Remodeling of the Tumor Microenvironment by a Chemokine/Anti-PD-L1 Nanobody Fusion Protein.

An optimal response to immune checkpoint blockade requires the presence of effector cells in the tumor microenvironment. We designed a PD-L1-targeted delivery strategy for chemokines, key molecules that drive leukocyte trafficking, to the tumor microenvironment, as a means of attracting the relevant leukocyte populations. This strategy combines a PD-L1-blocking single-domain antibody fragment (nanobody or VHH), a charge-engineered chemokine CCL21, and its subsequent characterization in a microfluidic device that resembles the tumor microenvironment. We show that the PD-L1-blocking VHH is a reliable fusion partner for the preparation of a functional chemokine fusion. Engineering the surface charge of CCL21 reduced its nonspecific binding to glycosaminoglycans, a property of chemokines that complicates their targeted delivery. Using a microfluidic assay, we show that it is possible to deliver a chemokine-VHH fusion to a PD-L1-positive environment and recruit effector cells.

Targeted antigen delivery by an anti-class II MHC VHH elicits focused αMUC1(Tn) immunity.

Unusual patterns of glycosylation on the surface of transformed cells contribute to immune modulation and metastasis of malignant tumors. Active immunization against them requires effective antigen presentation, which is complicated by a lack of access to tumor-specific posttranslational modifications through standard genetic approaches and by the low efficiency of passive antigen sampling. We found that antigen targeted to antigen presenting cells via class II MHC products can elicit a robust immune response against MUC1(Tn) bearing a defined tumor-associated glycoform, Tn. The two-component vaccine construct was prepared by sortase-mediated protein ligation of a synthetic MUC1(Tn) fragment to a class II MHC-binding single-domain antibody fragment (VHH7) as targeting moiety. We show that VHH7 targets antigen presenting cells in vivo, and when conjugated to MUC1(Tn) can elicit a strong αMUC1(Tn) immune response in mice. The resulting sera preferentially recognized the MUC1 epitope with the tumor-associated carbohydrate antigen Tn and were capable of killing cancer cells in a complement-mediated cytotoxicity assay. Immunoglobulin isotype analysis and cytokine release assays suggested a favorable Th1 response. A single boost 12 months after primary immunization triggered a recall response of the same quality, suggesting that long-term αMUC1(Tn) memory had been achieved.

Structurally Defined αMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma.

Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single-domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC-II and rendered "sortase-ready" for the introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.