Tri-specific tribodies targeting 5T4, CD3, and immune checkpoint drive stronger functional T-cell responses than combinations of antibody therapeutics.

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.

Fast-tracking antibody maturation using a B cell-based display system.

Affinity maturation, an essential component of antibody engineering, is crucial for developing therapeutic antibodies. Cell display system coupled with somatic hypermutation (SHM) initiated by activation-induced cytidine deaminase (AID) is a commonly used technique for affinity maturation. AID introduces targeted DNA lesions into hotspots of immunoglobulin (Ig) gene loci followed by erroneous DNA repair, leading to biased mutations in the complementary determining regions.

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

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.

Novel Bi-Specific Immuno-Modulatory Tribodies Potentiate T Cell Activation and Increase Anti-Tumor Efficacy.

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.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line.

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay.

Influenza A whole virion vaccine induces a rapid reduction of peripheral blood leukocytes via interferon-α-dependent apoptosis.

Infection with single strand RNA (ssRNA) viruses, such as influenza A virus, is known to induce protective acquired immune responses, including the production of neutralizing antibodies. Vaccination also causes a reduction in the number of peripheral blood leukocytes (PBL) shortly after inoculation, a result which may have undesirable adverse effects. The cellular mechanisms for this response have not been elucidated so far.

HIV-1 Nef impairs multiple T-cell functions in antigen-specific immune response in mice.

The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef.

Modulation of immunoglobulin gene conversion frequency and distribution by the histone deacetylase HDAC2 in chicken DT40.

Modifications of histones are reportedly associated with the regulation of immunoglobulin (Ig) gene diversification mechanisms, but the extent of their involvement in promoting sequence alterations at the Ig variable (V) regions still remains to be elucidated. We have previously demonstrated that Ig gene conversion in the B cell line DT40 is accompanied by the local hyperacetylation of histones, and that its frequency is highly increased in cells treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). In this report, we describe the enhancing effects of the homozygous deletion of HDAC2 (HDAC2-/-) on Ig gene conversion.

Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses.

The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine.

An ex vivo method for rapid generation of monoclonal antibodies (ADLib system).

Here, we describe a protocol for using the ADLib (Autonomously Diversifying Library) system to rapidly generate specific monoclonal antibodies using DT40, a chicken B-cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci. We previously developed the ADLib system on the basis of our finding that gene conversion in DT40 cells was enhanced by treatment of the cells with a histone deacetylase inhibitor, trichostatin A (TSA). TSA treatment evolves a diversified library of DT40 cells (ADLib), in which each cell has different surface IgM specificity.

Antigen-receptor genes of the agnathan lamprey are assembled by a process involving copy choice.

Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs.

Novel role of the Ras cascade in memory B cell response.

Engagement of the B cell antigen receptor (BCR) triggers the Ras cascade, but the biological role of the latter in B cell response is unknown. Here, we report that in T cell-dependent response, the role of the Ras cascade is confined to memory B cells and possibly marginal zone B cells. When Ras-dependent BCR signaling was impaired, the generation of IgG germinal center B cells was unaffected but the recruitment of high-affinity cells into the memory compartment and terminal differentiation were inhibited.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S).

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice.

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant.