In situ Vaccine Plus Checkpoint Blockade Induces Memory Humoral Response.

Claire C Baniel,Elizabeth G Sumiec,Ravi B Patel,Stephen D Gillies,Alexander L Rakhmilevich,Amy K Erbe,Raghava N Sriramaneni,Paul M Sondel,Alexander A Pieper,Zachary S Morris,Peter M Carlson,Won Jong Jin,Ciara N Schwarz,Anna Hoefges,Jaquelyn A Hank,Clinton M Heinze

doi:10.3389/fimmu.2020.01610

Claire C Baniel, Elizabeth G Sumiec + Show 14 more

Open Access

https://doi.org/10.3389/fimmu.2020.01610

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Abstract

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 106 GD2+ B78 MEL and treated at a target tumor size of ~200 mm3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 106 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response.

Highlights

Immunological memory is a critical component of adaptive immunity and may be essential for the long-term success of cancer therapies that prevent the growth of metastases and disease recurrence [1, 2]
In order to assess for a humoral memory response, we collected serum from mice cured of B78 melanoma by an RT + ITIC + anti-CTLA-4 in situ vaccine regimen and subsequently demonstrated immune memory by rejection of subcutaneous reengraftment of B78 cells 90 days after treatment
While our treatment regimen includes an exogenous antibody-based therapeutic targeting the GD2 antigen on these melanoma cells, the endogenous humoral response generated following this treatment is not specific to GD2 and recognizes antigens shared by B78 and the B16 melanoma line that is parental to B78 but lacks GD2

Summary

Introduction

Immunological memory is a critical component of adaptive immunity and may be essential for the long-term success of cancer therapies that prevent the growth of metastases and disease recurrence [1, 2]. One approach to minimize this risk has been to employ intratumoral (IT) routes of immunotherapy delivery [4] Such approaches make use of the capacity of a local adaptive immune system to result in a systemic anti-tumor response. Hu14.18-IL2 is a synthetic fusion protein consisting of an anti-GD2 tumor-specific antibody genetically fused with IL2, an immune-stimulating cytokine With this treatment regimen, we observed an in situ vaccination effect resulting in complete tumor regression in 71% of mice [9]. Mice that experienced complete tumor regression after treatment with our dual RT + IT-IC therapy demonstrated a tumor-specific memory T-cell response This T-cell response enabled rejection of the parental tumor lines that lacked the GD2 antigen targeted by IC, consistent with the generation of adaptive anti-tumor immunity [9]. The combination of this therapy with immune checkpoint inhibition (hu14.18-IL2 + RT + anti-CTLA4) further amplified anti-tumor responses and resulted in greater tumor regression and improved animal survival when compared to IC, RT or anti-CTLA-4 given alone, or dual combinations of: [1] RT + IC, [2] RT + anti-CTLA-4, or [3] IC + anti-CTLA-4 [9]

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