Bioconjugation is a critical step in the development of Antibody Drug Conjugates with two main strategies dominating the field. One approach employs covalent chemistry to randomly bind a drug to the accessible Lysines or Cysteines on an antibody. The other strategy is site-specific, using covalent chemistry to bind a drug ligand at defined amino acid positions within the antibody. AbYlinkTM is a novel regio-selective labeling method for antibodies, enabling a single-step covalent conjugation of payloads such as radionuclide chelators to the Fc domain of an antibody. Given the selectivity of this reaction for the Fc region of the immunoglobulin, the affinity of the antibody to the antigen isn’t compromised, unlike randomly labelled antibodies. The chelators that are covalently attached to the antibody enable radiolabeling with radioisotopes such as Gallium-68, Lutetium-177 and Indium-111 for molecular radiotherapy or molecular imaging purposes. The commercially available trastuzumab was labeled via regio-selective conjugation with a DOTA-GA chelating agent using the AbYlinkTM technology, then radiolabeled with Indium-111. The in vivo biodistribution of the HER2 radiolabeled bioconjugate was evaluated in mice bearing NCI-N87 gastric cancer tumors. Trastuzumab conjugation resulted in a homogeneous labeling of antibodies with a Degree of Conjugation (DoC) close to 2, conducted in a regio-selective manner. The biodistribution and imaging experiments demonstrated successful accumulation of the antibody within the NCI-N87 tumour implanted in mice. The radiolabeled conjugate remained stable in vitro and in vivo, demonstrating the AbYlink™ as a valuable tool for biologics conjugation. We have successfully validated in vivo the use of AbYlink™ technology in the context of Antibody Radio Conjugates. This regio-selective bioconjugation strategy holds potential for wider application across molecular radiotherapies or pharmacodynamic assessment of antibodies. Céline Mothes, Peggy Provent, Marie Ruch, Mathieu Moreau, Michael Claron, Alex Helbling, Viktoriia Postupalenko, Léo Marx, Patrick Garrouste, Eftychia Koumarianou. Pharmacological evaluation of bioconjugated trastuzumab using the AbYlinkTM regio-selective conjugation technology in gastric cancer expressing HER2+ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1825.

Antibody-drug conjugates (ADCs) are considered as promising cancer treatment modalities that combine the selectivity of antibodies and the cytotoxic properties of payloads using chemical linkers. However, despite their success, ADCs still suffer from drawbacks, e.g., systemic toxicity, which limit their potential clinical applications. The systemic toxicity of an ADC is mainly related to the stability of its linker, and to the selectivity of its antibody towards the targeted antigen expressed on cancer cells. Lu/BCAM (Lutheran/basal cell adhesion molecule) is a member of the immunoglobulin superfamily and is a receptor for laminin, a protein that facilitates cell adhesion, migration, and invasion. A growing number of studies show that BCAM plays an essential role in tumor progression and is overexpressed on epithelial cancers e.g., skin cancer. Here we describe GENA-111, a human monoclonal anti-BCAM IgG4 (S228P) antibody that binds to human BCAM with high affinity, and that is significantly internalized by BCAM-positive tumor cells. We also describe the GENA-111-auristatin F ADC, wherein the GENA-111 antibody has been armed with an auristatin F derivative using a new linker technology and a stabilized thiol maleimide conjugation. This new linker technology comprises a cleavable peptidic sequence that facilitates multidrug attachment and the production of ADCs with tailored drug-to-antibody ratios (DARs). Cytotoxic drugs are rapidly and selectively released from the linker by the carboxypeptidase activity of Cathepsin B. In vitro cytotoxicity examination showed the potent cytotoxic effects of this GENA-111-auristatin F ADC on BCAM-expressing tumor cells, with a positive correlation between cytotoxicity and BCAM expression. Moreover, this ADC was also shown to significantly reduce the growth of tumor cells, including A431, T47D, and Huh7. The GENA-111-auristatin F ADC was evaluated in a xenograft mouse model established by subcutaneous injection of A431 cells, a BCAM positive human skin cancer cell line. The results of this study will be presented and discussed. Taken together, our data suggest that an ADC targeting BCAM e.g., GENA-111-auristatin F, might be a promising treatment strategy for BCAM positive epithelial cancer patients. Citation Format: Hyunkyung Yu, Nathalie Bellocq, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Bu-Nam Jeon, Léo Marx, Mathilde Pantin, Hyunjin Yoo, Seungmin Byun, Joo-Yeon Chung, Mi Young Cha, Patrick Garrouste, Frédéric Lévy. The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1760.

