Increase In Tumor-infiltrating Lymphocytes Research Articles

Preclinical analysis of the efficacy of near-infrared photoimmunotherapy and anti-PD-1 antibody combination therapy for treating CD73-positive lung cancer.

e14521 Background: The CD73/adenosine pathway, which converts extracellular ATP to adenosine, is emerging as a promising target for immunotherapy in various cancers. In the tumor microenvironment, hypoxia triggers upregulation of adenosine-activating molecules, including CD73, a membrane-anchored enzyme expressed on cancer cells. CD73 induces the expression of PD-1 and CTLA4, and all of this leads to immunosuppression. Therefore, we focused utilizing near-infrared photoimmunotherapy (NIR-PIT) to kill CD73-expressing cells and alleviate immunosuppression. NIR-PIT is an innovative cancer treatment approach which involves irradiating NIR light after the administrated antibody-photosensitizer conjugate binds to the target cells. Methods: In vitro, anti-CD73 antibody-IR700 conjugate was prepared and added to the CMT167 and LLC cell lines, which were irradiated with NIR light under a microscope. In vivo, a mouse model was established using the CMT167 cell line. The mice were divided into 6 groups: 1) NIR-PIT group against CD73; 2) anti-PD-1 antibody group; 3) combined treatment group of 1) and 2); 4) control group; 5) anti-CD73 antibody-IR700 conjugate group; and 6) near-infrared light irradiation group. Tumor volume and survival rate were compared among these groups. Additionally, after 2 weeks of treatment, tumors in the four groups 1) to 4) above were pathologically evaluated using two different AI-equipped software programs. Results: Microscope observation revealed that NIR-PIT induced immunogenic cell death of CD73-positive mouse lung cancer cell lines in vitro. In vivo, the effect of NIR-PIT on this mouse model was also demonstrated. Although previous studies have shown the efficacy of treatment with anti-CD73 antibodies, the present study compared the CD73-IR700 conjugate group to the NIR-PIT to CD73 group, and the latter group showed better therapeutic outcomes. Additionally, the combined treatment group displayed the highest anti-tumor efficacy in terms of survival and tumor volume. Pathological evaluation of the tumor interior showed a significant increase in tumor-infiltrating lymphocytes compared to the other groups. Additionally, the percentage of tumor cells was significantly reduced, while the percentage of stroma, including fibrous tissue, was significantly increased. In line with other studies that have already reported the additive effect of combination therapy with anti-CD73 antibodies and immune checkpoint inhibitors, the present study showed that the combination therapy resulted in further activation of immune cells and enhanced the therapeutic effect. Conclusions: CD73 represents a promising target for NIR-PIT, with potential for enhanced effectiveness when used in combination with immune checkpoint inhibitors.

Abstract PO1-02-01: T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes

Abstract Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in breast cancer. We have previously reported the effect of pelareorep (pela), an intravenously delivered, non-engineered oncolytic reovirus, on a composite measurement of TILs and tumor cellularity (CelTIL) from the AWARE-1 window-of-opportunity study in patients with early breast cancer (eBC). These results showed treatment increased in CelTIL scores especially in the atezolizumab group. To confirm and extend these findings we applied T cell receptor sequencing of matched tumor tissue and whole blood pre and post-treatment from the AWARE-1 study to further explore the effects of pela therapy on TILs. Methods: Newly diagnosed HR+/HER2- eBC patients were enrolled into two cohorts: Cohort 1: pela + letrozole (n=10); and Cohort 2: pela + letrozole + atezolizumab (n=10). Pela was administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. Tumor biopsies (FFPE samples) collected pre-treatment (~D-23) and on day ~21 when tumors were surgically removed. T cell fraction analysis and T cell receptor sequencing were performed by Adaptive Biotechnology (Seattle, Washington) Immunoseq protocol. Results: As was shown for the CelTIL scores, an increase in the tumor T cell fraction, a direct measurement of TILs, was observed at 1-month post-treatment in both cohorts with a mean percent increase of 21% for Cohort 1 and 67% for Cohort 2. Through TCR-sequencing of tumor tissue, we identified TIL clones and tracked their differential abundance in tumor tissue and blood post-treatment. The results of this analysis at Day 21 showed a mean post-treatment expansion of 10 tumor specific clones in the blood for Cohort 1, and a mean post-treatment increase of 40 tumor specific clones for Cohort 2. Conclusions: These results confirm the previously reported CelTIL results from AWARE-1 on pela-induced increases in TILs and demonstrate the expansion of these clones in both tumor and blood as a consequence of pela therapy for HR+/HER2- breast cancer. Citation Format: Richard Trauger, Houra Loghmani, Matt Coffey, Fernando Salvador, Joaquín Gavilá, Luis Manso, Tomás Pascual, Aleix Prat, Thomas Heineman. T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-01.

Tumor-infiltrating lymphocytes in HER2-positive breast cancer treated with neoadjuvant chemotherapy and dual HER2-blockade.

