In early stage breast cancer, the degree of tumor-infiltrating lymphocytes (TILs) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, Tcell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared to

Background: In mTNBC, anti-PD-1/L1 monotherapy is most effective when administered early in the course of disease, with recent trials demonstrating overall response rates (ORR) of 23-26% in the first-line setting and 5-6% in later lines. This may reflect iatrogenic lymphopenia from preceding cytotoxic chemotherapy. Furthermore, curative-intent chemotherapy is associated with prolonged suppression of naïve CD4+ cells, a T-cell subset that may play a critical role in the generation of de novo anti-tumor immune responses. We present the final clinical results of a pilot study evaluating the safety and efficacy of combining pembrolizumab plus standard-of-care capecitabine in the first/second-line mTNBC setting. We also explore potential associations between clinical benefit and lymphopenia, preceding chemotherapy, and absolute naïve CD4+ counts. Methods: In a pilot study, we evaluated the tolerability and preliminary efficacy of concurrent pembro (200mg IV q21 day) plus investigator-selected 1st/2nd line paclitaxel (80mg/m2 IV weekly) or oral cape (2,000mg BID, weekly 1 on/1 off). The primary endpoint was tolerability, defined as the proportion of subjects receiving >6 weeks concurrent therapy without dose discontinuation with toxicities reported per CTCAE v4.0. The secondary endpoint was 12-week objective response rate (ORR) by RECIST1.1. Exploratory endpoints included peripheral blood cell enumeration by real-time flow cytometry and routine clinical laboratory. Naïve CD4+ cells were defined as CD45+ CD3+ TCRab+ CD4+ CD45RA+ CCR7+. Here, we report the results of the pilot phase of the cape cohort (NCT02734290). Results: Twelve of 14 subjects were treated in the first-line setting. All subjects (14/14, 100%) tolerated cape+pembro for >6 weeks, with toxicities consistent with monotherapy cape experience (diarrhea: grade I-II 50%, grade III 7%; hand-foot: grade I-II 71%) that improved with dose-reduction as needed. At 12 weeks, the ORR was 6/14 (42.9%), and the clinical benefit rate (ORR + stable disease) was 8/14 (57.1%). Depressed absolute lymphocyte count at baseline (ALC<1.0/uL: 33% CBR; ALC≥1.0/uL: 75% CBR) and recent exposure to cytotoxic chemotherapy (<6 months: 33% CBR; >6 months: 75% CBR) were associated with reduced clinical benefit. By flow cytometry, subjects experiencing clinical benefit had higher baseline absolute naïve CD4+ counts (average 283 cells/uL v. 93 cells/uL, p=.069). Conclusions: This study met the primary endpoint of safety for cape plus pembro in mTNBC, with encouraging clinical activity. These data are supportive of further studies evaluating combination chemotherapy plus anti-PD-1/L1 mTNBC. We observed greater clinical benefit in subjects with non-suppressed ALC, less exposure to recent chemo, and higher baseline naïve CD4+ counts, suggesting that iatrogenic immunosuppression can impair response to immune checkpoint therapy in mTNBC. These findings should be confirmed in ongoing randomized trials of immune checkpoint +/- chemotherapy in mTNBC, and should be considered in the design of future clinical trials. Citation Format: Page DB, Pucilowska J, Bennetts L, Kim I, Sanchez K, Martel M, Conlin A, Moxon N, Mellinger S, Acheson A, Kemmer K, Mitri Z, Vuky J, Ahn J, Abaya C, Manigault T, Basho R, Urba WJ, McArthur HL. Updated efficacy of first or second-line pembrolizumab (pembro) plus capecitabine (cape) in metastatic triple negative breast cancer (mTNBC) and correlations with baseline lymphocyte and naïve CD4+ T-cell count [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-03.

BackgroundChemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. MethodsWe report nal outcomes from a phase Ib trial evaluating pembrolizumab (200mg IV every 3 weeks) with either weekly paclitaxel (80mg/m 2 weekly) or at-dose capecitabine (2000mg orally twice daily for 7 days of every 14-day cycle) in the 1st /2nd line setting. The primary endpoint was safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥ 21-day delays). The secondary endpoint was e cacy (week 12 objective response rate). Exploratory aims were to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers. ResultsBoth regimens met the pre-speci ed safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate was 29% for pembrolizumab/paclitaxel and 43% for pembrolizumab/capecitabine. Partial responses were observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens were associated with signi cant peripheral leukocyte contraction over time. Response was associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor in ltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). ConclusionsPembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens were lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted.

