T Cell Immunoprofiling of Patients with Relapsed and/or Refractory Myeloma Who Receive Daratumumab Monotherapy: Longitudinal Analysis during 7 Cycle Follow-up of the Rebuild Phase 2 Study

Preliminary evidence for T cell receptor (TR) repertoire renewal and increased TR clonality has been reported by our group (Vlachonikola et al., ASH 2019) in multiple myeloma (MM) patients (pts) receiving daratumumab monotherapy within the context of the REBUILD study, an ongoing prospective, multicenter, non-comparative, open-label, phase II study in pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Herein, we report the results from the longitudinal analysis of the TR repertoire employing next generation sequencing (NGS) and multi-color flow cytometry in 24 pts who completed 3 cycles (n=24) and 6 cycles (n=11/24) of daratumumab monotherapy, in order to assess the immunomodulatory effects of daratumumab.

We assessed 59 peripheral blood samples collected at screening (SCR, n=24), on Day 1 of Cycle 4 (C4, n=24) and Day 1 of Cycle 7 (C7, n=11). Patients were grouped based on best responses at C4 into responders (i.e. pts with partial response [PR, n=7] and very good PR [VGPR, n=8]), and non-responders (i.e. pts with minimal response [MR, n=2], stable disease [SD, n=5], or progressive disease [PD, n=2]). TRBV-TRBD-TRBJ gene rearrangements were subjected to paired-end NGS and raw reads (n=13,886,646 | median 239,969/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=6,324,986 | median 100,738/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 325,789 distinct clonotypes (median 4,535 clonotypes/sample) were analyzed. The TR repertoire displayed clonal T cell expansions in both groups (responders/non-responders) in all pre/post-treatment timepoints. Clonality increased after treatment for both responders and non-responders in all assessed timepoints, with statistical significance at C4 in both groups (median cumulative frequency of the 10 most expanded T cell clonotypes/sample in responders: 31.6% pre-treatment vs 43% C4 post-treatment, p=0.009; and, in non-responders: 19.8% pre-treatment vs 39.6% C4 post-treatment, p=0.009). In both groups, the clonotype repertoire appeared to be renewed. Interestingly, in the responders' group a significant shift was noticed in the major clonotype repertoire at screening vs C4. In particular, the 10 most expanded clonotypes/sample at C4 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment. Additionally, although the major post-treatment clonotype at C4 also dominated at C7 in most cases, certain lower frequency clonotypes at C4 emerged among the top-10 at C7. Thirteen shared clonotypes were identified amongst the post-treatment repertoires of different patients but not in other entities in public databases, raising the possibility that they may be "MM-specific" and selected by common MM-associated antigens. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3⁺ T cells (median frequency at SCR 63% vs 78.7% at C4 | p=0.0045 and 87.4% at C7 | p=0.0009), driven mostly by the expansion of the CD8⁺ T cell compartment (median frequency at SCR 31.4% vs 45.3% at C4 | p=0.0045 and 53.8% at C7 | p=0.001) in both groups.

In conclusion, we document T cell clonal expansions and clonal drift after daratumumab treatment in MM. Our results suggest that daratumumab acts through renewing the greatest part of the pre-treatment TR clonotype repertoire, suggesting dynamic changes of the T cell compartment under treatment, a claim also supported by the significant increase in CD8⁺ cytotoxic T cell numbers overtime. The significant post-treatment expansion of certain low-frequency pre-treatment clones in responders raises the intriguing hypothesis that daratumumab treatment may have led to the outgrowth of anti-MM T cell clones, arguably contributing to clinical response.