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Combining immunogenic cell death-inducing chemotherapies and PD-1 blockade can generate remarkable tumor responses. It is now well established that TGF-β1 signaling is a major component of treatment resistance and contributes to the cancer-related immunosuppressive microenvironment. However, whether TGF-β1 remains an obstacle to immune checkpoint inhibitor efficacy when immunotherapy is combined with chemotherapy is still to be determined. Several syngeneic murine models were used to investigate the role of TGF-β1 neutralization on the combinations of immunogenic chemotherapy (FOLFOX: 5-fluorouracil and oxaliplatin) and anti-PD-1. Cancer-associated fibroblasts (CAF) and immune cells were isolated from CT26 and PancOH7 tumor-bearing mice treated with FOLFOX, anti-PD-1 ± anti-TGF-β1 for bulk and single cell RNA sequencing and characterization. We showed that TGF-β1 neutralization promotes the therapeutic efficacy of FOLFOX and anti-PD-1 combination and induces the recruitment of antigen-specific CD8

Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of both solid and hematological malignancies, rendering this receptor an attractive therapeutic target. Besides the only FDA-approved CXCR4 antagonist Plerixafor (AMD3100), multiple other classes of CXCR4-targeting molecules are under (pre-)clinical development. Nanobodies (Nb), small single variable domains of heavy-chain only antibodies from Camelids, have appeared to be ideal antibody-fragments for targeting a broad range of epitopes and cavities within GPCRs such as CXCR4. Compared to conventional antibodies, monovalent nanobodies show fast blood clearance and no effector functions. In order to further increase their binding affinities and to restore antibody-mediated effector functions, we have constructed three different bivalent nanobody Fc-fusion molecules (Nb-Fc), targeting distinct epitopes on CXCR4, via fusion of Nbs to a Fc domain of a human IgG1 antibody. Most Nb-Fc constructs show increased binding affinity and enhanced potency in CXCL12 displacement, inhibition of CXCL12-induced signaling and CXCR4-mediated HIV entry, when compared to their monovalent Nb counterparts. Moreover, Nb-Fc induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing CCRF-CEM leukemia cells and did not affect cells expressing low levels or no CXCR4. These highly potent CXCR4 Nb-Fc constructs with Fc-mediated effector functions are attractive molecules to therapeutically target CXCR4-overexpressing tumors. ispartof: BIOCHEMICAL PHARMACOLOGY vol:158 pages:413-424 ispartof: location:England status: published

TGF-β1, β2 and β3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-β inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-β1 production by Tregs with antibodies against GARP:TGF-β1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-β1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8+ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-β1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-β1 mAbs, by selectively blocking a single TGF-β isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer., Inhibiting TGF-β1 to increase immune responses against tumors bears the risk of tumor-promoting toxicity. Here the authors show that selectively blocking TGF-β1 produced by immunosuppressive cells is feasible with anti-GARP:TGF-β1 antibodies and improves the efficacy of PD-1 blockade immunotherapy.

Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review, we provide an extensive overview of mAbs, single-domain antibodies, and other antibody fragments targeting CXCR4 and ACKR3, formerly referred to as CXCR7. We discuss their unique properties and advantages over small-molecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on single-domain antibodies and scaffolds and their utilization in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT: Modulating the function of GPCRs, and particularly chemokine receptors, draws high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments, and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools, and use in the clinic are discussed.

The chemokine receptor CXCR4 and its ligand CXCL12 contribute to a variety of human diseases, such as cancer. CXCR4 is also a major co-receptor facilitating HIV entry. Accordingly, CXCR4 is considered as an attractive therapeutic target. Drug side effects and poor pharmacokinetic properties have been major hurdles that have prevented the implementation of CXCR4-directed inhibitors in treatment regimes. We evaluated the activity of a new and promising class of biologics, namely CXCR4-targeting nanobodies, with the purpose of identifying nanobodies that would preferentially inhibit HIV infection, while minimally disturbing other CXCR4-related functions. All CXCR4-interacting nanobodies inhibited CXCL12 binding and receptor-mediated calcium mobilization with comparable relative potencies. Importantly, the anti-HIV-1 activity of the nanobodies did not always correlate with their ability to modulate CXCR4 signaling and function, indicating that the anti-HIV and anti-CXCR4 activity are not entirely overlapping and may be functionally separated. Three nanobodies with divergent activity profiles (VUN400, VUN401 and VUN402) were selected for in depth biological evaluation. While all three nanobodies demonstrated inhibitory activity against a wide range of HIV (X4) strains, VUN402 poorly blocked CXCL12-induced CXCR4 internalization, chemotaxis and changes in cell morphology. Each of these nanobodies recognized distinct, although partially overlapping epitopes on CXCR4, which might underlie their distinct activity profiles. Our results demonstrate the potential of CXCR4-targeting nanobody VUN402 as a novel lead and starting point for the development of a more potent and selective anti-HIV agent.

The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound five different epitopes on the ECD of GCGR were derived from only two DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

Variable fragments of heavy-chain-only antibodies (VHH) found in camelids are valuable research tools in pharmacology and biotechnology and are being developed for the clinic to treat patients with autoimmune and infectious diseases or cancer. Their single-domain nature and biochemical properties greatly facilitate the development process. The most common technology to select single-domain antibody fragments is phage display following active immunization of llamas or other members of Camelidae family. Selection of VHH from immune phage libraries is a rapid approach to discover a broad panel of in vivo matured antigen-specific clones with comprehensive functionalities. In this chapter, we describe a detailed protocol for construction of VHH immune libraries and phage display selection against antigens in their native conformation.

© 2018 The Author(s). Taylor & Francis Group, LLC. Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50

Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor–b 1( TGF-b1). On the Treg cell surface, TGF-b 1i s in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-b 1b y human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137–139 within GARP/TGF-b1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-b 1p roduction by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.