PD-1 Blockade Therapy Research Articles

Single-Cell Polyfunctional Proteomic Profiling Reveals Temporal and Niche Differences in CD4 and CD8 T Cells in Acute Myeloid Leukemia Following PD-1 Blockade Therapy

Immune checkpoint blockade (ICB) has revolutionized solid tumor treatment by unleashing T cell cytotoxic potential, however, its effectiveness in leukemias such acute myeloid leukemia (AML) has been limited. Understanding the functional states of T cells in AML within the context of ICB is crucial to understanding its limited therapeutic potential. Here, we leveraged the IsoPlexis single cell secretome platform to assess T cell polyfunctional activity in relapsed/refractory (RelRef) AML patients treated on the phase 2 clinical trial with combination azacitidine and nivolumab (Aza/Nivo) (NCT02397720). We isolated CD4 and CD8 T cells by magnetic bead sorting from the peripheral blood (PB) and bone marrows (BMs) of 21 AML patients before (pretreatment) and after ICB (post-ICB). Isolated CD4 and CD8 cells were stimulated with CD3/CD28 agonism and functionally characterized by IsoPlexis, which measures T cell secreted cytokines, chemokines, and effector molecules at the single-cell level. In total, we characterized 52 patient samples (30 from PB and 22 from BMs). Functional CD4 and CD8 profiles were correlated with clinical and demographic characteristics of the patients. This enabled us to explore the temporal and niche differences in T cell functional states, specifically in the context of PD-1 blockade therapy, in a unique cohort of AML patients at both a pseudobulk and single-cell level. At the pseudobulk level, we observed baseline activity in the chemoattractive, effector, and regulatory functional groups was higher in both PB and BM CD8 cells compared to CD4 cells (Fig 1A), suggesting increased polyfunctionality in CD8 T cells prior to treatment. Analysis comparing pretreatment and post-ICB polyfunctionality revealed similar response patterns in CD4 and CD8 T cells in both the PB and BM spaces. Interestingly, CD4 T cells exhibited increased polyfunctional activity post-ICB in both PB and BM as compared to CD8 T cells. These findings point to an unexpected contribution of CD4 T cells with ICB therapy in AML. We employed an unbiased, unsupervised neighborhood analysis which leveraged the nearest cellular neighbor based on the log fold-change in secreted proteins between samples from distinct timepoints to reveal post-ICB CD4 and CD8 T cells aggregated into distinct cellular neighborhoods (Fig 1B). Both CD4 and CD8 analysis revealed distinct groups highlighting one group containing pre-ICB cells, and another group containing pre- and post-ICB cells. This suggests that T cell functionality is predetermined prior to ICB therapy. Comparative analysis at the single-cell level further identified higher expression of chemoattractive, inflammatory, regulatory, and stimulatory polyfunctional groups in non-responders (NR) in the BM for both CD4 and CD8 T cells, and higher expression of the chemoattractive group in NRs in the PB for CD4 cells. This suggests that BM polyfunctionality may be more critical in terms of ICB response. This study provides a novel single-cell functional analysis of T cells from both PB and BM compartments in RelRef AML patients treated with ICB. Our findings suggest the functional state of T cells in the circulating state and residence within the leukemia microenvironment are not equivalent and that there are dynamic changes of T cell functionality with ICB which has broad implications for correlative sample analysis in the ICB setting.

MiR-124 delivered by BM-MSCs-derived exosomes targets MCT1 of tumor-infiltrating Treg cells and improves ovarian cancer immunotherapy.

Metabolic rewiring of tumor cells leads to an enrichment of lactate in the tumor microenvironment (TME). This lactate-rich environment of solid tumors has been reported to support tumor-infiltrating regulatory T (Treg) cells. Therefore, agents that modify the lactate metabolism of Treg cells have therapeutic potential. Monocarboxylate transporter 1 (MCT1), which Treg cells predominantly express, plays an essential role in the metabolism of tumor-infiltrating Treg cells. In this study, we show that miR-124 directly targets MCT1 and reduces lactate uptake, eventually impairing the immune-suppressive capacity of Treg cells. Particularly, exosomal miR-124 derived from bone marrow mesenchymal stromal cells (BM-MSCs) slows tumor growth and increases response to PD-1 blockade therapy. These data indicate a potential treatment strategy for improving immune checkpoint blockade therapy using miR-124-carried BM-MSCs-derived exosomes.