Tumor-infiltrating lymphocytes (TILs) have been associated with outcomes in HER2-positive breast cancer patients treated with neoadjuvant chemotherapy and trastuzumab. However, it remains unclear if TILs could be a prognostic and/or predictive biomarker in the context of dual HER2-targeting treatment. In this study, we evaluated the association between TILs and pathological response (pCR) and invasive-disease free survival (IDFS) in 389 patients with stage II-III HER2 positive breast cancer who received neoadjuvant anthracycline-containing or anthracycline-free chemotherapy combined with trastuzumab and pertuzumab in the TRAIN-2 trial. Although no significant association was seen between TILs and pCR, patients with TIL scores ≥60% demonstrated an excellent 3-year IDFS of 100% (95% CI 100-100), regardless of hormone receptor status, nodal stage and attainment of pCR. Additionally, in patients with hormone receptor positive disease, TILs as a continuous variable showed a trend to a positive association with pCR (adjusted Odds Ratio per 10% increase in TILs 1.15, 95% CI 0.99-1.34, p = 0.070) and IDFS (adjusted Hazard Ratio per 10% increase in TILs 0.71, 95% CI 0.50-1.01, p = 0.058). We found no interactions between TILs and anthracycline treatment. Our results suggest that high TIL scores might be able to identify stage II-III HER2-positive breast cancer patients with a favorable prognosis.

Abstract 7527: Investigating biomarkers for immune-related adverse events following immune checkpoint inhibitor therapy in recurrent lung cancer patients

Abstract Background: The application of immune checkpoint inhibitors (ICIs) as adjuvant therapy post-lung cancer surgery is expected to confer significant benefits. However, the risk factors for severe immune-related adverse events (irAEs) remain incompletely understood, and managing irAEs may require long-term steroid use. The identification of biomarkers for irAEs in patients with surgically resected lung cancer is an urgent need. Methods: We analyzed 43 lung cancer patients who received ICI treatment for recurrence postoperatively. Macrodissection of lung cancer lesions was performed on FFPE specimens from resected lungs, from which total RNA was extracted. We conducted gene expression analysis of 770 immune-related genes using the PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA, USA). The relationship between gene expression changes and irAEs or the efficacy of ICI treatment was examined. For validation, immunostaining of proteins strongly associated with irAEs was performed to determine their expression status in tissue specimens. Results: Among the 43 patients (30 males, 13 females, median age 68), 28 had adenocarcinoma, and 15 had squamous cell carcinoma. PD-L1 positivity was observed in 36 patients. The treatment regimens included pembrolizumab monotherapy for 18 patients and combination immunotherapy for 25 patients, with 25 patients responding to treatment and 7 developing irAEs. There was a marked difference in gene expression between adenocarcinoma and squamous cell carcinoma, with a notably higher number of tumor-infiltrating lymphocytes (TILs) in adenocarcinoma. An increase in TILs was observed in cases with irAEs across both cancer types. Additionally, an increased CD8/Treg ratio and CD8/exhausted T cell ratio were noted in adenocarcinomas with irAEs. A strong correlation between IRF5 gene expression and the occurrence of irAEs in adenocarcinoma was also confirmed by immunohistochemistry. Conclusion: We have identified tumor immune microenvironments correlated with the onset of irAEs following ICI treatment in patients with postoperative recurrence of lung cancer. This discovery could contribute to perioperative treatment strategies and improve our understanding of tumor immune mechanisms. Citation Format: Yujin Kudo, Jun Matsubayashi, Satoshi Takahashi, Masaru Hagiwara, Masatoshi Kakihana, Toshitaka Nagao, Tatsuo Ohira, Norihiko Ikeda. Investigating biomarkers for immune-related adverse events following immune checkpoint inhibitor therapy in recurrent lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7527.

MRNA-based precision targeting of neoantigens and tumor-associated antigens in malignant brain tumors

BackgroundDespite advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized antigens expressed within brain tumors that can mediate tumor rejection; the low mutational burden of these tumors that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. The field of mRNA-based therapeutics has experienced a boon following the universal approval of COVID-19 mRNA vaccines. mRNA-based immunotherapeutics have also garnered widespread interest for their potential to revolutionize cancer treatment. In this study, we developed a novel and scalable approach for the production of personalized mRNA-based therapeutics that target multiple tumor rejection antigens in a single therapy for the treatment of refractory brain tumors.MethodsTumor-specific neoantigens and aberrantly overexpressed tumor-associated antigens were identified for glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (O.R.A.N). Personalized tumor antigen-specific mRNA vaccine was developed for each individual tumor model using selective gene capture and enrichment strategy. The immunogenicity and efficacy of the personalized mRNA vaccines was evaluated in combination with anti-PD-1 immune checkpoint blockade therapy or adoptive cellular therapy with ex vivo expanded tumor antigen-specific lymphocytes in highly aggressive murine GBM models.ResultsOur results demonstrate the effectiveness of the antigen-specific mRNA vaccines in eliciting robust anti-tumor immune responses in GBM hosts. Our findings substantiate an increase in tumor-infiltrating lymphocytes characterized by enhanced effector function, both intratumorally and systemically, after antigen-specific mRNA-directed immunotherapy, resulting in a favorable shift in the tumor microenvironment from immunologically cold to hot. Capacity to generate personalized mRNA vaccines targeting human GBM antigens was also demonstrated.ConclusionsWe have established a personalized and customizable mRNA-therapeutic approach that effectively targets a plurality of tumor antigens and demonstrated potent anti-tumor response in preclinical brain tumor models. This platform mRNA technology uniquely addresses the challenge of tumor heterogeneity and low antigen burden, two key deficiencies in targeting the classically immunotherapy-resistant CNS malignancies, and possibly other cold tumor types.

