Journal for Immunotherapy of Cancer最新文献

Background: Antigen escape is one of the leading causes of relapse following chimeric antigen receptor (CAR)-T therapy, particularly in multiple myeloma. A critical gap persists in understanding the tumor-intrinsic pathways that trigger antigen loss, insight essential for devising strategies to resensitize tumors to immune attack. We identify a previously uncharacterized post-translational mechanism centered on the metabolic enzyme ribonucleotide reductase subunit M2 (RRM2), termed trafficking-mediated antigen escape, to enhance cellular therapy efficacy.

Methods: We combined single-cell RNA sequencing analysis with multiplex immunofluorescence to identify a clinically relevant RRM2⁺ myeloma subpopulation exhibiting low MICA/B abundance. Functional validation included induced pluripotent stem cell-derived myeloma organoids monitored by real-time imaging and disseminated xenograft models to assess the effect of subtoxic osalmid treatment on NKG2D CAR-T cell activity. Co-immunoprecipitation, guanosine 5'-triphosphate pulldown, and confocal microscopy were used to investigate the underlying trafficking mechanism.

Results: Single-cell analysis uncovered a clinically prevalent RRM2⁺ myeloma subpopulation with profoundly reduced MICA/B surface abundance, which established tumor-intrinsic heterogeneity as one of fundamental causes of NKG2D CAR-T resistance. We further demonstrated RRM2's non-canonical role as a trafficking regulator that actively shuttles MICA/B toward lysosomal degradation via RAB7A activation while simultaneously blocking RAB11-mediated recycling. Therapeutic intervention using subtoxic osalmid, a clinically approved drug and previously characterized as an RRM2 inhibitor, successfully reversed this trafficking defect, restored MICA/B membrane presentation and synergized with NKG2D CAR-T cells to enhance their expansion, polyfunctional cytokine secretion, and stem-like properties. This combination strategy achieved durable tumor remission in vivo by sustaining T-cell fitness while reducing exhaustion, offering an immediately actionable solution to clinical antigen escape.

Conclusions: Our study establishes RRM2-driven trafficking as a novel and targetable mechanism of antigen escape in CAR-T therapy. By repurposing osalmid to restore MICA/B surface presentation, we provide a clinically translatable strategy that specifically potentiates NKG2D CAR-T cell efficacy in multiple myeloma and could potentially enhance the efficacy of CAR-T across diverse antigens. This work highlights the therapeutic potential of modulating intracellular trafficking to overcome resistance in cellular immunotherapy.

Background: The gut microbiota plays a critical role in regulating immune homeostasis and modulating responses to cancer immunotherapies. However, the impact of antibiotic-induced dysbiosis in patients with multiple myeloma (MM) treated with bispecific antibodies (BsAbs) remains unexplored. This multicenter, international study investigated whether antibiotic exposure prior to BsAb initiation alters the gut microbiome and affects clinical outcomes in patients with relapsed or refractory MM.

Methods: We retrospectively analyzed 237 adult patients with MM treated with CD3-engaging BsAbs across six academic institutions. Antibiotic exposure was defined as the administration of any broad-spectrum, non-prophylactic antibiotic within 30 days before BsAb initiation. Clinical outcomes included overall survival (OS), progression-free survival (PFS), and cumulative incidence of relapse, evaluated using Kaplan-Meier estimates, log-rank tests, and multivariable Cox and competing-risk regression models. Additionally, in a subset of 24 patients, peripheral blood samples were collected prior to BsAb infusion for immunophenotyping, cytokine profiling, and serum short-chain fatty acid (SCFA) quantification, while stool samples for 16S ribosomal RNA (rRNA) sequencing were collected in a subset of 19 patients.