Non-linear Modifications Enhance Prediction of Pathological Response to Pre-operative PD-1 Blockade in Lung Cancer: A Longitudinal Hybrid Radiological Model

Major pathologic remission (MPR, residual tumor <10%) is a promising clinical endpoint for prognosis analysis in patients with lung cancer receiving pre-operative PD-1 blockade therapy. Most of the current biomarkers for predicting MPR such as PD-L1 and tumor mutation burden (TMB) need to be obtained invasively. They cannot overcome the spatiotemporal heterogeneity or provide dynamic monitoring solutions. Radiomics and artificial intelligence (AI) models provide a practical tool enabling non-invasive follow-up observation of tumor structural information through high-throughput data analysis. Currently, AI-based models mainly focus on the single baseline scan or pipeline, namely sole radiomics or deep learning (DL). This work merged the delta-radiomics based on the slope of classic radiomics indexes within a time interval and the features extracted by deep networks from the subtraction between the baseline and follow-up images. The subtracted images describing the tumor changes were based on the transformation generated by registration. Stepwise optimization of components was performed by repeating experiments among various combinations of DL networks, registration methods, feature selection algorithms, and classifiers. The optimized model could predict MPR with a cross-validation AUC of 0.91 and an external validation AUC of 0.85. A core set of 27 features (eight classic radiomics, 15 delta-radiomics, one classic DL features, and three delta-DL features) was identified. The changes in delta-radiomics indexes during the treatment were fitted with mathematic models. The fitting results revealed that over half of the features were of non-linear dynamics. Therefore, non-linear modifications were made on eight features by replacing the original features with non-linear fitting parameters, and the modified model achieved an improved power. The dynamic hybrid model serves as a novel and promising tool to predict the response of lesions to PD-1 blockade, which implies the importance of introducing the non-linear dynamic effects and DL approaches to the original delta-radiomics in the future.

HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation

By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ Tcell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Pembrolizumab for the treatment of progressive multifocal leukoencephalopathy in China.

The aim of this study is to analyze the clinical characteristics and outcomes of Chinese patients with progressive multifocal leukoencephalopathy (PML) who were treated with programmed cell death protein 1 (PD1) blockade therapies. We retrospectively analyzed patients who were admitted to our hospital between October 1, 2020, and October 1, 2022, diagnosed with PML and treated with PD1 blockade therapies. Four patients with PML who were treated with PD1 blockade therapies were identified. All patients were male, and their ages ranged from 19 to 54 years old. One patient (Case 2) exhibited mild pleocytosis, while three patients (Cases 2-4) had markedly reduced T lymphocyte cell counts prior to treatment. The time interval between symptom onset and treatment initiation ranged from six to 54 weeks. All patients received pembrolizumab treatment, with a total of two to four doses administered. Three patients who responded to pembrolizumab treatment showed clinical improvement starting around 8 weeks after the initiation of therapy. Although one patient did not show clinical improvement, they ultimately survived until the last follow-up. None of the patients in this study exhibited immune-related adverse events or immune reconstitution inflammatory syndrome. PD1 blockade appears to be a promising novel therapeutic option for PML; additional prospective studies are necessary to confirm its efficacy.

Antitumor Immune Responses in B2M-Deficient Cancers.

β2-microglobulin (B2M) is a critical component of the MHC class I molecule and is required to present tumor antigens to T cells. Its loss results in acquired resistance to immune checkpoint blockade (ICB) therapies. However, there have been well-documented cases of B2M-inactivated tumors responding to ICB, justifying investigation of how an antitumor immune response can be generated to tumors without surface MHC class I. We knocked out B2M in three murine models with varying baseline MHC class I expression and sensitivity to anti-programmed death receptor (PD-1) therapy and analyzed the immune responses. MC38 and YUMMER2.1 without B2M responded to anti-PD-1 alone or with an IL2 agonist, and this was mediated by CD4+ T cells and natural killer (NK) cells. The more aggressive B16 without B2M expression only partially responded to the IL2 agonist, and this was dependent on NK cells. When analyzing nearly 300 pretreatment biopsies from patients with melanoma receiving PD-1 blockade-based therapies, we found infrequent B2M mutations or homozygous loss but more frequent LOH or copy-number gains. B2M LOH was enriched in biopsies from patients without response to therapy, and these biopsies were more frequently infiltrated by activated NK cells. We conclude that in the absence of B2M, activation of CD4+ T cells and NK cells can mediate responses to murine models of PD-1 blockade therapy. In addition, in human melanoma, the intratumoral presence of activated NK cells upon partial B2M loss likely selects against tumor escape through low surface MHC class I expression.

