Many approaches have been developed to improve the physical/chemical properties, potency and selectivity of antibody-drug conjugates (ADCs). Trastuzumab (Tz), a monoclonal antibody targeting HER2, was approved by the FDA in 1998 for the first-line treatment of HER2+ breast cancer patients. Due to its successful clinical application and important clinical treatment needs, it has become an important component of several ADC drugs (Kadcyla®, Enhertu®).
In addition to the two ADCs mentioned above, Tz is also widely used as a model antibody in the testing of new technologies/therapies in scientific research. The various model responses involved allow us to compare Tz in various linkers, payloads, drug-to-antibody ratio (DAR) and structure-activity relationship of conjugation methods. At the same time, the use of this model antibody also broadens the application of Tz outside the ADC field, such as antibody-nanogel conjugates (ANC) and antibody-epitope conjugates (AEC ).
Payload
Payload plays an important role in the ADC function, and the most widely used one is cytotoxic payload. The payloads used in early ADCs were mostly Methotrexate, Vinblastine, and Doxorubicin, etc., but their therapeutic effects were not ideal (IC50>10-8M). The currently used payloads are more cytotoxic, and the required cytotoxic activity values in various human tumor cell lines are usually in the subnanomolar range (IC50: 10-12 M~10-10 M). This higher cytotoxicity is critical for the efficacy of ADCs, reducing payload concentration limited by antigen expression. Currently clinically approved highly cytotoxic payloads include tubulin inhibitors, DNA damaging agents, and topoisomerase inhibitors.
Indolobenzodiazepine (IGN) and pyrrolobenzodiazepine (PBD) dimers are 100−1000 times more potent than conventional chemotherapy drugs. ADCT-502 is a high-efficiency Tz-ADC (DAR=1.7) based on PBD dimer, which exerts anti-tumor effect by cross-linking with DNA after specific internalization of the target antigen. Compared with T-DM1, ADCT-502 exhibited superior in vivo antitumor activity in various tumor xenograft models (HER2+FISH-BC/ESCA PDX). Unfortunately, ADCT-502 was terminated in 2018 due to significant clinical toxicity in lung tissue, but the development of PBD-based non-Tz-ADC drugs is still in progress. Another example that contains a new type of high-efficiency payload is SYD-985 [Trastuzumab duocarmazine (DAR=2.8)], developed by Byondis, using duocarmycin analogue (seco-DUBA) as the payload, which is coupled to Tz through VC-PABC linker. seco-DUBA acts as a prodrug and is only activated in tumors. In addition to tubulin inhibitors and DNA damage agents, topoisomerase inhibitors (DXd/Exatecan) occupy a place in ADC payload due to their high inhibitory activity, high permeability and bystander penetration effect. DS-8201a (DXd) can exhibit good antitumor activity against T-DM1 (Emtansine) insensitive/resistant tumors, which may be attributed to the cleavable linker and inhibitory activity of the payload.
With the deepening of research, the payload coupled with Tz is no longer limited to cytotoxic drugs, but has begun to expand to radionuclides, fluorescent dyes, Toll-like receptor agonists, etc. The application fields of the constructed conjugates are not limited to the anti-tumor level, but have begun to expand to the fields of cell imaging and immunodiagnosis (immunoPET).
Drug-to-Antibody Ratio (DAR)
DAR represents the average number of payloads conjugated to each antibody, and is currently calculated using a weighted average method. Although higher DAR values seem to be preferable for ADCs to exert therapeutic effects, increasing DAR values can pose many problems. For example, the production and accumulation of high-molecular-weight aggregates can inhibit the binding to receptor targets, may lead to toxicity, and may alter the in vivo metabolic pathway of the conjugate. The optimal DAR value is a very important consideration when using highly hydrophobic payloads. The physicochemical properties of payload were measured by total polar surface area (tPSA) and partition coefficient (LogP), and the change of DAR value was studied to improve ADC activity. In vivo studies showed that only DAR 4 of Tz-ADC (with PEG-24 spacer) could completely eliminate tumors, whereas DAR 1-3 resulted in DAR value-dependent tumor growth inhibition. This suggests that higher DAR values are preferred as long as DAR is kept within a certain range to prevent aggregation as much as possible.