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Background: Cytokines are being explored as a therapeutic strategy to modulate the tumor microenvironment and facilitate immunotherapy benefit in breast cancer. Here, we investigate a locoregional therapeutic approach whereby cytokines (IRX-2) are administered into the subcutaneous peri-areolar tissue (in an anatomic distribution similar to sentinel lymph node mapping) to facilitate immune cell recruitment/activation within the draining lymph nodes and tumor in ESBC. IRX-2 is derived from ex vivo phytohemagglutinin-stimulated lymphocytes and contains multiple cytokines including IL-1β, IL-2, TNF-α, IFN-γ, IL-6, IL-8, and GM-CSF, with stable concentrations from lot to lot. Preclinically, IRX-2 activates T-cells and natural killer (NK) cells, facilitates antigen presentation, and enhances activity of anti-PD-1/L1 in a SCC7 model. In a preceding head/neck squamous cell carcinoma phase I trial, perilymphatic IRX-2 was safe and increased TILs. Here, we report the final clinical results of a phase Ib trial evaluating the feasibility and immunologic activity of IRX-2 in ESBC. Methods: Beginning 21 days prior to surgical resection, enrolled operable patients with stage I-III ESBC (all subtypes) received the pre-operative IRX-2 regimen consisting of a single low-dose cyclophosphamide (300 mg/m2 to facilitate T-regulatory cell depletion), followed by 10 days of subcutaneous peri-areolar IRX-2 injections into the affected breast (1 mL × 2 at tumor axis and at 90°). Endpoints were feasibility (primary endpoint), stromal TIL (sTIL) count (pre-treatment versus post-treatment, blinded average of two pathologist reads using San Antonio H&E sTIL guidelines), PD-L1 expression (Nanostring) and enumeration of peripheral immune cells by flow cytometry. Results: All patients (n=16/16) completed and tolerated the regimen with no surgical delays or treatment-attributed grade III/IV toxicities. Common adverse events (occurring in >15% subjects) attributed to IRX-2 injections were: injection site reaction (grade 1, n=8/16), bruising (grade 1, n=7/16), and pain (grade 1, n=3/16). Common adverse events attributed to low-dose cyclophosphamide were: fatigue (grade 1, n=5/16) and nausea (grade 1/2, n=3/16). Treatment was associated with an increase in sTIL score (Wilcoxon signed-rank p=.04), with 4/10 sTIL-low tumors (0-10% score) re-categorized to sTIL-moderate (11-50% score). Increases in PD-L1 RNA expression were observed (Wilcoxon signed-rank p=.04) in 12/16 tumors (median 57% increase, range: -53% to 185% increase), as well as increases in Nanostring NK and Th1 cell signatures. In blood, increases in CD4 and CD8 effector T-cell activation (ICOS, HLA-DR, and CD38) and T-reg depletion were observed. Conclusions: IRX-2 was well tolerated with preliminary evidence of sTIL increase, PD-L1 upregulation, and peripheral lymphocyte activation. Based upon these data and preclinical evaluations demonstrating synergy with checkpoint inhibition, the IRX-2 regimen is being evaluated for clinical efficacy in conjunction with pembrolizumab and neoadjuvant chemotherapy (doxorubicin, cyclophosphamide, paclitaxel) in patients with stage II-III triple negative breast cancer. Citation Format: Pucilowska J, Egan JE, Berinstein NL, Moxon N, Aliabadi-Wahle S, Imatani JH, Conlin A, Acheson A, Massimino K, Martel M, Campbell M, Wu Y, Sun Z, Redmond W, Shah M, Urba WJ, Page DB. Perilymphatic IRX-2 cytokine therapy to enhance tumor infiltrating lymphocytes (TILs) and PD-L1 expression preceding curative-intent therapy in early stage breast cancer (ESBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-12.