutional review board and registered at ClinicalTrials.gov (NCT04372619). This was a prospective, open-label phase II trial for patients with advanced ICI-resistant melanoma with elevated sPD-L1 levels. Written informed consent was obtained from all participants prior to study enrollment. All data were collected in a deidentified fashion in a password-protected database. Inclusion criteria included age 18 or older, histologically confirmed melanoma, measurable disease per RECIST criteria, ECOG (Eastern Cooperative Oncology Group) status of 3 or better, sPD-L1 level 1.7 ng/mL or greater, and feasible vascular access. Exclusion criteria included contraindications to PD-(L)1 inhibitor ICI and unwillingness to undergo treatment and follow-up. Patients who consumed biotin supplementation were excluded due to the known interference of biotin supplements with accurate measurement of sPD-L1. 11 Key Inclusion criteria: Histological confirmation of melanoma Measurable or non-measurable disease Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤ 3 sPD-L1 levels > 1.7 ng/ml by enzyme-linked immunosorbent assay (ELISA) Negative pregnancy test done =< 7 days prior to radiation therapy, for women of childbearing potential only Provide written informed consent Willing to return to enrolling institution for follow-up (during the Active Monitoring Phase of the study) Willing to provide blood samples for correlative research purposes Recruitment Patients with metastatic melanoma progressing despite ICI treatment were approached in the Department of Medical Oncology or Department of Radiation Oncology at a single academic center between December 2020 and February 2023. Patients were screened for sPD-L1 levels using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (see Biomarker Assays). Patients with sPD-L1 levels ≥1.7 ng/mL were considered positive and eligible for the study. Treatment Consenting and eligible patients underwent 1 to 5 days of SBRT to between one and three metastatic sites. The majority of metastatic sites were not irradiated for each patient. Patients next underwent three daily sessions of TPE, performed using centrifugation-based cell separators, either the Fenwal Amicus (Fresenius KABI USA LLC, Lake Zurich, Illinois, USA) or the Spectra Optia (Terumo BCT, Lakewood, Colorado, USA). For each patient, a single plasma volume was exchanged at each session. Either peripheral intravenous or central line vascular access was used to perform the procedure. Removed plasma was discarded and replaced with albumin. Vascular access lines were removed after the last planned TPE session. After completing TPE, patients received PD-(L)1 ICI re-challenge per clinician preference. ICI cycle duration was determined per product label as standard of care. Assessments Clinical standard of care imaging was performed per clinician preference, generally comprising conventional computed tomography (CT) imaging with or without advanced molecular [¹⁸F]Fluorodeoxyglucose positron emission tomography (FDG PET) imaging every 3 months. Changes in size were reported as best response per Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 in unirradiated lesions assessed radiographically. 47 CR was defined as resolution of radiologically apparent disease on conventional imaging, PR as greater than 30% radiographic reduction on conventional imaging, SD as less than 30% radiographic reduction, and PD as 20% or greater increase in radiographically apparent disease. PET avidity was not used to determine response. For waterfall plots, at least two metastatic sites were reported for each patient commensurate with relative metastatic burden. Study endpoints The primary safety endpoint was the rate of adverse events (AE) measured using the revised National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The maximum grade for each type of AE was recorded for each patient. The primary efficacy endpoint was the kinetics of soluble sPD-L1 removal after plasma exchange. The secondary efficacy endpoint was response, defined as patients with complete response (CR), partial response (PR), or stable disease (SD) at 3 months. Correlative analyses comprised overall survival (OS), peripheral blood mononuclear cell (PBMC) subpopulations including prespecified T cell population changes, and plasma proteomic analysis as outlined below. Biomarker assays Soluble PD-L1 (sPD-L1) sPD-L1 was measured by enzyme-linked immunosorbent assay (ELISA) as previously published. 11 This assay comprises paired mouse IgG2 monoclonal antibody clones H1A (capture) and biotinylated B11 (detection) against extracellular human PD-L1 using a standard streptavidin-HRP method. Concentrations were determined by optical density (OD) measurements along a known standard curve of recombinant human PD-L1. Assays were performed in a blinded fashion in triplicate and reported in nanograms per mililiter (ng/mL). Extracellular vesicle-bound PD-L1 (evPD-L1) Extracellular vesicle-bound PD-L1 (evPD-L1) was measured by flow cytometry as previously published. 52 Plasma samples were centrifuged twice at 2000g to deplete platelets and analyzed using an A60-Micro Plus Nanoscale Flow Cytometer (Apogee FlowSystems) gating for mid-intensity light angle light scatter and markers of interest. Nanoscale flow cytometer calibration was performed using a standard reference bead mix. Clone H1A anti-PD-L1 antibodies were conjugated to fluorophores (Life Technologies) and incubated with samples prior to analysis. Concentrations were determined by gated counts using a standard reference bead mix. Assays were performed in a blinded fashion in triplicate and reported as number of particles. Olink proteomic profiling Soluble plasma markers were assessed using a commercially available platform (Olink) and compared to age-matched healthy controls. Plasma samples were labeled with oligonucleotide-labeled antibody probes and subjected to microfluidic PCR amplification using a Proximity Extension Assay (PEA) with NGS readout on Illumina instruments. Relative concentrations were determined on an arbitrary relative scale. Assays were performed in a blinded fashion in triplicate and reported in relative units. Peripheral blood mononuclear cell (PBMC) profiling Peripheral blood mononuclear cells (PBMC) were profiled for established cell types (see Supplemental Tables 1-2 ). We stained surface markers prior to intracellular markers. Flow cytometry data were collected on a Cytek Aurora (Cytek Biosciences). We analyzed flow cytometry data with FlowJo 10.10.0 (Tree Star). Changes in percent from baseline to next cycle of ICI re-challenge were compared and plotted. A prespecified analysis was performed for Granzyme B-positive, CX3CR1-positive, CD11a-high CD8-positive (GZMB + /CX3CR1 + /CD11a high or T TR ) CD8 + tumor-reactive T cells and CD4-positive, CD25-positive, FOXP3-positive (T reg ) regulatory T cells that are associated with response to immunotherapy. 