Abstract C079: Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy

Abstract Immuno-oncology has revolutionized cancer treatment by harnessing the immune system to target and eliminate tumor cells. In this study, we employed a combination of fluorescence-activated cell sorting (FACS) and Cytek full spectrum flow cytometry to investigate the impact of anti-PD1 (programmed cell death protein 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) treatments on the 4T1 breast cancer model. We comprehensively analyzed 25 surface markers on immune cells to gain insights into the dynamic changes induced by immunotherapy. The 4T1 murine breast cancer model is known for its aggressive and metastatic nature, resembling triple-negative breast cancer in humans. We established a syngeneic orthotopic tumor graft of 4T1 cells in immunocompetent mice and treated them individually with anti-PD1 or anti-CTLA4 antibodies and compared the results to the respective isotype controls. Using FACS, we characterized immune cell populations within the tumor microenvironment and spleenocytes at multiple time points (before treatment, day 5, and day 17) during the treatment course. The Cytek full spectrum flow cytometry platform allowed simultaneous measurement of 25 surface markers, enabling a detailed analysis of immune cell phenotypes and functional states in a single experiment. Our findings revealed significant alterations in the immune landscape following anti-PD1 or anti-CTLA4 treatments. We observed an increase in tumor-infiltrating lymphocytes (TILs) and specifically CD4+ as well as CD8+ T cell infiltration within the tumor, accompanied by upregulation of T cell activation markers, such as CD69, CD38, CD44, PD1 and CD25. The immune cell composition within 4T1 tumors included CD4+ and CD8+ TILs, as well as increased populations of monocytes and macrophages. Additionally, a decrease in regulatory T cells (TRegs) was observed, indicating a potential restoration of immune balance. Moreover, F4/80+ CD206+ tumor-associated macrophages (TAMs) were identified in the 4T1 tumors vs. the spleen. This was associated with increased granzyme B production by CD8+ T cells and a shift towards an effector memory phenotype, suggesting improved cytotoxic function. To gain insights into spatial infiltration patterns of immune cells, we performed Immunohistochemistry (IHC) stainings of various immune cell markers in the 4T1 tumors. In line with the FACS results, we observed increased immune cell infiltration in tumors treated with anti-PD1 and anti-CTLA4 antibodies. Our comprehensive immunophenotypic analysis, utilizing Cytek full spectrum flow cytometry and IHC sheds light on the dynamic interplay between immune cells and the 4T1 breast cancer model during anti-PD1 and anti-CTLA4 immunotherapy. These findings provide valuable insights into the mechanisms underlying the therapeutic efficacy of immune checkpoint blockade in this aggressive cancer model. Citation Format: Christos Nikolaou, Simon Heller, Stefan Kaulfuss, Carlo Stresemann, Alexandra Eichten, Oliver von Ahsen, Martin Lange. Comprehensive immunophenotypic profiling sheds light on the dynamic interplay between immune cells in the 4T1 breast cancer model upon anti-PD1 and anti-CTLA4 immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C079.

IMMU-33. PRECISION ANTIGEN-DIRECTED IMMUNOTHERAPY TREATMENT OF BRAIN TUMORS INCREASES TUMOR-INFILTRATING EFFECTOR LYMPHOCYTE RESPONSES

Abstract BACKGROUND Despite considerable advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized tumor antigens expressed within brain tumors that can mediate tumor rejection; the limited mutational burden within these cancers that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. Our OBJECTIVE is to develop a precision immunotherapy approach for brain tumor patients utilizing prospectively identified tumor-specific antigens using cancer immunogenomics and selective amplification of patients’ tumor-specific antigens for generating tumor antigen-specific lymphocyte therapy. METHODS We identified neoantigens and tumor-associated antigens that were uniquely expressed in preclinical glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (ORAN). A tumor antigen-specific RNA library was created for each tumor model and tumor-bearing animals were treated with ex vivo expanded tumor-specific lymphocytes and tumor antigen-specific RNA-loaded dendritic cell (tsRNA-DC) vaccines. In a different approach, animals were treated with a combination of tsRNA-DC vaccines and anti-PD-1 checkpoint-blockade therapy. Tumor progression and survival outcomes were determined. The immune cell response was evaluated using flow cytometry, TCR sequencing, and single-cell RNAseq. RESULTS Our results demonstrate the effectiveness of tsRNA-DC vaccines in eliciting robust anti-tumor immune responses in combination with adoptive cellular therapy and checkpoint blockade therapy. Additionally, our findings substantiate a robust increase in tumor-infiltrating lymphocytes characterized by enhanced effector function both intratumorally and systemically after tumor antigen-specific RNA-directed immunotherapies. CONCLUSION This powerful approach to generating immunotherapy recognizing a plurality of tumor antigens uniquely addresses the challenge of dealing with tumor antigenic heterogeneity and confronting the reality of patient-to-patient variation in antigen expression in the development of antigen targeting strategies.

