Titolo della tesi: B7-H3 redirected CAR T-cells for treatment of Medulloblastoma
Introduction:
Medulloblastoma (MB) is the most common malignant brain tumor of childhood, accounting for 20% of all pediatric brain malignancies.
MB is currently treated with multimodal therapy consisting of surgical resection of the tumor, chemotherapy, and cranio-spinal radiation; however, despite these aggressive treatments, the outcome of patients is still dismal (approximately 30% of patients still succumb to this disease) with long-term side effects that dramatically impacts on their quality of life.
For this reason, more effective and less toxic therapeutic strategies are needed. The identification of tumor-associated antigens (TAAs), such as B7-H3 (CD276), has led to the development of targeted cancer therapy such as Chimeric Antigen Receptor (CAR)-T cell therapy. B7-H3 [B7-homolog 3 or CD276] B7-H3 is a type I transmembrane protein, overexpressed in both hematologic and solid tumors, including pediatric CNS tumors such as Medulloblastoma. On the other hand, healthy tissues showed limited expression of B7-H3 making it an ideal candidate for CAR T cell therapy approaches.
Aim:
In our project we propose to develop an adoptive CAR-T cell-based immunotherapeutic approach targeting the B7-H3 (CD276) MB-associated antigen.
To this end, the following aims were pursued:
- Evaluation of B7-H3 expression in MB subgroups and its correlation with survival rate of patients
- Cloning two 3rd generation CAR targeting the B7-H3 antigen (B7-H3.CAR) carrying either CD28/41BB or CD28/OX40 as costimulatory domains (CAR.B7-H3.28.41BBζ and CAR.B7-H3.28.OX40ζ).
- Evaluation of in vitro functionality of both CAR.B7-H3.28.41BBζ and CAR.B7-H3.28.OX40ζ against MB tumor cell lines and patient-derived xenograft (PDX).
- Development of xenograft orthotopic mice model to assess the in vivo anti-tumor activity of B7-H3.CAR T cells towards MB cells.
Results:
To assess whether B7-H3 could be a suitable target for CAR T-cell therapy approach in MB, we first carried out a bioinformatic analysis across public MB RNAseq datasets. B7-H3 mRNA expression was found significantly overexpressed in MB samples compared to normal brain and normal cerebellum, making it a suitable target for a CAR-T cell therapy.
Thus, we generated two different 3rd generation CAR targeting the B7-H3 antigen (B7-H3.CAR) carrying either CD28/41BB or CD28/OX40 as costimulatory domains (B7-H3.41BB and B7-H3.OX40).
The cytotoxic activity of B7-H3.CAR T cells was tested in vitro by co-culture assays. Both B7-H3.41BB and B7-H3.OX40 CAR T-cells showed a near complete elimination of either D283 or DAOY MB cell lines, after 5 days of co-culture at Effector :Target (E.T) =1:1 ratio.
To further investigate anti-tumor activity in vitro, CAR T-cells were seeded at decreasing E:T ratios (from 1:1 to 1:32 E:T ratio). Both B7-H3.41BB and B7-H3.OX40 CAR T-cells showed an excellent anti-tumor activity in all experimental conditions.
Next, we tested the ability for B7-H3.CAR T-cells to control orthotopic MB xenografts using MED411 PDX. B7-H3.OX40 exerted a better tumor control compared to both B7-H3.41BB CAR T-cells and NT T-cells, inducing a rapid decrease of tumor bioluminescence and a better overall survival (OS) of treated mice.
Based on these experimental data, we decided to validate the potency of the most promising construct B7-H3.OX40 CAR T-cell –– in another MB model using D283 cell line.
Bioluminescence in MB-tumor-bearing mice, treated with B7-H3.OX40 CAR T-cells significantly decreased 7 days after T-cells infusion. At day +60 (ending point of the previous experiment), 50% of mice survived in the control cohorts, while B7-H3.OX40 CAR T-cells confirmed its strong anti-tumor activity with 100% of mice alive at selected time point. At later time-points, two mice receiving B7-H3.OX40 CAR T-cells died without any apparent sign of sufferance or disease relapse. Indeed, B7-H3.OX40 CAR T-cells induced a significantly better disease free survival (DFS) at day +94 relative to NT T-cells (0% vs 100% for mice treated with NT and B7-H3.OX40).
Conclusions
Overall, the significant in vivo reactivity, the high potency, the negligible toxicity in animals and the long persistence of B7-H3.OX40 CAR T-cells contribute to the value of this CAR design that will be tested in a clinical trial for pediatric patients with MB.