Macrophage-camouflaged epigenetic nanoinducers enhance chemoimmunotherapy in triple negative breast cancer.

Chemoimmunotherapy has been approved as standard treatment for triple-negative breast cancer (TNBC), but the clinical outcomes remain unsatisfied. Abnormal epigenetic regulation is associated with acquired drug resistance and T cell exhaustion, which is a critical factor for the poor response to chemoimmunotherapy in TNBC. Herein, macrophage-camouflaged nanoinducers co-loaded with paclitaxel (PTX) and decitabine (DAC) (P/D-mMSNs) were prepared in combination with PD-1 blockade therapy, hoping to improve the efficacy of chemoimmunotherapy through the demethylation of tumor tissue. Camouflage of macrophage vesicle confers P/D-mMSNs with tumor-homing properties. First, DAC can achieve demethylation of tumor tissue and enhance the sensitivity of tumor cells to PTX. Subsequently, PTX induces immunogenic death of tumor cells, promotes phagocytosis of dead cells by dendritic cells, and recruits cytotoxic T cells to infiltrate tumors. Finally, DAC reverses T cell depletion and facilitates immune checkpoint blockade therapy. P/D-mMSNs may be a promising candidate for future drug delivery design and cancer combination therapy in TNBC.

Low frequency of intracranial progression in advanced NSCLC patients treated with cancer immunotherapies.

Intracranial metastases are common in nonsmall-cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies compared with those treated without PD-1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD-1 blockade were suppressed. Accordingly, long-lived memory precursor effector T cells and antigen-specific T cells were increased by PD-1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long-term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD-1 blockade therapies, aggressive local therapies could be worthwhile.

Definitive treatment with PD-1 blockade and radiation therapy of unresected primary desmoplastic melanoma of the nose.

Two patients with primary desmoplastic melanoma of the nose were treated with definitive anti-PD-1 checkpoint inhibitor (anti-PD1) and radiation therapy. Both patients were technically resectable with partial rhinectomy but both declined for cosmetic reasons. The melanoma multidisciplinary team recommended that the patients receive PD1 blockade. The initial response to PD-1 blockade was temporary in the first case and partial in the second one. With the addition of high-dose radiotherapy, complete responses were obtained in both patients, with recurrence-free and good functional and cosmetic outcomes at a six-year and 22-month follow-up. Despite surgery being the gold standard, both cases illustrated that PD-1 blockade and radiation therapy can be a safe alternative option for desmoplastic melanoma where surgery is morbidly disfiguring.

18 F-FDG PET/CT predicts the role of neoadjuvant immunochemotherapy in the pathological response of esophageal squamous cell carcinoma.

This study aimed to investigate the predictive value of 18 F-FDG PET/CT for pathological response after neoadjuvant immunochemotherapy (NICT) in patients with esophageal squamous cell carcinoma (ESCC). The clinical data of 54 patients with ESCC who underwent two cycles of NICT followed by surgery were retrospectively analyzed. NICT consisted of PD-1 blockade therapy combined with chemotherapy. 18 F-FDG PET/CT scans were performed before and after NICT. The pathological results after surgery were used to assess the degree of pathological response. The scan parameters of 18 F-FDG PET/CT and their changes before and after NICT were compared with the pathological response. Among the 54 patients, 10 (18.5%) achieved complete pathological response (pCR) and 21 (38.9%) achieved major pathological response (MPR). The post-NICT scan parameters and their changes were significantly associated with the pathological response. In addition, the values of the changes in the scanned parameters before and after treatment can further predict the pathological response of the patient. 18 F-FDG PET/CT is a useful tool to evaluate the efficacy of NICT and predict pathological response in patients with ESCC. The post-NICT scan parameters and their changes can help identify patients who are likely to achieve pCR or MPR.