Antibodies are an attractive therapeutic modality for cancer treatment as they allow the increase of the treatment response rate and avoid the severe side effects of chemotherapy. Notwithstanding the strong benefit of antibodies, the efficacy of anti-cancer antibodies can dramatically vary among patients and ultimately result in no response to the treatment. Here, we have developed a novel means to regioselectively label the Fc domain of any therapeutic antibody with a radionuclide chelator in a single step chemistry, with the aim to study by SPECT/CT imaging if the radiolabeled antibody is capable of targeting cancer cells in vivo. A Fc-III peptide was used as bait to bring a carbonate electrophilic site linked to a metal chelator and to a carboxyphenyl leaving group in close proximity with an antibody Fc nucleophile amino acid (K317), thereby triggering the covalent linkage of the chelator to the antibody lysine, with the concomitant release of the carboxyphenyl Fc-III ligand. Using CHX-A''-DTPA, we radiolabeled trastuzumab with indium-111 and showed in biodistribution and imaging experiments that the antibody accumulated successfully in the SK-OV-3 xenograft tumour implanted in mice. We found that our methodology leads to homogeneous conjugation of CHX-A''-DTPA to the antibody, and confirmed that the Fc domain can be selectively labeled at K317, with a minor level of unspecific labeling on the Fab domain. The present method can be developed as a clinical diagnostic tool to predict the success of the therapy. Furthermore, our Fc-III one step chemistry concept paves the way to a broad array of other applications in antibody bioengineering.

Abstract Clinical development of therapeutic antibodies relies on robust and thorough preclinical evaluation of multiple pharmacodynamic and pharmacokinetic parameters of antibody candidates. While current methods are available for in vitro characterization of antibodies, in vivo determination of tissue distribution, tumor accumulation and retention and potential off-target effects cannot be performed non-invasively in vivo over a prolonged period of time. Here, we used AbYlinkTM, a novel site-selective labeling method of native antibodies, enabling the single-step covalent conjugation of a radionuclide chelator to the Fc domain of any IgG antibody isotype. Given the selectivity of this reaction for the Fc region of the immunoglobulin, labeling of the antibody at this location should not compromise binding to the antigen. Chelators that are covalently attached to the antibody allows for the labeling with radioisotopes such as 68Ga, 177Lu, 111In and 89Zr, and for the sensitive and quantitative molecular in vivo measurement by Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT). AbYlinkTM also permits the conjugation of fluorescent dyes to antibodies with the aim to measure antibody pharmacodynamic parameters by intravital microscopy. In this work, we have validated the technology by labeling several antibodies and ADCs with different payloads. Importantly, antibodies conjugated with AbYlinkTM retained the same affinity to the antigen as the original, unconjugated antibodies, contrary to the randomly labelled antibodies. We will also present data on the specific radiolabeling of commercially available trastuzumab with 111Indium and the SPECT images obtained after injection of mice bearing HER2-positive tumors with 111In-trastuzumab. The present data suggest that AbYlinkTM can be used to support preclinical drug discovery tools to assist in the selection of therapeutic antibody candidates and/or for pharmacodynamic assessment of commercial antibodies. Furthermore, the specificity of the chemical conjugation suggests that AbYlinkTM can help to control the chelator distribution on the antibody drug conjugates. Citation Format: Viktoriia Postupalenko, Léo Marx, David Viertl, Natalia Gasilova, Mathilde Plantin, Nadège Gsponer, Alexandre Johanssen, Thibaut Denoel, Gerrit Hagens, Jean-Manuel Segura, Frédéric Levy, Patrick Garrouste, John Prior, Margret Schottelius, Niklaus Schaefer, Origene Nyanguile. AbYlinkTM: A site-selective labeling method for preclinical imaging of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1304.

