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Abstract: Introduction: Immunotherapy is emerging as an efficacious treatment for some cancers, complementing traditional chemo-radiation therapies. Specific markers at the cell surface of cancer cells can be used as immunotherapy targets. However, many of these markers are defined by a patient’s genetic background, limiting their use across the human population. Methods: Here, we investigated the non-polymorphic antigen presenting molecule, CD1c, that is only expressed on subsets of mature hematopoietic cells, as a potential immunotherapy target with reduced risk of off-tumor on-target toxicity in healthy tissues. Results and discussion: We identified a T cell receptor (TCR) which recognises CD1c in a lipid independent manner and determined the crystal structure of the TCR-CD1c complex which revealed flexibility around the lipid binding region, and a new binding mechanism of auto-antigen recognition. We generated affinity enhanced variants of the TCR and fused them to an anti-CD3 antibody for T cell redirection. Lipidomic analysis revealed promiscuous lipid recognition of CD1c by the affinity enhanced TCR variants, with preference for larger lipid head group, a finding which is supported by the crystal structure. The bispecific molecule induced potent re-directed T cell killing of CD1c positive cell lines. These proof-of-concept findings demonstrate that CD1c targeting TCR bispecific engagers might be good candidates for the development of non-MHC restricted, universal therapeutics for the treatment of CD1c+ leukemias.

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Abstract: Introduction: Antibody Fc regions harbour the binding sites for receptors that mediate effector functions following antigen engagement by the Fab regions. An extended “hinge” region in IgG allows flexibility between Fab and Fc, but in both the most primitive antibody, IgM, and in the evolutionarily more recent IgE, the hinge is replaced by an additional domain pair in the homodimeric six-domain Fc region. This permits additional flexibility within the Fc region, which has been exploited by nature to modulate antibody effector functions. Thus, in pentameric or hexameric IgM, the Fc regions appear to adopt a planar conformation in solution until antigen binding causes a conformational change and exposes the complement binding sites. In contrast, IgE-Fc principally adopts an acutely bent conformation in solution, but the binding of different receptors is controlled by the degree of bending, and there is allosteric communication between receptor binding sites. Methods: We sought to trace the evolution of Fc conformational diversity from IgM to IgE via the intermediate avian IgY by studying the solution conformations of their Fc regions by small-angle X-ray scattering. We compared four extant proteins: human IgM-Fc homodimer, chicken IgY-Fc, platypus IgE-Fc, and human IgE-Fc. These are examples of proteins that first appeared in the jawed fish [425 million years ago (mya)], tetrapod (310 mya), monotreme (166 mya), and hominid (2.5 mya) clades, respectively. Results and discussion: We analysed the scattering curves in terms of contributions from a pool of variously bent models chosen by a non-negative linear least-squares algorithm and found that the four proteins form a series in which the proportion of acutely bent material increases: IgM-Fc < IgY-Fc < plIgE-Fc < huIgE-Fc. This follows their order of appearance in evolution. For the huIgM-Fc homodimer, although none are acutely bent, and a significant fraction of the protein is sufficiently bent to expose the C1q-binding site, it predominantly adopts a fully extended conformation. In contrast, huIgE-Fc is found principally to be acutely bent, as expected from earlier studies. IgY-Fc, in this first structural analysis of the complete Fc region, exhibits an ensemble of conformations from acutely bent to fully extended, reflecting IgY’s position as an evolutionary intermediate between IgM and IgE.

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Abstract: Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

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Abstract: Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

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Abstract: Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.