bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling
Authors: Landoni Elisa , Fucá Giovanni , Wang Jian , Chirasani Venkat R. , Yao Zhiyuan , Dukhovlinova Elena , Ferrone Soldano , Savoldo Barbara , Hong Lee K. , Shou Peishun , Musio Silvia , Padelli Francesco , Finocchiaro Gaetano , Droste Miriam , Kuhlman Brian , Shamshiev Abdijapar , Pellegatta Serena , Dokholyan Nikolay V. , Dotti Gianpietro
Published: April 2023

Abstract
Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.
Data from Targeted Deletion of CXCR2 in Myeloid Cells Alters the Tumor Immune Environment to Improve Antitumor Immunity
Authors: Yang Jinming , Yan Chi , Vilgelm Anna E. , Chen Sheau-Chiann , Ayers Gregory D. , Johnson Christopher A. , Richmond Ann
Published: April 2023

Abstract
Recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment (TME) contributes to cancer immune evasion. MDSCs express the chemokine receptor CXCR2, and inhibiting CXCR2 suppresses the recruitment of MDSCs into the tumor and the premetastatic niche. Here, we compared the growth and metastasis of melanoma and breast cancer xenografts in mice exhibiting or not exhibiting targeted deletion of Cxcr2 in myeloid cells (CXCR2myeΔ/Δ vs. CXCR2myeWT). Detailed analysis of leukocyte populations in peripheral blood and in tumors from CXCR2myeΔ/Δ mice revealed that loss of CXCR2 signaling in myeloid cells resulted in reduced intratumoral MDSCs and increased intratumoral CXCL11. The increase in intratumoral CXCL11 was derived in part from tumor-infiltrating B1b cells. The reduction in intratumoral MDSCs coupled with an increase in intratumoral B1b cells expressing CXCL11 resulted in enhanced infiltration and activation of effector CD8+ T cells in the TME of CXCR2myeΔ/Δ mice, accompanied by inhibition of tumor growth in CXCR2myeΔ/Δ mice compared with CXCR2myeWT littermates. Treatment of tumor-bearing mice with a CXCR2 antagonist (SX-682) also inhibited tumor growth, reduced intratumoral MDSCs, and increased intratumoral B1b cells expressing CXCL11, leading to an increase in activated CD8+ T cells in the tumor. Depletion of B220+ cells or depletion of CD8+ T cells reversed the tumor-inhibitory properties in CXCR2myeΔ/Δ mice. These data revealed a mechanism by which loss of CXCR2 signaling in myeloid cells modulates antitumor immunity through decreasing MDSCs and enriching CXCL11-producing B1b cells in the TME, which in turn increases CD8+ T-cell recruitment and activation in tumors.
Data from Targeting WEE1/AKT Restores p53-Dependent Natural Killer–Cell Activation to Induce Immune Checkpoint Blockade Responses in “Cold” Melanoma
Authors: Dinavahi Saketh S. , Chen Yu-Chi , Punnath Kishore , Berg Arthur , Herlyn Meenhard , Foroutan Momeneh , Huntington Nicholas D. , Robertson Gavin P.
Published: April 2023

Abstract
Immunotherapy has revolutionized cancer treatment. Unfortunately, most tumor types do not respond to immunotherapy due to a lack of immune infiltration or “cold” tumor microenvironment (TME), a contributing factor in treatment failure. Activation of the p53 pathway can increase apoptosis of cancer cells, leading to enhanced antigen presentation, and can stimulate natural killer (NK) cells through expression of stress ligands. Therefore, modulation of the p53 pathway in cancer cells with wild-type TP53 has the potential to enhance tumor immunogenicity to NK cells, produce an inflammatory TME, and ultimately lead to tumor regression. In this study, we report simultaneous targeting of the AKT/WEE1 pathways is a novel and tolerable approach to synergistically induce p53 activation to inhibit tumor development. This approach reduced the growth of melanoma cells and induced plasma membrane surface localization of the ER-resident protein calreticulin, an indicator of immunogenic cell death (ICD). Increase in ICD led to enhanced expression of stress ligands recognized by the activating NK-cell receptor NKG2D, promoting tumor lysis. WEE1/AKT inhibition resulted in recruitment and activation of immune cells, including NK cells, in the TME, triggering an inflammatory cascade that transformed the “cold” TME of B16F10 melanoma into a “hot” TME that responded to anti–programmed cell death protein 1 (anti–PD-1), resulting in complete regression of established tumors. These results suggest that AKT/WEE1 pathway inhibition is a potential approach to broaden the utility of class-leading anti–PD-1 therapies by enhancing p53-mediated, NK cell–dependent tumor inflammation and supports the translation of this novel approach to further improve response rates for metastatic melanoma.
Data from Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer
Authors: Mujal Adriana M. , Combes Alexis J. , Rao Arjun A. , Binnewies Mikhail , Samad Bushra , Tsui Jessica , Boissonnas Alexandre , Pollack Joshua L. , Argüello Rafael J. , Meng Maxwell V. , Porten Sima P. , Ruhland Megan K. , Barry Kevin C. , Chan Vincent , Krummel Matthew F.
Published: April 2023

Abstract
The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte–macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.
Data from Accounting for B-cell Behavior and Sampling Bias Predicts Anti–PD-L1 Response in Bladder Cancer
Authors: Dyugay Ilya A. , Lukyanov Daniil K. , Turchaninova Maria A. , Serebrovskaya Ekaterina O. , Bryushkova Ekaterina A. , Zaretsky Andrew R. , Khalmurzaev Oybek , Matveev Vsevolod B. , Shugay Mikhail , Shelyakin Pavel V. , Chudakov Dmitriy M.
Published: April 2023

Abstract
Cancer immunotherapy is predominantly based on T cell–centric approaches. At the same time, the adaptive immune response in the tumor environment also includes clonally produced immunoglobulins and clonal effector/memory B cells that participate in antigen-specific decisions through their interactions with T cells. Here, we investigated the role of infiltrating B cells in bladder cancer via patient dataset analysis of intratumoral immunoglobulin repertoires. We showed that the IgG1/IgA ratio is a prognostic indicator for several subtypes of bladder cancer and for the whole IMVigor210 anti–PD-L1 immunotherapy study cohort. A high IgG1/IgA ratio associated with the prominence of a cytotoxic gene signature, T-cell receptor signaling, and IL21-mediated signaling. Immunoglobulin repertoire analysis indicated that effector B-cell function, rather than clonally produced antibodies, was involved in antitumor responses. From the T-cell side, we normalized a cytotoxic signature against the extent of immune cell infiltration to neutralize the artificial sampling-based variability in immune gene expression. Resulting metrics reflected proportion of cytotoxic cells among tumor-infiltrating immune cells and improved prediction of anti–PD-L1 responses. At the same time, the IgG1/IgA ratio remained an independent prognostic factor. Integration of the B-cell, natural killer cell, and T-cell signatures allowed for the most accurate prediction of anti–PD-L1 therapy responses. On the basis of these findings, we developed a predictor called PRedIctive MolecUlar Signature (PRIMUS), which outperformed PD-L1 expression scores and known gene signatures. Overall, PRIMUS allows for reliable identification of responders among patients with muscle-invasive urothelial carcinoma, including the subcohort with the low-infiltrated “desert” tumor phenotype.
Data from Loss of LRRC33-Dependent TGFβ1 Activation Enhances Antitumor Immunity and Checkpoint Blockade Therapy
Authors: Jiang Aiping , Qin Yan , Springer Timothy A.
Published: April 2023

Abstract
TGFβ has multiple roles and gene products (TGFβ1, -β2, and -β3), which make global targeting of TGFβ undesirable. Expression of TGFβ requires association with milieu molecules, which localize TGFβ to the surface of specific cells or extracellular matrices. Here, we found that LRRC33 was specifically associated with TGFβ1, not TGFβ2 and TGFβ3, and was required for surface display and activation of TGFβ1 on tumor-infiltrating myeloid cells. Loss of LRRC33-dependent TGFβ1 activation slowed tumor growth and metastasis by enhancing innate and adaptive antitumor immunity in multiple mouse syngeneic tumor models. LRRC33 loss resulted in a more immunogenic microenvironment, with decreased myeloid-derived suppressor cells, more active CD8+ T and NK cells, and more skewing toward tumor-suppressive M1 macrophages. LRRC33 loss and PD-1 blockade synergized in controlling B16.F10 tumor growth. Our results demonstrate the importance of LRRC33 in tumor biology and highlight the therapeutic potential of dual blockade of the LRRC33/TGFβ1 axis and PD-1/PD-L1 in cancer immunotherapy.
Data from Integrin β7 Inhibits Colorectal Cancer Pathogenesis via Maintaining Antitumor Immunity
Authors: Zhang Youhua , Xie Ruting , Zhang Hailong , Zheng Yajuan , Lin Changdong , Yang Lei , Huang Mengwen , Li Man , Song Feifei , Lu Ling , Yang Muqing , Liu Ying , Wei Qing , Li Jiyu , Chen Jianfeng
Published: April 2023

