Part VIII · Therapeutic foundations · Chapter 47

Antibody-drug conjugates and targeted delivery

Three parts, six steps, and one unsolved sequencing problem.

1 · Antibody, linker, and payload design

A conjugate has three components and they are chosen together. Treating them as independent is the commonest error in reading this literature.

The antibody supplies address and pharmacokinetics. It needs differential expression rather than absolute expression, because normal tissue exposure is what limits dose. It does not need to bind a driver. Trop-2 is an epithelial surface antigen and is not an established driver in breast cancer, so an antibody against it works as a postcode.

The linker decides where the payload is released, and that decision determines almost everything downstream. A non-cleavable linker requires complete lysosomal proteolysis of the antibody. What emerges is a charged amino acid-linker-payload catabolite that does not readily cross membranes. A cleavable linker is cut by a lysosomal protease, by reduction, or by hydrolysis, and it can release free unmodified drug. Only the second route can produce a species that leaves the cell that took it up¹.

The payload has to be potent enough to kill at the intracellular concentration a saturated antigen can deliver, which is far lower than a conventional cytotoxic dose. Two classes dominate current practice. Microtubule agents include the maytansinoids and the auristatins. Topoisomerase I inhibitors include the exatecan derivative carried by the deruxtecan conjugates and the SN-38 carried by sacituzumab govitecan².

The design point worth holding is that membrane permeability of the released species is a payload and linker property jointly. The same payload attached through a different linker behaves differently, and that is demonstrated directly in Bystander killing and why it matters in heterogeneous tumors.

2 · Drug to antibody ratio and the therapeutic index

Drug-to-antibody ratio is the mean number of payload molecules carried per antibody in a preparation. It is a mean over a distribution, not a property of a molecule. Two preparations with the same mean can differ in how wide that distribution is, and the wide one contains species that behave differently in plasma.

The canonical experiment is worth stating in full because it is still misread. An anti-CD30 auristatin conjugate was purified into populations carrying two, four and eight drugs per antibody. Potency in vitro tracked loading, with the lowest half-maximal inhibitory concentrations in the eight-drug population. In vivo, at equal antibody dose, the four-drug population matched the eight-drug population while carrying half as much drug. The maximum-tolerated dose in mice ran the other way. The two-drug population was tolerated at least twice as high a dose as the four-drug population, which was tolerated at twice the dose of the eight-drug population. Plasma clearance also ran with loading, the eight-drug population clearing three times faster than the four-drug and five times faster than the two-drug population. Lowering the ratio raised the therapeutic index³.

The lesson is not that low ratios are better. It is that ratio trades efficacy against exposure and tolerability, and the trade is set by the payload, the linker and the conjugation chemistry together. Highly loaded conjugates are now in routine use and are among the better tolerated agents in this book, because the other variables were engineered around the loading.

3 · Target expression, internalization, and intracellular trafficking

Six things have to happen for a conjugate to kill through the internalisation route. The antibody must bind. The complex must be endocytosed. It must reach the lysosome. The linker or the antibody must be cleaved. The payload must reach the cytoplasm. The payload must engage its target and kill.

Immunohistochemistry answers the first step and none of the others. It measures protein at the membrane on one block at one time, through a particular antibody and a particular detection chemistry. Internalisation rate, receptor recycling and lysosomal delivery are not measured by any assay in clinical use.

The fifth step has a named requirement for one payload class. A short hairpin screen identified the lysosomal membrane protein SLC46A3 as necessary for the potency of non-cleavable antibody-maytansine conjugates, including trastuzumab emtansine. Non-cleavable conjugates carrying the structurally distinct auristatin monomethyl auristatin F were unaffected by the same attenuation⁴. A transporter requirement is therefore payload-specific rather than general, and loss of that transporter is a route to resistance⁵.

Target abundance still matters, and it matters as a gradient rather than a threshold. One prespecified biomarker analysis graded Trop-2 expression in a randomised trial of sacituzumab govitecan in metastatic triple-negative disease. Median progression-free survival was 6.9, 5.6 and 2.7 months in patients with high, medium and low expression. The corresponding figures on single-agent chemotherapy were 2.5, 2.2 and 1.6 months⁶. Trop-2 expression was measurable in 290 of 468 assessable patients, and the analysis was exploratory. Benefit was present across the whole gradient and it was not equal across it.

4 · Bystander killing and why it matters in heterogeneous tumors

Bystander killing is the death of a cell that never bound the antibody, caused by payload released from a neighbour that did. Three conditions must hold. The released species must cross membranes. It must remain cytotoxic after release. The antigen-negative cell must be within diffusion distance of an antigen-positive one.

The experiment that separates these from one another compared two HER2 conjugates. The exatecan derivative payload was shown to be highly membrane-permeable, while the lysine-linked maytansinoid catabolite of trastuzumab emtansine was not. In co-culture of HER2-positive and HER2-negative breast cancer lines, the deruxtecan conjugate killed both populations. Trastuzumab emtansine did not, and neither did a HER2 conjugate built with a low-permeability variant of the same payload family. In mice bearing mixed tumours, the deruxtecan conjugate suppressed the HER2-negative population⁷.