CTIM-33. EFFICACY AND SAFETY STUDY OF NEOADJUVANT EFINEPTAKIN ALFA (NT-I7) AND PEMBROLIZUMAB IN RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and aggressive form of brain tumor in adults. Despite maximal surgical resection, irradiation, and chemotherapy, median overall survival (OS) for recurrent GBM (rGBM) remains at only 30 weeks. GBM tumors have an immune-suppressive microenvironment with low numbers of tumor-infiltrating lymphocytes (TILs). Interleukin 7 (IL-7) plays a key role in T cell homeostasis and survival, with IL-7 inducing T cell expansion in lymphopenic conditions. Efineptakin alfa (NT-I7), a long-acting IL-7, has been shown to increase effector CD8 T cells and reduce suppressor T-regulatory cells in murine GBM models, with subsequently improved OS. We recently completed a Phase I study in newly diagnosed GBM patients which showed that NT-I7 is safe, well-tolerated, and increased absolute lymphocyte counts (ALCs), and determined maximum tolerated dose for Phase II. We hypothesized that NT-I7 may not only mitigate GBM treatment-associated immune suppression, but also potentiate the effect of immune checkpoint blockade, leading to an increase in TILs and improved survival. The purpose of this study is to evaluate the safety and efficacy of combining NT-I7 and pembrolizumab in rGBM patients. This is an open-label, single arm one-stage Phase II study of NT-I7 and pembrolizumab as neoadjuvant and adjuvant therapy with surgery for adult patients with rGBM. We plan to enroll 30 evaluable patients, including a 6-patient safety run-in. The primary endpoint is the 9-month OS rate. Secondary endpoints include progression-free survival, objective response rate, changes in ALC, and adverse events profile. The correlative endpoints include studies to assess the anti-glioma immune response, changes in T cell subsets and functionality, peripheral cytokine quantification, and immunohistochemical analysis of GBM tissue. Longitudinal cerebrospinal fluid proteomics is optional. This study opened in Jan 2023. Six (6) of the planned thirty (30) patients had been enrolled by time of submission.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma

Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Two doses of IT GLA-SE (5 μg and 10 μg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. ClinicalTrials.gov Identifier: NCT02180698.

Do tumor infiltrating lymphocytes (TILs) predict benefits from trastuzumab therapy for HER2 positive breast cancer? Meta-analysis of individual patient data from 4097 women in 5 trials.

508 Background: High TIL counts are associated with a lower risk of breast cancer recurrence, especially in women with ER negative, HER2 negative tumors and, possibly, greater benefit from trastuzumab in women with HER2 positive cancer: the FinHER trial reported a differential effect of trastuzumab based upon TIL status. We performed a meta-analysis of randomized trials of trastuzumab in early breast cancer to attempt to validate this finding. Methods: TILs were quantified in 4097 women in 5 randomized controlled trials (NSABP B-31, FinHER, HERA, Intergroup N9831, PACS-04). All trials contributed to the Early Breast Cancer Trialists' Collaborative Group individual patient data meta-analysis of trastuzumab for women with HER2 positive tumors which found a significant benefit for trastuzumab therapy. TILs were assessed using established International Guidelines, with HERA using digital TIL-scores. The primary outcome was time to first recurrence. Cox regression analyses, adjusted for trial, treatment allocation, and nodal status, were used to quantify the prognostic value of TILs; and standard stratified logrank tests were used to assess the differential effect of trastuzumab therapy. Results: The median percentage TILs was 13% (Interquartile Range 5-30), with fewer than 10% of patients exhibiting TILs >50%. The prognostic value of TILs was confirmed, with patients with higher TILs being at lower risk for recurrence (adjusted hazard ratio per 10% increase in TILs 0.87 (95% CI 0.84-0.90), p<.0001) with similar effects in both treatment groups. Outcomes improved steadily with increasing TILs, and unadjusted 10-year recurrence rates fell from 30% in women with TILs <10% to 15% in those with TILs 70% or greater. Consequently, analyses of the predictive effect of TILs were stratified into 5 groups (0-9, 10-19, 20-39, 40-59, 60+). Overall, there was a highly significant benefit of trastuzumab on recurrence (HR 0.62 (0.54-0.70) p<.0001), but there was no evidence of any interaction between TILs and the proportional reduction in recurrence (p=0.8 for heterogeneity and trend). Conclusions: While higher TILs are associated with lower recurrence rates, there was no indication that the proportional reduction in recurrence with trastuzumab varied by TILs, although the number of patients with high levels was limited. Owing to a lower underlying recurrence rate, absolute benefits from trastuzumab were lower, but still substantial, in women with high TIL tumors. Clinical trial information: NCT00045032 , ISRCTN76560285 , NCT00005970 , NCT00004067 , NCT00054587 . [Table: see text]

Efficacy and safety study of neoadjuvant efineptakin alfa (NT-I7) and pembrolizumab in recurrent glioblastoma.

TPS2085 Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor in adults. Despite maximal surgical resection, irradiation, and chemotherapy, median overall survival (OS) remains at only 30 weeks for recurrent GBM (rGBM) patients, emphasizing the need for novel treatments. GBM tumors are immunologically ‘cold,’ in that they have an immune-suppressive microenvironment with a low number of tumor-infiltrating lymphocytes (TIL). Interleukin 7 (IL-7) plays a key role in T cell development and survival. The physiological level of IL-7 increases to stimulate T cell expansion in lymphopenic condition. Previous studies have shown that lymphopenic GBM patients have shorter survival. However, their IL-7 levels are unexpectedly low. Efineptakin alfa (NT-I7), a long-acting IL-7, has been shown to increase effector CD8 T cells and reduce suppressor T-regulatory cells in murine GBM models, with subsequently improved OS. Further, we recently completed a Phase I study which showed that NT-I7 is safe, well- tolerated, and increased absolute lymphocyte counts (ALC) in newly diagnosed GBM patients. Phase II dosing was determined through this Phase I study reaching maximum tolerated dose. Thus, we hypothesized that NT-I7 may reverse GBM-associated immune suppression and potentiate the effect of an immune checkpoint inhibitor, leading to an increase in TILs and improved survival. Our preliminary data suggests that combining NT-I7 with pembrolizumab, an anti-PD-1 antibody, improves survival in murine glioma models. The purpose of this study is to evaluate the safety and efficacy of combining NT-I7 and pembrolizumab in rGBM patients. Methods: This is an open-label single arm one-stage Phase II study of NT-I7 and pembrolizumab as neoadjuvant and adjuvant therapy with surgery for adult patients with rGBM. We plan to enroll 30 evaluable patients, including a 6-patient safety run-in. Key eligibility criteria include candidates willing to undergo clinically indicated resection or biopsy, age ≥ 18 years, Karnofsky performance status ≥ 70, and dexamethasone dose > 2 mg/day ≤ 2 days prior to registration. The primary endpoint is a 9-month OS rate. Secondary endpoints include progression-free survival, objective response rate, changes in ALC, and adverse events profile. The correlative endpoints include studies to assess the anti-glioma immune response, changes in T cell subsets, T cell effector function, cytokine analysis, and immunohistochemical analysis of GBM tissue. Longitudinal cerebrospinal fluid proteomics is optional. This study opened on 1/20/2023. Two of the planned 30 patients had been enrolled by submission on 2/14/2023. Clinical trial information: NCT05465954 .

An engineered influenza virus to deliver antigens for lung cancer vaccination.

The development of cancer neoantigen vaccines that prime the anti-tumor immune responses has been hindered in part by challenges in delivery of neoantigens to the tumor. Here, using the model antigen ovalbumin (OVA) in a melanoma model, we demonstrate a chimeric antigenic peptide influenza virus (CAP-Flu) system for delivery of antigenic peptides bound to influenza A virus (IAV) to the lung. We conjugated attenuated IAVs with the innate immunostimulatory agent CpG and, after intranasal administration to the mouse lung, observed increased immune cell infiltration to the tumor. OVA was then covalently displayed on IAV-CPG using click chemistry. Vaccination with this construct yielded robust antigen uptake by dendritic cells, a specific immune cell response and a significant increase in tumor-infiltrating lymphocytes compared to peptides alone. Lastly, we engineered the IAV to express anti-PD1-L1 nanobodies that further enhanced regression of lung metastases and prolonged mouse survival after rechallenge. Engineered IAVs can be equipped with any tumor neoantigen of interest to generate lung cancer vaccines.

An increase in tumor-infiltrating lymphocytes after treatment is significantly associated with a poor response to neoadjuvant endocrine therapy for estrogen receptor-positive/HER2-negative breast cancers

BackgroundThe reason for the poor prognosis of estrogen receptor (ER) + /human epidermal growth factor receptor 2 (HER2)− breast cancer patients with high levels of tumor-infiltrating lymphocytes (TILs) is poorly understood. The association between TILs and response to neoadjuvant endocrine therapy (NET) was examined.MethodsWe recruited 170 patients with ER + /HER2− breast cancer who were treated with preoperative endocrine monotherapy. TILs were evaluated before and after NET, and their changes were noted. Furthermore, T cell subtypes were examined using CD8 and FOXP3 immunohistochemical analyses. Neutrophil and lymphocyte counts in the peripheral blood were analyzed with reference to TIL levels or changes. Responders were defined as Ki67 expression levels ≤ 2.7% after treatment.ResultsPost-treatment (p = 0.016), but not pre-treatment (p = 0.464), TIL levels were significantly associated with the response to NET. TIL levels increased significantly after treatment among non-responders (p = 0.001). FOXP3 + T cell counts increased significantly after treatment in patients with increased TILs (p = 0.035), but not in those without increased TILs (p = 0.281). Neutrophil counts decreased significantly after treatment in patients without increased TILs (p = 0.026), but not in patients with increased TILs (p = 0.312).ConclusionAn increase in TILs after NET was significantly associated with a poor response to NET. Given that FOXP3 + T-cell counts increased, and neutrophil counts did not decrease in patients with increased TILs after NET, the induction of an immunosuppressive microenvironment was speculated to play a role in the inferior efficacy. These data might partially indicate the involvement of the immune response in the efficacy of endocrine therapy.

Abstract 5114: Development of personalized RNA-based immunotherapeutics for glioblastom

Abstract Background: The development of successful immunotherapy targeting antigens in glioblastoma multiforme (GBM) remains a challenge owing to antigen heterogeneity and the low mutation burden in GBM. Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies for GBM. Our goal is to develop a personalized RNA-based therapeutic vaccine that simultaneously targets a selected pool of tumor rejection antigens including all neoantigens and tumor-associated antigens (TAAs) as identified using cancer immunogenomics. We evaluated the treatment efficacy of these RNA therapeutics in combination with adoptive cellular therapy and checkpoint inhibitors. Methods: We identified immunogenic neoantigens and TAAs that are aberrantly overexpressed in KR158-luc and GL261 murine GBM tumors using our cancer immunogenomics pipeline called the Open Reading Frame Antigen Network (O.R.A.N.). A tumor antigen-specific RNA library was created for each tumor using a novel gene enrichment strategy and validated by RNAseq. Tumor-bearing animals were treated with adoptively transferred ex vivo expanded lymphocytes and dendritic cells loaded with the tumor antigen-specific RNA as vaccines. Additionally, we treated animals with the tumor antigen-specific RNA vaccines in combination with anti-PD-1 checkpoint-blockade therapy. Tumor progression and survival outcomes were analyzed to evaluate the treatment efficacies. We determined the peripheral and tumor-infiltrating lymphocyte responses using flow cytometry, TCR sequencing, and single cell RNAseq. Results: The tumor antigen-specific RNA vaccines were significantly effective in slowing the progression of murine GBM tumors in combination with both the adoptive cellular therapy and the checkpoint blockade therapy and improved the outcome of these therapies as compared to monotherapy alone. Additionally, we identified antigen-specific T cells targeting several of our predicted antigens and an increase in tumor-infiltrating lymphocytes and memory T cells in the treated animals. Conclusion: Here, we developed an RNA-based immunotherapy that simultaneously targets numerous tumor-specific antigens and demonstrated its therapeutic efficacy in preclinical models of GBM. Citation Format: Vrunda Trivedi, Changlin Yang, Oleg Yegorov, Kelena Klippel, Graeme Fenton, Christina von Roemeling, Lan Hoang-Minh, Elizabeth Ogando-Rivas, Paul Castillo, Kyle Dyson, Ginger Moore, Duane Mitchell. Development of personalized RNA-based immunotherapeutics for glioblastom. [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 5114.

