Immune checkpoints are emerging as novel targets for cancer therapy, and antibodies against them have shown remarkable clinical efficacy with potential for combination treatments to achieve high therapeutic index. This work aims at providing a novel approach for the generation of several novel human immunomodulatory antibodies capable of binding their targets in their native conformation and useful for therapeutic applications.We performed a massive parallel screening of phage libraries by using for the first time activated human lymphocytes to generate large collections of single-chain variable fragments (scFvs) against 10 different immune checkpoints: LAG-3, PD-L1, PD-1, TIM3, BTLA, TIGIT, OX40, 4-1BB, CD27 and ICOS. By next-generation sequencing and bioinformatics analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment.As a proof of concept of the quality/potency of the binders identified by this approach, human IgGs from three of these collections (i.e., PD-1, PD-L1 and LAG-3) were generated and shown to have comparable or better binding affinity and biological activity than the clinically validated anti-PD-1 mAb nivolumab.The repertoires generated in this work represent a convenient source of agonistic or antagonistic antibodies against the ‘Checkpoint Immunome’ for preclinical screening and clinical implementation of optimized treatments.

The novel antibody-based immunotherapy in oncology exploits the activation of immune system mediated by immunomodulatory antibodies specific for immune checkpoints. Among them, the programmed death ligand-1 (PD-L1) is of particular interest as it is expressed not only on T-cells, but also on other immune cells and on a large variety of cancer cells, such as breast cancer cells, considering its high expression in both ErbB2-positive and Triple Negative Breast Cancers. We demonstrate here that PD-L1_1, a novel anti-PD-L1 T -cell stimulating antibody, inhibits PD-L1-tumor cell growth also by affecting the intracellular MAPK pathway and by activating caspase 3. Similar in vitro results were obtained for the first time here also with the clinically validated anti-PD-L1 mAb Atezolizumab and in vivo with another validated anti-mouse anti-PD-L1 mAb. Moreover, we found that two high affinity variants of PD-L1_1 inhibited tumor cell viability more efficiently than the parental PD-L1_1 by affecting the same MAPK pathways with a more potent effect. Altogether, these results shed light on the role of PD-L1 in cancer cells and suggest that PD-L1_1 and its high affinity variants could become powerful antitumor weapons to be used alone or in combination with other drugs such as the anti-ErbB2 cAb already successfully tested in in vitro combinatorial treatments.

Monoclonal antibodies have been approved by the Food and Drug Administration for the treatment of various human cancers. More recently, oligonucleotide aptamers have risen increasing attention for cancer therapy thanks to their low size (efficient tumor penetration) and lack of immunogenicity, even though the short half-life and lack of effector functions still hinder their clinical applications. Here, we demonstrate, for the first time, that two novel bispecific conjugates, consisting of an anti-epidermal growth factor receptor (EGFR) aptamer linked either with an anti-epidermal growth factor receptor 2 (ErbB2) compact antibody or with an immunomodulatory (anti-PD-L1) antibody, were easily and rapidly obtained. These novel aptamer–antibody conjugates retain the targeting ability of both the parental moieties and acquire a more potent cancer cell killing activity by combining their inhibitory properties. Furthermore, the conjugation of the anti-EGFR aptamer with the immunomodulatory antibody allowed for the efficient redirection and activation of T cells against cancer cells, thus dramatically enhancing the cytotoxicity of the two conjugated partners. We think that these bispecific antibody–aptamer conjugates could have optimal biological features for therapeutic applications, such as increased specificity for tumor cells expressing both targets and improved pharmacokinetic and pharmacodynamic properties due to the combined advantages of the aptamer and antibody.