Abstract
Immune cell infiltration is important for predicting the clinical outcomes of colorectal cancer. Integrin β7 (ITGB7), which is expressed on the surface of leukocytes, plays an essential role in the homing of immune cells to gut-associated lymphoid tissue and facilitating the retention of lymphocytes in gut epithelium; however, its role in colorectal cancer pathogenesis is poorly explored. Here, we found that the number of β7+ cells decreased significantly in tumor tissue compared with adjacent normal tissue. β7 expression decreased in tumor-derived compared with normal tissue–derived CD8+ T cells. With bulk RNA expression data from public platforms, we demonstrated that higher ITGB7 expression correlated with longer patient survival, higher cytotoxic immune cell infiltration, lower somatic copy-number alterations, decreased mutation frequency of APC and TP53, and better response to immunotherapy. The possible cell–cell interactions mediated by ITGB7 and its ligands MAdCAM-1, VCAM-1, and CDH1 were investigated using public single-cell RNA sequencing data. ITGB7 deficiency led to exaggerated tumorigenesis and progression in both Apcmin/+ spontaneous and MC38 orthotopic models of colorectal cancer, which could be due to a reduced infiltration of activated CD8+ T cells, effector memory CD8+ T cells, IFNγ+ CD8+ T cells, IFNγ+ natural killer cells, CD103+ dendritic cells, and other immune cell subsets that are essential players in antitumor immunity. In conclusion, our data revealed that ITGB7 could inhibit the tumorigenesis and progression of colorectal cancer by maintaining antitumor immunity.
Data from Glioblastoma Cell–Derived lncRNA-Containing Exosomes Induce Microglia to Produce Complement C5, Promoting Chemotherapy Resistance
Authors: Li Ziwei , Meng Xiangqi , Wu Pengfei , Zha Caijun , Han Bo , Li Lulu , Sun Nan , Qi Tengfei , Qin Jie , Zhang Yangong , Tian Kaifu , Li Shupeng , Yang Changxiao , Ren Lejia , Ming Jianguang , Wang Pandeng , Song Yifei , Jiang Chuanlu , Cai Jinquan
Published: April 2023

Abstract
Glioblastoma (GBM), the most common malignant primary brain cancer in adults, nearly always becomes resistant to current treatments, including the chemotherapeutic temozolomide (TMZ). The long noncoding RNA (lncRNA) TMZ-associated lncRNA in GBM recurrence (lnc-TALC) promotes GBM resistance to TMZ. Exosomes can release biochemical cargo into the tumor microenvironment (TME) or transfer their contents, including lncRNAs, to other cells as a form of intercellular communication. In this study, we found that lnc-TALC could be incorporated into exosomes and transmitted to tumor-associated macrophages (TAM) and could promote M2 polarization of the microglia. This M2 polarization correlated with secretion of the complement components C5/C5a, which occurred downstream of lnc-TALC binding to ENO1 to promote the phosphorylation of p38 MAPK. In addition, C5 promoted the repair of TMZ-induced DNA damage, leading to chemotherapy resistance, and C5a-targeted immunotherapy showed improved efficacy that limited lnc-TALC–mediated TMZ resistance. Our results reveal that exosome-transmitted lnc-TALC could remodel the GBM microenvironment and reduce tumor sensitivity to TMZ chemotherapy, indicating that the lnc-TALC–mediated cross-talk between GBM cells and microglia could attenuate chemotherapy efficacy and pointing to potential combination therapy strategies to overcome TMZ resistance in GBM.

See related Spotlight by Zhao and Xie, p. 1372.
Data from Exploiting Natural Killer Cell Engagers to Control Pediatric B-cell Precursor Acute Lymphoblastic Leukemia
Authors: Colomar-Carando Natalia , Gauthier Laurent , Merli Pietro , Loiacono Fabrizio , Canevali Paolo , Falco Michela , Galaverna Federica , Rossi Benjamin , Bosco Frédéric , Caratini Mélody , Mingari Maria Cristina , Locatelli Franco , Vivier Eric , Meazza Raffaella , Pende Daniela
Published: April 2023

Abstract
Natural killer (NK) cells represent a promising cell type in antitumor immunotherapy for efficacy and safety, particularly in the treatment of hematologic malignancies. NK cells have been shown to exert antileukemia activity in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Products have been developed to boost the activation of NK cells only when cross-linked by tumor cells, avoiding any off-target effect. Here, we tested the in vitro effect of different NK-cell engagers (NKCE), which trigger either NKp46 or NKp30 together with CD16A, and target either CD19 or CD20 to induce killing of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Target cells were NALM-16 and MHH-CALL-4 cell lines and four primary leukemias, while effector cells were resting NK cells derived from healthy donors and pediatric patients with leukemia after αβT/B-depleted haplo-HSCT. The NK cell–resistant MHH-CALL-4 was efficiently killed using all NKCEs. Boosting of NK activity against MHH-CALL-4 was also evident by degranulation and IFNγ production. Because of the lack of CD20 and high expression of CD19 on primary BCP-ALL, we focused on NKCEs targeting CD19. NKp46- and NKp30-based NKCEs displayed similar potency at inducing NK-cell activity, even when challenged with primary BCP-ALL blasts. Their efficacy was shown also using NK cells derived from transplanted patients. NKCE-induced activation against BCP-ALL can override HLA-specific inhibitory interactions, although the strongest response was observed by the alloreactive NK-cell subset. These data support the therapeutic use of NKp46/CD16A/CD19-NKCE to fight refractory/relapsed leukemia in pretransplantation or posttransplantation settings.
Data from Spatial Organization and Prognostic Significance of NK and NKT-like Cells via Multimarker Analysis of the Colorectal Cancer Microenvironment
Authors: Väyrynen Juha P. , Haruki Koichiro , Lau Mai Chan , Väyrynen Sara A. , Ugai Tomotaka , Akimoto Naohiko , Zhong Rong , Zhao Melissa , Dias Costa Andressa , Borowsky Jennifer , Bell Phoenix , Takashima Yasutoshi , Fujiyoshi Kenji , Arima Kota , Kishikawa Junko , Shi Shan-shan , Twombly Tyler S. , Song Mingyang , Wu Kana , Chan Andrew T. , Zhang Xuehong , Fuchs Charles S. , Meyerhardt Jeffrey A. , Giannakis Marios , Ogino Shuji , Nowak Jonathan A.
Published: April 2023

