Abstract
Simultaneous targeting of multiple tumor-associated antigens (TAAs) in cancer immunotherapy is presumed to enhance tumor cell selectivity and to reduce immune escape.The combination of B lymphoid marker CD19 and myeloid marker CD33 is exclusively present on biphenotypic B/myeloid leukemia cells. Triplebody 33-3-19 binds specifically to both of these TAAs and activates T cells as immune effectors. Thereby it induces specific lysis of established myeloid (MOLM13, THP-1) and B-lymphoid cell lines (BV173, SEM, Raji, ARH77) as well as of primary patient cells. EC50 values range from 3 pM to 2.4 nM. In accordance with our hypothesis, 33-3-19 is able to induce preferential lysis of double- rather than single-positive leukemia cells in a target cell mixture: CD19/CD33 double-positive BV173 cells were eliminated to a significantly greater extent than CD19 single-positive SEM cells (36.6% vs. 20.9% in 3 hours, p = 0.0048) in the presence of both cell lines. In contrast, equivalent elimination efficiencies were observed for both cell lines, when control triplebody 19-3-19 or a mixture of the bispecific single chain variable fragments 19-3 and 33-3 were used. This result highlights the potential of dual-targeting agents for efficient and selective immune-intervention in leukemia patients.
Highlights
In acute leukemia of ambiguous origin with B/ myeloid or trilineage phenotype and B-ALL or AML with aberrant antigen expression, the B lymphoid lineage marker CD19 and myeloid lineage marker CD33 are simultaneously displayed on the blast cell surface [1, 2]
The biological activity of 33-3-19, which was determined from its EC50-value in a standard redirected lysis (RDL) experiment against SEM target cells at different time points post production, weakened over time, in spite of stabilization attempts via a variety of formulation-buffers, disulfide stabilization and site-directed mutagenesis
We characterized a dualtargeting T cell-recruiting triplebody 33-3-19 that was designed for the selective lysis of CD19/CD33 doublepositive B/myeloid leukemia cells over CD19 singlepositive normal cells
Summary
In acute leukemia of ambiguous origin with B/ myeloid or trilineage phenotype (ca. 2 – 3% of all acute leukemias) and B-ALL or AML with aberrant antigen expression, the B lymphoid lineage marker CD19 and myeloid lineage marker CD33 are simultaneously displayed on the blast cell surface [1, 2]. There is no consensus regarding treatment protocols for mixed phenotype acute leukemias (MPAL) due to the rarity of these hematopoietic neoplasms and lack of clinical studies in this specific patient population [3, 5,6,7]. Both CD19 and CD33 are validated therapeutic targets. The co-expression of these two lineage markers may offer unique opportunities for selective, individualized immunotherapy with novel antibody-derived agents, because the leukemia cells are immunophenotypically distinct from the corresponding healthy cells. By targeting both tumor-associated antigens, i.e. CD19 and CD33, at the same time, selectivity of elimination may be achieved and immune escape will likely be reduced, because antigen double-negative leukemia cell clones are less likely to be selected than single-negative ones [8, 9]
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