Results: Broad-spectrum antibiotic exposure prior to BsAb therapy was associated with significantly inferior 1-year OS (60% (95% CI 44% to 81%) vs 77% (95% CI 71% to 83%), p=0.004) and PFS (26% (95% CI 14% to 47%) vs 53% (95% CI 46% to 61%), p<0.001), and higher relapse incidence (68% (95% CI 48% to 82%) vs 43% (95% CI 36% to 50%), p=0.004). In multivariable analyses, antibiotic exposure remained independently associated with poorer OS, PFS, and higher relapse risk. These associations were also observed within the subgroup of patients treated with CD3/B-cell maturation antibody-targeted BsAbs (n=155). Immunoprofiling revealed lower CD4⁺ T-cell counts (p=0.017) and reduced circulating cytokine levels among antibiotic-exposed patients. 16S rRNA sequencing demonstrated a marked depletion of SCFA-producing genera, including Roseburia and Eubacterium, accompanied by lower serum SCFA concentrations. Moreover, microbiota composition before BsAb treatment correlated with therapy response and treatment-related toxicity.

Conclusions: Antibiotic-induced dysbiosis prior to BsAb therapy is associated with impaired immune reconstitution and inferior clinical outcomes in MM. These findings underscore the importance of antibiotic stewardship and suggest that microbiota-preserving strategies could enhance the efficacy of BsAb therapy in MM.

Background: Mucosal melanomas (MM) arise from mucosal melanocytes at various anatomical sites. These tumors are rare, highly aggressive, and often associated with poor outcomes. Current treatments, including immune checkpoint inhibitors, show limited efficacy in advanced disease. Compared with cutaneous melanomas, there is a lack of data on the immunogenicity and interferon (IFN)-γ sensitivity of MM. In this study, we examined these features in sino-nasal melanomas (SN-MM) cell lines and clinical samples using microscopy and functional genomics.

Methods: The immune contexture of SN-MM was analyzed by immunohistochemistry on 48 tumor biopsies. RNA sequencing and mass spectrometry-based proteomic approaches were used to study the IFN-γ receptor (IFNGR) pathways in five patient-derived SN-MM cell lines. Moreover, their IFN-γ sensitivity, in terms of cell viability, IFNGR/JAK/STAT signaling pathway and IFN-γ inducible proteins, was evaluated by flow cytometry and immunoblots. Neoantigen prediction was performed through integrated whole exome sequencing and RNA-sequencing analysis using pVAC-Seq. Immune effector functions were evaluated in co-culture in vitro assays.

Results: SN-MM tumors are mainly immune "desert" with few tumor-infiltrating lymphocytes and contain immunosuppressive macrophages, features linked to poor prognosis; moreover, tumor cells are largely CD274/programmed death-ligand 1 negative. SN-MM cell lines express transcripts for melanocytic and cancer testis antigens; moreover, sequencing analysis identified a repertoire of high-confidence neoantigens, including candidates derived from recurrently mutated oncogenic drivers. Functional assays revealed that SN-MM cells are susceptible to NK cell-mediated killing. In terms of IFN-γ sensitivity, SN-MM cells show normal surface expression of IFNGR and maintain the integrity of the IFNGR/JAK/STAT signaling pathway. Transcriptomic and proteomic analyses demonstrate that SN-MM cell lines, as a group, respond to IFN-γ by upregulating genes involved in immune recognition and antigen presentation. In 60% of SN-MM lines, IFN-γ also induces cytotoxic and anti-proliferative effects, the release of CXCL10 and upregulation of CD274/PD-L1. The remaining SN-MM cell lines, characterized by poor differentiation, show refractoriness to these effects.

Conclusions: SN-MM displays an immune-desert phenotype yet retains intrinsic immunogenicity. Most tumors preserve functional IFN-γ signaling, while poorly differentiated cells show resistance to IFN-γ-mediated effects. These findings underscore heterogeneity in immune responsiveness and support functional immune profiling to refine immunotherapy strategies in MM.

