Development of CD63-based Bispecific ADCs

Bispecific antibody-drug conjugates (ADCs) are designed to be stable in circulation and to release potent cytotoxic drugs intracellularly following antigen-specific binding, uptake, and degradation in tumor cells. Efficient internalization and routing to lysosomes where proteolysis can take place are therefore essential. Based on the well-established ADC and bispecific antibody (bsAb) development platforms, Creative Biolabs offers customized bispecific ADCs development services to achieve your goals.

The Overview of CD63

The CD63 gene is located on human chromosome 12q13 and is the first characterized tetraspanin. Originally, CD63 was discovered as a protein present on the cell surface of activated blood platelets, known as platelet glycoprotein 40 and in early stage human melanoma cells, where it was known as melanoma antigen 491. CD63, being a tetraspanin, interacts with many different proteins either directly or indirectly. Interaction partners include integrins, other tetraspanins, cell surface receptors, kinases, and adaptor proteins.

CD63 is a ubiquitously expressed protein localized within the endosomal system and at the cell surface. In most cells, the major pool of CD63 resides is in late endosomes and lysosomes, and this is why it is also referred to as a lysosomal membrane protein. Lysosomal membrane proteins that exit the trans Golgi network (TGN) can travel to lysosomes via either a direct TGN-to-endosome pathway or an indirect route, involving passage over the plasma membrane and subsequent endocytosis. In general, the sorting of lysosomal membrane proteins depends on tyrosine and dileucine-based consensus motifs within their cytosolic tails. CD63 bears a YXXØ consensus motif in its carboxy terminal cytoplasmic domain with an essential tyrosine residue, 2 hydrophobic XX residues, and the Ø residue being a bulky hydrophobic amino acid. The YXXØ motif is required for endocytosis at the plasma membrane but has also been implicated in direct TGN to lysosome targeting.

The possible pathway of CD63 trafficking. Fig.1 The possible pathway of CD63 trafficking. (Plos, 2009)

Functions of CD63 in Protein Internalization

Many studies performed in different cell types implicate a role for CD63 in intracellular transport of other proteins. In gastric parietal cells, the co-expression of CD63 causes a redistribution of the H, K-ATPase β-subunit from the cell surface to intracellular compartments. This occurs by CD63-mediated linkage of the H, K-ATPase β-subunit to AP-2, thereby facilitating its clathrin-mediated endocytosis. These results suggest that CD63 might be involved in the recycling of the H, K-ATPase pump between the plasma membrane and intracellular storage compartments.

What Can We Do for You?

CD63, as a high-turnover surface protein, was used in bispecific ADC development. For example, in a HER2 x CD63 bispecific ADC, the binding affinity of the CD63 arm was limited to reduce monovalent target binding and prevent uptake by healthy tissues, thus relatively increasing the targeting properties of the bsAb to tumor cells that co-express HER2 and CD63. The bispecific ADC was armed using lysine conjugation with antimitotic agent duostatin-3 attached to a lysosomal protease-cleavable linker. It demonstrated potent cytotoxicity against HER2-positive tumors in vitro and in vivo, which was not observed with monovalent HER2- and CD63-specific ADCs.

Creative Biolabs is a well-recognized custom ADC development services provider. Our Ph.D. level experts have rich knowledge and extensive experience in generating CD63-based bispecific ADCs. We are confident in providing one-stop service from experiment design, antibody preparation to bio-conjugation to fit your specific demands. If you are interested in our services, please contact us for more details.

Reference

  1. Pols M.; et al. Trafficking and function of the tetraspanin CD63. Experimental Cell Research, 2009, 315(9): 1584-1592.

For Research Use Only. NOT FOR CLINICAL USE.


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