Abstract
Although tumor-infiltrating T cells hold a beneficial prognostic role in colorectal cancer, other lymphocytic populations are less characterized. We developed a multiplexed immunofluorescence assay coupled with digital image analysis and machine learning to identify natural killer (NK) cells (NCAM1+CD3−), natural killer T-like (NKT-like) cells (NCAM1+CD3+), and T cells (NCAM1−CD3+) within the PTPRC+ (CD45+) cell population and to measure their granzyme B (GZMB; cytotoxicity marker) and FCGR3A (CD16a; NK-cell maturity marker) expression. We evaluated immune cell densities and spatial configuration in 907 incident colorectal carcinoma cases within two prospective cohort studies. We found that T cells were approximately 100 times more abundant than NK and NKT-like cells. Overall, NK cells showed high GZMB expression and were located closer to tumor cells than T and NKT-like cells. In T and NKT-like cells, GZMB expression was enriched in cells in closer proximity to tumor cells. Higher densities of both T and NKT-like cells associated with longer cancer-specific survival, independent of potential confounders (Ptrend < 0.0007). Higher stromal GZMB+ and FCGR3A+ NK-cell densities associated with longer cancer-specific survival (Ptrend < 0.003). For T and NKT-like cells, greater proximity to tumor cells associated with longer cancer-specific survival (Ptrend < 0.0001). These findings indicate that cytotoxic NCAM1+CD3−GZMB+ NK cells and NCAM1+CD3+ NKT-like cells are relatively rare lymphocytic populations within the colorectal cancer microenvironment and show distinct spatial configuration and associations with patient outcome. The results highlight the utility of a quantitative multimarker assay for in situ, single-cell immune biomarker evaluation and underscore the importance of spatial context for tumor microenvironment characterization.
Data from Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression
Authors: Nirschl Christopher J. , Brodkin Heather R. , Hicklin Daniel J. , Ismail Nesreen , Morris Kristin , Seidel-Dugan Cynthia , Steiner Philipp , Steuert Zoe , Sullivan Jenna M. , Tyagi Ethika , Winston William M. , Salmeron Andres
Published: April 2023

Abstract
IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment.

See related Spotlight by Silva, p. 544.
Data from Hematopoietic Prostaglandin D2 Synthase Controls Tfh/Th2 Communication and Limits Tfh Antitumor Effects
Authors: Mary Romain , Chalmin Fanny , Accogli Théo , Bruchard Mélanie , Hibos Christophe , Melin Joséphine , Truntzer Caroline , Limagne Emeric , Derangère Valentin , Thibaudin Marion , Humblin Etienne , Boidot Romain , Chevrier Sandy , Arnould Laurent , Richard Corentin , Klopfenstein Quentin , Bernard Antoine , Urade Yoshihiro , Harker James A. , Apetoh Lionel , Ghiringhelli François , Végran Frédérique
Published: April 2023

Abstract
T follicular helper (Tfh) cells are a subset of CD4+ T cells essential in immunity and have a role in helping B cells produce antibodies against pathogens. However, their role during cancer progression remains unknown. The mechanism of action of Tfh cells remains elusive because contradictory data have been reported on their protumor or antitumor responses in human and murine tumors. Like Tfh cells, Th2 cells are also involved in humoral immunity and are regularly associated with tumor progression and poor prognosis, mainly through their secretion of IL4. Here, we showed that Tfh cells expressed hematopoietic prostaglandin D2 (PGD2) synthase in a pSTAT1/pSTAT3-dependent manner. Tfh cells produced PGD2, which led to recruitment of Th2 cells via the PGD2 receptor chemoattractant receptor homologous molecule expressed on Th type 2 cells (CRTH2) and increased their effector functions. This cross-talk between Tfh and Th2 cells promoted IL4-dependent tumor growth. Correlation between Th2 cells, Tfh cells, and hematopoietic PGD2 synthase was observed in different human cancers and associated with outcome. This study provides evidence that Tfh/Th2 cross-talk through PGD2 limits the antitumor effects of Tfh cells and, therefore, could serve as a therapeutic target.
Data from Circadian Regulator CLOCK Drives Immunosuppression in Glioblastoma
Authors: Xuan Wenjing , Hsu Wen-Hao , Khan Fatima , Dunterman Madeline , Pang Lizhi , Wainwright Derek A. , Ahmed Atique U. , Heimberger Amy B. , Lesniak Maciej S. , Chen Peiwen
Published: April 2023

