In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Summary

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Abstract

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

Introduction

Therapeutic antibodies are recombinant monoclonal antibodies (mAbs) developed for the treatment of different pathologies, including cancers, autoimmune and chronic diseases, neurologic disorders, and others¹. Currently, the FDA has granted approval to more than 40 therapeutic mAbs, and more are expected to reach the market in the following years.

Rituximab is a high-affinity chimeric monoclonal IgG1 antibody approved for the treatment of CD20⁺ B-cell non-Hodgkin's lymphoma (NHL), CD20⁺ follicular NHL, chronic lymphocytic leukemia, and rheumatoid arthritis^2,3. The recognition of CD20, which is overexpressed in B cells, by rituximab induces apoptosis; complement activation; and antibody-dependent cell mediated cytotoxicity (ADCC)³. The patents of this drug expired in Europe and in the U.S. in 2013 and 2016, respectively. Thus, pharmaceutical companies worldwide are developing rituximab biosimilars. As in any other drug for human consumption, biosimilars require approval from regulatory agencies. International guidelines indicate that for mAbs, biosimilarity should be demonstrated by comparing the physicochemical characteristics, pharmacokinetics, efficacy, and safety of the new and reference products⁴.

Accordingly, the methodologies used in such comparisons must assess the structural and functional characteristics of the mAbs, especially those with clinical relevance. To that end, in vitro assays show several advantages over in vivo experiments (reviewed in Chapman et al.)⁵: i) in vitro studies are more sensitive to differences between the proposed biosimilar and the reference product; ii) in vivo studies must be performed in relevant species, which for many mAbs are non-human primates; and iii) since the mechanism of action, the preclinical toxicology, and the clinical effects of the reference product are well known, in vivo studies with biosimilars may not provide additional useful information. Accordingly, the European Union's Guidance for biosimilars allows candidates to enter clinical trials based on robust in vitro data alone⁶.

Here, we present two fast, economic, and simple assays that evaluate the biological activity of rituximab using CD20⁺ cultured cells. These assays can be included as part of the comparability exercise for rituximab biosimilar candidates.

Protocol

1. Evaluation of Target Binding by Flow Cytometry

1. Preparation of biological materials and reagents
   1. Make 500 mL of RPMI culture medium supplemented with 10% heat-inactivated fetal bovine serum (H-IFBS).
   2. Culture Daudi Burkitt's Lymphoma (Daudi) cells and Daudi GFP⁺ cells using RPMI and 75-cm² culture flasks. Maintain the cultures at 37 °C in a 5% CO₂ humidified atmosphere until they reach 6 - 9 x 10⁵ cells/mL.
   3. Make 50 mL of staining buffer by diluting 1/100 H-IFBS in PBS; this buffer is stable at 2 - 8 °C for at least one month.
   4. Prepare the test solutions for the reference and biosimilar mAbs. Make ten 1:2 serial dilutions (500 µL each) in staining buffer, starting from 5 µg/mL.
   5. Use staining buffer to dilute human IgG (isotype control) to 5 µg/mL and PE-Cy5 mouse anti-human IgG (secondary antibody) to the concentration suggested by the manufacturer.
   6. Prepare 4% paraformaldehyde in PBS (fixation buffer).
2. Target binding
   1. Collect the Daudi and Daudi GFP⁺ cell suspensions from the 75-cm² culture flasks and transfer them to a 15-mL centrifuge tube. Centrifuge at 400 x g for 5 min.
   2. Wash the cells by adding 5 mL of PBS and centrifuging the cell suspension at 400 x g for 5 min.
   3. Resuspend the cells in PBS and perform a cell count and viability analysis with trypan blue. Use cultures with cell viability levels ≥ 95% for the analysis.
   4. Dilute the cell suspension to 4 x 10⁶ cells/mL with cold staining buffer.
   5. In 1.5-mL microcentrifuge tubes, add 50 µL of the cell suspension to 100 µL of the different test concentrations of the reference or biosimilar mAbs. Include replicates for each experimental condition.
   6. Prepare additional tubes for the isotype control (human IgG1 instead of rituximab) and negative control (secondary antibody without primary antibody).
   7. Incubate at 4 °C for 20 - 30 min.
   8. Wash the cells by adding 1 mL of PBS and centrifuging the cell suspension at 400 x g for 5 min at 10 °C. Discard the supernatant.
   9. Suspend the cells in 100 µL of the secondary antibody and incubate for 20 - 30 min at 4 °C, protected from light.
   10. Wash the cells twice with PBS and suspend them in 200 µL of fixation buffer.
   11. Analyze the cells on a flow cytometer.
       NOTE: The signal remains stable for several days if the samples are stored at 4 °C and protected from light.
3. Data acquisition
   1. Open two dot-plots on a worksheet of the flow cytometer operating software. Set the FSC-A versus FSC-H in the first and the FSC-A versus SSA-A in the second. Open a histogram for the PE-Cy5 channel.
   2. In the FSC-A versus FSC-H plot, make a gate (R1) selecting singlet events (Figure 1A).
   3. Set the R1 population in the FSC-A versus SSA-A dot-plot and then make a new gate (R2) selecting target cells (Figure 1B). Set the R2 population in the PE-Cy5 intensity histogram to view the frequency distribution of the cells.
   4. Adjust the lower fluorescence intensity (FI) limit for the PE-Cy5 channel using the negative and isotype control (Figure 1C).
   5. Acquire 10,000 events within R2 from the sample with the higher concentration of the reference product. FI of this sample should be the highest expected (Figure 1C).
   6. Acquire the rest of the samples.
   7. For each sample, get the median fluorescence intensity (MFI) in the PE-Cy5 channel.
   8. For samples with the reference or biosimilar mAb, calculate the difference between sample MFI and that of the isotype control (ΔMFI).

