Cytotoxicity Assays

Cytotoxicity Assays

Clean Cells has developed an innovative cytotoxicity potency assay, compliant with the Regulatory Authorities requirements. The method could be used for in vitro characterization of ADCC, CDC and/or apoptosis activities of the classical cytolytic antibodies.

Specificities and Benefits of the Clean Cells’ assay

1. Consideration and Benefits of the Clean Cells’ Assay

All our assays are developed based on the Regulatory guidelines and recommendations:

2. An innovative non-adioactive cytotoxicity measurement method

Standardized and biologically relevant potency assays are required by Antibody Developers and Regulators for the characterization of therapeutic antibodies, including apoptosis, ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity) potency assays. In the case of antitumor antibodies such as MabThera® or Herceptin®, the critical point to achieve biological relevance is to measure the target cell death.

Although the 51Cr-release method is considered as specific and sensitive for evaluating cytotoxicity, it is very difficult to implement in an industrial context because of radioactivity-related constraints. Alternative non-radioactive methods (e.g., calcein or LDH methods) suffer from a lack of sensitivity, reproducibility and/or specificity. Regardless of the read-out method, cytotoxicity assays (as other bioassays) are usually considered to have limited performances in standardization.

Based on these observations, we developed a non-radioactive luminescent method:

  • specific of the mechanism of action of cytolytic antibodies (i.e., measuring the target cell lysis);
  • exhibiting improved standardization potential via a reporter molecule stably expressed by target cells (no recurrent staining step required);
  • easy to implement via the use of widely available equipments (luminometers).

The method is based on the genetic transformation of target cells to express a reporter protein, which is released in the culture supernatant during cell death. As illustrated by the figure 1, the method gives a linear and highly sensitive signal, allowing to use less than 1000 target cells/well. Moreover, the reporter protein expression is highly stable over target cell passages.

Figure 1. (A) Maximum and spontaneous release signals are proportional to the number of target cells (Raji or SKOV-3) (mean±SD of triplicates and linear regression). Maximum lysis is obtained by incubation with Triton X-100. (B) High and reproducible signal/background (S/B) ratios can be achieved using 500 to 3000 target cells /well. (C) The stability of the reporter protein expression was monitored over 50 passages (the dotted lines represent the median ± 40%, respectively).

Furthermore, when apoptosis is induced in the transformed target cells by a chemical agent (staurosporine) or by UV exposure, the release of the reporter protein is fully correlated with the actual cell death measured by flow cytometry, as shown on Figure 2. This demonstrates that the method is fully representative of the target cell death.

Fig2

Figure 2. Transformed Raji cells were cultured in the presence of increasing doses of staurosporine (red) or exposed to increasing doses of UV (blue). In each well, the percentage of dead cells was calculated by measuring the luminescent signal in the supernatant and, in parallel, via flow cytometry on the cell pellets. The dotted line indicates the linear regression calculated from the data (n=2 independent experiments).

3. Specially developed standardized ADCC effector cells

ADCC assays are generally considered as very variable assays. The major source of variability is the effector cell origin. By using human peripheral blood mononuclear cells (PBMCs), the assay reproducibility will be strongly impacted by inter-individual variability, involving genetic polymorphisms (e.g., the FCGR3A-158 polymorphism). On the other hand, NK cell lines (e.g. NK92) may suffer from culture condition-linked variability and often exhibit a high non-specific background lysis because of their NK receptors.

Thanks to a collaboration with INSERM¹, Clean Cells produces standardized ADCC effector cells, consisting of Human cytotoxic T cells expressing Human CD16 (FcγRIIIa, with a valine in position 158). These cells are able to mediate ADCC² with the following benefits:

¹INSERM UMR892, Nantes (France), under licence from Inserm, Université d’Angers, Université de Nantes and Centre National de la Recherche Scientifique

²Clémenceau et al. Blood 2006. 107:4669-4677.

