Targeting multiple myeloma with nanobody-based heavy chain antibodies, bispecific killer cell engagers, chimeric antigen receptors, and nanobody-displaying AAV vectors

Abstract

Nanobodies are well suited for constructing biologics due to their high solubility. We generated nanobodies directed against CD38, a tumor marker that is overexpressed by multiple myeloma and other hematological malignancies. We then used these CD38-specific nanobodies to construct heavy chain antibodies, bispecific killer cell engagers (BiKEs), chimeric antigen receptor (CAR)-NK cells, and nanobody-displaying AAV vectors. Here we review the utility of these nanobody-based constructs to specifically and effectively target CD38-expressing myeloma cells. The promising results of our preclinical studies warrant further clinical studies to evaluate the potential of these CD38-specific nanobody-based constructs for treatment of multiple myeloma.

Keywords: AAV; BiKE; CAR; CD38; VHH; heavy chain antibodies; multiple myeloma; nanobody.

Figure 1

Schematics of nanobody-based biologics (A) Conventional antibodies (yellow) contain light and heavy chains that pair via hydrophobic regions (black bars) in the framework of the VL and VH domains and by a disulphide bond (light black line) between the CL and CH1 domains. The complementarity-determining regions (red) of VH and VL together form the antigen-binding paratope. A recombinant antigen-binding module, i.e. a single chain variable fragment (scFv), can be derived from a conventional antibody by genetically fusing the VH and VL domains via a peptide linker. Heavy chain antibodies (hcAb, olive) derived from llamas lack the light chain and the CH1 domain. A hydrophilic patch (dashed bar) of the VHH domain in place of the corresponding hydrophobic region of a VH domain accounts for the excellent solubility of recombinant VHH domains, i.e. nanobodies (Nb), that allows easy fusion to other proteins and/or Nbs. (B) Nanobodies can be converted into hcAbs of any isotype by genetic fusion to hinge, CH2 and CH3 domains, e.g. of human IgG1. Genetic fusion to a second nanobody that recognizes either a second epitope on the same target antigen or a distinct target (orange), results in a biparatopic or a bispecific hcAb, respectively. (C) Genetic fusion of one or two nanobodies to a transmembrane domain and to cytosolic costimulatory domains yields a monomeric, biparatopic or bispecific chimeric antigen receptor (CAR) that can be transduced into T cells or NK cells. (D) Fusion of one or two tumor marker-specific nanobodies to a nanobody recognizing the Fc-receptor of NK cells (CD16, light blue) yields bispecific or trispecific killer cell engagers (nano-BiKEs or nano-TriKEs). As an alternative to a second tumor-specific nanobody, IL-15 can be fused to a tumor marker-specific nanobody and a CD16-specific nanobody to generate a nano-TriKE. Half-life extension (HLE) of these molecules can be achieved by genetic fusion to an albumin-specific nanobody (magenta). (E) Genetic fusion of a membrane protein-specific nanobody into an exposed surface loop of the VP1 capsid protein yields a targeted nanobody-displaying adeno-associated viral vector (AAV) that specifically transduces cells expressing the target antigen.

Figure 2

Schematic illustration of the mode of action of the CD38-specific nanobody-based hcAbs, BiKEs, CARs and nanobody-displaying AAVs. (A) Antibody-dependent cellular cytotoxicity (ADCC) by an NK cell against a myeloma cell is mediated by a CD38-specific nanobody-based heavy chain antibody. A nanobody-based CD38-specific heavy chain antibody (hcAb) bound to a tumor-antigen (CD38, olive) on the plasma membrane of a multiple myeloma cell is recognized by an Fc-receptor (CD16, blue) of an NK cell. Cross-linking of CD16 on the NK cell induces the release of perforins, which form pores and kill the myeloma cell. (B) A half-life extended (HLE)-nano-BIKE crosslinks CD38 on the tumor cell and CD16 on the NK cell, causing release of perforins and killing of the myeloma cell. A third, albumin-specific nanobody (magenta) binds to albumin in the plasma and thereby extends the half-life of the construct by slowing renal filtration. (C) An NK cell transduced with a biparatopic nanobody-based chimeric antigen receptor (CAR) binds to CD38. Cross-linking of multiple nano-CARs on the NK cell surface triggers the release of perforins and lysis of the myeloma cell. (D) A membrane protein-specific CD38-specific nanobody inserted in the capsid of an AAV mediates specific transduction of tumor cells expressing the cognate target. Expression of the transgene encoding a pro-inflammatory cytokine (e.g. IL-12) and/or a checkpoint blocking nanobody dimer (e.g. α-PDL-1) helps to convert an immunosuppressive into an inflammatory tumor microenvironment.