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Rational Design of a Humanized Antibody Inhibitor of Cathepsin B.

ABSTRACT: Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into heavy chain complementarity-determining region 3 (CDR3H) of Herceptin that is used in the clinic for the treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 h for this anti-CTSB antibody inhibitor, comparable to that of the parental Herceptin scaffold. This study demonstrates a new approach for the efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.

SUBMITTER: Dai Z

PROVIDER: S-EPMC7184884 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.

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Rational Design of a Humanized Antibody Inhibitor of Cathepsin B.

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