44–46 Additional exploratory analyses were performed for each cell subtype. Flow cytometry was performed in a blinded fashion and reported as a percent of total live PBMCs. Statistical analysis A total of 20 patients (17 evaluable) was estimated to be needed to assess primary efficacy and safety endpoints. All patients who were eligible were included in the intention to treat (ITT) analysis regardless of treatment completion. Overall survival was estimated by the Kaplan-Meier method and groups were further compared by Cox proportional hazards or log rank tests as indicated. Adverse events were reported by maximum grade in tabular form. Wilcoxon signed- rank test was used to assess changes in levels over time across the different timepoints of interest. Student’s t test was used for additional comparisons as indicated. Declarations Financial support: Lawrence W. And Marilyn W. Matteson Fund at Mayo Clinic (JJO and SSP), National Cancer Institute (R21-CA259236; JJO, JLW, and SSP) COI: Intellectual property has been filed addressing discoveries disclosed in this manuscript. J.J.O. reports support and/or research and consulting support from the Prostate Cancer Foundation, NaNotics LLC, Genentech, and Partner Therapeutics. L.K. reports consultant activities for Immunocore. M.S.B. reports institutional research support from Alkermes, Bristol-Myers Squibb, Genentech, Merck, nFerence, Pharmacyclics, Regeneron, Sorrento, TILT Biotherapeutics, Transgene, and Viewpoint Molecular Therapeutics; as well as consultant activities for Sorrento Therapeutics, TILT Biotherapeutics, and Viewpoint Molecular Targeting. A.S.M. reports consulting activities with AbbVie, AstraZeneca, BMS, Genentech, Janssen, Takeda Oncology, Sanofi Genzyme, Gilead, Johnson & Johnson Global Services, Chugai Pharmaceutical Co., Ltd. (Roche), TRIPTYCH Health Partners, Ideology Health LLC, Intellisphere LLC, Answers in CME, and Immunocore; as well as study funding subsequent publication processing fees from Bristol Myers Squibb; travel support from Roche; was a non-remunerated director of the Mesothelioma Applied Research Foundation and is a non-remunerated director of the Friends of Patan Hospital. S.N.M. reports grant funding, clinical trial for BMS and grant funding, PI for Sorrento Pharma. K.O. reports honorarium from UptoDate. D.O. reports research support from AstraZeneca, and Varian; as well as honorarium from UptoDate. D.M. reports intellectual property from EXACT Sciences, InSitu Biologics, and CurrentHealth. F.L. reports receiving research support to institution from Nanotics LLC; serving as scientific consultant for Mursla Bio; receiving royalties from Early is Good. J.W. reports non-remunerated director of ASFA and ISFA. DATA SHARING STATEMENT All data, including redacted individual patient data, and a data dictionary defining each field will be made available in Data File 1. Study protocol and informed consent form will also be available with publication. FUNDING: Lawrence W. And Marilyn W. Matteson Fund at Mayo Clinic (JJO and SSP), National Cancer Institute (R21-CA259236; JJO, JLW, and SSP) ACKNOWLEDGEMENTS We thank the brave and selfless patients and their families who agreed to participate in this clinical trial. We thank clinical staff and study coordinators. Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science (CCaTS). Funding for the study was provided by the Lawrence W. And Marilyn W. Matteson Fund at Mayo Clinic and the National Cancer Institute (R21-CA259236). References Dong H, Strome SE, Salomao DR et al (2002) Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. 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Immunology 123(4):538–546 Oaks MK, Hallett KM, Penwell RT, Stauber EC, Warren SJ, Tector AJ (2000) A native soluble form of CTLA-4. Cell Immunol 201(2):144–153 Botticelli A, Zizzari IG, Scagnoli S et al (2021) The Role of Soluble LAG3 and Soluble Immune Checkpoints Profile in Advanced Head and Neck Cancer: A Pilot Study. J Pers Med ;11(7) Wang Q, He Y, Li W et al (2022) Soluble Immune Checkpoint-Related Proteins in Blood Are Associated With Invasion and Progression in Non-Small Cell Lung Cancer. Front Immunol 13:887916 Möller-Hackbarth K, Dewitz C, Schweigert O et al (2013) A Disintegrin and Metalloprotease (ADAM) 10 and ADAM17 Are Major Sheddases of T Cell Immunoglobulin and Mucin Domain 3 (Tim-3)*. J Biol Chem [Internet]. ;288(48):34529–44. https://www.sciencedirect.com/science/article/pii/S0021925820554495 Kashyap S, Singh MK, Kumar N et al (2023) Implications of LAG3 and CTLA4 immune checkpoints beyond PD-1/PD-L1 as a potential target in determining the prognosis of uveal melanoma patients. Br J Ophthalmol ; 21;108(6):903–912 Orme JJ, Enninga EAL, Lucien-Matteoni F et al (2020) Therapeutic plasma exchange clears circulating soluble PD-L1 and PD-L1-positive extracellular vesicles. J Immunother Cancer [Internet]. ;8(2). http://www.ncbi.nlm.nih.gov/pubmed/32817395 Gicobi JK, Mao Z, DeFranco G et al (2023) Salvage therapy expands highly cytotoxic and metabolically fit resilient CD8 + T cells via ME1 up-regulation. Sci Adv [Internet]. ;9(46):eadi2414. https://doi.org/10.1126/sciadv.adi2414 Zhang H, Orme JJ, Abraha F et al (2021) Phase II evaluation of Stereotactic Ablative Radiotherapy (SABR) and immunity in 11 C-Choline-PET/CT-identified oligometastatic castration-resistant prostate cancer. Clin Cancer Res [Internet]. ;clincanres.2510.2021. http://clincancerres.aacrjournals.org/lookup/doi/ 10.1158/1078-0432.CCR-21-2510 Lucien F, Kim Y, Qian J et al (2022) Tumor-Derived Extracellular Vesicles Predict Clinical Outcomes in Oligometastatic Prostate Cancer and Suppress Antitumor Immunity. Int J Radiat Oncol Biol Phys [Internet]. [cited 2022 Sep 25]; https://pubmed.ncbi.nlm.nih.gov/35671867/ Eisenhauer EA, Therasse P, Bogaerts J et al (2009) New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 45(2):228–247 Yan Y, Cao S, Liu X et al (2018) CX3CR1 identifies PD-1 therapy-responsive CD8 + T cells that withstand chemotherapy during cancer chemoimmunotherapy. JCI Insight ;3(8) Gibbons RM, Liu X, Pulko V et al (2012) B7-H1 limits the entry of effector CD8(+) T cells to the memory pool by upregulating Bim. Oncoimmunology 1(7):1061–1073 Wen T, Barham W, Li Y et al (2022) NKG7 Is a T-cell-Intrinsic Therapeutic Target for Improving Antitumor Cytotoxicity and Cancer Immunotherapy. Cancer Immunol Res 10(2):162–181 VanderWalde A, Bellasea SL, Kendra KL et al (2023) Ipilimumab with or without nivolumab in PD-1 or PD-L1 blockade refractory metastatic melanoma: a randomized phase 2 trial. Nat Med 29(9):2278–2285 Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations Yes there is potential Competing Interest. Support and/or research and consulting support from the Prostate Cancer Foundation, NaNotics LLC, Genentech, and Partner Therapeutics. Supplementary Files SupplementalData.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":159011,"visible":true,"origin":"","legend":"\u003cp\u003eTPE removes sPD-L1 and SBRT, TPE, and ICI re-challenge resensitize melanomas to immunotherapy.(A) In the primary efficacy endpoint, TPE removed a mean 78% of sPD-L1 (Wilcoxon p=0.0002).(B) In a secondary efficacy endpoint, patients with CR, PR, or SD experienced superior OS (p=0.002). See also Supplemental Figure 3. (C) A blinded radiologist assessed up to five lesions per patient for SUV change on treatment. SUV for each lesion at each timepoint was calculated by mean blood pool SUV. Irradiated (\u003cem\u003ered\u003c/em\u003e) and unirradiated (\u003cem\u003eyellow\u003c/em\u003e) index lesions are plotted. 63% of lesions had an SUV reduction greater than 50% (78% of irradiated lesions, 54% of unirradiated lesions) on treatment. (D) A blinded radiologist assessed up to five lesions per patient for size change on treatment. Irradiated (\u003cem\u003ered\u003c/em\u003e) and unirradiated (\u003cem\u003eyellow\u003c/em\u003e) index lesions are plotted. 78% of lesions reduced in size (91% of irradiated lesions, 70% of unirradiated lesions) on treatment. (E) One patient experienced complete response with irradiation of one lesion (red arrow). Unirradiated lesions are also marked (yellow arrow). (F) Patient survival over time is plotted and marked by treatment response. Patients marked with “x” died at the timepoint specified. TPE = therapeutic plasma exchange, SBRT = stereotactic body radiotherapy, ICI = immune checkpoint inhibitor, OS = overall survival, HR = hazard ratio. CR = complete response, PR = partial response, SD = stable disease, PD = progressing disease, SUV = standardized uptake value (SUV). See also Supplemental Fig 3-5.