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Background: The IRX-2 biologic is a subcutaneous injectable immunotherapy composed of IL-2 and other cytokines derived from stimulated lymphocytes. Preclinically, IRX-2 activates T cells, natural killer cells, macrophages, and dendritic cells, and facilitates maturation of antigen-presenting cells.Tumor-infiltrating lymphocytes (TILs) are associated with improved outcomes in many cancers including early stage breast cancer (ESBC) and head and neck squamous cell carcinoma (HNSCC). We report data on TIL recruitment associated with pre-operative IRX-2 in a phase Ib ESBC trial, as well as phase Ib and IIa HNSCC trials.

Methods: The pre-operative IRX-2 regimen was evaluated in both ESBC and HNSCC trials for safety and immunologic activity. Beginning 21 days prior to surgical resection, enrolled operable patients with resectable stage I-III ESBC and stage II-IVA HNSCC received single low-dose intravenous cyclophosphamide (300 mg/m2 to facilitate T-regulatory cell depletion), followed by 10 days of subcutaneous injections of IRX-2 (1mL × 2 directed to regional peri-lymphatic space, 230IU/day). Endpoints included feasibility, TIL count by H&E blinded pathology review, and Nanostring RNA analysis.

Results: In the ESBC trial, 16 patients were enrolled and evaluable for TIL analysis, whereas in the HNSCC trials, 40 patients were enrolled and 36 patients were evaluable. In both trials, all patients received all planned injections with no treatment-related surgical delays, complications, or treatment-related grade III/IV toxicities. Treatment was associated with a mean 116% relative increase in TILs (range –36% to +1275%, p = 0.02) in ESBC and a mean 58% relative increase (range -57 to +452%, p=0.01) in HNSCC. Treatment was associated with PD-L1 RNA upregulation in EBSC (mean +54%, range –53% to +185%, p=0.04) but not HNSCC, however PD-L1 was higher at baseline in HNSCC. RNA analysis in ESBC and HNSCC revealed concordant increases in cytokine gene expression, including CXCL2, CCL4, CXCR4, and CXCL12 as well as transcription factors including FOS, ETS1, NFKB, EGR1/2 which are involved in T-cell activation and differentiation. We also note augmentation of ITGAE (CD103), a known marker of memory T-cell activation in EBSC cohort.

Conclusions: Pre-operative IRX-2 was well tolerated in both tumor histologies with statistically significant TIL recruitment, as well as PD-L1 upregulation in ESBC. Future directions include an evaluation of neoadjuvant IRX-2 with anti-PD-1 and chemotherapy in stage II-III TNBC, ongoing follow-up of a randomized phase IIb trial of neoadjuvant IRX-2 regimen in HNSCC to ascertain clinical benefit, and trials evaluating efficacy of IRX-2/anti-PD-1 combination across various metastatic cancers.

Trial Registration: NCT02950259, NCT02609386

Publication Date


Clinical Institute





Earle A. Chiles Research Institute


cancer, immunotherapy, T cells, tumor infiltrating lymphocytes, TILs, cytokines, breast cancer, head and neck squamous cell carcinoma, HNSCC, clinical trial




Poster presented by Joanna Pucilowska at Society for Immunotherapy of Cancer Annual Meeting, Washington, D.C., November 7 – 11, 2018

Tumor infiltrating lymphocyte recruitment after peri-lymphatic IRX-2 cytokine immunotherapy in resectable breast cancer and head and neck carcinoma

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