Abstract
The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSC) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK–BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via hypoxia-inducible factor 1-alpha (HIF1α) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162 and polarizes infiltrating microglia toward an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK–OLFML3–HIF1α–LGMN–CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T-cell infiltration, activation, and cytotoxicity, and synergizes with anti–programmed cell death protein 1 (anti–PD-1 therapy). In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial abundance and poor prognosis. Together, these findings uncover the CLOCK–OLFML3–HIF1α–LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for patients with GBM.
Data from Visualizing Spatial and Stoichiometric Barriers to Bispecific T-Cell Engager Efficacy
Authors: You Ran , Artichoker Jordan , Ray Arja , Gonzalez Velozo Hugo , Rock Dan A. , Conner Kip P. , Krummel Matthew F.
Published: April 2023

Abstract
Bispecific T-cell engager (BiTE) molecules are biologic T cell–directing immunotherapies. Blinatumomab is approved for treatment of B-cell malignancies, but BiTE molecule development in solid tumors has been more challenging. Here, we employed intravital imaging to characterize exposure and pharmacodynamic response of an anti-muCD3/anti-huEGFRvIII mouse surrogate BiTE molecule in EGFR variant III (EGFRvIII)-positive breast tumors implanted within immunocompetent mice. Our study revealed heterogeneous temporal and spatial dynamics of BiTE molecule extravasation into solid tumors, highlighting physical barriers to BiTE molecule function. We also discovered that high, homogeneous EGFRvIII expression on cancer cells was necessary for a BiTE molecule to efficiently clear tumors. In addition, we found that resident tumor-infiltrating lymphocytes (TIL) were sufficient for optimal tumor killing only at high BiTE molecule dosage, whereas inclusion of peripheral T-cell recruitment was synergistic at moderate to low dosages. We report that deletion of stimulatory conventional type I DCs (cDC1) diminished BiTE molecule–induced T-cell activation and tumor clearance, suggesting that in situ antigen-presenting cell (APC) engagements modulate the extent of BiTE molecule efficacy. In summary, our work identified multiple requirements for optimal BiTE molecule efficacy in solid tumors, providing insights that could be harnessed for solid cancer immunotherapy development.
Data from CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1
Authors: Kantari-Mimoun Chahrazade , Barrin Sarah , Vimeux Lene , Haghiri Sandrine , Gervais Claire , Joaquina Sandy , Mittelstaet Joerg , Mockel-Tenbrinck Nadine , Kinkhabwala Ali , Damotte Diane , Lupo Audrey , Sibony Mathilde , Alifano Marco , Dondi Elisabetta , Bercovici Nadège , Trautmann Alain , Kaiser Andrew D. , Donnadieu Emmanuel
Published: April 2023

Abstract
Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1–dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.
Data from Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma
Authors: Riedel Angela , Helal Moutaz , Pedro Luisa , Swietlik Jonathan J. , Shorthouse David , Schmitz Werner , Haas Lisa , Young Timothy , da Costa Ana S.H. , Davidson Sarah , Bhandare Pranjali , Wolf Elmar , Hall Benjamin A. , Frezza Christian , Oskarsson Thordur , Shields Jacqueline D.
Published: April 2023

Abstract
Communication between tumors and the stroma of tumor-draining lymph nodes (TDLN) exists before metastasis arises, altering the structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRC), the dominant stromal population of lymph nodes, has revealed that FRCs in TDLNs are reprogrammed. However, the tumor-derived factors driving the changes in FRCs remain to be identified. Taking an unbiased approach, we have shown herein that lactic acid (LA), a metabolite released by cancer cells, was not only secreted by B16.F10 and 4T1 tumors in high amounts, but also that it was enriched in TDLNs. LA supported an upregulation of Podoplanin (Pdpn) and Thy1 and downregulation of IL7 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we found that tumor-derived LA altered mitochondrial function of FRCs in TDLNs. Thus, our results demonstrate a mechanism by which a tumor-derived metabolite connected with a low pH environment modulates the function of fibroblasts in TDLNs. How lymph node function is perturbed to support cancer metastases remains unclear. The authors show that tumor-derived LA drains to lymph nodes where it modulates the function of lymph node stromal cells, prior to metastatic colonization.
Data from CD137 Costimulation Counteracts TGFβ Inhibition of NK-cell Antitumor Function
Authors: Cabo Mariona , Santana-Hernández Sara , Costa-Garcia Marcel , Rea Anna , Lozano-Rodríguez Roberto , Ataya Michelle , Balaguer Francesc , Juan Manel , Ochoa Maria C. , Menéndez Silvia , Comerma Laura , Rovira Ana , Berraondo Pedro , Albanell Joan , Melero Ignacio , López-Botet Miguel , Muntasell Aura
Published: April 2023

