Abstract

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Highlights

  • Introduction of theE345R HexaBodyMutation Enhances the complement-dependent cytotoxicity (CDC) Potency of CD38-Specific heavy chain antibody (hcAb)Combining Nanobodies Directed to TwoDistinct Epitopes on CD38 in a Biparatopic hcAb Induces Potent CDCIt has been shown that certain amino acid substitutions in the C1q binding face of daratumumab enhance the tendency of daratumumab to spontaneously form hexamers [18]

  • Recombinant VHH domain or nanobody (Nb) is highly soluble and does not show any tendency to associate with light chains or any other hydrophobic proteins

  • We further exploited the inherent solubility of VHHs to generate biparatopic hcAbs by fusing two distinct

Read more

Summary

Introduction

Introduction of theE345R HexaBodyMutation Enhances the CDC Potency of CD38-Specific hcAbsCombining Nanobodies Directed to TwoDistinct Epitopes on CD38 in a Biparatopic hcAb Induces Potent CDCIt has been shown that certain amino acid substitutions in the C1q binding face of daratumumab enhance the tendency of daratumumab to spontaneously form hexamers [18]. Numbering of binding epitopes is according to Fumey et al [38] Such nanobody dimers can be fused to the hinge, CH2, and CH3 domains of human IgG1 to generate tetravalent bispecific or biparatopic hcAbs [32]. In order to determine whether the potent CDC induction capacity of certain hcAb combinations could be combined into a single molecule, we constructed biparatopic hcAbs containing two nanobodies that recognize distinct epitopes of CD38. These biparatopic hcAbs were produced at high yield as soluble proteins in transiently transfected HEK-6E cells. The results of CDC assays reveal that biparatopic hcAbs induce potent CDC as single reagents (Figure 5)

Objectives
Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call