Nanobodies in cell-mediated immunotherapy: On the road to fight cancer

Abstract

The immune system is essential in recognizing and eliminating tumor cells. The unique characteristics of the tumor microenvironment (TME), such as heterogeneity, reduced blood flow, hypoxia, and acidity, can reduce the efficacy of cell-mediated immunity. The primary goal of cancer immunotherapy is to modify the immune cells or the TME to enable the immune system to eliminate malignancies successfully. Nanobodies, known as single-domain antibodies, are light chain-free antibody fragments produced from Camelidae antibodies. The unique properties of nanobodies, including high stability, reduced immunogenicity, enhanced infiltration into the TME of solid tumors and facile genetic engineering have led to their promising application in cell-mediated immunotherapy. They can promote the cancer therapy either directly by bridging between tumor cells and immune cells and by targeting cancer cells using immune cell-bound nanobodies or indirectly by blocking the inhibitory ligands/receptors. The T-cell activation can be engaged through anti-CD3 and anti-4-1BB nanobodies in the bispecific (bispecific T-cell engagers (BiTEs)) and trispecific (trispecific T-cell engager (TriTEs)) manners. Also, nanobodies can be used as natural killer (NK) cell engagers (BiKEs, TriKEs, and TetraKEs) to create an immune synapse between the tumor and NK cells. Nanobodies can redirect immune cells to attack tumor cells through a chimeric antigen receptor (CAR) incorporating a nanobody against the target antigen. Various cancer antigens have been targeted by nanobody-based CAR-T and CAR-NK cells for treating both hematological and solid malignancies. They can also cause the continuation of immune surveillance against tumor cells by stopping inappropriate inhibition of immune checkpoints. Other roles of nanobodies in cell-mediated cancer immunotherapy include reprogramming macrophages to reduce metastasis and angiogenesis, as well as preventing the severe side effects occurring in cell-mediated immunotherapy. Here, we highlight the critical functions of various immune cells, including T cells, NK cells, and macrophages in the TME, and discuss newly developed immunotherapy methods based on the targeted manipulation of immune cells and TME with nanobodies.

Keywords: BiKE; BiTE; CAR; cancer immunotherapy; immune cell therapy; immune checkpoint; nanobodies; single domain antibodies.

Figure 1

Overview on application of nanobodies in cell-based immunotherapy. Nanobodies participate in anti-cancer immune cell-mediated therapies through (A) bridging between tumor and immune cells, (B) targeting tumor cells with immune cell-bound nanobodies, and (C) blocking inhibitory receptors.

Figure 2

Schematic structures of conventional and heavy chain antibodies and their derivatives. HCAb: heavy chain antibody, Fab: antigen-binding fragment, scFv: single chain variable fragment, Nb, nanobody.

Figure 3

Nanobody-based T cell immunotherapy. (A) T cells are activated through synapsing CD3-TCR/MHC/tumor peptide antigen, CD28/CD80 and 4-1BB/4-1BB ligand. (B) T cells are activated against tumor cells using 4-1BB/CD3 BiTEs, 4-1BB/CD3 TriTEs, and anti-CD3-Nanobody (aCD3-Nb). (C) T cells target tumor antigens through monospecific CAR, bispecific CAR, oligoclonal CAR, biCAR, and monospecific-/bispecific-universal CAR-T cells.

Figure 4

Nanobody-based NK cell immunotherapy. (A) NK cells are activated through synapsing Fc and CD16 in physiological conditions. (B) BiKE, TriKE, and TetraKE target tumor antigen(s) and IL-15R, another functional element on the NK cell membrane, e.g., CD16. (C) CAR-NK cell recognizes the tumor antigen and exerts anti-tumor effects.

Figure 5

Approaches of nanobody/antibody-based targeting of Macrophages. Direct targeting strategies include: (A) M2 repolarization: CD40 and TLR7/8 agonists, the blockades of MACRO, CSF1R, MMR, and blocking of ‘don’t eat me’ signals such as PD1, SIRP-a, LILRB1/and 2, and SIGLEC10 on TAMs lead to repolarization of M2 to M1 phenotype in the TME. As a result, tumor migration, invasion, and Treg induction decline, whereas phagocytosis, T-cell activation, and tumor infiltration increase. (B) TAMs depletion using anti-MMR conjugated clodronate, as a bisphosphonate, increase reprogramming of immunosuppression of TME and T-cell infiltration. Indirect targeting strategies include: (C) blocking of immunosuppressive signaling pathways: inhibition of CSF-1 ligand secreted by tumor cells, blocking of don’t eat me signals like CD47 and PDL1/2 on the tumor cells, as well as blocking EGFR on the tumor cells prevent scape of the tumors from phagocytosis and decrease metastasis, tumor migration, and invasion. (D) Inhibition Macrophage recruitment: Chemokine of MCP-1 on the tumor cells, soluble chemoattractants such as CCL2, CLL5, CXCL12, CSF-1, VEGF-A has secreted by tumor cells and bystander cells to recall macrophages to the TME. Anti-VEGF-A nanobody/antibody reduces angiogenesis. Nanobodies/antibodies against these chemokines prevent the diapedesis of monocytes into the TME and then their differentiation to the immunosuppressive TAMs. Also, the blockade of the CXCL12/CXCL4 axis reduces metastasis.

Figure 6

Nanobody-based Immune checkpoint Inhibition. (A) Immune checkpoints in physiological conditions. (B) Immune checkpoints in the tumor microenvironment, nanobodies can inhibit immune checkpoints by binding to them or their ligands, work as the binding domain in CAR-T cells, or be secreted in the tumor microenvironment by CAR-T cells.

Figure 7

Different stimuli involved in the activation of different subsets of the inflammasome family and the canonical role of ASC in inflammasome assembly. Four key inflammasomes, namely NLRP1, NLRP3, NLRC4, and AIM2, have been best characterized. Following the activation of inflammasomes, inflammatory caspases are converted from the pro-active to the active form. The main consequence of this event is the creation of the active form of the pro-inflammatory cytokine interleukin 1 beta, which is one of the main drivers of inflammation. Apoptosis inhibitory proteins (NAIPs) function as direct receptors for bacterial flagellin and the needle and rod subunits. NLRP3 activation also requires NIMA-related kinase 7 (NEK7), which binds to the NLRP3 leucine-rich repeats (LRRs) and is required for its oligomerization. Many stimuli, such as bacterial lipopolysaccharide (LPS), crystals, extracellular ATP, and dsDNA, can activate the inflammasome complex. In addition to creating the active form of gasdermins, the consequence of inflammasome activation is the secretion of significant amounts of pro-inflammatory cytokines, such as IL-1β and IL-6, by macrophages, which are the main drivers of inflammatory events during CRS.