One control in that study carries more weight than the main result. The same conjugate had no effect on a HER2-negative tumour implanted on the opposite flank⁷. The effect is local. It is a diffusion radius, not a systemic drug level.

That is why spatial arrangement rather than percentage is the variable that matters. A two-centimetre unamplified region is not reached. Single unamplified cells scattered among amplified ones are. The clinical demonstration across the HER2 expression range, including disease scored immunohistochemistry 0, is developed in Implications for conjugate payload delivery and the bystander effect and is not repeated here. The eligibility boundary that DESTINY-Breast04 created is an assay boundary rather than a biological one⁸, and the argument for that reading is in HER2-low and ultralow reframed as a heterogeneity problem.

5 · Extracellular payload release and immunological effects

The internalisation model understates two things, and both matter for how these agents are used.

The first is extracellular release. Linkers cleavable by proteases present outside cells, or hydrolysable at the pH of the tumour interstitium, can release payload before any internalisation has occurred. Bystander killing by some conjugates does not require antigen-dependent uptake at all¹. The practical consequence is that activity in low-antigen disease is not evidence that internalisation was efficient. It may be evidence that release was not internal.

The second is immunological activity. An immunocompetent mouse model was built with human HER2-expressing colon carcinoma cells. The deruxtecan conjugate increased tumour-infiltrating dendritic cells and their CD86 expression, and increased tumour-infiltrating CD8 T cells. It also raised MHC class I and PD-L1 on the tumour cells. Cured mice rejected rechallenge with antigen-negative cells as well as antigen-positive ones. Combination with an anti-PD-1 antibody controlled tumours better than either agent alone⁹. Those are mouse data and should be labelled as such.

A mechanistic route to the same conclusion exists in human breast cancer tissue. Topoisomerase I payloads create S-phase DNA damage, and S-phase-specific DNA damage activates STING-dependent innate immune signalling in breast cancer¹⁰.

6 · Payload class, cross-resistance, and sequencing between conjugates

Three separable things in a conjugate each carry their own cross-resistance. The antibody target is one. The payload class is a second. The cell's handling of that payload, meaning efflux, lysosomal transport and repair of the lesion, is a third. A patient can lose one and retain the other two.

The current problem is that payload classes are not diverse. Trastuzumab deruxtecan, sacituzumab govitecan and datopotamab deruxtecan all carry topoisomerase I inhibitors^11,2,12. A patient with metastatic breast cancer may receive two or three conjugates in sequence that differ in target and share a lesion.

The preclinical evidence says that linker and payload class, rather than the conjugate as a unit, carry the cross-resistance. Breast cancer lines selected under a trastuzumab-maytansinoid conjugate became roughly 250-fold and 16-fold resistant in two models. Both were cross-resistant to other non-cleavable conjugates. Both retained sensitivity to conjugates carrying cleavable auristatin payloads, and both showed minimal resistance to unconjugated maytansine¹³.

The clinical evidence is thinner and it is consistent with partial rather than complete cross-resistance. Eighty-five patients received both trastuzumab deruxtecan and sacituzumab govitecan, and progression-free survival on the second conjugate was shorter than on the first in 75.2% of them. Fourteen of the 85 were still on treatment at the data cutoff, and the source does not state which denominator the percentage is taken on. Read it as about three patients in four. Longer progression-free survival on the first predicted longer progression-free survival on the second. PTEN loss was associated with de novo resistance to trastuzumab deruxtecan, with a hazard ratio of 3.20 and a 95% confidence interval of 1.47 to 6.97. It was not associated with resistance to sacituzumab govitecan, where the hazard ratio was 1.18 with an interval of 0.54 to 2.56¹⁴.

This section should end without an answer, because there is not one. No randomised trial has compared conjugate sequences. No assay in clinical use predicts which patient retains sensitivity after a topoisomerase I payload. No study has shown that switching payload class recovers activity in patients who progressed on the previous class. The retrospective series above is the best available evidence and it is 85 patients treated off protocol. This is the single most consequential unanswered question in current metastatic practice, and the honest position is that sequencing is being done on a rationale rather than on a result. The clinical ordering as it currently stands is set out in General principles of metastatic management and Cross-resistance between classes and what it means for sequencing.

7 · Mechanisms of conjugate resistance

Each of the six steps in Target expression, internalization, and intracellular trafficking is a place to fail, and the experimental evidence shows that the failures are genuinely independent.

Two trastuzumab emtansine-resistant breast cancer lines failed by unrelated routes. One showed reduced HER2 with upregulation of the efflux transporter MDR1. The other showed loss of SLC46A3 together with PTEN deficiency. An MDR1 inhibitor reversed the first and a PI3K inhibitor reversed the second⁵. Two further models selected against a trastuzumab-maytansinoid conjugate showed increased ABCC1 in one and reduced HER2 in the other, and both retained sensitivity to unconjugated payload and to standard chemotherapeutics¹³. The conjugate failed. The payload did not.

Antigen loss under treatment is real and is developed in HER2 loss as an acquired resistance mechanism. It is the only one of these mechanisms that a routine assay detects.

