Tissue assessment of therapeutic responses to neoadjuvant short course radiotherapy with and without anti-CD40 immunotherapy sotigalimab (sotiga) in rectal cancer.
Authors: Aguilera Todd Anthony , Elghonaimy Eslam , Sanford Nina Niu , Kazmi Syed Mohammad Ali , Pogacnik Javier Salgado , Kardosh Adel , Paulman Brendan , Mufich Cassandra , Goodyear Shaun , Huang Emina , Jones Amy Little , Karagkounis Georgios , Kainthla Radhika , Ali Fadwa , Al Mutar Salwan , Sanjeevaiah Aravind , Yang Xiaodong , Timmerman Robert D. 
Published: January 2023

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Background: There has been tremendous progress in the management of rectal cancer in the neoadjuvant setting and in non-operative management (NOM). However, there is a critical need to personalize systemic therapy and radiotherapy to achieve responses conducive for NOM. This study was initiated to profile immune changes to therapy in serial tumor tissue, blood, and stool from the INNATE trial (NCT04130854). Methods: INNATE, is a multi-institutional Phase II randomized trial for patients with Stage III or high-risk Stage II rectal cancer. Patients were randomized 3:2 to receive daily short course radiotherapy (SCRT- 5 Gy x 5) and 6 cycles of FOLFOX +/- 6 infusions of sotiga, an anti-CD40 agonist, followed by surgery. The primary end point is the pathologic complete response rate and additional clinical endpoints are being assessed. Biopsies of tumor and adjacent tissue before and 2 weeks after SCRT +/- sotiga prior to chemotherapy were obtained from consenting patients. Fresh tissue was digested into single cell suspension for scRNAseq with proteomic and immune repertoire analysis. Fixed tissue was processed for histology and multiplexed immunofluorescence. Here, we report integrated and batch effect removed data from 9 patients pre- and post-therapy and analyzed patients as their own control. Results: A total of 25 of 30 patients were enrolled at UTSW, 21 of whom underwent pre- and post-SCRT biopsies. The study population was diverse: 11 patients (37%) were Hispanic and 15 (50%) uninsured receiving care at the Dallas County hospital, Parkland. The median age was 55 (27-75) years and 13 (43.3%) were diagnosed prior to age 50. From integrated scRNAseq, we observed a significant induction of genes associated with M1- and M2-like macrophages, and dendritic cell antigen presentation in the SCRT alone group. This was accompanied by a robust Th1 and B cell response, but not T cell mediated cytotoxicity. In the SCRT + sotiga group there was a greater induction of genes associated with M1-like macrophages and dendritic cell antigen presentation, with no change in M2 genes. In lymphocytes, sotiga treatment led to an induction of genes related to Th1 and B cell response. Unlike SCRT, the combination therapy group had evidence of induced T cell mediated cytotoxicity. To better understand individual responses to therapy, we analyzed pre- and post-treatment samples for each patient. This enabled identification of features associated with poor and robust response in each treatment group. Conclusions: On treatment biopsies during neoadjuvant therapy for rectal cancer enables deep assessment of therapeutic response, provides insight into action of investigational agents, and may identify therapy specific predictive biomarkers. Our data shows that SCRT can induce immune responses and adaptive immune responses can be further induced by sotiga.