A platform trial of neoadjuvant and adjuvant antitumor vaccination alone or in combination with PD-1 antagonist and CD137 agonist antibodies in patients with resectable pancreatic adenocarcinoma

A neoadjuvant immunotherapy platform clinical trial allows for rapid evaluation of treatment-related changes in tumors and identifying targets to optimize treatment responses. We enrolled patients with resectable pancreatic adenocarcinoma into such a platform trial (NCT02451982) to receive pancreatic cancer GVAX vaccine with low-dose cyclophosphamide alone (Arm A; n = 16), with anti-PD-1 antibody nivolumab (Arm B; n = 14), and with both nivolumab and anti-CD137 agonist antibody urelumab (Arm C; n = 10), respectively. The primary endpoint for Arms A/B - treatment-related change in IL17A expression in vaccine-induced lymphoid aggregates - was previously published. Here, we report the primary endpoint for Arms B/C: treatment-related change in intratumoral CD8+ CD137+ cells and the secondary outcomes including safety, disease-free and overall survivals for all Arms. Treatment with GVAX+nivolumab+urelumab meets the primary endpoint by significantly increasing intratumoral CD8+ CD137+ cells (p = 0.003) compared to GVAX+Nivolumab. All treatments are well-tolerated. Median disease-free and overall survivals, respectively, are 13.90/14.98/33.51 and 23.59/27.01/35.55 months for Arms A/B/C. GVAX+nivolumab+urelumab demonstrates numerically-improved disease-free survival (HR = 0.55, p = 0.242; HR = 0.51, p = 0.173) and overall survival (HR = 0.59, p = 0.377; HR = 0.53, p = 0.279) compared to GVAX and GVAX+nivolumab, respectively, although not statistically significant due to small sample size. Therefore, neoadjuvant and adjuvant GVAX with PD-1 blockade and CD137 agonist antibody therapy is safe, increases intratumoral activated, cytotoxic T cells, and demonstrates a potentially promising efficacy signal in resectable pancreatic adenocarcinoma that warrants further study.

Elevated nuclear PIGL disrupts the cMyc/BRD4 axis and improves PD-1 blockade therapy by dampening tumor immune evasion.

To improve the efficacy of lenvatinib in combination withprogrammed death-1 (PD-1) blockade therapy for hepatocellular carcinoma (HCC), we screened the suppressive metabolic enzymes that sensitize HCC to lenvatinib and PD-1 blockade, thus impeding HCC progression. After analysis of the CRISPR‒Cas9 screen, phosphatidylinositol-glycan biosynthesis class L (PIGL) ranked first in the positive selection list. PIGL depletion had no effect on tumor cell growth in vitro but reprogrammed the tumor microenvironment (TME) in vivo to support tumor cell survival. Specifically, nuclear PIGL disrupted the interaction between cMyc/BRD4 on the distant promoter of target genes and thus decreased the expression of CCL2 and CCL20, which are involved in shaping the immunosuppressive TME by recruiting macrophages and regulatory T cells. PIGL phosphorylation at Y81 by FGFR2 abolished the interaction of PIGL with importin α/β1, thus retaining PIGL in the cytosol and facilitating tumor evasion by releasing CCL2 and CCL20. Clinically, elevated nuclear PIGL predicts a better prognosis for HCC patients and presents a positive correlation with CD8 + T-cell enrichment in tumors. Clinically, our findings highlight that the nuclear PIGL intensity or the change in PIGL-Y81 phosphorylation should be used as a biomarker to guide lenvatinib with PD-1 blockade therapy.

Effect of MDSC-targeted TFF2-MSA with PD-1 blockade therapy in advanced gastric cancer models.

e16037 Background: Recent studies revealed chemotherapy increases anti-PD1 response of gastric cancer (GC) by reducing myeloid-derived suppressor cells (MDSCs). However, a more specific MDSC-targeted treatment is needed to further improve anti-PD1 efficacy in advanced GC. Trefoil factor family 2 (TFF2), a partial agonist of CXCR4 and a secreted anti-inflammatory peptide, can suppress MDSC expansion. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 in syngeneic GC mouse models. Methods: Murine serum albumin (MSA) was fused to murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice then received either TFF2-MSA or anti-PD-1 antibody or both when tumors reached 150 mm3. At the endpoint, flow cytometry analysis was performed to examine immune profiles. In parallel, a group of the s.c. tumor bearing mice were followed up for survival differences in response to the treatments. In the orthotopic model, ACKP-luc cells were implanted to stomach submucosa, and recipient mice received the same treatments started after 1 week. In the metastasis model, subcutaneous flank tumors were resected in ACKP-grafted mice once tumors reached 500 mm3 to permit spontaneous lung metastasis, and mice then received the same treatments. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p&gt;0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p&lt;0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) in a synergistic manner. The combination therapy efficiently reduced tumor HDC-GFP+ MDSCs by 67%, and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells by 18 fold. The myeloid/lymphoid progenitor and output ratio in the bone marrow were decreased by the combination, to a similar level of tumor-free mice. While standard chemotherapy (5-fluorouracil and oxaliplatin) showed no efficacy, adding chemotherapy to the combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, in the orthotopic model, the TFF2-MSA/PD-1 antibody combination eradicated GC in 80% mice without recurrence compared to 0% with either monotherapy. Finally, the combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p&lt;0.0001), compared to minimal inhibition with either monotherapy (p&gt;0.05). Conclusions: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of gastric cancer.

