Gamma delta TCR anti-CD3 bispecific molecules (GABs) as novel immunotherapeutic compounds

Abstract

Backgroundγ9δ2 T cells hold great promise as cancer therapeutics because of their unique capability of reacting to metabolic changes with tumor cells. However, it has proven very difficult to translate this promise into clinical success.MethodsIn order to better utilize the tumor reactivity of γ9δ2T cells and combine this with the great potential of T cell engager molecules, we developed a novel bispecific molecule by linking the extracellular domains of tumor-reactive γ9δ2TCRs to a CD3-binding moiety, creating gamma delta TCR anti-CD3 bispecific molecules (GABs). GABs were tested in vitro and in vivo for ability to redirect T lymphocytes to a variety of tumor cell lines and primary patient material.ResultsGABs utilizing naturally occurring high affinity γ9δ2TCRs efficiently induced αβT cell mediated phosphoantigen-dependent recognition of tumor cells. Reactivity was substantially modulated by variations in the Vδ2 CDR3-region and the BTN2A1-binding HV4-region between CDR2 and CDR3 of the γ-chain was crucial for functionality. GABs redirected αβT cells against a broad range of hematopoietic and solid tumor cell lines and primary acute myeloid leukemia. Furthermore, they enhanced infiltration of immune cells in a 3D bone marrow niche and left healthy tissues intact, while eradicating primary multiple myeloma cells. Lastly, GABs constructed from natural high affinity γ9δ2TCR sequences significantly reduced tumor growth in vivo in a subcutaneous myeloma xenograft model.ConclusionsWe conclude that GABs allow for the introduction of metabolic targeting of cancer cells to the field of T cell engagers.

Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present-Future

“…LYT-210 is a mAb directed towards the Vδ1 + TCR with the aim of eliminating these pathogenic cells ( Table 2 ). Gamma-delta TCR bispecific molecules (GABs) combine the extracellular domain of the Vγ9Vδ2 TCR fused with a CD3 binding domain, allowing conventional T-cells to recognize the presence of pAg on tumor cells ( 108 ). In the presence of GABs, αβ T-cells recognized and killed the squamous cell carcinoma cell line SCC9 in a pAg dependent manner and produced increased amounts of IFNγ when exposed to patient-derived AML blasts but not with healthy hematopoietic cells indicating preferential recognition of tumor cells.…”

γ9δ2 T-Cell Expansion and Phenotypic Profile Are Reflected in the CDR3δ Repertoire of Healthy Adults

“…Here we expand the evidence on the role of CDR3δ in γ9δ2TCR-mediated responses. We show that in vivo γ9δ2T cell expansion and phenotypic maturation are associated with repertoire enrichment in the CDR3δ determinants of sensing pAg-induced changes ( 12 , 48 , 49 ), presumably equating to the concentration of the γ9δ2TCRs with higher binding affinity and signal strength ( 13 , 14 ). We find that the number of the highly common public TCRδ sequences that combine all known pAg reactivity features, known to diminish with the transition from infant to adult repertoires, diminishes even further within an individual upon T-cell maturation, despite optimal signaling.…”

“…Researchers exploring these areas should consider the fact that donors with the highest numbers of γ9δ2T cells in the periphery, which are likely to be selected for in vitro experiments, as it is easy to get sufficient cell numbers, will frequently harbor cells whose phenotype is largely skewed toward terminal differentiation and whose repertoire, in turn, will be skewed to near-oligoclonality, where potentially poorly pAg-reactive clonotypes may take prominent positions. Concerning the design of the high-affinity γ9δ2TCR-based therapies ( 14 , 24 ), CDR3δ features such as the AA5 and CDR3 length are essential, while J1 region usage might reflect an advantage of the shorter length. Lastly, as a high-affinity γ9δ2TCR will influence the net NKR signaling through upregulation of inhibitory receptors, it is worth modifying this second signal in favor of the activating arm.…”

“…Nevertheless, despite the unique antigen specificity and recognition mode, the γ9δ2TCR repertoire equally acquires imprints of selection and expansion throughout the human lifetime ( 7 , 8 ), starting with a wave of polyclonal expansion immediately after birth ( 9 , 10 ) and ultimately leading to a characteristic “selected,” highly private pAg-reactive γ9δ2TCR repertoire in adults [reviewed in ( 11 )]. While the TCRγ9 chain harbors motifs that are critical for binding the non-polymorphic BTN ligand, the TCRδ2 chain contains the yet unresolved clue to pAg reactivity ( 4 , 12 ), possibly through the influence of the CDR3δ on the γ9δ2TCR binding affinity in this multiple-ligand system ( 13 , 14 ). Therefore, the most dramatic repertoire changes are seen in the CDR3δ repertoire from T-cell generation to maturity.…”

“…In addition, the expression of certain NKRs has been associated with a CDR3δ bias ( 22 ). Considering the growing interest in γδT cells and their TCRs as therapeutics ( 23 ), and in reliance on a model in which the γ9δ2TCR diversity is assumed to underpin a range of ligand-binding affinities ( 13 , 14 ), the question of whether the repertoire changes are related to the phenotypic changes is particularly relevant, since it clears the way for isolating the TCRs with the highest affinity to the antigen and the T-cell populations most potent for therapeutic use ( 14 , 24 ). In this study, we deconstruct the adult CDR3δ repertoire in relation to the phenotype of the γ9δ2T cells in an attempt to understand the functional role of the γ9δ2TCR diversity in adults.…”