Background: Immune-related hepatitis (ir-hepatitis) ranks among the most frequent adverse events of immune checkpoint inhibitors (ICIs). Limited knowledge exists regarding the incidence, characteristics, and treatment of patients having an inadequate response to initial therapy with steroids. Characterizing ir-hepatitis phenotypes and treatment responses can provide valuable insights for guiding treatment decisions and prognosis.

Methods: This is a prospective study including patients treated with ICIs experiencing grade 3-4 ir-hepatitis. All patients received methylprednisolone 2 mg/kg for at least 72 hours and underwent liver biopsy. Ursodeoxycholic acid was added in mixed and cholestatic drug-induced liver injury (DILI) phenotypes, and mycophenolate mofetil (MMF) was added in patients with inadequate response to steroids. Multiplex immunohistochemistry (mIHC) for CD3, CD8, FoxP3, CD20, and CD56/NKp46 was used on liver biopsies, and single-cell RNA sequencing of peripheral blood samples was employed to characterize the immunological response.

Results: A total of 34 patients with ir-hepatitis were included, of which 20 patients (59%) responded to steroids. Six patients (18%) were steroid-unresponsive and needed MMF. Eight patients (23%) had steroid-dependent ir-hepatitis; they had an initial response to steroids but relapsed during tapering. Patients with steroid-unresponsive and steroid-dependent ir-hepatitis were treated with significantly higher accumulated steroid doses. Alcohol consumption and male sex were significantly related to inadequate response to steroids. Patients with mixed DILI had the highest steroid response rates (72%), while only half of the patients with hepatocellular and cholestatic DILI responded. Patients with cholestatic DILI had the worst prognosis concerning management of ir-hepatitis, risk of cancer progression and death.MIHC of liver biopsies revealed significantly increased T cell infiltration in ir-hepatitis, including cytotoxic, helper and regulatory T cells. Single-cell RNA sequencing of blood samples showed CD8+ effector T cell clonal expansion in a patient with steroid-unresponsive ir-hepatitis than in a steroid responder.

Conclusion: Nearly half of patients developing ir-hepatitis had an inadequate response to steroids and needed MMF as a secondary immunosuppressant. Patients with mixed DILI were more likely to respond to steroids, while alcohol consumption was associated with inadequate steroid response. Immune analyses showed high T cell infiltration in the liver among patients with ir-hepatitis.

Trial registration number: ClinicalTrials.gov ID number NCT04810156 and EudraCT no. 2020-004483-26.

Background: Anti-programmed cell death protein 1 (PD-1) immunotherapy has revolutionized the treatment of stage III and IV melanoma. Real-world data on its resistance is needed to facilitate the development of combinatorial approaches to overcome anti-PD-1 resistance.

Objectives: To characterize anti-PD-1 resistance and assess whether progressive disease assigned by clinicians is concordant with scan data assessed by independent central reviewers (ICR).

Methods: A retrospective chart review was conducted in adult patients with stage III/IV melanoma who initiated anti-PD-1 therapy from January 2018 until 12 months before the start of data collection at 22 sites across six countries. Primary resistance and late relapse in the adjuvant setting, and primary, secondary resistance, and late progression in the advanced setting were assigned using Society for Immunotherapy of Cancer definitions. Demographic and clinical characteristics by type of resistance were compared with appropriate univariate tests. Time to resistance (TTR) and overall survival were analyzed using Kaplan-Meier. To compare the concordance of progression assigned by clinicians and ICR, the positive predictive value (PPV) was calculated in a subset of patients.

Results: Of 981 eligible patients, 738 were included. In the adjuvant setting (n=240), 53 (22.1%) patients developed primary resistance and 60 (25.0%) experienced late relapse. In the advanced setting (n=498), 222 (44.6%), 50 (10.0%), and 64 (12.9%) patients developed primary, secondary resistance, and late progression. Type of resistance significantly differed by country, race, type of BRAF mutation, and PD-L1 expression in both settings; and by sex, disease stage and tumor thickness in the adjuvant setting only (p<0.05). Mean (SD) TTR was 47.7 (1.3) and 24.2 (1.0) months in the adjuvant and advanced setting, respectively. Patients with primary resistance had the poorest overall survival. The PPV of progression assigned by clinicians was 87.2% (95% CI 72.6% to 95.7%).