PTEN deserves separating carefully, because it sits on two arguments at once. PTEN loss predicts resistance to trastuzumab itself¹⁵, so the clinical association with trastuzumab deruxtecan failure¹⁴ cannot be attributed to payload handling without further evidence. The preclinical association runs through lysosomal transport instead⁵. Both may be true and they are not the same claim.

Repair capacity should matter for topoisomerase I payloads, because the lesion is a DNA lesion resolved by defined repair pathways DNA repair, replication stress, and genomic instability. The preclinical reasoning is coherent. There is no clinical evidence in breast cancer that repair capacity predicts benefit from a conjugate carrying this payload class.

8 · Toxicity mechanisms, interstitial lung disease, ocular, and mucosal

Conjugate toxicity is mostly off-target and payload-driven. The evidence for that statement is comparative. Agents sharing a payload class resemble each other in toxicity more than agents sharing a target do.

Trastuzumab emtansine carries a maytansinoid and produces thrombocytopenia and transaminase elevation¹⁶. Trastuzumab deruxtecan carries a topoisomerase I inhibitor against the same target and produces neutropenia, nausea and interstitial lung disease¹¹. Same antibody, different dominant toxicity. Sacituzumab govitecan and datopotamab deruxtecan both target Trop-2 with a topoisomerase I payload, and their dominant toxicities still differ. Sacituzumab govitecan produced grade 3 or higher neutropenia in 51% of patients against 33% with single-agent chemotherapy, and grade 3 or higher diarrhoea in 10% against under 1%². Datopotamab deruxtecan produced stomatitis of any grade in 50% of patients and grade 3 or higher in 6.4%. Its overall rate of grade 3 or higher treatment-related events was lower than that of investigator's choice chemotherapy, at 20.8% against 44.7%¹². Linker chemistry and normal tissue handling separate them.

Interstitial lung disease is the toxicity that changes management most. A pooled analysis of nine trastuzumab deruxtecan monotherapy studies included 1150 patients across several tumour types. Adjudicated drug-related interstitial lung disease or pneumonitis occurred in 15.4% of them, and was grade 5 in 2.2% of them. Among the patients who had an event, 77.4% had only grade 1 or grade 2 disease, and 87.0% had their first event within 12 months of the first dose. Median time to first event was 5.4 months. A stepwise model identified several baseline factors associated with higher risk: age under 65 years, enrolment in Japan, dose above 6.4 mg per kilogram, oxygen saturation under 95%, moderate or severe renal impairment, pre-existing lung disease, and more than four years since diagnosis¹⁷. In the randomised comparison against trastuzumab emtansine, adjudicated drug-related interstitial lung disease or pneumonitis occurred in 10.5% of patients on trastuzumab deruxtecan and 1.9% on trastuzumab emtansine¹⁸.

One finding in the pooled analysis is a measurement result rather than a biological one. Independent adjudication placed onset earlier than the investigator had, for 53.2% of events, with a median difference of 43 days¹⁷. Investigators were recognising this six weeks late.

Ocular surface events and oral mucositis are recognised adverse events of special interest for the Trop-2 deruxtecan conjugate, with published prophylaxis and monitoring recommendations¹⁹. The mechanism is consistent across both. Corneal epithelium and oral mucosa renew rapidly and are directly exposed, so a payload that reaches them kills the dividing compartment whether or not the target antigen is abundant there.

9 · Combination strategies with immunotherapy and DNA repair agents

Two rationales for combining a conjugate with checkpoint blockade are mechanistically distinct. The first is that the conjugate raises antigen presentation and T cell infiltration, demonstrated in an immunocompetent mouse model with the combination outperforming either agent⁹. The second is that a topoisomerase I payload creates the S-phase DNA damage that drives STING-dependent innate signalling in breast cancer tissue¹⁰. Both predict benefit. Neither establishes it. No randomised trial in breast cancer has shown that adding checkpoint blockade to a conjugate improves survival, and the immunotherapy arguments are developed in Immunotherapy.

The DNA repair combination rests on the same lesion from the other side. A topoisomerase I payload leaves trapped cleavage complexes that become double-strand breaks at the replication fork, which is the substrate PARP inhibition exploits PARP trapping and synthetic lethality. The obstacle is not the rationale. Both classes suppress the marrow, so the combination is constrained by overlapping myelosuppression rather than by mechanism.

The most informative combination in early disease is not a combination of two drugs. In KATHERINE, patients with residual invasive HER2-positive disease after neoadjuvant therapy received trastuzumab emtansine or trastuzumab. Invasive disease or death occurred in 91 of 743 patients on the conjugate and 165 of 743 on trastuzumab. The hazard ratio was 0.50, with a 95% confidence interval of 0.39 to 0.64. Three-year freedom from invasive disease was 88.3% against 77.0%²⁰. The conjugate was combined with a selection rule rather than with an agent, and the selection rule was observed response. That logic belongs to Principles of multidisciplinary treatment and its HER2-specific instance to Deciding whether to treat before surgery.

References

All 20 references

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