Abstract 700: Induction of cancer-specific T-cell responses in patients immunized with P10s-PADRE vaccine

Abstract Introduction: HR+/HER2− breast cancer is the most common form of breast cancer in the United States. HR+/HER2− tumors are known for a low number of tumor-infiltrating lymphocytes (TILs) and considered less immunogenic than other breast cancer subtypes. We have demonstrated that vaccination with P10s-PADRE, a carbohydrate-mimetic-based peptide, cancer vaccine in combination with standard-of-care chemotherapy in HR+/HER2− breast cancer patients led to an increase in TILs and stromal CD3+ T cells. The current study was performed to determine the vaccine specificity and anti-tumor functionality of T cells in treated patients. Methods: Peripheral blood mononuclear cells (PBMCs) collected at the baseline and after vaccination were used in T-cell assays by multi-color ELISpot, examining Th1, Th2, and cytotoxic responses. PBMCs were also interrogated for the vaccine-induced changes in immune gene expression by next-generation RNA-seq analysis. Results: We observed a significant increase in IFN-g, but not in IL-5 or IL-10, responses in post-immune PBMCs after stimulation with P10s, P10s-PADRE and polyclonal stimulation of T cells by anti-CD3/CD28 antibodies. Stimulation with cancer cell lysate resulted in a strikingly high IFN-g response in post-immune samples. Determining the trio of IFN-g, GzB, and IL-2 together with CD4/CD8 cell proliferation assays of consequent post vaccination specimens established the dynamics of effector T-cell populations. RNA-seq data clearly distinguished post-treatment samples by the increase in immune cell activation, cytokine response, and antigen presentation. Conclusions: The data indicate that immunization of HR+/HER2− breast cancer patients with P10s-PADRE in combination with chemotherapy leads to specific activation of T-cells that recognize breast cancer cells. Improving immunogenicity of such immunologically cold tumors could increase the effectiveness of standard therapeutic approaches. Citation Format: John J. Lee, Bernice C. Nounamo, Fariba Jousheghany, Issam Makhoul, Eric R. Siegel, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Induction of cancer-specific T-cell responses in patients immunized with P10s-PADRE vaccine [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 700.

Abstract P2-23-05: Trastuzumab-Induced Tumor Microenvironment Changes in Early HER2+ Breast Cancer

Abstract Background Trastuzumab (H) is a critical component of therapy (tx) for HER2+ breast cancers (BCs) and can induce anti-tumor immune responses that as part of its mechanism of action. Pre-clinical studies have suggested that antibody-dependent cell-mediated cytotoxicity (ADCC) through NK cells is a major driver of the immune response. However, there is also evidence that macrophages and dendritic cells (DCs) can play an important role in H immune response through antibody-dependent cellular phagocytosis (ADCP). Here, we present pre- and post-tx gene expression and immunohistochemistry (IHC) data to characterize the tumor microenvironment (TME) changes in response to a single dose of H in patients (pts) with early HER2+ BC. Methods Pts with Stage I-III HER2+ BC with minimum 1 cm tumors were eligible. Pts received 8 mg/kg IV H followed by tissue collection. There was a minimum of 14 days between (btw) H and post-tx tissue collection. Pre- and post-tx tissues were assessed for tumor-infiltrating lymphocytes (TILs), immune-related gene expression, and AE1/3, CD31, CD4, CD8, CD11b, CD68, and CD56 expression through IHC. Results Thirteen pts were enrolled and evaluable. 5/13 were estrogen-receptor positive (ER+). TILs were assessed independently by two pathologists and the results averaged. Tx with H resulted in an increase in TILs in the post-tx tissue (p < 0.01). Pre-tx TILs ranged from 2-80% (median 6%) and post-tx TILs ranged from 2-80% (median 20%). 6/13 pts had immune infiltration (Inf), defined as a TIL increase of greater than 1 decile btw pre- and post-tx samples. 2/6 pts with Inf were ER+. Pts with Inf had a significantly higher DC immune signature (ISS) compared to pts without Inf (p < 0.01, univariate analysis). No other ISS reached significance. 6 pts had paired pre- and post-tx samples evaluated by IHC. 4/6 pts had Inf per prior definition. Pts with Inf had an increase in CD11b+ (includes macrophages and some DCs) and CD56+ cells (includes NK cells) after H tx within the tumor compartment (p < 0.05). Pts with Inf had an increase in CD11b+, CD56+, and CD44+ cells in the stromal compartment (p < 0.05). Conclusions Innate immune cells such as macrophages and DCs are key players in H-induced immune response in the early HER2+ BC TME. This data is consistent with prior pre-clinical findings. Citation Format: Laura C. Kennedy, Rebeca Alvarez, Suzanne Dintzis, Vijayakrishna K. Gadi. Trastuzumab-Induced Tumor Microenvironment Changes in Early HER2+ Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-05.

