Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo

Abstract

Background Immunotherapy based on Bi-specific T Cell Engagers (TCE) represents one of the most attractive strategy to treat cancers resistant to conventional therapies. TCE are antibody-like proteins that simultaneously bind with one arm to a Tumor Associated Antigen (TAA) on cancer cells and with the other one to CD3 complex on a T-cell to form a TCR-independent immune synapse and circumvent Human Leucocyte Antigen restriction. Among them, the tribodies, such as Tb535H, a bi-specific molecule, made up of a Fab and a scFv domain both targeting 5T4 and another scFv targeting CD3, have demonstrated anti-tumor efficacy in preclinical studies. Methods Here, we generated five novel tri-specific and multi-functional tribodies, called 53X tribodies, composed of a 5T4 binding Fab arm and a CD3 binding scFv, but differently from the parental Tb535H, they contain an additional scFv derived from an antibody specific for an immune checkpoint, such as PD-1, PD-L1 or LAG-3. Results Compared with the parental Tb535H bi-specific T cell engager targeting 5T4, the novel 53X tribodies retained similar binding properties of Tb535H tribody, but showed enhanced anti-tumor potency due to the incorporation of the checkpoint inhibitory moiety. In particular, one of them, called 53L10, a tri-specific T cell engager targeting 5T4, CD3 and PD-L1, showed the most promising anti-tumor efficacy in vitro and led to complete tumor regression in vivo. Conclusions The novel tribodies have the potential to become strong and safe therapeutic drugs, allowing to reduce also the cost of production as one single molecule contains three different specificities including the anti-TAA, anti-CD3 and anti-IC binding arms.

A Comparison of the Antitumor Efficacy of Novel Multi-Specific Tribodies with Combinations of Approved Immunomodulatory Antibodies

“…This goal can be achieved with the construction of bispecific antibodies (BsAbs). Most developed BsAbs bind with an arm to a tumor-specific antigen and, with the second one, bind to immune cell receptors such as CD3 or CD16 to stimulate the immune response against cancer cells [23,24]. Three BsAbs are currently clinically approved and marketed, and more than 85 clinical trials targeting different tumor-associated antigens (TAAs) are in progress, demonstrating the validity of this approach.…”

“…Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors using a Greiner Leucosep ® tube (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's instructions, as previously reported [16,19,[23][24][25], and frozen in 90% FBS plus 10% dimethyl sulfoxide (DMSO) solution until use. Cryopreserved cell vials were gently thawed using RPMI 1640 medium supplemented with 1% L-glutamine, 1% CTLWash™ (Immunospot by Cellular Technology Limited, Shaker Heights, Cleveland, OH, USA) and 100 U/mL Benzonase (Merck Millipore, Darmdstadt, Germany) and washed via centrifugation.…”

Comparative Analysis of a Human Neutralizing mAb Specific for SARS-CoV-2 Spike-RBD with Cilgavimab and Tixagevimab for the Efficacy on the Omicron Variant in Neutralizing and Detection Assays

“…After washes, D3, Tixagevimab, Cilgavimab or the human anti-Spike-RBD commercial Ab (used as positive control) was added in parallel at a concentration of 30 nM, and the plates were incubated for 2 h at RT. Then, the plates were washed and the signals detected by using an anti-Fab HRP-conjugated antibody, as previously described [ 32 , 33 , 34 , 35 ].…”

Stepping forward: T-cell redirecting bispecific antibodies in cancer therapy

“…Some proteins with significant expression differences in solid tumors are intracellular proteins, but traditional antibodies cannot penetrate the cells 26 , 27 . These intracellular proteins require recognition through HLA class I molecules, such as ImmTACs or TCR-like antibodies 28 , 29 , 30 . TCR-like CD3 BsAbs still exhibit on-target-off-tumor side effects in clinical trials.…”