A symposium report on synthetic strategies which provide a synthetic entry to a new generation of template assembled synthetic proteins (TASP). [on SciFinder (R)]

We report here the synthesis and activity of HIV protease inhibitors. In the first stage hydrophobic compounds incorporating a ‘carba’ bond surrogate or a beta-homologated residue were synthesized. Secondly, we synthesized cyclic compounds in which we incorporated 2-quinoline carboxylic acid in the P3 position and the amino-hydroxyindane moiety in the P3. The last part of this work was dedicated to a structure/activity study of a peptide substrate. These modifications allowed us to work up the synthesis of new pseudopeptide bonds: amino-amide and hydroxy-amide. Compounds with activity in the micromolar range were actually a starting point for the synthesis of new protease inhibitors.

(S-2-amino-5-(aminooxy)pentanoic acid (L-homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L-homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. (C) 1998 European Peptide Society and John Wiley Br Sons, Ltd.

The sequential condensation of peptide loops onto selectively addressable templates immobilised on solid supports allows the rapid and efficient synthesis of protein mimetics applying the TASP (Template Assembled Synthetic Protein) concept. As an example of the versatility of this methodology, we describe here the solid phase assembly of a representative 3-loop receptor mimetic.

Angewandte Chemie International Edition in EnglishVolume 35, Issue 13-14 p. 1482-1485 Communication Template Assembled Synthetic Proteins (TASP) as Functional Mimetics of Proteins† Prof. Dr. Manfred Mutter, Corresponding Author Prof. Dr. Manfred Mutter Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Pascal Dumy, Dr. Pascal Dumy Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Patrick Garrouste, Dr. Patrick Garrouste Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Christian Lehmann, Dr. Christian Lehmann Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Marc Mathieu, Dipl.-Chem. Marc Mathieu Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Cristina Peggion, Dipl.-Chem. Cristina Peggion Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Stéphane Peluso, Dipl.-Chem. Stéphane Peluso Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Alain Razaname, Dr. Alain Razaname Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Gabriele Tuchscherer, Dr. Gabriele Tuchscherer Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this author Prof. Dr. Manfred Mutter, Corresponding Author Prof. Dr. Manfred Mutter Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Pascal Dumy, Dr. Pascal Dumy Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Patrick Garrouste, Dr. Patrick Garrouste Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Christian Lehmann, Dr. Christian Lehmann Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Marc Mathieu, Dipl.-Chem. Marc Mathieu Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Cristina Peggion, Dipl.-Chem. Cristina Peggion Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDipl.-Chem. Stéphane Peluso, Dipl.-Chem. Stéphane Peluso Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Alain Razaname, Dr. Alain Razaname Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this authorDr. Gabriele Tuchscherer, Dr. Gabriele Tuchscherer Institute for Organic Chemistry, University of Lausanne BCH-Dorigny, CH-1015 Lausanne (Switzerland) Fax: Int. code +(21)692-4015 e-mail: [email protected]Search for more papers by this author First published: July 1996 https://doi.org/10.1002/anie.199614821Citations: 82 † This work was supported by the Swiss National Science Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract The bare essentials of a functional protein are all that are retained when the structural domain of a native protein is replaced by a synthetic template molecule (see picture below). New strategies for the synthesis of this novel type of template-assembled synthetic peptide (TASP) provide functional mimetics of receptors and antibodies. References 1 G. Tuchscherer, M. Mutter, J. Biotechnol. 1995, 41, 197. 2 G. Tuchscherer, B. Dörner, U. Sila, B. Kamber, M. Mutter, Tetrahedron 1993, 49, 3559. 3 J. W. Bryson, S. F. Betz, H. S. Lu, D. J. Suich, H. X. Zhou, K. T. O'Neil, W. F. DeGrado, Science 1995, 270, 935. 4 J. P. Schneider, J. W. Kelly, Chem. Rev. 1995, 95, 2169. 5 M. Mutter, S. Vuilleumier, Angew. Chem. 1989, 101, 551; Angew. Chem. Int. Ed. Engl. 1989, 28, 535. 6 M. Mutter, G. Tuchscherer, C. Miller, K. H. Altmann, R. I. Carey, D. F. Wyss, A. M. Labhardt, J. E. Rivier, J. Am. Chem. Soc. 1992, 114, 1463. 7 G. Tuchscherer, M. Mutter, J. Peptide Sci. 1995, 1, 3. 8 G. Tuchscherer, C. Servis, G. Corradin, U. Blum, J. Rivier, M. Mutter, Protein Sci. 1992, 1, 1377. 9 K. Rose, J. Am. Chem. Soc. 1994, 116, 30. 10 P. E. Dawson, S. B. H. Kent, J. Am. Chem. Soc. 1993, 115, 7263. 11 G. Tuchscherer, Tetrahedron Lett. 1993, 34, 8419. 12 A. Nefzi, X. Sun, M. Mutter, Tetrahedron Lett. 1994, 36, 229. 13 I. Ernest, J. Kalvoda, C. Sigel, G. Rihs, H. Fritz, M. J. J. Blommers, F. Raschdorf, E. Francotte, M. Mutter, Helv. Chim. Acta 1993, 76, 1539. 14 P. Dumy, I. M. Eggleston, S. E. Cervigni, U. Sila, X. Sun, M. Mutter, Tetrahedron Lett. 1995, 36, 1255. 15 P. Dumy, I. M. Eggleston, G. Esposito, S. Nicula, M. Mutter, Biopolymers 1996, in press. 16 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, J. H. Cuervo, Nature 1991, 354, 84. 17 According to cyclization theory, the cyclization equilibrium constant K is proportional to the volume density W(r) of the head to tail vectors 〈r〉 (where 〈〉 denotes the statistical mechanical average of r over all accessible loop conformations). Assuming in a first approximation a random coil conformation of the flexible loop sequence and a Gaussian distribution of 〈r〉, W(r) at a fixed distance r1 of the reacting chain ends (r1 corresponds to the distance between the loop attachment sites on the template) approaches a maximal value for r1 ≈ 〈r〉. Consequently, the tendency for ring closure in loop condensation reactions (where r1 → 〈r〉) is predicted to be higher than in regular cyclization reactions (where r1 → 0). see: M. Mutter, J. Am. Chem. Soc. 1977, 99, 8307. 18 Y. Satow, G. H. Cohen, E. A. Padlan, D. R. Davies, J. Mol. Biol. 1986, 593, 190. 19 G. B. Bloomberg, D. Askin, A. R. Gargaro, M. J. A. Tanner, Tetrahedron Lett. 1993, 34, 4709. 20 R. W. Woody, Peptides, (NY) 1985, 7, 15. 21 These studies will be subject of a separate publication. 22 J. M. Stewart, J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical, Rockford, IL, 1984. 23 P. R. Gerber, Biopolymers 1992, 32, 1003. 24 P. R. Gerber, K. Müller, J. Comput. Aided Mol. Design 1995, 9, 251. 25 P. J. Flory, Statistical Mechanics of Chain Molecules, Interscience, New York, NY, 1969. 26 G. Fransson, B. K. S. Lundberg, Acta Chem. Scand 1972, 26, 3969. Citing Literature Volume35, Issue13-14July 1996Pages 1482-1485 ReferencesRelatedInformation