Abstract No. 6 Treatment of HCC by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant

To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination (ISV) approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). A bilateral, subcutaneous HCC tumor model was established using RIL-175 cells in male C57BL/6 mice. Contralateral, untreated tumors were monitored for an abscopal effect. Mice were randomized into one of four groups: phosphate-buffered saline injected (Control), cryoablation only (Cryo), CPMV-treated only (CPMV), and cryoablation plus CPMV-treated (Cryo+CPMV), (N = 10-12/group). Ten days after tumor inoculation, CPMV was administered intratumorally (day 0) and then on days 3, 6 and 9. Cryoablation was performed on day 3. Tumor growth was measured with digital calipers. Systemic chemokine/cytokine concentrations were measured from tail bleed serum samples. Two mice from each group were sacrificed at 1 week post treatment initiation, and tumors and spleens were harvested for immunohistochemistry and flow cytometry, using innate and adaptive immune cell panels. One-way ANOVA was performed for all statistical comparisons among treatment groups. A P value of < 0.05 served as the threshold for statistical significance. At 2 weeks post treatment, Cryo+CPMV outperformed solo treatment arms and Control. For the treated tumor, Cryo+CPMV significantly delayed and inhibited tumor progression compared with tumors in the Control group (1.6- ± 0.9-fold vs 6.3- ± 0.5-fold; P< 0.0001). For the untreated tumor, only the Cryo+CPMV group showed statistically significant tumor growth reduction compared with Control (9.2- ± 0.9-fold vs 17.8- ± 2.1-fold; P = 0.01). This was associated with a transient increase in IL-10 and a significantly lower concentration of CXCL1 in the Cryo+CPMV group. Flow cytometry revealed an enrichment of natural killer (NK) cells, activated CD4+ and CD8+ T cells, and an increase in PD-1 expression on both CD4+ and CD8+ T cells in untreated tumors and/or spleens of the Cryo+CPMV group. Immunohistochemistry demonstrated a robust increase in tumor-infiltrating lymphocytes in the Cryo+CPMV group, especially into the center of the tumors. Cryoablation combined with intratumoral CPMV as a multimodal ISV approach had potent efficacy against this aggressive HCC tumor model, both to the treated tumor and to the contralateral untreated tumor, indicating an abscopal effect.

EXTH-44. PERSONALIZED IMMUNOTHERAPY VACCINES FOR GLIOBLASTOMA AND THEIR TREATMENT EFFICACY

Abstract BACKGROUND Glioblastoma multiforme (GBM) remains a disease with debilitating survival outcomes. Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies for brain malignancies. Using a cancer immunogenomics approach that we developed, called the Open Reading Frame Antigen Network (O.R.A.N.), we identified the immunogenic neoantigens and tumor-associated antigens (TAAs) including cancer-testis and developmental antigens, that are aberrantly overexpressed in murine models of GBM. The aim of this study is to evaluate the immunologic effects and safety of immunotherapy vaccines targeting neoantigens and TAAs in preclinical models of GBM. METHODS RNAseq and WES were performed for murine glioblastoma tumors- KR158-Luc and GL261, and the results were fed to the O.R.A.N pipeline for antigen prediction. A tumor antigen-specific RNA library was created for each tumor using a gene enrichment strategy and validated by RNAseq. Tumor-bearing animals were treated with adoptively transferred ex vivo expanded lymphocytes and dendritic cell vaccines loaded with the tumor antigen-specific RNA. Tumor volume, and thus progress, was determined using in vivo luciferase imaging technique and the survival outcomes were noted. We also evaluated the efficacy of the tumor vaccines in combination with checkpoint blockade therapy by treating tumor-bearing animals with dendritic cell vaccines loaded with the tumor antigen-specific RNA combined with an anti-PD-1 antibody. RESULTS The dendritic cell vaccines loaded with tumor antigen-specific RNA were significantly effective in slowing the progression of murine GBM tumors in combination with both the adoptive cellular therapy as well as checkpoint blockade therapy. Additionally, we identified antigen-specific T cells targeting several of our predicted antigens and an increase in tumor-infiltrating lymphocytes and memory T cells in the treated animals. CONCLUSION We developed a dendritic cell-based vaccination approach targeting neoantigens and TAAs identified as being tumor-specific and evaluated the efficacy of immunotherapy vaccines in preclinical models of GBM.

Assessing the Relationship between Residual Cancer Burden and the Tumor Immune-Microenvironment in Early-Stage, Hormone Receptor-Positive Breast Cancer Following Preoperative Radiation Therapy in MC1732 Clinical Trial

