Golcadomide Extends Survival of Lymphoma GEMM Mice in Combination with a CD20xCD3 T-Cell Bispecific through Increased T-Cell Activation and Infiltration

Background: Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease of malignant B cells with accelerated growth kinetics and known to suppress immunity in the tumor microenvironment (TME) through a variety of mechanisms. Understanding the mechanisms of this immunosuppressive TME is critical to developing additional effective therapies. Golcadomide (golca), a CRBN modulator which degrades transcription factors Aiolos and Ikaros, is being developed for the treatment of R/R DLBCL in combination with CD20 targeting T cell engagers (TCE). Herein, we utilized a novel in vivo disease model to characterize the effects of golca on immune TME by combining multiplex immunofluorescence (mIF) and spatial transcriptomics (ST) to analyze the interaction and function of cells in TME to support the combination strategy for golca and TCE.

Methods: We developed an in vivo mouse model of DLBCL using eµ-myc mice backcrossed with hCRBN mice by adoptive transfer of eµ-myc/hCRBN splenocytes into recipient hCRBN mice. Mice were treated with the vehicle or golca from day 5 to 8. On day 8, spleen was harvested and analyzed for ST using Visium 10X system as well as by mIF. In the ST data, cell fractions were inferred following spot deconvolution with BayesPrism, spots were clustered using gene expression with Seurat, and gene-set scores were obtained with ssGSEA. In a subsequent set of experiments, mice were treated with isotype or TCE alone or in combination with golca. In combination studies, efficacy was measured as survival benefits. Additionally, peripheral blood and splenic tissue were harvested and assessed by mIF and flow cytometry in satellite cohorts to measure pharmacodynamic effects of the treatments.

Results: Eµ-myc/hCRBN transplanted mice developed large B cell lymphoma like disease in spleen. Spleens from diseased mice revealed that vehicle treated spleens contained increased tumor cells compared to golca treated spleens. These observations were qualified with H&E and mIF staining. ST analysis further identified 14 clusters based on spot-level gene expression data. These clusters were evaluated in the context of the cell fractions obtained by spot deconvolution. Two clusters of interest were prioritized for further analysis; cluster 5 (C5) was enriched for cycling B cells and eµ-myc tumor cells and cluster 6 (C6) was enriched for cytolytic T and NK cells. The relative fraction of C5 is the highest in vehicle group and is decreased in response to golca treatment, validating prior observations of direct anti-tumor activity of golca. Conversely, C6 abundance is enhanced in mice treated with golca compared to vehicle controls, indicating increased levels of CD8+ T cells through proliferation or trafficking. We next examined pathways more abundantly expressed in tumor rich areas, which included myc targets and G2M checkpoint signaling. The expression of genes associated with these pathways were decreased in golca treated mice compared to vehicle. The reduction of the tumor-associated pathway expression patterns by golca suggests a reversion of disease toward a normal state.

Based on these observations, we examined the ability of golca to enhance TCE effects with a murine CD20xCD3 TCE in our in vivo model. Flow cytometric analysis of day 11 peripheral blood and spleens demonstrated that both the TCE and golca alone and in combination decreased naive T-cells and increased central/effector memory populations compared to isotype/vehicle controls. mIF performed on diseased spleens demonstrated increased abundance of T cells and caspase 3 expression, decreased Pax5+ tumor cells in the combination arm compared to single agents. The combination treatment also decreased splenic hypertrophy compared to single agents and vehicle controls. Finally, compared to isotype control, the TCE monotherapy extended survival of the mice by an additional week (21 days vs 14 days) with the combination of golca/TCE further extending survival by two additional days.

Conclusion: We utilized a hCRBN/eµ-myc mouse model to demonstrate that the combination of golca with a CD20XCD3 TCE can enhance T-cell activation, cytotoxic capacity and extend survival compared to single agents. ST and mIF analysis of immune components within the TME provide mechanistic evidence of the dual mechanism of action for golca (direct anti-tumor activity and enhanced immune activity) further supporting this combination in on-going clinical trials.