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/9d1818a877ab910967933944.png"},{"id":67878868,"identity":"017c12ce-cbda-4842-895a-45656435552d","added_by":"auto","created_at":"2024-10-30 16:39:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186496,"visible":true,"origin":"","legend":"\u003cp\u003eTumor effector T cell changes predict outcomes to immunotherapy re-challenge after radiotherapy and TPE. (A) PBMCs were isolated from each patient and subpopulations were measured by a broad flow cytometry panel and changes in percent of each subpopulation is reported for each patient on a log scale. (B) Cox proportional hazards regression was performed for changes in each subpopulation. Hazard ratios with 95% confidence intervals and are shown. (C) Overall Survival (OS) is predicted by change in GZMB\u003csup\u003e+\u003c/sup\u003eCX3CR1\u003csup\u003e+\u003c/sup\u003eCD11a\u003csup\u003ehigh\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e (T\u003csub\u003eTR\u003c/sub\u003e) T cells from baseline to the second cycle of ICI re-challenge (log rank p=0.01; HR 0.16 [95% CI 0.03-0.8], p=0.026). (D) Overall Survival (OS) is predicted by change in CD25\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+ \u003c/sup\u003e(T\u003csub\u003ereg\u003c/sub\u003e) T cells from baseline to the second cycle of ICI re-challenge (log rank p=0.0035; HR 7.87 [95% CI 1.6-38.7], p=0.01). (E) Overall Survival (OS) is predicted by change in NKG7\u003csup\u003e+\u003c/sup\u003eCD11a\u003csup\u003ehigh\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells from baseline to the second cycle of ICI re-challenge (log rank p=0.019; HR 0.19 [95% CI 0.04-0.88], p=0.03). (F) Overall Survival (OS) is predicted by change in Bim\u003csup\u003e+ \u003c/sup\u003eT cells from baseline to the second cycle of ICI re-challenge (log rank p=0.003; HR 6.8 [95% CI 1.58-29.32], p=0.01). CI = confidence interval, GZMB = Granzyme B, HR = Cox proportional hazard ratio, PBMCs = peripheral blood mononuclear cells. See also Supplemental Tables 1-4, Supplemental Figs 6-7.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/cded92790253dd41ce6321ab.png"},{"id":67878869,"identity":"73286234-02dc-40d4-9582-ffc13ae5e485","added_by":"auto","created_at":"2024-10-30 16:39:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231353,"visible":true,"origin":"","legend":"\u003cp\u003eSoluble mediators of immunosuppression are elevated in patients with ICI-refractory melanoma, are reduced by TPE, and rapidly reaccumulate. (A) Soluble factors at baseline in patients with ICI-refractory melanoma and matched healthy controls were measured by O-link multiplex assay. The log difference in mean level (x axis) and p value of the difference by Student’s \u003cem\u003et \u003c/em\u003etest (y axis) is shown for each soluble factor. Soluble immunosuppressive molecules are shown in red. (B) sPD-1 was significantly reduced (p=0.0001) by TPE and quickly recovered (p=0.0003) before the second cycle of re-challenge ICI. (C) LAG3 was significantly reduced (p=0.0001) by TPE and quickly recovered (p=0.0003) before the second cycle of re-challenge ICI. (D) TIM3 was significantly reduced (p=0.0002) by TPE and quickly recovered (p=0.0004) before the second cycle of re-challenge ICI. (E) CTLA4 was not significantly affected by TPE in our cohort. (F) Prostaglandin E synthase 2 (PTGES2), a marker of systemic inflammatory response, increased significantly after TPE (p=0.0004) and recovered by cycle 2 of ICI re-challenge (p=0.016). See also Data File 1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/bb2c0c0148f114b00111039b.png"},{"id":67878872,"identity":"3f759370-dafe-4934-9281-77a2f0dc2232","added_by":"auto","created_at":"2024-10-30 16:39:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181401,"visible":true,"origin":"","legend":"\u003cp\u003eSoluble mediators of immunosuppression predict overall survival. (A-B) High post-TPE sPD-L1 and sTIM3 levels predicted inferior OS (p=0.033 and 0.007, respectively). (C-D) High second-cycle sPD-L1 and sTIM3 levels did not predict OS (p=0.23 and 0.059, respectively).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/51c9cbd5782c5274d1b2f9dc.png"},{"id":78502403,"identity":"3dd60f29-9c53-44ec-bcdc-8aa1a94076e8","added_by":"auto","created_at":"2025-03-14 07:06:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1819505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/8339366a-a252-4750-86d8-e0e0b07f488c.pdf"},{"id":67878871,"identity":"51025a22-104d-426e-a937-44a0115a6a37","added_by":"auto","created_at":"2024-10-30 16:39:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1689830,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalData.docx","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/0e3fc073bdb9ce21564a7e82.docx"},{"id":67879516,"identity":"0d9ed0bc-6937-4a7f-9f2e-9c8fa2b8ec06","added_by":"auto","created_at":"2024-10-30 16:47:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19990,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4542220/v1/c33abe575d5a59c9ed498fef.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nSupport and/or research and consulting support from the Prostate Cancer Foundation, NaNotics LLC, Genentech, and Partner Therapeutics.","formattedTitle":"Reduction of soluble PD-L1 by plasma exchange and radiation therapy in patients with refractory melanoma re-sensitizes to immunotherapy.","fulltext":[{"header":"MAIN","content":"\u003cp\u003eProgrammed death-ligand 1 (PD-L1) on the surface of cancer cells engages receptor Programmed cell death protein 1 (PD-1) on the surface of immune cells to prevent anti-tumor immunity.\u003csup\u003e1\u003c/sup\u003e Immune checkpoint inhibitor therapies (ICI) blocking PD-1/PD-L1 and other immunosuppressive ligand-receptor interactions are an important tool in the fight against many cancers, including melanoma.\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e Unfortunately, the use of ICI in most metastatic cancers does not lead to a cure, as these cancers eventually develop resistance. Overcoming acquired ICI resistance is a major unmet need in melanoma and other cancers.\u003c/p\u003e\n\u003cp\u003eOne promising method to address ICI resistance has been the combined use of multiple classes of ICI. For example, an anti-PD-1 ICI such as nivolumab may achieve partial immune cell activation but fail to fully mobilize anti-tumor immunity. In some patients, adding an anti-Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) ICI such as ipilimumab may provide the necessary added activation to completely control a tumor. Indeed, the combination of nivolumab and ipilimumab leads to a robust immune response in multiple cancers, including melanoma, and represents an important clinical tool.\u003csup\u003e5,6\u003c/sup\u003e Unfortunately, cancers employ many defenses and, while the number of available ICI classes has grown, it may not be practical to combine more than a few such treatments in a clinical setting due to overlapping and expanding toxicities, including treatment-related death.\u003csup\u003e6,7\u0026nbsp;\u003c/sup\u003eFurthermore, successive ICI regimens in later lines of therapy have shown mixed results in clinical trials.\u003csup\u003e6,8\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThis challenge is explained in part by the presence of soluble immunosuppressive factors that prevent the therapeutic effect of ICI through multiple mechanisms. The most well-studied of these factors is soluble PD-L1 (sPD-L1). Produced by proteolytic cleavage or by alternative splicing, sPD-L1 binds PD-1 directly on the surface of T cells to dampen anti-tumor immunity despite ICI treatment.\u003csup\u003e9\u0026ndash;12\u003c/sup\u003e These experimental observations have been confirmed in clinical studies showing that high plasma sPD-L1 predicts ICI resistance in multiple cancers.\u003csup\u003e13\u0026ndash;19\u003c/sup\u003e Unsurprisingly, sPD-L1 impairs circulating tumor-reactive (T\u003csub\u003eTR\u003c/sub\u003e) CD8\u003csup\u003e+\u003c/sup\u003eCD11a\u003csup\u003ehigh\u003c/sup\u003e T cells that are critical for ICI response.\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e Malignant cells also release exosomal PD-L1 (exPD-L1) to drive a similar immunosuppression.\u003csup\u003e23\u0026ndash;26\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWhile sPD-L1-mediated resistance is formidable on its own, evidence is emerging that similar properties are shared by other cancer-derived soluble factors. These include soluble forms of B7H3 (CD276),\u003csup\u003e27,28\u003c/sup\u003e CD27,\u003csup\u003e29\u003c/sup\u003e CD80 (B7.1),\u003csup\u003e29\u0026ndash;31\u003c/sup\u003e CD137,\u003csup\u003e32,33\u003c/sup\u003e CTLA-4 (sCD152),\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e Lymphocyte-activation gene 3 (LAG3),\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e major histocompatibility complex I chain-related molecule (MIC),\u003csup\u003e40,41\u003c/sup\u003e PD-1,\u003csup\u003e34\u003c/sup\u003e PD-L2,\u003csup\u003e29\u003c/sup\u003e and T-cell immunoglobulin and mucin domain proteins (sTIM1, 3, and 4).