  • Complete absence of non-specific background lysis (no NK receptor expression);
  • No inter-individual variability in ADCC response because all batches come from the same original cell source;
  • Completely homogeneous cell batches produced in a thaw-and-use format and highly reproducible ADCC activity between consecutive batches produce very consistent and reproducible results over time;

As shown on this short movie, the cells are very active in the presence of the anti-CD20 MabThera (right panel) as compared to an irrelevant antibody (left panel) [the blue to green to red color shifts represent increasing calcium influx into the cells]. The effectors “screen” the target cell layer, fix on a given target and then activate the cytolytic process, resulting in the shrinkage and death of the target cell.

FILM

These ADCC effector cells are perfect candidates for any kind of ADCC assay, regardless of your read-out system, e.g., 51Cr, LDH, calcein, Europium, proliferation assay, and so on. Of course, they are perfectly well adapted to Clean Cell’s luminescent method !

Method performance and comparison with  51Cr

The experimental procedures associated to both methods are compared on the following flowchart.

Schema

This first shows that the Luminescent method requires less manipulation, decreasing the risk of variability, and less experimental time.

By comparing both methods in parallel, the luminescence-based method showed fully comparable lysis to the reference 51Cr-release assay, as illustrated by the curves in Figure 3 and by the 4-parameter logistic regression data shown in Table 1. In combination with Clean Cells’ ADCC effector cells, the assay achieves a very good intermediate precision (CV of EC50).

Fig3

Figure 3. ADCC (upper panel) and CDC (lower panel) activities were measured using in parallel the 51Cr-release (blue symbols) or the luminescence-based (green symbols) method. The data are from 3 independent assays (mean ± SD).

Essai Modèle Méthode S/B ratio Emin (moyenne) Emax (moyenne) EC50 (ng/mL) [CV]
ADCC Raji 51Cr 5.6 -1.2 % 75.7 % 55.7 [29.3%]
Lumi. 17.1 -1.2 % 73.6 % 31.8 [22.8%]
SKOV-3 51Cr 4.4 0.4 % 41.2 % 387.7 [38.4%]
Lumi. 13.6 0.1 % 35.1 % 191.6 [7.2%]
CDC Raji 51Cr 3.0 3.0 % 88.6 % 156.7 [19.2%]
Lumi. 5.7 -0.3 % 95.6 % 73.1 [13.5%]
CHO/TNF 51Cr 4.4 -0.5 % 47.9 % 142.7 [18.6%]
Lumi. 7.9 -2.2 % 46.5 % 152.5 [15.0%]

Table 1. The mean values of S/B ratios, Emin, Emax, and EC50 with corresponding coefficient of variation (CV), are summarized for both the ADCC and CDC assays using 51Cr or luminescence readouts. The data were obtained from 3 independent experiments.

Conclusion

  • The novel method gives completely comparable results to 51Cr-release readout, but in an easy-to-use and cost effective Its high sensitivity allows to use very low amounts of cells (500 to 1000 target cells/well), compatible with high throughput screening (HTS).
  • Associated with Clean Cells’ exclusive ADCC standardized effector cells, the method demonstrates an impressive standardization potential for such kind of bioassays, with improved performances compared to 51Cr.
  • The method is representative of mAbs mechanisms of action. Overall, it is compliant with Regulatory requirements (e.g., EMA/CHMP/BWP/157653/07) and fully usable for ADCC, CDC and apoptosis
  • Available as outsourced service at Clean Cells (development and validation of customized assays…) or by technology transfer to customer’s lab. The available models are listed below (any other model may be developed on demand):
Lignées cibles disponibles Antigènes cibles testés Exemples d’anticorps Testé en ADCC

Testé en CDC

Testé en apoptose
Raji (lymphome B) CD19, CD20 MabThera® Oui Oui Oui

SKOV-3 (ovaire)

SKBR-3 (sein)

EGFR, HER2 Erbitux®, Herceptin® Oui

Non

(Résistante à la lyse)

Oui
CHO exprimant le TNF-α membranaire TNF-α Humira®, Remicade® Oui Oui Oui

EXAMPLES OF USE

  • Comparison of antibody modifications (e.g., fucose level or changes in production process) on cytotoxic activity over time;
  • Testing of lot-to-lot consistency during development or production processes;
  • Assessment of biosimilarity;
  • Development of GMP-validated QC and/or lot-release potency assays for innovator or biosimilar antibodies.

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