2. Assessment of CDC

1. Preparation of biological materials and reagents
   1. Prepare cell culture medium and culture Daudi and Daudi GFP⁺ cells as described above (steps 1.1.1 - 1.1.2).
      NOTE: Additionally, the CDC assay requires serum-free RPMI.
   2. Dilute normal human serum complement (NHSC) 1:2 with serum-free RPMI. Prepare 2.5 mL.
   3. Prepare 1 mL of heat-inactivated (30 min/56 °C) NHSC diluted 1:2 with RPMI.
   4. Prepare sets of tests solutions for the reference and biosimilar mAbs in serum-free RPMI. Make ten dilutions (200 µL each) from 1 to 0.025 µg/mL.
2. CDC assay
   1. Collect the Daudi and Daudi GFP⁺ cells from the cultures and quantify the cell viability (see steps 1.2.1 - 1.2.3).
   2. Prepare a cell suspension with 4 x 10⁵ cells/mL in serum-free RPMI.
   3. Add 50 µL of cell suspension to 50 µL of each reference or biosimilar mAb test concentration in 96-well conical (V)-bottom microplates. Include replicates for each experimental condition.
   4. Prepare additional wells for the negative control (i.e., without mAb), basal death control (i.e., heat-inactivated NHSC in the presence of mAb), and staining positive control (i.e., cells exposed to 50 µL of 70% EtOH).
   5. Incubate the cells for 20 - 30 min at 37 °C in a 5% CO₂ humidified atmosphere.
   6. Add 50 µL of NHSC (diluted 1:2) to each well and incubate the opsonized cells for 2.5 h at 37 °C in a 5% CO₂ humidified atmosphere. Use heat-inactivated NHSC in the basal death control wells.
   7. Centrifuge at 400 x g for 5 min at 10 °C. Discard the supernatant.
   8. Wash the cells by adding 150 µL of PBS and centrifuging the cell suspension for 5 min at 400 x g and 10 °C. Discard the supernatant.
   9. Stain the samples with 7-aminoactinomycin (7-AAD), as previously described^7,8.
   10. Analyze the cells on a flow cytometer on the same day.
3. Data acquisition
   1. Open two dot-plots on a worksheet of the flow cytometer operating software. Set those plots as in steps 1.3.1 - 1.3.3 (Figure 2A-B). Create a third plot that is a dot-plot for GFP versus 7-AAD on the R2 population.
   2. Define the adequate FI limits using the Daudi cells, Daudi GFP⁺ cells, and death positive control (Figure 2C).
   3. For each sample, measure the percentage of 7-AAD⁺ target cells. Acquire at least 5,000 events from R2.
   4. Calculate the specific mAb-induced cytotoxicity by subtracting the percentage of 7-AAD⁺ in the basal death control from the percentage found in samples with different concentrations of mAbs (Figure 2D).