In order to seek higher DAR values, Mersana has developed novel Dolaflexin and Dolasynthen platform technologies. Mersana constructed a Tz-PHF-vinca conjugate with a DAR value of 20 based on the Dolaflexin platform. The binding of the Tz-ADC to the antigen was not affected, and the inhibitory activity in in vitro cell experiments was at the nanomolar level. In the mouse xenograft model can cause effective tumor killing. The Dolasynthen platform technology can construct ADCs with a DAR range of 2-24 after optimization of scaffold and coupling technology. The ADC constructed by this technology is progressing rapidly at present is XMT-1592 (DAR 6)/1660, which has entered clinical phase I test.
ADC Linker
When the ADC is circulating in the blood, the ideal linker must remain stable to limit off-target toxicity and ensure that the payload is only released in the target cells. Linker not only plays the role of attaching payload to the antibody, but also plays an important role in the PK/PD characteristics of ADC. The linkers of ADC drugs approved so far are diverse, including cleavable linkers, non-cleavable linkers, acid-sensitive linkers, and protease-sensitive linkers, all of which play key roles in the mechanism of action of ADCs.
For the Tz-ADC constructed by DM1 and its analogs, by comparing four different disulfide bonds and non-reducible SMCC linker, the influence of steric hindrance in linker design on the stability of disulfide bonds was analyzed. Tz-SMCC-DM1 and Tz-SSNPP-DM4 have nearly identical mouse serum stability, with >70% of the conjugate remaining after 7 days of incubation. In contrast, the least sterically hindered Tz-SPDP-DM1 (no methyl group) was completely cleaved within 3 days, Tz-SPP-DM1 (monomethyl group) was also almost completely cleared within 7 days, while Tz-SSNPP- DM3 (dimethyl) showed moderate clearance. In vivo experiments showed that Tz-SMCC-DM1 and Tz-SSNPP-DM3 showed the greatest therapeutic effect, while Tz-SPP-DM1 and Tz-SSNPP-DM4 produced only slight therapeutic effect. In terms of toxicity test, the toxicity produced by Tz-SMCC-DM1 was not significantly different from that of the control group, while Tz-SPP-DM1 showed significant weight loss, which was related to the level of free drug level in blood circulation. In view of the stability of Tz-SMCC-DM1 and its in vivo efficacy and low toxicity, SMCC linker was selected as the best linker for research and eventually clinical success Kadcyla®. This example illustrates the toxicity reduction effect of a non-cleavable linker over a well-controlled cleavable linker. Non-cleavable linkers used in current scientific research also include AMAS, GMBS, EMCS, MBS, SMPB, LC-SMCC, SMPH, KMUS, SM-(PEG)n, etc.
ADC Conjugation
Based on the above problems encountered in the development of ADCs, researchers focused on the development of site-specific antibody conjugation technology. One that has made great strides in this regard is Genentech. The introduction of engineered cysteine through genetic engineering technology provides a reactive sulfhydryl group (-SH) for homogeneous coupling. This method is called THIOMAB technology, which has the characteristics of uniform DAR value and does not destroy the disulfide bond between antibody molecular chains. By comparing the Tz-ADCs constructed by engineering cysteines at three different sites (LC-V205C, HC-A114C and HC-S396C), it was found that Tz generated by low solvent accessibility and partially positively charged coupling sites – ADC has higher plasma stability. This environment can promote the hydrolysis of the succinimide ring in the linker to form a more stable linker and prevent the generation of reverse Michael addition. Using this method, Tz-MPEO-DM1 (DAR=1.8) was constructed using BMPEO [bismaleimide poly(ethylene oxide)] linker. Compared with Tz-MCC-DM1 (DAR=3.3), Tz-MPEO-DM1 has similar cell permeability, target binding and cell killing effects on SK-BR-3 cell line. In in vivo experiments, Tz-MPEO-DM1 was more effective than Tz-MCC-DM1 when DM1 dose was similar.
References
- Li, W. et al. Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy. Molecules. 2017, 22: 1281.
- Bryant, P. et al. In Vitro and In Vivo Evaluation of Cysteine Rebridged Trastuzumab-MMAE Antibody Drug Conjugates with Defined Drug-to-Antibody Ratios. Mol Pharm. 2015, 12(6): 1872-9.
- Nicolas, J. et al. Antibody-Drug Conjugates: The Last Decade. Pharmaceuticals. 2020, 13, 245.