\u003csup\u003e42\u003c/sup\u003e Although their mechanisms and contributions to tumor cell immune checkpoint evasion vary, each of these soluble factors has the potential to overwhelm ICI therapy and prevent anti-tumor immunity in cancer. There is a critical need to address this Gordian knot of soluble factors mediating immunosuppression and ICI resistance in cancer.\u003c/p\u003e\n\u003cp\u003eTherapeutic plasma exchange (TPE) is a safe and widely available clinical procedure in which patient blood is filtered or centrifuged to remove deleterious substances. We previously demonstrated that TPE removes sPD-L1 and exosomes from circulation.\u003csup\u003e43\u003c/sup\u003e In our pilot study, TPE removed 70.8% of circulating sPD-L1 from the plasma. In other studies, we have demonstrated that stereotactic body radiotherapy (SBRT) to metastatic sites of disease can expand critical T cell populations that drive ICI response.\u003csup\u003e26,44,45\u0026nbsp;\u003c/sup\u003eBased on these observations, we proposed clinical use of SBRT and TPE to re-sensitize ICI-refractory cancers.\u003c/p\u003e\n\u003cp\u003eIn the present study (NCT04581382), we explored sequential limited SBRT, TPE, and ICI re-challenge in the setting of ICI-refractory metastatic melanoma with high (\u0026ge;1.7 ng/ml) sPD-L1 (\u003cstrong\u003eSupplemental Fig 1\u003c/strong\u003e). Each patient received SBRT to three or fewer (and never all) sites of metastasis, three daily sessions of TPE, and finally ICI re-challenge. The primary safety endpoint of this study was adverse events (AE) per version 5.0 of the revised National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). The primary efficacy endpoint was sPD-L1 removal after TPE. Secondary efficacy endpoints were the number of patients achieving complete response (CR), partial response (PR), or stable disease (SD) based on unirradiated lesions and overall survival (OS). Correlative analysis included peripheral blood mononuclear cell (PBMC) subpopulations (including a prespecified change in tumor-reactive CD8+ [T\u003csub\u003eTR\u003c/sub\u003e] and regulatory T cells),\u003csup\u003e44\u0026ndash;46\u003c/sup\u003e extracellular vesicle (EV) kinetics, and exploratory plasma proteomic analysis.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003ch2\u003ePatients and baseline characteristics\u003c/h2\u003e\n\u003cp\u003ePatients with metastatic melanoma progressing on any PD-1 ICI were eligible for this study. Potentially eligible patients provided informed consent prior to a baseline screening blood draw. Patients were screened from December 2020 to February 2023 with an accrual goal of between 17 and 20 evaluable patients.\u003c/p\u003e\n\u003cp\u003eThirty-four (34) patients were screened for eligibility (see CONSORT diagram in \u003cstrong\u003eSupplemental Fig 2\u003c/strong\u003e). Sixteen (16) patients were excluded due to low sPD-L1 (n=8), poor venous access (n=2), unwillingness to undergo treatment or follow-up (n=2), contraindications to ICI (n=2), or death before sPD-L1 level could be assessed (n=2).\u003c/p\u003e\n\u003cp\u003eEighteen (18) patients were eligible (\u003cstrong\u003eTable 1\u003c/strong\u003e). Mean age of patients in the study was 63 years (SD 13), and 7 (39%) were female. Mean baseline sPD-L1 measured by ELISA was 26.31 ng/mL. All patients had previously received at least one PD-1 inhibitor that had failed, and fifteen (83%) had previously received at least one ICI doublet (\u003cem\u003ee.g.\u003c/em\u003e, ipilimumab and nivolumab) that had failed. Half of patients had received two prior lines of therapy, and an additional one third of patients had received four or more lines of therapy before enrollment in the study.\u003c/p\u003e\n\u003cp\u003eAll eligible patients (n=18) underwent treatment (\u003cstrong\u003eSupplemental Fig 1 and 2\u003c/strong\u003e). Treatment comprised 1-5 days of stereotactic body radiotherapy (SBRT) to at least one\u0026mdash;but not all\u0026mdash;sites of disease seen on imaging. This was followed by three consecutive days of one session each of TPE, followed by re-initiation of ICI therapy. ICI therapy was the same as previously received in 16 patients (89%). One patient was unable to complete the third TPE treatments due to a line infection. All patients were included in the intent to treat analysis regardless of treatment completion.\u003c/p\u003e\n\u003ch2\u003eSBRT, TPE, and ICI re-challenge is safe and well-tolerated.\u003c/h2\u003e\n\u003cp\u003eThe primary safety endpoint of this trial was to assess the adverse events (AE) observed during SBRT, TPE, and ICI re-challenge in patients with melanoma receiving PD-1 immunotherapy (\u003cstrong\u003eTable 2\u003c/strong\u003e). AEs were collected and reported according to CTCAE V.5.0. In a pre-planned stopping rule, if 3 of the first 10 patients were to experience grade 4 or greater adverse events attributable to the intervention, the study would be halted.\u003c/p\u003e\n\u003cp\u003eIn total, one patient experienced AE grade 3 and 4 thromboembolic event and sepsis, respectively, secondary to an infection of a vascular access device. This patient required hospitalization with line removal and intravenous antibiotics. Other AEs were grade 1-2 and included fatigue and arthralgias/myalgias. No deaths were attributable to therapy.\u003c/p\u003e\n\u003ch2\u003eTherapeutic plasma exchange removes soluble PD-L1 with varying reconstitution rates.\u003c/h2\u003e\n\u003cp\u003eThe primary efficacy endpoint of the study was the reduction of sPD-L1 levels by TPE (\u003cstrong\u003eFig 1A, Supplemental Figure 3A\u003c/strong\u003e). Relative and absolute levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p=6.1*10\u003csup\u003e-5\u003c/sup\u003e). Mean recovery rate of sPD-L1 at the second cycle of ICI re-challenge was 21.4% of pre-TPE levels.\u003c/p\u003e\n\u003ch2\u003eSBRT, TPE, and ICI re-challenge resensitized melanomas to immunotherapy.\u003c/h2\u003e\n\u003cp\u003eA secondary efficacy endpoint of the study was the proportion of patients achieving CR (defined as absence of radiologically apparent disease), PR (defined as greater than 30% radiographic reduction), or SD (defined as less than 30% radiographic reduction) as a best response per Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 in unirradiated lesions assessed radiographically.\u003csup\u003e47\u003c/sup\u003e Response was assessed by a trained radiologist. Two patients (11.1%) experienced CR, one (5.6%) PR, three (16.7%) SD, and 12 (66.7%) progressive disease (PD) (\u003cstrong\u003eTable 3\u003c/strong\u003e). Notably, there was a deeper absolute sPD-L1 reduction with TPE in patients who experienced CR or PR (p=0.02, \u003cstrong\u003eSupplemental Fig 3B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eA further secondary endpoint of the study was overall survival (OS). Median overall survival for the cohort by Kaplan-Meier estimate was 18.9 months (95% CI 15.1-NR) (\u003cstrong\u003eSupplemental Fig 4\u003c/strong\u003e). CR, PR, or SD response to treatment predicted superior overall survival (OS) (p=0.002) (\u003cstrong\u003eFig 1B, Supplemental Fig 5\u003c/strong\u003e). Median OS for patients experiencing PD was 15 months. Median OS for responders was not reached.\u003c/p\u003e\n\u003cp\u003eRepresentative irradiated and unirradiated lesions were selected and measured by a blinded radiologist across time in approximately 3-month intervals and assessed for standardized uptake value (SUV) and tumor size. The majority of measured irradiated and unirradiated lesions showed a greater than 50% reduction in SUV (\u003cstrong\u003eFig 1C\u003c/strong\u003e). Approximately one third of measured irradiated and unirradiated lesions showed a greater than 50% reduction in size (\u003cstrong\u003eFig 1D)\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAn example FDG PET scan image of irradiated and non-irradiated lesions in a patient experiencing CR is shown in \u003cstrong\u003eFig 1E\u003c/strong\u003e. A swimmer plot of all patients is shown in \u003cstrong\u003eFig 1F\u003c/strong\u003e.