Abstract
Enhancing natural killer (NK) cell–based cancer immunotherapy by overcoming immunosuppression is an area of intensive research. Here, we have demonstrated that the anti-CD137 agonist urelumab can overcome TGFβ-mediated inhibition of human NK-cell proliferation and antitumor function. Transcriptomic, immunophenotypic, and functional analyses showed that CD137 costimulation modified the transcriptional program induced by TGFβ on human NK cells by rescuing their proliferation in response to IL2, preserving their expression of activating receptors (NKG2D) and effector molecules (granzyme B, IFNγ) while allowing the acquisition of tumor-homing/retention features (CXCR3, CD103). Activated NK cells cultured in the presence of TGFβ1 and CD137 agonist recovered CCL5 and IFNγ secretion and showed enhanced direct and antibody-dependent cytotoxicity upon restimulation with cancer cells. Trastuzumab treatment of fresh breast carcinoma–derived multicellular cultures induced CD137 expression on tumor-infiltrating CD16+ NK cells, enabling the action of urelumab, which fostered tumor-infiltrating NK cells and recapitulated the enhancement of CCL5 and IFNγ production. Bioinformatic analysis pointed to IFNG as the driver of the association between NK cells and clinical response to trastuzumab in patients with HER2-positive primary breast cancer, highlighting the translational relevance of the CD137 costimulatory axis for enhancing IFNγ production. Our data reveals CD137 as a targetable checkpoint for overturning TGFβ constraints on NK-cell antitumor responses.
Data from Overcoming CAR-Mediated CD19 Downmodulation and Leukemia Relapse with T Lymphocytes Secreting Anti-CD19 T-cell Engagers
Authors: Blanco Belén , Ramírez-Fernández Ángel , Bueno Clara , Argemí-Muntadas Lidia , Fuentes Patricia , Aguilar-Sopeña Óscar , Gutierrez-Agüera Francisco , Zanetti Samanta Romina , Tapia-Galisteo Antonio , Díez-Alonso Laura , Segura-Tudela Alejandro , Castellà Maria , Marzal Berta , Betriu Sergi , Harwood Seandean L. , Compte Marta , Lykkemark Simon , Erce-Llamazares Ainhoa , Rubio-Pérez Laura , Jiménez-Reinoso Anaïs , Domínguez-Alonso Carmen , Neves Maria , Morales Pablo , Paz-Artal Estela , Guedan Sonia , Sanz Laura , Toribio María L. , Roda-Navarro Pedro , Juan Manel , Menéndez Pablo , Álvarez-Vallina Luis
Published: April 2023

Abstract
Chimeric antigen receptor (CAR)–modified T cells have revolutionized the treatment of CD19-positive hematologic malignancies. Although anti-CD19 CAR-engineered autologous T cells can induce remission in patients with B-cell acute lymphoblastic leukemia, a large subset relapse, most of them with CD19-positive disease. Therefore, new therapeutic strategies are clearly needed. Here, we report a comprehensive study comparing engineered T cells either expressing a second-generation anti-CD19 CAR (CAR-T19) or secreting a CD19/CD3-targeting bispecific T-cell engager antibody (STAb-T19). We found that STAb-T19 cells are more effective than CAR-T19 cells at inducing cytotoxicity, avoiding leukemia escape in vitro, and preventing relapse in vivo. We observed that leukemia escape in vitro is associated with rapid and drastic CAR-induced internalization of CD19 that is coupled with lysosome-mediated degradation, leading to the emergence of transiently CD19-negative leukemic cells that evade the immune response of engineered CAR-T19 cells. In contrast, engineered STAb-T19 cells induce the formation of canonical immunologic synapses and prevent the CD19 downmodulation observed in anti-CD19 CAR-mediated interactions. Although both strategies show similar efficacy in short-term mouse models, there is a significant difference in a long-term patient-derived xenograft mouse model, where STAb-T19 cells efficiently eradicated leukemia cells, but leukemia relapsed after CAR-T19 therapy. Our findings suggest that the absence of CD19 downmodulation in the STAb-T19 strategy, coupled with the continued antibody secretion, allows an efficient recruitment of the endogenous T-cell pool, resulting in fast and effective elimination of cancer cells that may prevent CD19-positive relapses frequently associated with CAR-T19 therapies.
Data from Tumor Necrosis Factor-α–Induced Protein 8-Like 2 Fosters Tumor-Associated Microbiota to Promote the Development of Colorectal Cancer
Authors: Lou Yunwei , Song Miaomiao , Han Meijuan , Zhong Jiateng , Tian Xueqin , Ren Yahan , Song Yaru , Duan Liangwei , Zhao Peiqing , Song Xiangfeng , Zhang Wen , Chen Youhai H. , Wang Hui
Published: April 2023

