A Superglue for ADCs: Connecting the Cytsteine Residues of a Tumor-sensing Antibody to a Payload

A new technology enables a simple way of connecting the cytsteine residues (SH) of a tumor-sensing antibody (yellow) to the cytotoxic payload. The emerging linker is highly stable during blood circulation and enables therefore a safe transport to the tumor side. Image courtesy: Barth van Rossum/FMP
A new technology enables a simple way of connecting the cytsteine residues (SH) of a tumor-sensing antibody (yellow) to the cytotoxic payload. The emerging linker is highly stable during blood circulation and enables therefore a safe transport to the tumor side. Image courtesy: Barth van Rossum/FMP

The Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and the Ludwig-Maximilians-Universität München (LMU) hve developed an innovative technology designed to make it possible to treat cancer more selectively and effectively.

The process the scientists at FMP and LMU have developed, transforms proteins and antibodies into stable, highly functional drug transporters, with which tumor cells can be detected and killed.

Traditional, classic, chemotherapy for the treatment of cancer is based on toxic substances that are particularly effective for rapidly dividing cells. However, since healthy tissue also depends on cell division, treatment with chemotherapeutic substances is often accompanied by severe side effects.

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A dose sufficient to completely remove the tumor, would in many cases be too toxic to administer to a diseased person. With more modern approaches, it is now possible to transport active pharmaceutical agents, the drugs, in the body selectively to the site of action, for example by linking a drug with an antibody that can differentiate cancer cells from healthy tissue through changes on the surface of the cell.

Five such antibody-drug conjugates or ADCs are already on the market.

However, antibody-drug conjugates may lose some of their toxic payload en route to the cancer cell. In that case, the drug attached to the ADC is released into the bloodstream and dangerous, sometimes life-threatening adverse effects can occur.

And this is, in part, because the underlying bioconjugation reactions for the synthesis of antibody–drug conjugates (ADCs) are exceptionally demanding, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product.

A more stable linker chemistry
A stable link between drug and antibody would therefore be highly desirable. And this is precisely what the researchers, led by Professor Christian Hackenberger Ph.D at The Leibniz-Forschungsinstitut für Molekulare Pharmakologie and Professor Heinrich Leonhardt, Ph.D, at the Ludwig-Maximilians-Universität München’s Biocenter – have focused on.

Their study results have been published in Angewandte Chemie. In two consecutive articles, the team of scientists describe the development of methods and the application of these methods to selective drug transport are presented. [1][2]

The new drug transporters enable lower doses and less severe side effects.

“We have developed an innovative technology that makes it possible to link native proteins and antibodies to complex molecules, such as fluorescent dyes or drugs more easily and with better stability than ever before,” explained Marc-André Kasper, a researcher in Christian Hackenberger’s group.

The researchers discovered the outstanding properties of unsaturated phosphorus (V) compounds and took advantage of those.

These phosphonamidates connect a desired modification – for example, a cancer-fighting agent – exclusively to the amino acid cysteine, in a protein or antibody. Since cysteine is a very rare natural occurring amino acid, the number of modifications per protein can be controlled quite effectively, which is essential for the construction of drug conjugates. In addition, phosphonamidates can easily be incorporated into complex chemical compounds.

“The greatest achievement of the new method, however, is that the resulting bond is also stable during blood circulation,” Kasper added.

The currently approved and commercially available ADCs cannot achieve this.

Comparison
To test the applicability on targeted drug delivery, the researchers compared their technology directly with brentuximab vedotin (Adcetris®; Seattle Genetics/Takeda), an antibody-drug conjugate used to treat patients with relapsed or refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma and one of the currently FDA-approved ADCs.

The scientists re-created brentuximab vedotin as precisely as possible with the same antibody and active agent. However, instead of using the linker chemistry used in brentuximab vedotin, the scientists used their own innovative phosphonamidate linkage. When applied to blood serum, the researchers observed that the modified ADC lost significantly less active ingredient over a period of days. They also used the new technology in experiments with mice to combat Hodgkin’s lymphoma. The preparation proved to be more effective than the commercially available, approved, version of brentuximab vedotin.

“From our results, we conclude that phosphonamidate-linked drug transporters can be administered in lower doses. This approach is expected to greatly reduced adverse events. Thus the technology has great potential to replace current methods in order to develop more effective and safer ADCs in the future,” commented Christian Hackenberger.

Future developments
In the next step, the research groups will continue their efforts in the development of ADCs based on phosphonamidates. Tubulis, start-up company generating uniquely matched protein-drug conjugates by combining proprietary novel technologies with disease-specific biology, is currently conducting preclinical studies designed as a first step to expand the therapeutic potential of antibody-drug conjugates. The company was awarded the Leibniz Start-Up Prize last year, functions as a platform for the further development to market maturity.

Reference
[1] Kasper MA, Glanz M, Stengl A, Penkert M, Klenk S, Sauer T, Schumacher D, Helma J, Krause E, Cardoso MC, Leonhardt H, Hackenberger CPR Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations. Angew Chem Int Ed Engl. 2019 Aug 19;58(34):11625-11630. doi: 10.1002/anie.201814715. Epub 2019 Apr 29. [Pubmed][Article]
[2] Kasper MA, Stengl A, Ochtrop P, Gerlach M, Stoschek T, Schumacher D, Helma J, Penkert M, Krause E, Leonhardt H, Hackenberger CPR. Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates. Angew Chem Int Ed Engl. 2019 Aug 19;58(34):11631-11636. doi: 10.1002/anie.201904193. Epub 2019 Jul 18. [Pubmed][Article]