\u003c/p\u003e\n\u003ch2\u003eTumor-reactive effector T cell changes predict outcomes to immunotherapy re-challenge after radiotherapy and TPE.\u003c/h2\u003e\n\u003cp\u003ePeripheral blood mononuclear cell (PBMC) subpopulations act as liquid biomarkers and as critical substrates of ICI immunotherapy. Changes in these subpopulations reflect the effects of therapy on anti-tumor immunity.\u003c/p\u003e\n\u003cp\u003eWe isolated PBMCs and measured subpopulations by flow cytometry according to a broad panel (\u003cstrong\u003eSupplemental Tables 1-2, Supplemental Fig 6-7\u003c/strong\u003e). We compared subpopulations from pre-treatment to the beginning of the second cycle of ICI re-challenge. A heatmap of changes is shown (\u003cstrong\u003eFig 2A).\u0026nbsp;\u003c/strong\u003eWe further evaluated how these changes correlated with overall survival in our cohort by Cox proportional hazards regression (\u003cstrong\u003eFig 2B, Supplemental Tables 3-4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eOur group previously identified tumor-reactive CD8+ T cells (T\u003csub\u003eTR\u003c/sub\u003e) with high expression of CD11a, Granzyme B, and CX3CR1 (GZMB\u003csup\u003e+\u003c/sup\u003e/CX3CR1\u003csup\u003e+\u003c/sup\u003e/CD11a\u003csup\u003ehigh\u003c/sup\u003e or T\u003csub\u003eTR\u003c/sub\u003e).\u003csup\u003e44\u0026ndash;46\u003c/sup\u003e In a prespecified analysis, peripheral blood T\u003csub\u003eTR\u003c/sub\u003e changes from baseline to the next cycle of ICI re-challenge predicted overall survival (OS) with a Cox proportional hazard ratio (HR) of 0.16 (95% confidence interval [CI] 95% CI 0.03-0.8, p=0.026). We plotted survival by T\u003csub\u003eTR\u003c/sub\u003e change using Kaplan-Meier estimates (\u003cstrong\u003eFig 2C\u003c/strong\u003e, log rank p=0.01). When T\u003csub\u003eTR\u003c/sub\u003e levels were analyzed dichotomously as increasing versus decreasing (cutoff ratio=1), the results were identical.\u003c/p\u003e\n\u003cp\u003eRegulatory T cells marked by CD4, CD25, and FOXP3 expression (T\u003csub\u003ereg\u003c/sub\u003e) are well-established predictors of poor response to immunotherapy. In a prespecified analysis, peripheral blood T\u003csub\u003ereg\u003c/sub\u003e changes from baseline to the next cycle of ICI re-challenge predicted OS (HR 7.87 [95% CI 1.6-38.7], p=0.01). We plotted survival by T\u003csub\u003ereg\u003c/sub\u003e change using Kaplan-Meier estimates (\u003cstrong\u003eFig 2D\u003c/strong\u003e, log rank p=0.004). When T\u003csub\u003ereg\u003c/sub\u003e levels were analyzed dichotomously as increasing versus decreasing, these cells did not predict a significant difference in OS (HR 2.89 [95% CI 0.74-11.3], p=0.13).\u003c/p\u003e\n\u003cp\u003eWe previously showed that NKG7 marks resilient T cells that are critical to immunotherapy response.\u003csup\u003e44,48\u003c/sup\u003e In an exploratory analysis, changes in peripheral blood CD11a\u003csup\u003ehigh\u003c/sup\u003e CD8+ T cells with NKG7 expression from baseline to the next cycle of ICI re-challenge predicted superior OS (HR 0.19 [95% CI 0.04-0.88], p=0.03). We plotted survival by NKG7-positive T cell change using Kaplan-Meier estimates (\u003cstrong\u003eFig 2E\u003c/strong\u003e, log rank p=0.02).\u003c/p\u003e\n\u003cp\u003eIn addition, we previously reported that Bim (BCL-2-interacting mediator of cell death) is upregulated in T cells by PD-L1/PD-1 engagement and that T cell Bim expression drives poor anti-tumor immunity.\u003csup\u003e44,49\u003c/sup\u003e Persistent CD8+ T cell Bim expression despite ICI predicts poor responses to immunotherapy.\u003csup\u003e50\u003c/sup\u003e In an exploratory analysis, changes in peripheral blood CD11a\u003csup\u003ehigh\u003c/sup\u003e CD8+ T cells with Bim expression from baseline to the next cycle of ICI re-challenge predicted inferior OS (HR 6.8 [95% CI 1.58-29.32], p=0.01). We plotted survival by Bim\u003csup\u003e+\u003c/sup\u003e T cell change using Kaplan-Meier estimates (\u003cstrong\u003eFig 2F\u003c/strong\u003e, log rank p=0.003).\u003c/p\u003e\n\u003ch2\u003eSoluble mediators of immunosuppression are elevated in patients with ICI-refractory melanoma, are reduced by TPE, and rapidly reaccumulate.\u003c/h2\u003e\n\u003cp\u003eAs noted above, soluble factors beyond sPD-L1 contribute to ICI resistance.\u003csup\u003e27\u0026ndash;42\u003c/sup\u003e We next sought to determine the effect of TPE on these soluble factors in systemic circulation. We measured a large library of soluble factors in sufficient plasma samples from patients on the study and compared these to matched healthy controls using a high-throughput O-link multiplex assay. A variety of immunosuppressive soluble factors were elevated at baseline in the blood of patients with ICI-refractory melanoma versus healthy controls (\u003cstrong\u003eFig 3A, Data File 1\u003c/strong\u003e), including sCTLA-4, sLAG3, sPD-1, and sTIM-3.\u003c/p\u003e\n\u003cp\u003eWe next measured reduction and rebound of these factors in the plasma of patients from SBRT to TPE, pre- to post-TPE, and at the second cycle of ICI re-challenge (see \u003cstrong\u003eData File 1\u003c/strong\u003e). TPE significantly reduced exemplary immunosuppressive factors sPD-1, sLAG3, and sTIM3 (HAVCR2) (\u003cstrong\u003eFig 3B-D\u003c/strong\u003e). Notably, sCTLA4, which was only moderately elevated in this cohort versus healthy controls, was unchanged (\u003cstrong\u003eFig 3E\u003c/strong\u003e). Conversely, Prostaglandin E synthase 2 (PTGES2), a marker of systemic inflammatory response, increased significantly after TPE (p=0.0004) and recovered by cycle 2 of ICI re-challenge (p=0.016) (\u003cstrong\u003eFig 3F\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGiven the direct connection between soluble immunosuppressive factors and ICI resistance, we sought to determine whether the level of these factors at the time of ICI re-challenge (\u003cem\u003ei.e.\u003c/em\u003e, post-TPE) could predict overall survival. Level of sPD-L1 (log rank p=0.003) and sTIM3 (log rank p=0.007) after TPE predicted OS (\u003cstrong\u003eFig 4A-B\u003c/strong\u003e). Conversely, OS was not predicted by sPD-1, sLAG3, or sCTLA-4 in this cohort.\u003c/p\u003e\n\u003cp\u003eAs outlined above, we observed rapid reconstitution of most factors prior to second cycle ICI re-challenge. We next sought to determine whether levels of these factors at the second cycle of ICI re-challenge could predict outcomes. These factors did not predict OS in our cohort (\u003cstrong\u003eFig 4C-D\u003c/strong\u003e).\u003c/p\u003e\n\u003ch2\u003eExosomal PD-L1 was not significantly changed on treatment.\u003c/h2\u003e\n\u003cp\u003eWe and others have previously showed that malignant cells release exosomes or extracellular vesicles bearing PD-L1 that can drive immunosuppression and can release sPD-L1 by proteolytic cleavage.\u003csup\u003e23\u0026ndash;26\u0026nbsp;\u003c/sup\u003eRadiation can cause tumor exosome shedding by multiple mechanisms, including cell death.\u003csup\u003e26\u0026nbsp;\u003c/sup\u003eHowever, exosomal PD-L1 (exPD-L1) was previously reduced by TPE in a pilot study.\u003csup\u003e43\u0026nbsp;\u003c/sup\u003eThus, it is unclear whether exPD-L1 would be expected to be reduced or increased after both SBRT and TPE. We measured\u0026nbsp;exPD-L1 by flow cytometry before and after TPE in the present cohort\u0026nbsp;(\u003cstrong\u003eSupplemental Fig 8A\u003c/strong\u003e). Circulating exPD-L1 was not significantly changed. However, change in exPD-L1 after TPE predicted improved OS (HR 0.28 [95% CI 0.08-0.98], p=0.047) in the cohort (\u003cstrong\u003eSupplemental Fig 8B\u003c/strong\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSoluble immunosuppressive factors present a significant challenge to ICI therapy. While PD-1 inhibitors and sPD-L1 are the best studied classes of ICI and resistance-inducing soluble immunosuppressive factors, respectively, they are archetypal of other ICI treatments and the multi-layered evasions employed by cancer cells. Current ICI therapies can be overwhelmed by the volume and variety of these factors in the blood of patients with ICI-resistant malignancies.