Abstract
Although increasing evidence links the gut microbiota with the development of colorectal cancer, the molecular mechanisms for microbiota regulation of tumorigenesis are not fully understood. Here, we found that a member of the TNFα-induced protein 8 (TNFAIP8) family called TIPE2 (TNFAIP8-like 2) was significantly upregulated in murine intestinal tumors and in human colorectal cancer, and colorectal cancer with high expression of Tipe2 mRNA associated with reduced survival time of patients. Consistent with these findings, TIPE2 deficiency significantly inhibited the development of colorectal cancer in mice treated with azoxymethane/dextran sodium sulfate and in Apcmin/+ mice. TIPE2 deficiency attenuated the severity of colitis by successfully resolving and restricting colonic inflammation and protected colonic myeloid cells from death during colitis. Transplantation of TIPE2-deficient bone marrow into wild-type mice successfully dampened the latter's tumorigenic phenotype, indicating a hematopoietic-specific role for TIPE2. Mechanistically, restricting the expansion of Enterobacteriaceae/Escherichia coli (E. coli) decreased intestinal inflammation and reduced the incidence of colonic tumors. Collectively, these data suggest that hematopoietic TIPE2 regulates intestinal antitumor immunity by regulation of gut microbiota. TIPE2 may represent a new therapeutic target for treating colorectal cancer.
Data from Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer
Authors: Tsuji Shingo , Reil Katherine , Nelson Kinsey , Proclivo Veronica H. , McGuire Kathleen L. , Giacalone Matthew J.
Published: April 2023

Abstract
Emerging clinical evidence indicates that the combination of local administration of immunotherapy with systemic immune-checkpoint blockade targeting the PD-1/PD-L1 pathway improves response rates in select solid tumor indications; however, limited clinical experience with this approach exists in advanced bladder cancer patients. VAX014 is a novel bacterial minicell-based, integrin-targeted oncolytic agent undergoing clinical investigation for intravesical (IVE) treatment of nonmuscle-invasive bladder cancer. Here, we demonstrated that the antitumor activity of VAX014 following IVE administration was dependent upon CD4+ and CD8+ T cells in two syngeneic orthotopic bladder tumor models (MB49 and MBT-2). PD-L1 upregulation was found to be an acquired immune-resistance mechanism in the MB49 model, and the combination of VAX014 with systemic PD-L1 blockade resulted in a significant improvement in bladder tumor clearance rates and development of protective antitumor immunologic memory. Combination treatment also led to enhanced systemic antitumor immune responses capable of clearing distal intradermal tumors and controlling pulmonary metastasis. Distal tumors actively responding to combination therapy demonstrated a phenotypic shift from regulatory T cell to Th1 in intratumoral CD4+ T cells, which was accompanied by a higher percentage of activated CD8+ T cells and higher IFNγ. Finally, VAX014’s target integrins α3β1 and α5β1 were overexpressed in tumor biopsies from advanced-stage bladder cancer patients, as well as in both the MB49 and MBT-2 orthotopic mouse models of bladder cancer. These collective findings provide a rationale for the clinical investigation of VAX014 and systemic PD-1/PD-L1 blockade in advanced-stage bladder cancer.
Data from cDC1 Vaccines Drive Tumor Rejection by Direct Presentation Independently of Host cDC1
Authors: Ferris Stephen T. , Ohara Ray A. , Ou Feiya , Wu Renee , Huang Xiao , Kim Sunkyung , Chen Jing , Liu Tian-Tian , Schreiber Robert D. , Murphy Theresa L. , Murphy Kenneth M.
Published: April 2023

