A multimodal assay identifies optimized binders for a bicistronic CAR against GPC2 OR CD276 to overcome heterogeneity in Neuroblastoma
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA752052 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15341776.fastq.gz | Fastqsanger.gz | |||
| SRR15341777.fastq.gz | Fastqsanger.gz | |||
| SRR15341778.fastq.gz | Fastqsanger.gz | |||
| SRR15341779.fastq.gz | Fastqsanger.gz | |||
| SRR15341780.fastq.gz | Fastqsanger.gz |
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Project description:Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and remains deadly in patients with high-risk disease. Single chimeric antigen receptor T-cell therapies (CAR-T) for solid tumor have shown poor efficacy in clinical trials due, in part, to T cell exhaustion and uneven expression of target antigens in tumors. Here, we attempted to target Glypican-2(GPC2) and CD276(B7-H3), both highly but heterogeneously expressed on NB cell surface, to overcome these challenges in single CAR T- therapies. First, to identify optimal binders for CAR T- cell targeting each antigen, we made individual CAR constructs using multiple binders against these antigens and utilized a multimodal approach including simultaneous protein and transcriptome measurement at single cell level to characterize each individual CAR T- cell in a pooled strategy. Combined with cell expansion and cytotoxicity assays, we identified the most effective CAR construct for each target.
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Project description:Poor tumor trafficking and the immunosuppressive tumor microenvironment (TME) limit chimeric antigen receptor (CAR) T cell efficacy in solid tumors, such as neuroblastoma. We previously optimized GPC2 CARs in human neuroblastoma xenografts leading to clinical translation, however, there have not been preclinical studies using immunocompetent models. Thus, here we generated murine GPC2 CAR T cells using the D3-GPC2-targeting single-chain variable fragment being utilized clinically (NCT05650749) and tested them in neuroblastoma syngeneic allografts. Immune profiling of GPC2 CAR T cell-treated tumors revealed significant reprogramming of the TME, most notably poor intra-tumor CAR T cell persistence being associated with increased recruitment of myeloid-derived suppressor cells (MDSCs), along with MDSC-recruiting CXCL1/2 chemokines. These tumor-infiltrating MDSCs directly inhibited GPC2 CAR T cell activation and proliferation ex vivo. To both capitalize on this chemokine gradient and mitigate MDSC-tumor trafficking, we engineered GPC2 CAR T cells to express the CXCL1/2 receptor, CXCR2. CXCR2-armored GPC2 CAR T cells migrated towards CXCL1/2 gradients, enhanced anti-neuroblastoma efficacy, and reduced the level of MDSCs in the TME. Together, these findings suggest CAR T cell studies in immunocompetent models are imperative to define mechanisms of solid tumor immune escape and rationally design armoring strategies that will lead to durable clinical efficacy.
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Project description:Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.
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Project description:Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients with high-risk features, such as MYCN aberrations, and relapsed/refractory disease have poor clinical outcomes. This underscores the urgent need for new therapies. Glypican-2 (GPC2) is a MYCN-regulated and recently discovered oncofetal antigen. Given that GPC2 is also expressed in brain tumors, we evaluated the preclinical activity of our GPC2-CAR (CT3-CD28HTM-BBζ) against MB and compared it to two existing CARs targeting GD2 and B7-H3. A newly generated patient-derived xenograft (PDX) MAF1433, with MYCN aberrations, were used to test CAR T-cells in vivo. Single-cell RNA-sequencing were used for mechanistic studies. MB patient samples express intermediate to high levels of GPC2 and can be targeted with a GPC2-CAR, leading to significant in vivo tumor regression in orthotopic tumor models. GPC2-CAR T-cells had equivalent activity to the B7-H3-CAR and enhanced activity compared to the GD2-CAR in vivo. T-cell kinetic studies revealed that GPC2-CAR T-cells home to the TME, expand, and upregulate genes critical for cytotoxicity and T-cell homing to induce MB cell death. CT3-CD28HTM-BBζ GPC2-CAR can regress GPC2+ MB with MYCN aberrations in preclinical studies, providing a preclinical rationale for including children with GPC2+ MB in our upcoming clinical GPC2-CAR T-cell trial.
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Project description:70-100% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7-H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.
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