Key factors associated with response in the treatment of liver metastatic melanoma by cryoablation combined with PD-1 blockade therapy: The Cryo-Fire-001 study.

e21500 Background: Liver metastasis remains a challenge in the treatment of advanced stage melanoma through immunotherapy. Although our previous study showed cryoablation could enhance the efficacy of PD-1 blockade therapy in the treatment of melanoma patients with liver metastasis, the key factors correlated with survival and treatment response remains unknown. Methods: Between 2018 and 2022, 45 melanoma patients receiving combined PD-1 blockade therapy and cryoablation of liver metastasis were retrospectively analyzed. The technical parameters of cryoablation, the sequence of the combined treatment, tumor characteristics, important immune markers and their correlations with treatment response were evaluated. The associations between key factors and overall survival (OS) were determined by Cox regression. Results: Of the 45 patients, the 3-month overall response rate (RECIST 1.1 criteria) is 26.7%. Multivariate analysis showed the number of metastatic organs ( &gt; 2 vs ≤2; HR 3.54; 95% CI 1.32-9.49; P = 0.012), pretreatment LDH level ( &gt; 300 vs ≤300 U/L; HR 4.59; 95% CI 1.78-11.85; P = 0.002) and diameter of ablated lesion ( &gt; 30 vs ≤30 mm; HR 3.27; 95% CI 1.17-9.15; P= 0.024) were independent unfavorable prognostic factors for OS. Further analysis showed patients with larger intrahepatic tumors ablated had larger tumor size (P &lt; 0.001). A scatter plot by the two key variables showed patients with intrahepatic tumor size less than 45 mm and size of ablated lesion less than 30 mm had a marginally higher rate of 3-month response (10/28, 35.7%) compared to those not (2/17, 11.7%; P = 0.076). Large diameter of ablated lesion ( &gt; 30 mm) require more ablation needles and cause greater elevation in the level of IL-6, CD8+T cell to Treg cell ratio and neutrophil to lymphocyte ratio after cryoablation while these changes disappeared when tested one month later. Conclusions: Fine selection based on baseline characteristics of patients and size of intrahepatic lesion for cryoablation are essential to achieve a high response rate during cryoablation-PD-1 combination treatment for liver metastatic melanoma.

Targeting programmed cell death protein 1 (PD-1) for treatment of non-small-cell lung carcinoma (NSCLC); the recent advances.

The immune system uses various immune checkpoint axes to adjust responses, support homeostasis, and deter self-reactivity and autoimmunity. Nevertheless, non-small-cell lung carcinoma (NSCLC) can use protective mechanisms to facilitate immune evasion, which leads to potentiated cancer survival and proliferation. In this light, many blocking anti-bodies have been developed to negatively regulate checkpoint molecules, in particular, programmed cell death protein 1 (PD-1) / PD-ligand 1 (L1), and bypass these immune suppressive mechanisms. Meanwhile, anti-PD-1 anti-bodies such as nivolumab, pembrolizumab, cemiplimab, and sintilimab have shown excellent competence in successfully inspiring immune responses versus NSCLC. Accordingly, the United States Food and Drug Administration (FDA) has recently approved nivolumab (alone or in combination with ipilimumab) and pembrolizumab (alone or in combination with chemotherapy) as first-line treatment for advanced NSCLC patients. However, PD-1 blockade monotherapy remains inefficient in more than 60% of NSCLC patients, and many patients don't respond or acquire resistance to this modality. Also, toxicities related to anti-PD-1 anti-body have been progressively identified in clinical trials and oncology practice. Herein, we will outline the clinical benefits of PD-1 blockade therapy alone or in combination with other treatments (e.g., chemotherapy, radiotherapy, anti-angiogenic therapy) in NSCLC patients. Moreover, we will take a glimpse into the recently identified predictive biomarkers to determine patients most likely to suffer serious adverse events to decrease untoward toxicity risk and diminish treatment costs.