Abstract
As a cell-based cancer vaccine, dendritic cells (DC), derived from peripheral blood monocytes or bone marrow (BM) treated with GM-CSF (GMDC), were initially thought to induce antitumor immunity by presenting tumor antigens directly to host T cells. Subsequent work revealed that GMDCs do not directly prime tumor-specific T cells, but must transfer their antigens to host DCs. This reduces their advantage over strictly antigen-based strategies proposed as cancer vaccines. Type 1 conventional DCs (cDC1) have been reported to be superior to GMDCs as a cancer vaccine, but whether they act by transferring antigens to host DCs is unknown. To test this, we compared antitumor responses induced by GMDCs and cDC1 in Irf8 +32–/– mice, which lack endogenous cDC1 and cannot reject immunogenic fibrosarcomas. Both GMDCs and cDC1 could cross-present cell-associated antigens to CD8+ T cells in vitro. However, injection of GMDCs into tumors in Irf8 +32–/– mice did not induce antitumor immunity, consistent with their reported dependence on host cDC1. In contrast, injection of cDC1s into tumors in Irf8 +32–/– mice resulted in their migration to tumor-draining lymph nodes, activation of tumor-specific CD8+ T cells, and rejection of the tumors. Tumor rejection did not require the in vitro loading of cDC1 with antigens, indicating that acquisition of antigens in vivo is sufficient to induce antitumor responses. Finally, cDC1 vaccination showed abscopal effects, with rejection of untreated tumors growing concurrently on the opposite flank. These results suggest that cDC1 may be a useful future avenue to explore for antitumor therapy.

See related Spotlight by Hubert et al., p. 918
Data from Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition
Authors: Alvarez-Breckenridge Christopher , Markson Samuel C. , Stocking Jackson H. , Nayyar Naema , Lastrapes Matt , Strickland Matthew R. , Kim Albert E. , de Sauvage Magali , Dahal Ashish , Larson Juliana M. , Mora Joana L. , Navia Andrew W. , Klein Robert H. , Kuter Benjamin M. , Gill Corey M. , Bertalan Mia , Shaw Brian , Kaplan Alexander , Subramanian Megha , Jain Aarushi , Kumar Swaminathan , Danish Husain , White Michael , Shahid Osmaan , Pauken Kristen E. , Miller Brian C. , Frederick Dennie T. , Hebert Christine , Shaw McKenzie , Martinez-Lage Maria , Frosch Matthew , Wang Nancy , Gerstner Elizabeth , Nahed Brian V. , Curry William T. , Carter Bob , Cahill Daniel P. , Boland Genevieve Marie , Izar Benjamin , Davies Michael A. , Sharpe Arlene H. , Suvà Mario L. , Sullivan Ryan J. , Brastianos Priscilla K. , Carter Scott L.
Published: April 2023

Abstract
Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.
Data from Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor–Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors
Authors: Kim Sanghyun P. , Vale Nolan R. , Zacharakis Nikolaos , Krishna Sri , Yu Zhiya , Gasmi Billel , Gartner Jared J. , Sindiri Sivasish , Malekzadeh Parisa , Deniger Drew C. , Lowery Frank J. , Parkhurst Maria R. , Ngo Lien T. , Ray Satyajit , Li Yong F. , Hill Victoria , Florentin Maria , Masi Robert V. , Paria Biman C. , Levin Noam , Bera Alakesh , Hedges Elizabeth A. , Choi Agnes , Chatani Praveen D. , Parikh Anup Y. , Levi Shoshana , Seitter Samantha , Lu Yong-Chen , Zheng Zhili , Prickett Todd D. , Jia Li , Hernandez Jonathan M. , Hoang Chuong D. , Robbins Paul F. , Goff Stephanie L. , Sherry Richard M. , Yang James C. , Rosenberg Steven A.
Published: April 2023

Abstract
Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo–expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53–reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02–restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers.

See related Spotlight by Klebanoff, p. 919
Data from Preclinical Characterization of Relatlimab, a Human LAG-3–Blocking Antibody, Alone or in Combination with Nivolumab
Authors: Thudium Kent , Selby Mark , Zorn Julie A. , Rak Gregory , Wang Xi-Tao , Bunch Roderick Todd , Hogan Jason M. , Strop Pavel , Korman Alan J.
Published: April 2023

Abstract
Novel therapeutic approaches combining immune-checkpoint inhibitors are needed to improve clinical outcomes for patients with cancer. Lymphocyte-activation gene 3 (LAG-3) is an immune-checkpoint molecule that inhibits T-cell activity and antitumor immune responses, acting through an independent mechanism from that of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Here, we describe the development and preclinical characterization of relatlimab, a human antibody that binds to human LAG-3 with high affinity and specificity to block the interaction of LAG-3 with the ligands MHC II and fibrinogen-like protein-1, and to reverse LAG-3–mediated inhibition of T-cell function in vitro. Consistent with previous reports, in mouse models, the combined blockade of LAG-3 and PD-1 with surrogate antibodies resulted in enhanced antitumor activity greater than the individual blockade of either receptor. In toxicity studies in cynomolgus monkeys, relatlimab was generally well tolerated when combined with nivolumab. These results are consistent with findings from the RELATIVITY-047 phase II/III trial showing that relatlimab combined with nivolumab is a well-tolerated regimen that demonstrates superior progression-free survival compared with nivolumab monotherapy in patients with unresectable or metastatic melanoma.