Antibody-drug conjugates (ADCs) using DNA Topoisomerase I inhibitor DXd/SN-38 have transformed cancer treatment, yet more effective ADCs are needed for overcoming resistance. We have designed an ADC class using a novel self-immolative T moiety for traceless conjugation and release of exatecan, a more potent Topoisomerase I inhibitor with less sensitivity to multidrug (MDR) resistance. Characterized by enhanced therapeutic indices, higher stability and improved intra-tumoral pharmacodynamic response, antibody-T moiety-exatecan conjugates targeting HER2, HER3, TROP2 overcome intrinsic or treatment-resistance of equivalent DXd/SN-38 ADCs in low-target expression, large-size and MDR+ tumors. T moiety-exatecan ADCs display durable antitumor activity in PDX and organoid models representative of unmet clinical needs including EGFR-del19/T790M/C797S triple mutation lung cancer and BRAF/KRAS–TP53 double-mutant colon cancer, and show synergy with PARP/ATR inhibitor and anti-PD-1 treatment. High tolerability of T moiety-exatecan ADC class in non-human primate supports its potential to expand responding patient population and tumor types beyond current ADCs.

Antibodies are essential for elucidating gene function. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 monoclonal antibodies (mAbs) against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic antibody feature. Distinctive combinations of 10,000 to 20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-protein pairs were found for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for membrane proteins and chromatin immunoprecipitation–sequencing mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues identified internalizing tumor antigens for antibody-drug conjugates. By finding high-affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

Peach (Prunus persica) is a typical climacteric fruit that produces ethylene rapidly during ripening, and its fruit softens quickly. Stony hard peach cultivars, however, do not produce large amounts of ethylene, and the fruit remains firm until fully ripe, thus differing from melting flesh peach cultivars. To identify the key proteins involved in peach fruit ripening, an antibody-based proteomic analysis was conducted. A mega-monoclonal antibody (mAb) library was generated and arrayed on a chip (mAbArray) at a high density, covering~4950 different proteins of peach. Through the screening of peach fruit proteins with the mAbArray chip, differentially expressed proteins recognized by 1587 mAbs were identified, and 33 corresponding antigens were ultimately identified by immunoprecipitation and mass spectrometry. These proteins included not only important enzymes involved in ethylene biosynthesis, such as ACO1, SAHH, SAMS, and MetE, but also novel factors such as NUDT2. Furthermore, protein-protein interaction analysis identified a metabolon containing SAHH and MetE. By combining the antibody-based proteomic data with the transcriptomic and metabolic data, a mathematical model of ethylene biosynthesis in peach was constructed. Simulation results showed that MetE is an important regulator during peach ripening, partially through interaction with SAHH.

4Antibodies are essential for elucidating the roles of genes decoded by genome 5 sequencing. However, affordable technology for proteome-scale antibody 6 generation does not exist. To address this, we developed the Proteome Epitope 7 Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 mAbs 8 against 15,199 peptides from diverse proteomes. PETAL harbors binders for a 9 great multitude of proteins in nature due to antibody multispecificity, an intrinsic 10 feature of an antibody. Distinctive combinations of 10,000-20,000 mAbs were 11 found to target specific proteomes by array screening. Phenotype-specific mAb-12 target pairs were discovered for maize and zebrafish samples. 13 Immunofluorescence and flow cytometry mAbs for human membrane proteins 14 and ChIP-seq mAbs for transcription factors were identified from respective 15 proteome-binding PETAL mAbs. Differential screening of cell surface proteomes 16 of tumor and normal tissues discovered internalizing tumor antigens for antibody-17 drug conjugates. By discovering high affinity mAbs at a fraction of current time 18 and cost, PETAL enables proteome-scale antibody generation and target 19 discovery.20 21 22 Keywords: 1 antibody; proteome-scale; multispecificity; antibody microarray; target discovery; 2 antibody-drug conjugate (ADC) 3 1Facilitated by the ever-growing capability of current DNA sequencing 2 technologies, more than 1,300 genomes of animals and 496 genomes of plants 3 and many others have already been sequenced, representing millions of genes, 4 and the number will increase faster from projects such as G10K, i5k and so on 1 . 5To understand the roles of these genes, the functions of the gene-coded proteins 6 need to be explored, and antibodies, especially renewable monoclonal antibodies 7 (mAbs) generated at a proteome-scale, are urgently needed. mAbs produced by 8 hybridoma technologies for human proteins have long been recognized as the 9 most direct tools for diagnostic and therapeutic target discovery 2 . Classic 10 therapeutic targets Sialyl Lewis Y, prostate-specific membrane antigen (PSMA) 11 and, more recently, a novel target for multiple myeloma were discovered by 12 mAbs for cell surface proteins 3-6 . 13Despite the power of mAbs and mAb-based discovery, large-scale 14 generation of mAbs remains difficult since traditional hybridoma development is 15 time-consuming (4-6 months starting from antigens), expensive ($3,000-8,000 16 per antigen), and challenging to scale. Further, mAb generation by immunization 17 typically requires a milligram of purified antigens, a significant challenge for many 18 protein targets, especially for membrane proteins of primary research interest. 19Even for human proteins, a majority of the 6,000 membrane proteins have not 20 been directly explored as diagnostic or therapeutic targets due to a lack of high-21 quality antibodies for applications such as flow cytometry (FACS) and 22 immunofluorescence (IF) 7, 8 . 23The Human Protein Atlas (HPA) offers an alternative approach for proteome-1 sca...

HER3 is a unique member of the epidermal growth factor receptor family of tyrosine kinases, which is broadly expressed in several cancers, including breast, lung, pancreatic, colorectal, gastric, prostate, and bladder cancers and is often associated with poor patient outcomes and therapeutic resistance. U3-1402/Patritumab-GGFG-DXd is the first successful HER3-targeting ADC molecule with clinical efficacy in non-small cell lung cancer (NSCLC). However, over 60% of patients are non-responsive to U3-1402 due to low target expression levels and responses tend to be in patients with higher target expression levels. U3-1402 is also ineffective in more challenging tumor types such as colorectal cancer. AMT-562 was generated by a novel anti-HER3 antibody Ab562 and a modified self-immolative PABC spacer (T800) to conjugate exatecan. Exatecan showed higher cytotoxic potency than its derivative DXd. Ab562 was selected due to its moderate affinity for minimizing potential toxicity and improving tumor penetration purposes. Both alone or in combination therapies, AMT-562 showed potent and durable antitumor response in low HER3 expression xenograft and heterogeneous patient-derived xenograft/organoid (PDX/PDO) models, including digestive system and lung tumors representing of unmet needs. Combination therapies pairing AMT-562 with therapeutic antibodies, inhibitors of CHEK1, KRAS and TKI showed higher synergistic efficacy than Patritumab-GGFG-DXd. Pharmacokinetics and safety profiles of AMT-562 were favorable and the highest dose lacking severe toxicity was 30 mg/kg in cynomolgus monkeys. AMT-562 has potential to be a superior HER3-targeting ADC with a higher therapeutic window that can overcome resistance to generate higher percentage and more durable responses in U3-1402-insensitive tumors.