Conclusions: This study showed that a substantial proportion of patients with melanoma receiving anti-PD-1 therapy in the adjuvant (47.1%) and advanced (67.5%) settings developed resistance or late relapse/progression, highlighting an unmet medical need. Real-world clinical practice provided a reliable assessment of progression. Factors associated with different types of resistance were identified. Further study is warranted to evaluate their impact on patient risk stratification. (Graphical abstract).

Background: In murine studies, we demonstrated that a DNA vaccine encoding the androgen receptor (pTVG-AR) given prior to androgen deprivation elicited prostate tumor-infiltrating lymphocytes and an antitumor response. The current trial evaluated this approach, with or without programmed cell death protein-1 (PD-1) blockade, in patients with high-risk newly diagnosed prostate cancer.

Methods: In the first stage of a two-stage protocol, 24 patients were randomized to treatment with (1) degarelix alone (n=6); (2) pTVG-AR followed by degarelix (n=9); or (3) pTVG-AR, degarelix and nivolumab (n=9), each delivered over 12 weeks prior to prostatectomy. The primary objectives were safety and pathological complete response or minimal residual disease (MRD). Secondary endpoints were residual cancer burden (RCB) <0.25 cm³ and 1-year prostate-specific antigen (PSA) progression-free survival.

Results: Adverse events were almost exclusively in Arm 3, attributed to nivolumab. One patient achieved MRD (Arm 2), and three patients had an RCB <0.25 cm³ (all in Arm 2). At 1 year after surgery, the PSA progression-free survival rate was 33% (2/6) in Arm 1, 89% (8/9) in Arm 2, and 33% (3/9) in Arm 3 (p=0.039). Tissue analysis at prostatectomy demonstrated reduced CD4+cells with a regulatory phenotype in patients in Arm 2.

Conclusion: In this first stage of a pilot study that awaits confirmation with larger numbers of patients, our results suggest that vaccination targeting AR given prior to androgen deprivation therapy might improve outcome for patients with high-risk prostate cancer. Contrary to our initial hypothesis, this was not improved with the addition of PD-1 blockade, possibly due to the activation of regulatory CD4+T cells.

Trial registration number: NCT04989946.

Background: The efficacy of combined chemotherapy and programmed cell death protein-1 (PD-1) immune checkpoint blockade (ICB) is constrained by the collateral cytotoxicity of chemotherapy toward proliferating tumor-specific CD8⁺ T (T_TST) cells, a population indispensable for antitumor immunity. This study aimed to overcome this limitation by targeting a potential chemotherapy-resistant immune cell reservoir.

Methods: By using the murine acutely resolved lymphocytic choriomeningitis virus (LCMV) infection and tumor models (colorectal cancer and melanoma), we characterized the susceptibility of CD8⁺ T_TST and virus-specific bystander memory CD8⁺ T (T_BYS) cells to platinum-based chemotherapy-induced cytotoxicity. We next evaluated the antitumor efficacy and underlying mechanisms of combined chemotherapy and T_BYS cell-targeted oncolytic virus therapy (OV-BYTE) in tumor-bearing mice with prior LCMV infection or SARS-CoV-2 vaccination. Finally, we assessed the antitumor efficacy of the triple combination regimen (including OV-BYTE, chemotherapy, and PD-1 ICB) in both murine colorectal cancer model and patient-derived colorectal cancer organoid.