3. Biosimilarity Analysis

1. Enter the concentration and response values into a graphing software.
2. Generate graphs and calculate non-linear regressions with the following considerations: i) use the log-transformation of the mAb concentration as "X"; ii) use the variable slope mathematical model (Y = minimum response + (maximal response - minimum response)/ 1+10^((LogEC₅₀-X)*Hill slope)); and iii) constrain the bottom values to zero, since the basal response has been subtracted.
   NOTE: Curves with a symmetrical sigmoidal shape are expected.
3. Compare both non-linear fits with a global fit using an F-test (many graphing software programs include this feature).
   NOTE: Such tests establish as the null hypothesis that the maximal response, logEC₅₀, and the Hill slope are the same for the two datasets, which matches the biological question intended to be addressed.

Results

Using the protocols described above, target binding and the CDC induction of reference rituximab were compared in parallel with those of a biosimilar rituximab produced and commercially available in Asia.

In Daudi cells, both mAbs bound CD20 in a concentration-dependent manner (Figure 1D). Non-linear regressions of binding data displayed an r² of 0.978 and 0.848 for reference and biosimilar rituximab,...

Discussion

The patent expiration of a therapeutic mAb is promoting the development of biosimilars. Thus, there is a need for simple methods that can identify differences in clinically relevant activities of these products. CD20⁺ cultured cells were employed for the evaluation of two key functional characteristics of rituximab: target binding and CDC induction. The former activity requires the recognition of CD20 by the Fab region of the mAb, while the latter depends mainly on the interaction of the Fc region with its com...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-1640 medium	ATCC	30-2001	Modify the culture depending on the cell line
Trypan Blue solution	Sigma	T8154	0.4%, liquid, sterile-filtered, suitable for cell culture
Daudi Burkitt's Lymphoma Cells	ATCC	CCL-213	You can modify the cell line depending on the antibody of interest
Fetal bovine serum (FBS)	GIBCO	16000-044	You can modify the source of serum depending of requirements of the cell line
Normal Human Serum Complement	Quidel	A113	It is therefore appropriate for use in biocompatibility experiments including drug development, biomaterials testing and other applications
7AA-D	BDPharmigen	559925	You can use broad range of color options, compatible with most instrument configurations for to analyze viability.
PECy5 Mouse Anti-human IgG	BDPharmigen	551497	Change fluorochrome depending on the filter and laser of your flow cytometer
Human IgG Isotype Control	ThermoFisher Scientific	07-7102	Change depending to mAb
BDCytofix	BDPharmigen	554655	Flow Cytometry Fixation Buffer (1 - 4% formaldehyde or paraformaldehyde )
PBS pH 7.4 10x (Phosphate buffer saline)	GIBCO	70011-044	Phosphatebuffer without Ca2+/Mg2+ [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4] and endotoxin free.
Cell culture plates 96 well, V-bottom	Corning	29442-068	12 x 75 mm round bottom test tubes or 96-well V- or U-bottom microtiter plates
MabThera (Rituximab)	Roche	—	Reference product
Rituximab	Indian	—	Biosimilar product
15- or 50-mL conical centrifuge tubes	Corning	430290 or 430052	—
Pipette Tips	Eppendorf	—	Multiple volume configurations are necessary
Pipettes	Eppendorf	—	Adjustable-volume pipettes are necessary
Centrifuge 5430/ 5430R model	Eppendorf	—	Refrigerated variable-speed centrifuge (4 to 25 °C) with speeds ranging from 10 to 30,130 × g
Flow cytometer	BD Dickinson	—	BD FACSAria III or other flow cytometer
Olympus optical and light microscope	Olympus	—	To quantify and evaluate cell growth
Incubator	SANYO	—	Incubatorfor temperature and CO2 control to culture cells
Biological Safety Cabinet	CHC BIOLUS	—	Biological safety cabinet that is used to protect the researcher, product and environment.