\u003c/p\u003e\n\u003cp\u003eThis study provides the first clinical evidence that a combination of limited SBRT, TPE, and ICI re-challenge can restore responsiveness to immune checkpoint inhibitors (ICIs) in patients with metastatic melanoma who had previously progressed on the same ICI regimen. We observed a disease control rate (DCR) of 33% among these patients, meeting our prespecified secondary endpoint. This DCR is remarkable in such a heavily pre-treated population. 89% of patients had previously received multiple lines of therapy that had failed. 83% had previously received at least one ICI doublet that had failed in a prior line of therapy. 89% of patients on the study received the same PD-1 ICI that had failed in a prior line of therapy.\u003c/p\u003e\n\u003cp\u003eThis study met its primary safety and efficacy endpoints. AEs were commensurate with other studies of immunotherapy. TPE effectively reduced plasma sPD-L1, although the level of sPD-L1 and other soluble factors known to limit anti-tumor immunity rebounded rapidly by the second cycle of ICI in this study. The study also provides provocative correlative markers that may help inform future approaches. First, treatment induced significant changes in peripheral circulating immune cells. Some of these changes, including tumor reactive CD8+ T cells that express CD11, Granzyme B, and CX3CR1 and classical regulatory T cells, predicted clinical outcomes despite the limited number of patients in the study. A favorable shift in the balance of anti-tumor immunity and tolerance provides evidence that ICI-resistant tumors remain potentially sensitive to immunotherapy. Furthermore, these changes reflect the accepted mechanism of treatment response and may help guide future studies as biomarkers. Second, TPE significantly reduced soluble mediators of immunosuppression. The reduction of several of these factors also predicted clinical outcomes in this cohort. While soluble mediators of ICI resistance will vary widely from patient to patient and from cancer to cancer, TPE offers a broad way to address many such mediators. In all, this study suggests that SBRT and\u0026nbsp;TPE may \u0026ldquo;reset\u0026rdquo; systemic immunity and restore immunotherapy sensitivity in ICI-refractory cancers.\u003c/p\u003e\n\u003cp\u003eThe recent SWOG study S1616 provides important context to our findings and an alternative approach to ICI resistance. In this study, patients with anti-PD-1 ICI-refractory melanoma were randomized to anti-CTLA-4 ipilimumab monotherapy or the ipilimumab and nivolumab doublet in the second line (NCT03033576).\u003csup\u003e6\u003c/sup\u003e Anti-CTLA-4 ICI monotherapy achieved a 9% response rate in this study as compared with 28% in the doublet arm, showing that anti-PD-(L)1 ICI resistance can be addressed clinically. A small minority of these patients had previously received an ICI doublet. It is unknown whether a combination of multiple ICI modalities and TPE could further improve response.\u003c/p\u003e\n\u003cp\u003eSeveral limitations to this study should be acknowledged. First, this study combined three sequential treatments\u0026mdash;SBRT, TPE, and PD-(L)1 ICI re-challenge\u0026mdash;in an open label, single arm trial. While biomarkers were measured at multiple timepoints in the study, the individual contribution of SBRT and TPE to overcoming ICI resistance is uncertain. Although SBRT was highly effective against individual metastases in this study, most lesions were not irradiated. Of these unirradiated lesions, most showed a reduction in both size and FDG avidity. However, our observations must not be taken as evidence for a purported \u0026ldquo;abscopal\u0026rdquo; effect\u0026mdash;meaning involution of unirradiated tumors after radiation of distant metastases\u0026mdash;because this trial included neither an SBRT-free control nor a TPE-free comparator arm from which to draw such conclusions. A potential synergy of SBRT and TPE is that SBRT may reduce the \u003cem\u003ede novo\u003c/em\u003e production of sPD-L1 from tumor sites and TPE may reduce the products (including sPD-L1) directly from circulation.\u003c/p\u003e\n\u003cp\u003eWhile the clinical activity observed in this study\u0026mdash;particularly against individual melanoma lesions\u0026mdash;is favorable and warrants further study, most patients experienced disease progression and death. Even in patients with a favorable response, there was rapid re-accumulation of many immunosuppressive factors in the bloodstream. It is unclear whether a more sustained suppression of these factors would improve the robustness of ICI re-challenge response. Our approach was limited to a single three-day course of TPE. It is possible that serial TPE prior to each ICI cycle may be more effective, but this cannot be determined from the present study. Similarly, it is unclear to what extent SBRT affects these changes. This study did not include novel ICI classes. Thus, additional clinical trials are needed to determine how best to leverage SBRT and TPE in ICI resistance.\u003c/p\u003e\n\u003cp\u003eAs a related limitation, SBRT and TPE are medical procedures with attributes that may limit their use. First, while the current cost of each TPE session is approximately one sixth of the cost of each immunotherapy injection, the costs of immunotherapy are expected to decrease in the coming years through a combination of formulation changes and alternatives.\u003csup\u003e51\u0026nbsp;\u003c/sup\u003eConversely, the costs of SBRT and TPE are inelastic. This may limit the number of patients who are able to obtain the procedures. Further, while SBRT is widely available, TPE requires specialized equipment and expertise that are currently available only in limited quantity in major medical centers. Lastly, TPE requires wide-bore peripheral venous access. This increases risks of infection, hypotension, and hematoma formation.\u003c/p\u003e\n\u003cp\u003eTaken together, this study suggests that SBRT, TPE, and ICI re-challenge provide a promising method to overcome ICI resistance in melanoma. Future studies are warranted to validate and optimize this approach as a tool to overcome ICI resistance in melanoma and other malignancies. These studies may include multiple cycles of TPE to achieve effective suppression of immunosuppressive factors.\u003c/p\u003e"},{"header":"METHODS","content":"\u003ch2\u003eStudy design and participants\u003c/h2\u003e\n\u003cp\u003eWe conducted the ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma) study under a protocol approved by the local institutional review board and registered at ClinicalTrials.gov (NCT04372619). This was a prospective, open-label phase II trial for patients with advanced ICI-resistant melanoma with elevated sPD-L1 levels. Written informed consent was obtained from all participants prior to study enrollment. All data were collected in a deidentified fashion in a password-protected database.\u003c/p\u003e\n\u003cp\u003eInclusion criteria included age 18 or older, histologically confirmed melanoma, measurable disease per RECIST criteria, ECOG (Eastern Cooperative Oncology Group) status of 3 or better, sPD-L1 level 1.7\u0026thinsp;ng/mL or greater, and feasible vascular access. Exclusion criteria included contraindications to PD-(L)1 inhibitor ICI and unwillingness to undergo treatment and follow-up. Patients who consumed biotin supplementation were excluded due to the known interference of biotin supplements with accurate measurement of sPD-L1.