Results: We first demonstrated that within the tumor microenvironment, CD8⁺ T_TST cells are highly susceptible to platinum-based chemotherapy, whereas CD8⁺ T_BYS cells constitute a quiescent, chemo-resistant population. Leveraging this, CD8⁺ T_BYS-targeted OV-BYTE therapy synergized with chemotherapy to control tumorigenesis in multiple murine models. Mechanistically, this dual combination directly engaged the CD8⁺ T_BYS cell reservoir for tumor killing, which was accompanied by the restoration of CD8⁺ T_TST cell function via reduced apoptotic susceptibility and acquisition of a polyfunctional, effector-like state. Consequently, integrating OV-BYTE into the standard chemo-PD-1 ICB regimen resulted in improved antitumor efficacy in both preclinical and patient-derived tumor models.

Conclusions: Our study establishes the chemotherapy-resistant CD8⁺ T_BYS cell niche as a pivotal therapeutic target. By engaging this target, OV-BYTE emerges as a potent combinatorial agent that successfully circumvents a core limitation of standard chemo-immunotherapy, thus offering a rationally designed and translatable strategy to advance combination cancer therapy.

Background: Antibody therapeutics have revolutionized cancer treatment, but their use is increasingly associated with adverse events. Among these, anaphylaxis is particularly concerning due to its severity and unpredictability. Our previous studies demonstrated that repeated administration of anti-programmed death-ligand 1 antibodies to tumor-bearing mice induces antidrug antibodies (ADAs) and anaphylaxis. However, the specific characteristics of antibody therapeutics responsible for this effect and the underlying mechanism of ADA production remain poorly understood. This study aimed to identify the immunological and molecular determinants of ADA-associated anaphylaxis following antibody therapeutics in tumor-bearing hosts.

Methods: CT26 and 4T1 tumor-bearing mice were repeatedly administered various therapeutic antibodies with differing affinities for Fcγ receptors (FcγRs). Anaphylaxis symptoms, body temperature, and mortality were evaluated. Serum ADA levels were quantified using ELISA. Antibody affinity for mouse FcγR was determined using surface plasmon resonance. Antibody distribution in the spleen was assessed via immunofluorescence staining, and antibody glycosylation was analyzed by liquid chromatography-mass spectrometry. Immune cell populations were examined using flow cytometry.

Results: Repeated administration of antibodies with high affinities for FcγRs to tumor-bearing mice induced robust ADA production and anaphylaxis, whereas antibodies with low affinities for FcγRs against the same target elicited only minimal ADA responses and did not trigger anaphylaxis. We identified this difference as being attributed to the ability of tumor-associated monocytic-macrophage lineage cells to capture antibodies via FcγR, altering antibody biodistribution in the spleen, thereby facilitating antigen presentation and activating humoral immunity. Pretreatment with FcγR blocking antibodies attenuated this response, reducing anaphylaxis severity and improving survival. Analysis of clinical therapeutic antibodies also showed that those with a high affinity for FcγRs have a higher risk of inducing anaphylaxis, whereas neutralizing/blocking antibodies with a low or no affinity for FcγRs have a lower risk.

Conclusions: High affinities for FcγRs were identified as a critical determinant of anaphylaxis and reveal a mechanism linking FcγR-mediated antibody capture by tumor-associated myeloid cells to ADA induction. This contributes to the mechanistic foundation of AllergoOncology, an emerging interdisciplinary field exploring the interplay between cancer pathology and hypersensitivity reactions to therapeutic agents and providing insights for improving the safety and design of antibody therapeutics.

Background: Immune checkpoint inhibitor therapy (ICI) with nivolumab+ipilimumab is a first-line (1L) standard for metastatic clear cell renal cell carcinoma (ccRCC), yet outcomes remain heterogeneous. Increasing evidence suggests that host factors influence the tumor microenvironment and response to ICI. Although higher body mass index (BMI) has been associated with improved outcomes in several malignancies, BMI is an imprecise surrogate for underlying adipose and muscle compartments. We evaluated the association between body composition and outcomes with 1L nivolumab+ipilimumab in metastatic ccRCC.