Auf das Wesentliche zurückgeführt wird ein natives Protein, wenn man dessen Strukturteil durch ein synthetisches Templat ersetzt (siehe Bild unten). Der funktions‐bestimmende Teil bleibt also erhalten, und das Templat‐assoziierte synthetische Protein (TASP) sollte beispielsweise als Rezeptor‐oder Antikörpermimeticum fungieren. Neue Strategien zur Synthese von derartigen TASP‐Verbindungen wurden nun erarbeitet. magnified image

The synthesis of chiral N-protected tetramic acid derivatives which are important precursors of β-hydroxy γ-amino acid under mild conditions is described. Reaction of urethane-N-carboxyanhydrides (UNCAs) with Meldrum's acid in the presence of a tertiary amine, followed by subsequent cyclisation produced tetramic acid derivatives. This procedure is applicable to Boc-, Fmoc- and Z- N-carboxyanhydrides.

A facile synthesis of a wide variety of N-benzyloxycarbonyl-amino acid-tert-butyl ester derivatives under mild conditions is described. N-protected amino acids were esterified with tert-butyl bromide in dimethylacetamide as solvent, in the presence of benzyltriethylammonium chloride (BTEAC)and a large excess of potassium carbonate. Many amino Z-acid-Tert-butyl esters that might be difficult to prepare by other methods have been synthesized in high yields by this procedure. The reaction is simple, unexpansive, easily scaled up, and proceeds without observable racemization.