\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eKey Inclusion criteria:\u003c/h3\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eHistological confirmation of melanoma\u003c/li\u003e\n \u003cli\u003eMeasurable or non-measurable disease\u003c/li\u003e\n \u003cli\u003eEastern Cooperative Oncology Group (ECOG) performance status (PS) \u0026le; 3\u003c/li\u003e\n \u003cli\u003esPD-L1 levels \u0026gt; 1.7 ng/ml by enzyme-linked immunosorbent assay (ELISA)\u003c/li\u003e\n \u003cli\u003eNegative pregnancy test done =\u0026lt; 7 days prior to radiation therapy, for women of childbearing potential only\u003c/li\u003e\n \u003cli\u003eProvide written informed consent\u003c/li\u003e\n \u003cli\u003eWilling to return to enrolling institution for follow-up (during the Active Monitoring Phase of the study)\u003c/li\u003e\n \u003cli\u003eWilling to provide blood samples for correlative research purposes\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch3\u003eRecruitment\u003c/h3\u003e\n\u003cp\u003ePatients with metastatic melanoma progressing despite ICI treatment were approached in the Department of Medical Oncology or Department of Radiation Oncology at a single academic center between December 2020 and February 2023. Patients were screened for sPD-L1 levels using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (see Biomarker Assays). Patients with sPD-L1 levels \u0026ge;1.7 ng/mL were considered positive and eligible for the study.\u003c/p\u003e\n\u003ch3\u003eTreatment\u003c/h3\u003e\n\u003cp\u003eConsenting and eligible patients underwent 1 to 5 days of SBRT to between one and three metastatic sites. The majority of metastatic sites were not irradiated for each patient.\u003c/p\u003e\n\u003cp\u003ePatients next underwent three daily sessions of TPE, performed using centrifugation-based cell separators, either the Fenwal Amicus (Fresenius KABI USA LLC, Lake Zurich, Illinois, USA) or the Spectra Optia (Terumo BCT, Lakewood, Colorado, USA). For each patient, a single plasma volume was exchanged at each session. Either peripheral intravenous or central line vascular access was used to perform the procedure. Removed plasma was discarded and replaced with albumin. Vascular access lines were removed after the last planned TPE session.\u003c/p\u003e\n\u003cp\u003eAfter completing TPE, patients received PD-(L)1 ICI re-challenge per clinician preference. ICI cycle duration was determined per product label as standard of care.\u003c/p\u003e\n\u003ch3\u003eAssessments\u003c/h3\u003e\n\u003cp\u003eClinical standard of care imaging was performed per clinician preference, generally comprising conventional computed tomography (CT) imaging with or without advanced molecular [\u0026sup1;⁸F]Fluorodeoxyglucose positron emission tomography (FDG PET) imaging every 3 months. Changes in size were reported as best response per Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 in unirradiated lesions assessed radiographically.\u003csup\u003e47\u0026nbsp;\u003c/sup\u003eCR was defined as resolution of radiologically apparent disease on conventional imaging, PR as greater than 30% radiographic reduction on conventional imaging, SD as less than 30% radiographic reduction, and PD as 20% or greater increase in radiographically apparent disease. PET avidity was not used to determine response. For waterfall plots, at least two metastatic sites were reported for each patient commensurate with relative metastatic burden.\u003c/p\u003e\n\u003ch2\u003eStudy endpoints\u003c/h2\u003e\n\u003cp\u003eThe primary safety endpoint was the rate of adverse events (AE) measured using the revised National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The maximum grade for each type of AE was recorded for each patient.\u003c/p\u003e\n\u003cp\u003eThe primary efficacy endpoint was the kinetics of soluble sPD-L1 removal after plasma exchange. The secondary efficacy endpoint was response, defined as patients with complete response (CR), partial response (PR), or stable disease (SD) at 3 months. Correlative analyses comprised overall survival (OS), peripheral blood mononuclear cell (PBMC) subpopulations including prespecified T cell population changes, and plasma proteomic analysis as outlined below.\u003c/p\u003e\n\u003ch2\u003eBiomarker assays\u003c/h2\u003e\n\u003ch3\u003eSoluble PD-L1 (sPD-L1)\u003c/h3\u003e\n\u003cp\u003esPD-L1 was measured by enzyme-linked immunosorbent assay (ELISA) as previously published.\u003csup\u003e11\u003c/sup\u003e This assay comprises paired mouse IgG2 monoclonal antibody clones H1A (capture) and biotinylated B11 (detection) against extracellular human PD-L1 using a standard streptavidin-HRP method. Concentrations were determined by optical density (OD) measurements along a known standard curve of recombinant human PD-L1. Assays were performed in a blinded fashion in triplicate and reported in nanograms per mililiter (ng/mL).\u003c/p\u003e\n\u003ch3\u003eExtracellular vesicle-bound PD-L1 (evPD-L1)\u003c/h3\u003e\n\u003cp\u003eExtracellular vesicle-bound PD-L1 (evPD-L1) was measured by flow cytometry as previously published.\u003csup\u003e52\u0026nbsp;\u003c/sup\u003ePlasma samples were centrifuged twice at 2000g to deplete platelets and analyzed using an A60-Micro Plus Nanoscale Flow Cytometer (Apogee FlowSystems) gating for mid-intensity light angle light scatter and markers of interest. Nanoscale flow cytometer calibration was performed using a standard reference bead mix. Clone H1A anti-PD-L1 antibodies were conjugated to fluorophores (Life Technologies) and incubated with samples prior to analysis. Concentrations were determined by gated counts using a standard reference bead mix. Assays were performed in a blinded fashion in triplicate and reported as number of particles.\u003c/p\u003e\n\u003ch3\u003eOlink proteomic profiling\u003c/h3\u003e\n\u003cp\u003eSoluble plasma markers were assessed using a commercially available platform (Olink) and compared to age-matched healthy controls. Plasma samples were labeled with oligonucleotide-labeled antibody probes and subjected to microfluidic PCR amplification using a Proximity Extension Assay (PEA) with NGS readout on Illumina instruments. Relative concentrations were determined on an arbitrary relative scale. Assays were performed in a blinded fashion in triplicate and reported in relative units.\u003c/p\u003e\n\u003ch3\u003ePeripheral blood mononuclear cell (PBMC) profiling\u003c/h3\u003e\n\u003cp\u003ePeripheral blood mononuclear cells (PBMC) were profiled for established cell types (see \u003cstrong\u003eSupplemental Tables 1-2\u003c/strong\u003e). We stained surface markers prior to intracellular markers. Flow cytometry data were collected on a Cytek Aurora (Cytek Biosciences). We analyzed flow cytometry data with FlowJo 10.10.0 (Tree Star). Changes in percent from baseline to next cycle of ICI re-challenge were compared and plotted. A prespecified analysis was performed for Granzyme B-positive, CX3CR1-positive, CD11a-high CD8-positive (GZMB\u003csup\u003e+\u003c/sup\u003e/CX3CR1\u003csup\u003e+\u003c/sup\u003e/CD11a\u003csup\u003ehigh\u003c/sup\u003e or T\u003csub\u003eTR\u003c/sub\u003e) CD8\u003csup\u003e+\u003c/sup\u003e tumor-reactive T cells and CD4-positive, CD25-positive, FOXP3-positive (T\u003csub\u003ereg\u003c/sub\u003e) regulatory T cells that are associated with response to immunotherapy.\u003csup\u003e44\u0026ndash;46\u003c/sup\u003e Additional exploratory analyses were performed for each cell subtype. Flow cytometry was performed in a blinded fashion and reported as a percent of total live PBMCs.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eA total of 20 patients (17 evaluable) was estimated to be needed to assess primary efficacy and safety endpoints. All patients who were eligible were included in the intention to treat (ITT) analysis regardless of treatment completion. Overall survival was estimated by the Kaplan-Meier method and groups were further compared by Cox proportional hazards or log rank tests as indicated. Adverse events were reported by maximum grade in tabular form. Wilcoxon signed- rank test was used to assess changes in levels over time across the different timepoints of interest. Student\u0026rsquo;s \u003cem\u003et\u0026nbsp;\u003c/em\u003etest was used for additional comparisons as indicated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFinancial support: \u003c/h2\u003e\n\u003cp\u003eLawrence W. And Marilyn W. Matteson Fund at Mayo Clinic (JJO and SSP), National Cancer Institute (R21-CA259236; JJO, JLW, and SSP)\u003c/p\u003e\n\u003cp\u003eCOI: Intellectual property has been filed addressing discoveries disclosed in this manuscript. J.J.O. reports support and/or research and consulting support from the Prostate Cancer Foundation, NaNotics LLC, Genentech, and Partner Therapeutics.