PD-1 blockade potentiates neoadjuvant chemotherapy in NSCLC via increasing CD127+ and KLRG1+ CD8 T cells

The combination of PD-1 blockade with neoadjuvant chemotherapy (NAC) has achieved unprecedented clinical success in non-small cell lung cancer (NSCLC) compared to NAC alone, but the underlying mechanisms by which PD-1 blockade augments the effects of chemotherapy remain incompletely elucidated. Single-cell RNA sequencing was performed on CD45+ immune cells isolated from surgically resected fresh tumors of seven NSCLC patients receiving NAC or neoadjuvant pembrolizumab and chemotherapy (NAPC). Multiplex fluorescent immunohistochemistry was performed on FFPE tissues before and after NAC or NAPC from 65 resectable NSCLC patients, and results were validated with GEO dataset. NAC resulted in an increase only of CD20+ B cells, whereas NAPC increased the infiltration of CD20+ B cells, CD4+ T cells, CD4+CD127+ T cells, CD8+ T cells, CD8+CD127+ and CD8+KLRG1+ T cells. Synergistic increase in B and T cells promotes favorable therapeutic response after NAPC. Spatial distribution analysis discovered that CD8+ T cells and their CD127+ and KLRG1+ subsets were in closer proximity to CD4+ T/CD20+ B cells in NAPC versus NAC. GEO dataset validated that B-cell, CD4, memory, and effector CD8 signatures correlated with therapeutic responses and clinical outcomes. The addition of PD-1 blockade to NAC promoted anti-tumor immunity through T and B cells recruitment in the tumor microenvironment and induced tumor-infiltrating CD8+ T cells skewed toward CD127+ and KLRG1+ phenotypes, which may be assisted by CD4+ T cells and B cells. Our comprehensive study identified key immune cell subsets exerting anti-tumor responses during PD-1 blockade therapy and that may be therapeutically targeted to improve upon existing immunotherapies for NSCLC.

MYC Induces Immunotherapy and IFNγ Resistance Through Downregulation of JAK2.

Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

Abstract 5088: MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer

Abstract Aims: Recent studies revealed myeloid-derived suppressor cell (MDSC) in the tumor microenvironment as an appealing therapeutic target to sensitize intestinal-type gastric cancer (GC) to PD-1 blockade therapy. However, whether MDSC inhibition can improve survival in the more aggressive diffuse-type GC is unknown. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, suppresses MDSC expansion partly via CXCR4 receptor. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 to prolong survival in syngeneic diffuse GC mouse models. Methods: Murine serum albumin (MSA) was appended to the murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the more immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice subsequently received either TFF2-MSA or anti-PD-1 antibody or both. Further, to assess treatment efficacy in inhibiting spontaneous lung metastasis, subcutaneous flank tumors were resected in ACKP cell-grafted mice once tumors reached 500 mm3, and mice then received treatment with TFF2-MSA or anti-PD-1 or the combination. At the endpoint, flow cytometry and histopathological analyses were performed to examine immune profiles and tumor metastasis. In another model, ACKP-luc cells were implanted orthotopically to stomach submucosa, and recipient mice received the same treatments started from 1 week later to assess their efficacy. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p&amp;gt;0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p&amp;lt;0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) of host mice in a synergistic manner. The combination therapy efficiently reduced HDC-GFP+ MDSCs (by 67% vs. control, p&amp;lt;0.01), and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells (by 18 fold, p&amp;lt;0.001). Further, while standard chemotherapy (5-fluorouracil and oxaliplatin) showed limited efficacy in this model, addition of chemotherapy to the TFF2-MSA/PD-1 antibody combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p&amp;lt;0.0001), in contrast to minimal inhibition observed with either monotherapy (p&amp;gt;0.05). Finally, in the orthotopic model, the combo regimen eradicated GC in 50% mice compared to 0% in either monotherapy treatment. Conclusion: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of diffuse-type GC. Citation Format: Jin Qian, Sandra Ryeom, Bruce Daugherty, Seth Lederman, Timothy C. Wang. MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5088.

