A presentation on assessing the target specificity of biotherapeutics by Diogo Rodrigues Ferreirinha, European Business Development Manager, Retrogenix Limited
Silvia Hnatova highlights a screening technology for assessing the target specificity of biotherapeutics presented by Diogo Rodrigues Ferreirinha, European Business Development Manager at Retrogenix Limited.
Mr. Ferreirinha’s talk focused on a novel cell microarray technology developed by Retrogenix to screen for assessing the target specificity of biotherapeutics. The main use of cell microarrays is to identify the primary receptor ID or secondary/off-target receptor ID, or both at the same time.
Retrogenix’s cell microarray contains a large set of expressed human proteins (6,200+) that can be screened in live or fixed cells.
The protein library contains all human proteins, both plasma membrane and secreted proteins, that can be screened as targets or off-targets of the candidate molecule.
Many proteins are present with multiple isoforms and miscategorized proteins are excluded from the library.
The array works by using expression vectors with cDNA embedded in gelatin in fixed positions, using green fluorescent protein as a reporter.
The slides undergo reverse transcription, and the expression of the target protein, at which point they can be tested against the molecule’s targets.
The readout can be customized based on the application. The main value is that the proteins are expressed in the cells, leading to a native protein preserving posttranslational modifications.
Diving deeper into how the technology works, Mr. Ferreirinha explained that the test molecule is always pre-screened to confirm whether there is a background binding of the molecule in HEK293 cells, to determine the signal: background ratio, and to reduce the costs if this ratio is not optimal.
If passing the initial step, the molecule is then tested against the library of proteins, identifying library hits that are then tested, further validated, including the option to validate these in live cells.
Building upon the previous explanation of the cell microarray, Mr. Ferreirinha presented several case studies.
Using the technology, a key malarial receptor was identified to bind to the human receptor for PfEMP1 (the malaria parasite protein) which is associated with a severe form of childhood malaria.
The His-tagged version of PfEMP1 was screened, leading to potential hits, and subsequent validation led to the identification of EPCR, the endothelial protein receptor.
Another case study used the microarray for identifying post-alloHSCT antibodies for the treatment of chronic lymphocytic leukemia (Chang et al., 2018). Siglec-6 was found as a CLL-specific antigen and a promising immunotherapy target.
The microarray can also be used for specificity screening of antibody off-targets, aiding in safety or candidate selection screening.
The screening can be used to aid Investigational New Drug submissions to the FDA, EMA, and equivalent agencies, improving the acceptance rate.
A case study of camrelizumab was presented, explaining how the toxicity profile was improved by identifying off-targets, informing the reengineering of the original antibody through understanding the side effects associated with the previous antibody.
Ultimately, an antibody with an improved safety profile was successfully produced.
The cell microarray was also used to screen for highly specific anti-ROR1 and anti-ROR2 monoclonal antibodies, to test Antibody Drug Conjugates (ADC) to carry out specificity screens, and to identify the off-targets of CAR-T cells.
An interesting case study on KITE-585 targeting CAR-T cells was presented, identifying proteins known to be involved in immunological synapse formation, and confirming the specificity to the targets.
BCMA was the only hit identified by antibody clone RD-1 in the additional confirmation and specificity screen.
B-cell maturation antigen (BCMA) was confirmed as the specific target of the clinical candidate KITE-585 and no functional off-target was identified.
Mr. Ferreirinha finalized his talk by highlighting that the cell microarray technology can also be used to screen other cell types, including Jurkat cells.
During the Q&A session, there was an interesting discussion about the sensitivity of the technology, concluding that certain kinds of molecules can have more off-targets than others.