\u0026nbsp;L.K. reports consultant activities for Immunocore.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eM.S.B. reports institutional research support from Alkermes, Bristol-Myers Squibb, Genentech, Merck, nFerence, Pharmacyclics, Regeneron, Sorrento, TILT Biotherapeutics, Transgene, and Viewpoint Molecular Therapeutics; as well as consultant activities for Sorrento Therapeutics, TILT Biotherapeutics, and Viewpoint Molecular Targeting.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eA.S.M. reports consulting activities with AbbVie, AstraZeneca, BMS, Genentech, Janssen, Takeda Oncology, Sanofi Genzyme, Gilead, Johnson \u0026amp; Johnson Global Services, Chugai Pharmaceutical Co., Ltd. (Roche), TRIPTYCH Health Partners, Ideology Health LLC, Intellisphere LLC, Answers in CME, and Immunocore; as well as study\u0026nbsp;funding subsequent publication processing fees from Bristol Myers Squibb; travel support from Roche; was a non-remunerated director of the Mesothelioma Applied Research Foundation and is a non-remunerated director of the Friends of Patan Hospital. S.N.M. reports grant funding, clinical trial for\u0026nbsp;BMS and grant funding, PI for Sorrento Pharma. K.O. reports honorarium from UptoDate. D.O. reports research support from AstraZeneca, and Varian; as well as honorarium from UptoDate. D.M. reports intellectual property from EXACT Sciences,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eInSitu Biologics, and CurrentHealth.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eF.L. reports receiving research support to institution from Nanotics LLC; serving as scientific consultant for Mursla Bio; receiving royalties from Early is Good. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eJ.W. reports non-remunerated director of ASFA and ISFA. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDATA SHARING STATEMENT\u003c/h2\u003e\n\u003cp\u003eAll data, including redacted individual patient data, and a data dictionary defining each field will be made available in Data File 1. Study protocol and informed consent form will also be available with publication.\u003c/p\u003e\n\u003ch2\u003eFUNDING:\u003c/h2\u003e\n\u003cp\u003eLawrence W. And Marilyn W. Matteson Fund at Mayo Clinic (JJO and SSP), National Cancer Institute (R21-CA259236; JJO, JLW, and SSP)\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\n\u003cp\u003eWe thank the brave and selfless patients and their families who agreed to participate in this clinical trial. We thank clinical staff and study coordinators. Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science (CCaTS). Funding for the study was provided by the Lawrence W. And Marilyn W. Matteson Fund at Mayo Clinic and the National Cancer Institute (R21-CA259236).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDong H, Strome SE, Salomao DR et al (2002) Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. \u003cem\u003eNat Med\u003c/em\u003e [Internet]. 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Nat Med 29(9):2278\u0026ndash;2285\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Immunotherapy resistance, Therapeutic Plasma Exchange, Programmed Cell Death 1 Receptor","lastPublishedDoi":"10.21203/rs.3.rs-4542220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4542220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e: Immune checkpoint inhibitors (ICI) are an essential systemic therapy for advanced melanoma. However, most melanomas develop resistance to ICI. Tumor-derived soluble PD-L1 (sPD-L1) and other soluble immunosuppressive factors drive checkpoint inhibitor resistance and correlate with inferior survival. We previously showed that therapeutic plasma exchange (TPE) removes sPD-L1 from circulation. Thus, we hypothesized that TPE-mediated removal of sPD-L1 and other immunosuppressive factors could overcome immunotherapy resistance in refractory melanoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e In this clinical trial (NCT04581382), we prospectively enrolled eighteen (18) patients with widely metastatic melanoma with progression despite anti-PD-1 ICI and elevated sPD-L1 by ELISA (≥1.7ng/mL). Each patient received radiotherapy to between one and three metastatic lesions (at least two unirradiated lesions) followed by three TPE sessions on consecutive days and re-challenge with checkpoint inhibitor. The primary safety and efficacy endpoints of the study were adverse events (AEs) and sPD-L1 reduction by TPE, respectively. Secondary endpoints included RECIST-based response in unirradiated lesions and overall survival. Correlative studies included kinetics of sPD-L1 and soluble immonsuppressive factors and dynamics of peripheral immune cell phenotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Mean age was 62 (SD 13) and seven of eighteen (39%) were female. Mean baseline sPD-L1 was 26.31 ng/mL (40.01). The treatment was well-tolerated with one (6%) patient with grade 3 and 4 adverse events from a central line infection after TPE. Levels of sPD-L1 were significantly reduced by TPE (mean 80.2% reduction, p\u0026lt;0.0001). Two patients (11.1%) experienced complete response (CR), one (5.6%) partial response, three (16.7%) stable disease, and 12 (66.7%) progressive disease. In one case, immunotherapy was discontinued after two years due to no detectable lesions. Changes in tumor-reactive (T\u003csub\u003eTR\u003c/sub\u003e) GZMB\u003csup\u003e+\u003c/sup\u003e/CX3CR1\u003csup\u003e+\u003c/sup\u003e/CD11a\u003csup\u003ehigh\u003c/sup\u003e and other peripheral immune cell populations predicted overall survival in this cohort. In addition to sPD-L1, other soluble mediators of ICI resistance were also reduced by TPE and predicted overall survival in this cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSummary/Conclusion\u003c/strong\u003e: sPD-L1 and other soluble immunoregulatory signaling molecules are important mediators of ICI resistance. SBRT and TPE can resensitize ICI-refractory melanoma by removing these factors. Patients with persistently elevated or rapid rebound of sPD-L1 following TPE experienced poor response and overall survival. Serial monitoring of sPD-L1 may predict response to ICI and multiple courses of TPE may be necessary. Our findings may apply in other ICI-resistant cancers with elevated sPD-L1. ClinicalTrials.gov registration: NCT04581382, ReCIPE-M1 (Rescuing Cancer Immunotherapy with Plasma Exchange in Melanoma 1).\u003c/p\u003e","manuscriptTitle":"Reduction of soluble PD-L1 by plasma exchange and radiation therapy in patients with refractory melanoma re-sensitizes to immunotherapy.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-30 16:39:24","doi":"10.21203/rs.3.rs-4542220/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bcb2754a-cc2e-4655-8320-9b048d9b958f","owner":[],"postedDate":"October 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33262589,"name":"Biological sciences/Immunology/Immunotherapy"},{"id":33262590,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2025-03-14T07:06:30+00:00","versionOfRecord":{"articleIdentity":"rs-4542220","link":"https://doi.org/10.1038/s41467-025-57865-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-13 04:00:00","publishedOnDateReadable":"March 13th, 2025"},"versionCreatedAt":"2024-10-30 16:39:24","video":"","vorDoi":"10.1038/s41467-025-57865-9","vorDoiUrl":"https://doi.org/10.1038/s41467-025-57865-9","workflowStages":[]},"version":"v1","identity":"rs-4542220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4542220","identity":"rs-4542220","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}