Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies.

The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells has shown satisfactory results in preclinical cancer models. Even so, the approach's translation into the clinic will require incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE), consisting of an EGFR-specific single-domain VHH antibody fused to a CD3-specific scFv. We generated two LiTEs with the anti-EGFR VHH and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers are, therefore, suitable for future applications in gene-based immunotherapy approaches.

Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.

Efficient production of single-chain fragment variable-based N-terminal trimerbodies in Pichia pastoris.

BACKGROUND: Recombinant antibodies are highly successful in many different pathological conditions and currently enjoy overwhelming recognition of their potential. There are a wide variety of protein expression systems available, but almost all therapeutic antibodies are produced in mammalian cell lines, which mimic human glycosylation. The production of clinical-grade antibodies in mammalian cells is, however, extremely expensive. Compared to mammalian systems, protein production in yeast strains such as Pichia pastoris, is simpler, faster and usually results in higher yields. RESULTS: In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for the human carcinoembryonic antigen (CEA), was expressed in human embryonic kidney 293 cells and in Pichia pastoris. Mammalian- and yeast-produced anti-CEA trimerbody molecules display similar functional and structural properties, yet, the yield of trimerbody expressed in P. pastoris is about 20-fold higher than in human cells. CONCLUSIONS: P. pastoris is an efficient expression system for multivalent trimerbody molecules, suitable for their commercial production.

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity.

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy.

The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies.

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.

The coming of age of engineered multivalent antibodies.

The development of monoclonal antibody (mAb) technology has had a profound impact on medicine. The therapeutic use of first-generation mAb achieved considerable success in the treatment of major diseases, including cancer, inflammation, autoimmune, cardiovascular, and infectious diseases. Next-generation antibodies have been engineered to further increase potency, improve the safety profile and acquire non-natural properties, and constitute a thriving area of mAb research and development. Currently, a variety of alternative antibody formats with modified architectures have been generated and are moving fast into the clinic. In fact, the bispecific antibody blinatumomab was the first in its class to be approved by the US Food and Drug Administration (FDA) as recently as December 2014. Here, we outline the fundamental strategies used for designing the next generation of therapeutic antibodies, as well as the most relevant results obtained in preclinical studies and clinical trials.

Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications.

Gene therapy to achieve in vivo secretion of recombinant anti-CD3 x anti-tumor bispecific antibodies in cancer patients is being explored as a strategy to counterbalance rapid renal elimination, thereby sustaining levels of bispecific antibodies in the therapeutic range. Here, we performed a comparative analysis between single- and two-chain configurations for anti-CD3 x anti-CEA (carcinoembryonic antigen) bispecific antibodies secreted by genetically-modified human cells. We demonstrate that tandem single-chain variable fragment (scFv) antibodies and two-chain diabodies are expressed as soluble secreted proteins with similar yields. However, we found significant differences in their biological functionality (i.e., antigen binding) and in their ability to induce non-specific T cell activation. Whereas single-chain tandem scFvs induced human T cell activation and proliferation in an antigen-independent manner, secreted two-chain diabodies exerted almost no proliferative stimulus when human T cells were cultured alone or in co-cultures with CEA negative cells. Thus, our data suggest that two-chain diabodies are preferable to single-chain tandem scFvs for immunotherapeutic strategies comprising in vivo secretion of bispecific antibodies aiming to recruit and activate anticancer specific lymphocytic effector T cells.

Immunotherapeutic organoids: a new approach to cancer treatment.

Therapeutic monoclonal antibodies have revolutionized the treatment of cancer and other diseases. However, several limitations of antibody-based treatments, such as the cost of therapy and the achievement of sustained plasma levels, should be still addressed for their widespread use as therapeutics. The use of cell and gene transfer methods offers additional benefits by producing a continuous release of the antibody with syngenic glycosylation patterns, which makes the antibody potentially less immunogenic. In vivo secretion of therapeutic antibodies by viral vector delivery or ex vivo gene modified long-lived autologous or allogeneic human mesenchymal stem cells may advantageously replace repeated injection of clinical-grade antibodies. Gene-modified autologous mesenchymal stem cells can be delivered subcutaneously embedded in a non-immunogenic synthetic extracellular matrix-based scaffold that guarantees the survival of the cell inoculum. The scaffold would keep cells at the implantation site, with the therapeutic protein acting at distance (immunotherapeutic organoid), and could be retrieved once the therapeutic effect is fulfilled. In the present review we highlight the practical importance of living cell factories for in vivo secretion of recombinant antibodies.

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors.

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CAR(v2)) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CAR(v2) fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.Molecular Therapy-Nucleic Acids (2013) 2, e93; doi:10.1038/mtna.2013.19; published online 21 May 2013.