<h3>Purpose/Objective(s)</h3> Tumor-infiltrating lymphocytes (TILs) and ectopic lymphoid formations known as tertiary lymphoid structures (TLS) are prognostically significant in triple-negative breast cancer (TNBC). However, there is a paucity of data on this matter in hormone receptor-positive (HR+) breast cancer and whether these immunological parameters are impacted by preoperative radiation therapy (RT). The purpose of this work is to assess the relationship between residual cancer burden (RCB) and TILs/TLS in HR+ early-stage breast cancer following preoperative radiation therapy. <h3>Materials/Methods</h3> During 2019-2020, patients with cT0-T2, N0, M0 breast cancer were enrolled in a clinical trial (MC1732) in which they received hypofractionated whole-breast RT in 25 Gy in 5 fractions 4-8 weeks prior to breast-conserving surgery (BCS). RCB (determined per MD Anderson calculator), estimated % cellularity, TILs, and TLS were assessed in the surgical tissues. Low TIL was defined as < 10%, intermediate TIL as 11-49%, and high TIL as > 50%. The relationships between RCB class, numerical RCB values, and immunological parameters were explored using Kruskal-Wallace/Wilcoxon Rank Sum Test, linear regression, and Kendall's tau correlations. TILs and TLS were log-transformed to overcome extreme distributions. Lastly, a predictive model for pathologic outcomes was developed using backward elimination. <h3>Results</h3> Among the 22 patients analyzed for RCB, all were ER/PR positive. One patient was HER2 positive, and 13/22 had Grade 1 disease. Pathologic complete response (pCR) was noted in 18%, RCB-I in 60%, and RCB-II in 22%. In post-treatment samples, low, intermediate, and high TIL were found in 77%, 18%, and 5% of patients, respectively. Approximately 36% of patients had at least one TLS present after radiation. Kruskal-Wallace/Wilcoxon Rank Sum Test indicated a significant association between RCB class and % TILs and TLS (p = 0.010 and 0.003, respectively.) Kendall's tau correlation indicated a statistically significant positive correlation between RCB values and % TILs (p = 0.02) and TLS (p = 0 .001). Linear regression revealed that for every one-unit increase in RCB value, there was a statistically significant 4.3 unit increase in the % TILs (p <0.001), and a 2.48 unit increase in TLS (p <0.001). Finally, per the backward selection model, the number of TLS was associated with increased % cellularity at the time of surgery. <h3>Conclusion</h3> Our data suggest that increases in TILs and TLS following preoperative radiation are associated with increased residual disease in HR+ early-stage breast cancer. Given the fact that there are multiple types of potential immune infiltrates, further work is needed to elucidate the mechanisms behind these findings and specific types of immune infiltration.

Tumor-infiltrating lymphocytes and PD-L1 expression in pre- and post-treatment ovarian cancer during neoadjuvant chemotherapy (254)

Objectives: To investigate the dynamic changes of tumor immune microenvironment (TME) by evaluating lymphocyte infiltration before and after neoadjuvant chemotherapy (NACT) in patients with advanced-stage ovarian cancer. Methods: We conducted whole-transcriptome sequencing coupled with immunohistochemistry of 147 patients, including 140 pairs of serial tumor samples collected before NACT and at the time of interval debulking surgery (IDS). PD-L1 expression was determined by using a combined positive score (CPS). Tumor-infiltrating lymphocytes (TIL) were assessed by standardized methods according to the guidelines of the International TIL working group. Results: All the patients had high-grade serous carcinoma and were treated with platinum-based chemotherapy. High pre-NACT PD-L1 level (CPS ≥ 10%) was associated with worse progression-free survival (PFS, p < 0.001) and overall survival (OS, p = 0.038). High pre-NACT TIL level (>20%) was associated with better PFS (p = 0.039) and trend of better OS (p = 0.077). Post-NACT PD-L1 and TIL levels were not associated with survival outcomes. PD-L1 level (p = 0.001) and TIL level (p <0.001) were significantly increased before and after NACT. An increase in TIL in response to NACT was associated with shorter PFS compared to patients with decreased TIL levels (p = 0.020). A change in the PD-L1 level had no impact on the prognosis. In whole-transcriptome sequencing, stromal score (p = 8.6e-0.7), immune score (p = 4e-0.6), and CYT score (p = 2.1e-0.5) were significantly increased after NACT. Conclusions: Pre-NACT TIL level was associated with a survival benefit in advanced-stage ovarian cancer. By contrast, increased TIL level was an adverse prognostic factor for survival outcomes. Further research is needed to find a patient group that can benefit from immunotherapy in the NACT setting. Objectives: To investigate the dynamic changes of tumor immune microenvironment (TME) by evaluating lymphocyte infiltration before and after neoadjuvant chemotherapy (NACT) in patients with advanced-stage ovarian cancer. Methods: We conducted whole-transcriptome sequencing coupled with immunohistochemistry of 147 patients, including 140 pairs of serial tumor samples collected before NACT and at the time of interval debulking surgery (IDS). PD-L1 expression was determined by using a combined positive score (CPS). Tumor-infiltrating lymphocytes (TIL) were assessed by standardized methods according to the guidelines of the International TIL working group. Results: All the patients had high-grade serous carcinoma and were treated with platinum-based chemotherapy. High pre-NACT PD-L1 level (CPS ≥ 10%) was associated with worse progression-free survival (PFS, p < 0.001) and overall survival (OS, p = 0.038). High pre-NACT TIL level (>20%) was associated with better PFS (p = 0.039) and trend of better OS (p = 0.077). Post-NACT PD-L1 and TIL levels were not associated with survival outcomes. PD-L1 level (p = 0.001) and TIL level (p <0.001) were significantly increased before and after NACT. An increase in TIL in response to NACT was associated with shorter PFS compared to patients with decreased TIL levels (p = 0.020). A change in the PD-L1 level had no impact on the prognosis. In whole-transcriptome sequencing, stromal score (p = 8.6e-0.7), immune score (p = 4e-0.6), and CYT score (p = 2.1e-0.5) were significantly increased after NACT. Conclusions: Pre-NACT TIL level was associated with a survival benefit in advanced-stage ovarian cancer. By contrast, increased TIL level was an adverse prognostic factor for survival outcomes. Further research is needed to find a patient group that can benefit from immunotherapy in the NACT setting.