Antibody-Drug Conjugates: Internalisation, Trafficking & the Bystander Effect
An antibody\u2013drug conjugate is not a targeted drug. It is a delivery problem with an antibody bolted to the front of it. Binding is the easy part and almost never the part that fails. What decides whether a conjugate works is what happens in the ninety minutes after binding: how fast the receptor is pulled into a coated pit, whether the complex is recycled back to the surface or committed to the lysosome, whether the lumen acidifies enough to activate the proteases, and whether the released payload can actually cross a membrane once it is free. Each of those steps is a property of the receptor, not of the antibody \u2014 which is why TROP2 supports two approved conjugates that behave nothing like each other, and why HER2-low tumours respond to one HER2 conjugate and not to another. Click any protein for the matching Assay Genie antibody, biosimilar or ELISA kit.
Binding is not delivery. This is the fact the map is built around. An ADC that binds beautifully and is recycled intact has delivered nothing while consuming antigen occupancy, and that failure is invisible in every binding assay. The parameter that separates conjugates is internalisation rate, and it belongs to the receptor: CD79b works at modest copy number because it internalises constitutively, while abundant, slowly internalising antigens have repeatedly disappointed. CD71 is the reference point at one extreme \u2014 continuous, rapid uptake \u2014 and also the warning, since continuous recycling returns much of what it takes in. The quantity worth measuring early in a programme is not affinity but the fraction of bound conjugate that reaches LAMP1-positive compartments.
The sorting decision is where dose is silently lost. Past EEA1 the conjugate is still fully armed and nothing has happened. Two routes then diverge. Rab11 and FcRn return it to the surface \u2014 the same FcRn mechanism that gives an ADC its long circulating half-life also rescues conjugate out of the tumour cell that just internalised it, which is an uncomfortable design tension rather than a bug. Rab5-to-Rab7 conversion commits it instead to degradation, which on this pathway is the desired outcome. Caveolin-1 adds a third possibility that is easy to misread: a conjugate taken up by the caveolar route can reach a compartment that never acidifies properly, giving high measured uptake and low potency. That result looks like a payload problem and is a routing problem, and the way to tell them apart is a V-ATPase block, which abolishes potency without touching binding.
Release chemistry decides the bystander effect, and the bystander effect decides the clinic. A cleavable valine-citrulline linker cut by cathepsin B frees membrane-permeable MMAE, which diffuses into neighbouring cells whether or not they carry the antigen \u2014 this is why brentuximab vedotin and enfortumab vedotin work in heterogeneous tumours. A non-cleavable linker yields a charged lysine-linker catabolite that cannot cross a membrane, and needs the lysosomal transporter SLC46A3 even to reach the cytosol of the cell that took it up. Losing SLC46A3 stops trastuzumab emtansine while leaving trastuzumab deruxtecan working: resistance that is invisible at the antigen, at the receptor, and in the lysosome. Legumain is where the next generation of linkers is going, for the straightforward reason that it is lysosomal and tumour-enriched. And P-glycoprotein is the price of permeability \u2014 a payload that can diffuse into a neighbour can also be pumped straight back out.
What the reagents on this map are, precisely. The antigen layer is served by the naked antibody backbones of ten clinical conjugates \u2014 trastuzumab, sacituzumab govitecan, datopotamab, mirvetuximab, zolbetuximab, tusamitamab, polatuzumab, brentuximab vedotin, gemtuzumab ozogamicin and enfortumab \u2014 supplied as research-grade biosimilars of the antibody without the payload. They carry their clinical identity in the tooltip and deliberately do not carry the blue In Vivo dot, which is reserved for the functional-grade range. Stated plainly: only 5 of the 37 nodes are blockable in vivo \u2014 EGFR, CD71, EpCAM, CD16 (clone 3G8) and mouse CD16/CD32. That last one earns its place: without Fc\u03b3 receptor blockade, uptake by host myeloid cells in a xenograft is scored as antigen-specific delivery. Everything between the door and the cytosol is measurable here rather than blockable, and on a trafficking pathway that is the correct division of labour \u2014 the experiment is to follow the conjugate, not to neutralise the machinery carrying it. For research use only; not for use in diagnostic or therapeutic procedures.
Every protein node links to a product — ELISA kit, In Vivo antibody or research antibody.