Dysfunctional Hyper-Expanded Clonotypes and Lack of TCR Clonal Replacement Predict Resistance to T Cell Engagers in Multiple Myeloma

T cell engagers (TCE) with dual specificities against CD3 and MM specific antigens (BCMA, GPRC5D and FcRL5) have demonstrated unprecedented efficacy in refractory patients. However, the cellular and molecular predictors of response are not fully defined as well as the mediators of resistance remain elusive. T cell trafficking to tumor sites with local T cell proliferation are prerequisites of responses to TCE. Therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of exhausted T cells coupled with lack of T cells homing to tumor sites may dictate clinical responses.

In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood (PB) and bone marrow (BM) T cells in responders (R) and non-responders (NR) MM patients treated with TCE. Using single cell paired TCR sequencing coupled with 5' scRNA and CITEseq we longitudinally tracked (pre- and post-TCE) the expansion of variable T cell subsets and analyzed their TCR repertoires (clonality and diversity) to identify the causes of resistance.

Methods: BM aspirates (n=29) with matched PB samples (n=21) were longitudinally collected from 15 patients treated with TCEs, prior and post initiation of therapy and at relapse. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein of sorted CD3⁺ T cells was performed (10x Genomics Single cell VDJ solution). Cell Ranger VDJ pipeline was used for sample de-multiplexing, barcode processing and grouping of T cells into clonotypes with shared TCR α/β sequences. TCR sequences were also coupled with scRNA 5' sequencing and CITEseq to track and characterize the T cells populations harboring these clonotypes. Further data processing, contigs filtering and clonotypes overlay was performed with Seurat, scRepertoire and Immunarch.

Results: The parallel measurement of transcripts and cell surface proteins of CD3⁺ T cells identified canonical CD8⁺ and CD4⁺ T cell subsets including precursor and terminally exhausted T cells within the BM and PB compartment. Longitudinal tracking of T cells using their CDR3 TCR sequences as barcodes revealed preferential expansion of cytotoxic CD8⁺ cells as opposed to CD4+ T cells post TCE therapy, corroborating the notion that TCE treatment preferentially expands cytotoxic CD8⁺ T cells. Within the CD8+ T compartment, T naive and memory CD8 cells were enriched in PB and BM of R while an higher proportion of exhausted T cells (Tex) was present in NR. To define T cells clonal dynamics in response to TCE, we mapped hyperexpanded (hyperExp) clonotypes (TCR > 0.01), predicted to be tumor reactive, to the different CD8 T cell clusters. These hyperExp clonotypes mapped to the Temra, memory, effector memory and Tex cells. Of interest, comparing R and NR, hyperExp clonotypes exclusively mapped to the dysfunctional (Tex.) T cells in NR patients, while mapping to effector memory and cytotoxic CD8 T cells in R. Analysis of TCR repertoires clonality and diversity also demonstrated a reduced richness and increased clonality of CD8 TCRs in R when compared to NR suggesting a non-random but rather selective expansion of tumor-reactive TCRs following TCE therapies. To this extent, in R we observed a dramatic clonotypic TCR expansion (10-40% of TCRs) of non-virally reactive clonotypes with known CDR3 reactivity to tumoral antigens (NY-ESO1, WT1, MLNA, BST2) in some. Functional validation of these expanded TCRs is ongoing. Lastly, clonal overlay analysis of CD8 hyperExp TCRs based on source (PB vs BM) revealed differential patterns of clonal expansion with hyperExp TCRs exclusively overlaying Tex in both the BM and PB compartments of NR while mapping to memory non-exhausted T cells (Tm/Tem) in the PB (and to lesser extent in the BM) of R. These findings are consistent with a TCE mediated clonal replacement of BM exhausted clonotypes by non-dysfunctional PB clonotypic cytotoxic CD8⁺ T cells in R.

Conclusions: Combined single cell features profiling with paired TCR sequencing of PB and BM T cells from MM patients receiving TCE therapies revealed in responders a selective expansion of clonotypic CD8 T cells with a clonal replacement of BM exhausted clonotypes by non-dysfunctional circulating CD8⁺ T cells. Dramatic expansion of non-virally reactive clonotypic T cells was also noted in responders consistent with epitope spreading and selective expansion of tumor reactive TCRs.

Neri:Janssen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Sanofi-Aventis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Raab:Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Bahlis:GSK: Consultancy, Other; Takeda: Consultancy; Karyopharm Therapeutics: Consultancy, Honoraria; Forus: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Genentech: Consultancy; AbbVie: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.