Functional Profiling of B Cell Targeting Therapeutic Antibodies

B cell targeting monoclonal antibodies provide their clinical efficacy by three different mechanisms i) antibody-dependent cell-mediated cytotoxicity (ADCC), ii) complement-dependent cytotoxicity and iii) induction of direct apoptosis. This functional profile is pivotal for the characterisation of the mode-of-action, potency and for the selection of antibody-producing clones.

Together with adequate target cell lines IBR Inc. offers reproducible ADCC, CDC and apoptosis testing as a package of cell based assays dedicated to functionally profile clones of anti-B cell therapeutic antibodies. To further improve reproducible ADCC testing a protocol has been introduced that enables fast and accurate propagation of large numbers of CD56 and CD16-expressing NK-cells from heparinised blood. Furthermore, a CD16a-expressing NK effector cell line is being developed to provide blood donor independent ADCC testing.

ADCC (Antibody-dependent Cell-mediated Cytotoxicity)
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a prominent mode-of-action of B cell targeting Therapeutic Antibodies: the Natural Killer (NK-) and other specialised immune cells attack B cells by binding to immune complexes on the surface of targeted B cells. ADCC is triggered via Fcy RIIIA (CD16a) recognising the activated Fc region of complexedIgG on target cells leading to their perforin-mediated lysis.

ADCC assays can be performed with peripheral blood mononuclear cells (PBMC) isolated from heparinised blood or further purified to primary NK-cells expressing the phenotype CD56+/ CD16a+/ CD3-/TCR-. Unfortunately, the limited numbers of NK-cells present in blood limits productivity and effectiveness of ADCC assays.

To overcome this limitation and the inherent donor-to-donor variability, IBR Inc. makes use of a protocol that allows in-vitro propagation of large numbers of CD56+/CD16+ NK-cells and combines them with proprietary CD20-expressing B-cell clones on the target cell side.

CDC (Complement-dependent cytotoxicity)
CDC is known to contribute to the therapeutic efficacy of CD20 antibodies. These therapeutic antibodies drive CDC due to their ability to bind the complement component C1q that by triggering the complement cascade leads to the assembly of the terminal pore-forming C5-9 complex. Based on their ability to redistribute into lipid rafts on target cells, CD20 antibodies are defined as either Type I or Type II with CDC activity most prominent in Type I antibodies. The proprietary B cell clones of IBR Inc. provide the cellular basis of a sensitive CDC assay. In the presence of human serum as source of complement (C), lysis depends on the concentration of the bound antibody.

Antibody-mediated Apoptosis
In addition to ADCC and CDC assays especially anti-CD20 antibodies provide direct apoptosis after binding to target cells. IBR Inc. offers determination of apoptosis by measuring Annexin IV by flow cytofluorometry in addition to different caspase assays.

If you would like more information about testing of B cell targeting Therapeutic Antibodies and need advice about how they can benefit your own products or processes, please contact IBR Inc. directly.