Bi-specific antibody engagers for cancer immunotherapy.

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains ( VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

Controlling Antigen Fate in Therapeutic Cancer Vaccines by Targeting Dendritic Cell Receptors.

Antigen-presenting cells (APCs) orchestrate immune responses and are therefore of interest for the targeted delivery of therapeutic vaccines. Dendritic cells (DCs) are professional APCs that excel in presentation of exogenous antigens toward CD4+ T helper cells, as well as cytotoxic CD8+ T cells. DCs are highly heterogeneous and can be divided into subpopulations that differ in abundance, function, and phenotype, such as differential expression of endocytic receptor molecules. It is firmly established that targeting antigens to DC receptors enhances the efficacy of therapeutic vaccines. While most studies emphasize the importance of targeting a specific DC subset, we argue that the differential intracellular routing downstream of the targeted receptors within the DC subset should also be considered. Here, we review the mouse and human receptors studied as target for therapeutic vaccines, focusing on antibody and ligand conjugates and how their targeting affects antigen presentation. We aim to delineate how targeting distinct receptors affects antigen presentation and vaccine efficacy, which will guide target selection for future therapeutic vaccine development.

Efficient targeting of NY-ESO-1 tumor antigen to human cDC1s by lymphotactin results in cross-presentation and antigen-specific T cell expansion.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. METHODS: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. RESULTS: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. CONCLUSION: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Anti-CTLA-4 therapy requires an Fc domain for efficacy.

Ipilimumab, a monoclonal antibody that recognizes cytotoxic T lymphocyte antigen (CTLA)-4, was the first approved "checkpoint"-blocking anticancer therapy. In mouse tumor models, the response to antibodies against CTLA-4 depends entirely on expression of the Fcγ receptor (FcγR), which may facilitate antibody-dependent cellular phagocytosis, but the contribution of simple CTLA-4 blockade remains unknown. To understand the role of CTLA-4 blockade in the complete absence of Fc-dependent functions, we developed H11, a high-affinity alpaca heavy chain-only antibody fragment (VHH) against CTLA-4. The VHH H11 lacks an Fc portion, binds monovalently to CTLA-4, and inhibits interactions between CTLA-4 and its ligand by occluding the ligand-binding motif on CTLA-4 as shown crystallographically. We used H11 to visualize CTLA-4 expression in vivo using whole-animal immuno-PET, finding that surface-accessible CTLA-4 is largely confined to the tumor microenvironment. Despite this, H11-mediated CTLA-4 blockade has minimal effects on antitumor responses. Installation of the murine IgG2a constant region on H11 dramatically enhances its antitumor response. Coadministration of the monovalent H11 VHH blocks the efficacy of a full-sized therapeutic antibody. We were thus able to demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect.