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.

One of the most promising cancer immunotherapies is based on bi-specific T-cell engagers (BiTEs) that simultaneously bind with one arm to a tumor-associated antigen on tumor cells and with the other one to CD3 complex on T cells to form a TCR-MHC independent immune synapse. We previously generated four novel tri-specific tribodies made up of a Fab targeting 5T4, an oncofetal tumor antigen expressed on several types of tumors, a scFv targeting CD3 on T cells, and an additional scFv specific for an immune checkpoint (IC), such as PD-1, PD-L1 or LAG-3. To verify their advantages over the combinations of BiTEs (CD3/TAA) with IC inhibitors, recently used to overcome tumor immunosuppressive environment, here we tested their functional properties in comparison with clinically validated mAbs targeting the same ICs, used alone or in combination with a control bi-specific devoid of immunomodulatory scFvs, called 53 P. We found that the novel tri-specific tribodies activated human peripheral blood mononuclear cells more efficiently than clinically validated mAbs (atezolizumab, pembrolizumab, and relatlimab) either used alone or in combination with 53 P, leading to a stronger tumor cytotoxicity and cytokines release. In particular, 53L10 tribody targeting PD-L1 displayed much more potent effects than the combination of 53 P with all the clinically validated mAbs and led to complete tumor regression in vivo, showing much higher efficacy than the combination of 53 P and atezolizumab. We shed light on the molecular basis of this potentiated anti-tumor activity by evidencing that the insertion of the anti-PD-L1 moiety in 53L10 led not only to stronger binding of the tri-specific to tumor cells but also efficiently blocked the effects of increased PD-L1 on tumor cells, induced by IFNγ secretion also due to T-cell activation. These results are important also for the design of novel T-cell engagers targeting other tumor antigens.

Cancer immunotherapy has already shown significant improvements by combining different antibodies specific for distinct immune checkpoints, such as Ipilimumab and Nivolumab. Here, we tested combinatorial treatments of immunomodulatory antibodies, previously generated in our laboratory, for their effects on hPBMC activation, either upon stimulation with SEB or in co-cultures with tumor cells by cytokine secretion assays. We found that some of them showed additive or synergistic effects, and on the basis of these observations, we constructed, for the first time, four novel bispecific tribodies (TR), made up of a Fab derived from one anti-IC mAb and two scFvs derived from another mAb targeting a different IC. All four TRs cotargeting either programmed cell death protein 1 (PD-1) and Lymphocyte Activating 3 (LAG-3) or programmed death-ligand 1 (PD-L1) and LAG-3 retained binding affinity for their targets and the antagonistic effects of their parental mAbs, but some of them also showed an increased ability to induce lymphocyte activation and increased in vitro cytotoxicity against tumor cells compared to parental antibodies used either alone or in combinatorial treatments. Furthermore, none of the tribodies showed significant increased cytotoxicity on human cardiomyocytes. Considering that the tribody format reduces production costs (as only one construct provides the inhibitory effects of two antibodies), has an intermediate molecular size (100 kDa) which is well suited for both tumor penetration and an acceptable half-life, we think that these novel immunomodulatory TRBs have the potential to become precious tools for therapeutic applications, particularly in monotherapy-resistant cancer patients.

Many advances in antitumor therapies have been achieved with antagonistic antibodies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1); however, many cancer patients still develop resistance to anti–PD-1/PD-L1 treatments often associated with the upregulation of other immune checkpoints such as Lymphocyte Activation Gene-3 (LAG-3). In order to verify whether it is possible to overcome these limits, we analyzed and compared the effects of combinations of the clinically validated anti-LAG-3 mAb (Relatlimab) with anti-PD-1 (Pembrolizumab) or anti-PD-L1 (Atezolizumab) monoclonal antibodies (mAbs) with those of novel bispecific tribodies (TRs), called TR0304 and TR0506, previously generated in our lab by combining the binding moieties of novel human antibodies targeting the same ICs of the mentioned mAbs. In particular, TR0304, made up of a Fab derived from an anti-PD-L1 mAb and two single-chain variable fragments (scFvs) derived from an anti-LAG-3 mAb, was tested in comparison with Relatlimab plus Atezolizumab, and TR0506, made up of an antigen-binding fragment (Fab) derived from the same anti-LAG-3 mAb and two scFvs derived from an anti-PD-1 mAb, was tested in comparison with Relatlimab and Pembrolizumab. We found that the two novel TRs showed similar binding affinity to the targets with respect to validated mAbs, even though they recognized distinct or only partially overlapping epitopes. When tested for their functional properties, they showed an increased ability to induce lymphocyte activation and stronger in vitro cytotoxicity against tumor cells compared to combinatorial treatments of clinically validated mAbs. Considering that tribodies also have other advantages with respect to combinatorial treatments, such as reduced production costs and lower dose requirements, we think that these novel immunomodulatory TRs could be used for therapeutic applications, particularly in monotherapy-resistant cancer patients.

Delta-like 1 homolog (DLK1), a non-canonical Notch ligand, is highly expressed in various malignant tumors, especially in hepatocellular carcinoma (HCC). CBA-1205 is an afucosylated humanized antibody against DLK1 with enhanced antibody-dependent cellular cytotoxicity (ADCC). The binding characteristics of CBA-1205 were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting assay. The ADCC activity of CBA-1205 was assessed. The anti-tumor efficacy of CBA-1205 was evaluated in xenograft mouse models, and toxicity and toxicokinetic profiles of CBA-1205 were evaluated in cynomolgus monkeys. CBA-1205 selectively bound to DLK1 among the Notch ligands and only to monkey and human DLK1. The binding epitope was between epidermal growth factor-like domains 1 and 2 of DLK1, which are not involved in any known physiological functions. The ADCC activity of CBA-1205 was confirmed using human peripheral blood mononuclear cells as effector cells. CBA-1205 as a single agent and in combination with lenvatinib demonstrated long-lasting anti-tumor efficacy, including tumor regression, in two liver cancer xenograft models. The toxicity and toxicokinetic profiles of CBA-1205 in cynomolgus monkeys were favorable. These findings suggest that CBA-1205 has the potential to be a useful therapeutic option for drug treatment in HCC. A phase 1 study is ongoing in patients with advanced cancers (jRCT2080225288, NCT06636435).

Delta-like 1 homolog (DLK1), a non-canonical Notch ligand, is highly expressed in various malignant tumors, especially in hepatocellular carcinoma (HCC). CBA-1205 is an afucosylated humanized antibody against DLK1 with enhanced antibody-dependent cellular cytotoxicity (ADCC). The binding characteristics of CBA-1205 were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting assay. The ADCC activity of CBA-1205 was assessed. The anti-tumor efficacy of CBA-1205 was evaluated in xenograft mouse models, and toxicity and toxicokinetic profiles of CBA-1205 were evaluated in cynomolgus monkeys. CBA-1205 selectively bound to DLK1 among the Notch ligands and only to monkey and human DLK1. The binding epitope was between epidermal growth factor-like domains 1 and 2 of DLK1, which are not involved in any known physiological functions. The ADCC activity of CBA-1205 was confirmed using human peripheral blood mononuclear cells as effector cells. CBA-1205 as a single-agent and in combination with lenvatinib demonstrated long-lasting anti-tumor efficacy, including tumor regression, in two liver cancer xenograft models. The toxicity and toxicokinetic profiles of CBA-1205 in cynomolgus monkeys were favorable. These findings suggest that CBA-1205 has the potential to be a useful therapeutic option for drug treatment in HCC. A phase 1 study is ongoing in patients with advanced cancers (jRCT2080225288).