Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel... Show moreIdentification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium-and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level. Show less
Wagner, K.; Apai, D.; Kasper, M.; McClure, M.; Robberto, M. 2022
Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-021-21176-6, published online 10 February 2021.The original version of this Article contained an error in Fig. 1b, in which the... Show moreCorrection to: Nature Communicationshttps://doi.org/10.1038/s41467-021-21176-6, published online 10 February 2021.The original version of this Article contained an error in Fig. 1b, in which the units incorrectly read ‘AU,’ instead of the correct ‘au’. This has been corrected in both the PDF and HTML versions of the Article. Show less
Close, L.M.; Males, J.; Long, J.D.; Gorkom, K. van; Hedglen, A.D.; Kautz, M.; ... ; Doyon, R. 2020
Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form... Show moreTargeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3 epsilon. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues. Show less