Abstract 704: MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models

Abstract Aims: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can suppress MDSC expansion and activate tumor immunity in part through agonism of the CXCR4 receptor. The aim of this study is to investigate whether a novel TFF2 - albumin fusion peptide (TFF2-MSA) can improve survival in anti-PD-1 treated syngeneic colorectal cancer (CRC) mouse models. Methods: Two syngeneic colon carcinoma mouse models were developed using cell lines grafted subcutaneously into mice. MC38 CRC cells were engrafted into C57BL/6 mice while CT26.wt CRC cells were implanted into BALB/C mice We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing dose frequency. Mice subsequently received either TFF2-MSA or anti-PD-1 antibody (clone 29F.1A12) or both, and tumor volume, and survival were measured. At the endpoint, flow cytometry was performed to examine treatment-induced effects on immune profiles. Results: In the MC38 model, administration of TFF2-MSA suppressed tumor growth (TGI 38%), the combination of TFF2-MSA and anti-PD-1 had an additive effect and suppressed tumor growth dramatically (TGI 74%). The combination also exhibited a survival rate of 90% after 50 days, while vehicle and single TFF2-MSA therapy were 30% and 65%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination treatment, as measured by flow cytometry using antibodies against LAG3, TIM3 and PD-1. In the CT26.wt model, administration of TFF2-MSA alone exhibited little effect, but the combination of anti-PD-1 and TFF2-MSA showed a profound effect. In the CT26.wt model, administration of TFF2-MSA suppressed tumor growth (TGI 17%), anti-PD-1 alone (TGI 43%) and the combination of TFF2-MSA and anti-PD-1 (TGI 54%). Conclusion: Targeting MDSCs using TFF2-MSA fusion protein synergizes well with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of colorectal cancer. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer. Citation Format: Bruce L. Daugherty. MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 704.

Abstract 4064: PD-1 blockade therapy enhances the antitumor effects of lymphodepletion and adoptive T cell transfer

Abstract Background: Lymphodepleting cytotoxic regimens, such as chemotherapy and radiotherapy, have shown the ability to enhance antitumor immunity. Although the mechanisms of antitumor effects augmentation in tumor-bearing hosts after lymphodepletion and T cell transfer have been intensively investigated, the influence of lymphodepletion followed by T cell transfer on immune checkpoint molecules (ICMs) expressed on tumor cells and immune cells remains to be elucidated. In the current study, we evaluated the effects of lymphodepletion and adoptive T cell transfer on ICMs in the tumor microenvironment. In addition, we investigated whether lymphodepletion and adoptive transfer of T cells augments antitumor effects of anti-PD-1 blockade therapy. Methods: B6 mice were inoculated subcutaneously with the MCA205 murine fibrosarcoma. On day 7, mice were lymphodepleted by sublethal irradiation with 500 cGy and were reconstituted intravenously with spleen cells from normal mice as a source of naïve T cells. These mice were injected intraperitoneally with anti-PD-1 mAb on days 7 and 14. On day 21, tumor tissues were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: The expressions of ICMs, including PD-L1, PD-1, TIGIT, TIM-3 and LAG-3, were upregulated on recipient CD4+ and CD8+ T cells in the tumor microenvironment. In contrast, these ICMs were significantly reduced in transferred donor T cells. Administration of anti-PD-1 antibodies after lymphodepletion and adoptive transfer of T cells significantly inhibited tumor progression. Furthermore, transfer of both donor CD4+ and CD8+ T cells was responsible for this augmentation of antitumor effects. Conclusions: Our observations suggest that a possible mechanism of the antitumor effect augmentation mediated by lymphodepletion followed by T cell transfer is the prevention of donor T cell exhaustion and dysfunction. Blockade of PD-1 significantly enhanced the antitumor effects of lymphodepletion and T cell transfer. Citation Format: Satoshi Watanabe, Miho Takahashi, Ryo Suzuki, Masashi Arita, Ko Sato, Toshiya Fujisaki, Aya Ohtsubo, Satoshi Shoji, Kunihiro Shono, Takaaki Masuda, Tomoki Sekiya, Koichiro Nozaki, Tomohiro Tanaka, Rie Kondo, Yu Saida, Satoshi Hokari, Toshiyuki Koya, Toshiaki Kikuchi. PD-1 blockade therapy enhances the antitumor effects of lymphodepletion and adoptive T cell transfer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4064.