Methods: We retrospectively analyzed patients with mccRCC treated with 1L nivolumab+ipilimumab at MD Anderson Cancer Center between June 2015 and February 2024. Body composition was measured using an artificial intelligence segmentation tool at the L3 vertebra from pretreatment CT scans obtained within 45 days prior to starting therapy. Endpoints included real-world progression-free survival (PFS) and overall survival (OS). Multivariable Cox regression models, guided by directed acyclic graphs, evaluated associations between continuous body composition measures and outcomes, incorporating non-linear cubic splines. An exploratory analysis used single-cell RNA sequencing from 12 treatment-naïve patients with metastatic ccRCC, stratified by median Skeletal Muscle Mass Index (SMMi) and Subcutaneous Adipose Tissue Index (SATi) values.

Results: Among 309 patients (80.3% male, median age 61.9 years; 61.8% intermediate-risk and 28.5% poor-risk), increasing SMMi and SATi were independently associated with shorter PFS (HR 1.50, 95% CI 1.05 to 2.15 per 3-unit increase; and HR 1.39, 95% CI 1.01 to 1.90 per 10-unit increase). The associations of BMI, visceral adiposity, and skeletal muscle density with PFS were inconclusive. OS associations for all body-composition measures were likewise indeterminate. In the single-cell cohort, low SMMi was associated with numerically higher T-cell fractions (p=0.064), fewer myeloid cell proportions (p=0.10), and higher IDO1 expression.

Conclusions: Greater subcutaneous adiposity and skeletal muscle mass were associated with shorter PFS among patients with metastatic ccRCC treated with 1L nivolumab+ipilimumab. These findings support the concept that host body composition influences the heterogeneous clinical benefit observed with ICI in metastatic ccRCC.

Background: B-cell maturation antigen (BCMA) is the main target for chimeric antigen receptor (CAR)-T cells in multiple myeloma (MM), demonstrating promising outcomes. However, unlike what happens with CART19 in lymphoblastic leukemia and non-Hodgkin's lymphoma, a high proportion of patients will relapse after CAR-T BCMA therapy due to insufficient antigen expression, low CAR-T cell persistence and/or T-cell exhaustion. In other B cell malignancies, second-generation anti-CD19 4-1BB CARs with CD28-transmembrane domain (TMD) have shown high efficacy and a favorable toxicity profile. We have developed a second-generation CD8α-TM BCMA-4-1BBζ CAR-T product, ARI0002h (Cesnicabtagene-autoleucel) for patients with relapsed/refractory MM. We hypothesized that replacing the TMD of ARI0002h with a CD28-TMD could increase efficacy and reduce tumor escape while maintaining a tolerable toxicity profile.

Methods: We generated CAR-T cells using T-cells isolated from buffy coats and evaluated the efficacy and fitness of CAR-Ts at day 8-10 of expansion against several MM cell lines. In vitro analyses included cytotoxicity, proliferation, cytokine secretion, T-cell subset markers, activation and exhaustion profiling, metabolomic assays, and RNA-seq after multiple tumor challenges. In in vivo xenograft studies using NSG mice, with tumor cells expressing GFP-ffLuc, disease progression was monitored weekly via bioluminescence imaging.

Results: Despite showing similar in vitro performance regarding cytotoxicity, proliferation and cytokine production, ARI2h-TM28 outperforms ARI0002h in a low BCMA expression setting and achieves superior in vivo tumor control and survival in relapse models with antigen downregulation. Furthermore, ARI2h-TM28 showed an optimized metabolic profile, more oxidative and energetic compared with ARI0002h, with downregulation of proinflammatory genes in CD8 T cells, contributing altogether both to reduced exhaustion and increased persistence of the CARs, improving their efficacy in preclinical models.

Conclusions: Incorporating a CD28-TMD into the ARI0002h CAR enhances tumor control even in relapse models with downregulation of the target antigen, offering improved long-term disease management. This modification increases potency against MM tumor cell lines with both normal and reduced BCMA expression, demonstrating superior metabolic endurance and in vivo activity.