TITLE: Test-Luciferase-Based Cytotoxicity Assay with bispecific antibodies
AUTHORS: Gülfem Öner-Ziegler, Christiane Stadler

[DESCRIPTION]
The purpose of this assay is to determine the cytotoxic potential of bispecific antibodies.

This protocol describes the preparation and analysis of an in vitro assay with human target cells, human  effector cells and bispecific antibodies (bsAb), which may be secreted into cell culture supernatants, found  in mouse plasma/serum or prepared as a chromatographically-purified protein.




[GUIDELINES]
If not specified differently all procedures are performed under sterile conditions at RT. 
As primary human  material is used as effector cells, all work needs to meet biosafety level 2 requirements. 

[STEPS]
SECTION: Preparation
1. Required Supplies
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             It is necessary to obtain stock solutions and sample material before beginning an experiment.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
SECTION: Preparation
2. Preparation of Effector Cells

The total number of human effector cells required is determined during planning of the experiment. The  effectors can be used after being freshly isolated from buffy coats, pre-cultured or directly after thawing.
If fresh cells are used, an adequate number of buffy coat bags needs to be ordered at least 48 h in
advance  from the blood bank.  On average, one buffy coat bag yields approx. 1x108 PBMC. The buffy coats are  stored at RT. No particular HLA-type or CMV status is required.

SECTION: Preparation
2.1. Isolation of effector cells 
Human PBMC are isolated according to Ficoll gradient method. If purified T cells are required, they are isolated from bulk PBMCs via MACS separation. All effector cell counts need to be determined.
SECTION: Preparation
2.2. Thawing of effector cells 
Prior to thawing, thawing medium need to be prepared, for which 10x CTL-wash is diluted 1:10 with RPMI 1640 medium in an appropriate tube. 2 µL DNAse I  solution (1 mg/mL) is added per milliliter medium, resulting in 2 µg/mL DNAse I in the thawing medium. 9 mL thawing medium is needed per cryo-tube; the latter can be pooled for thawing if more than one. The cryo-tube/tubes are thawed in a 37 °C water bath.  The cell suspension is aspirated with a 2 mL or 5 mL transfer pipette and added dropwise to the thawing  medium. The tube is inverted und centrifuged at 300 xg, 8 min, RT. Finally, the cell count and viability are  determined .The viability must be >80 %, below this the assay is not performed.

SECTION: Preparation
2.3. Determination of T-cell populations after isolation 
The percentage of CD4+ as well as CD8+ T-cells in a PBMC population, the T-cell purity after MACS  separation or the T-cell activation status is determined via FACS analysis. 
For staining, 5x105 cells per sample tube (pelleted at 460 xg, 5 min, RT program) are resuspended in FACS staining buffer at a 50 µL final volume containing the following antibodies:
3.0 μL α-CD4-FITC (ID043) 
1.5 μL α-CD8-APC (ID044) 
1.5 μL α-CD25-PE (ID047) 
5.0 μL α-CD69-PerCP (ID117) 
0.125 μL eFluor 506 
38.875 μL staining buffer 
A master mix is prepared if multiple samples are to be analyzed.
Since the negative control cells are stained only with the live/dead dye eFluor 506 without all other  antibodies, the FACS antibody volume is replaced with staining buffer (11 µL per sample). All samples are  vortexed for 3 s at moderate speed (level 4) and then incubated for 30 min at 2 – 8 °C in the dark.
Afterwards, 2 mL DPBS is distributed to all samples with a suitable transfer pipette and vortexed shortly.  The sample tubes are then centrifuged, the DPBS supernatant discarded and the washing step repeated once more. With an appropriate pipette or dispenser, 100 µL fixative solution  is added to each tube and the pellet dislodged/broken by quick vortexing. The sample tubes are then  sealed e.g. with Parafilm® M and stored protected from light at 2 – 8 °C for max. three days.  
The FACS sample acquisition is performed.
All gates must be adapted to the individual PBMC sample; an unchangeable template is therefore not  applicable. Exclusion criteria for effectors with respect to the CD4+/CD8+ percentage or their activation  status do not apply due to the need for FACS analysis, which is performed mostly after the assays  themselves. The purity of T-cell populations after MACS isolation usually reaches >95 %; otherwise, the assay eventually needs to be repeated.
SECTION: Preparation
2.4. Determination of T-cell populations after thawing cryopreserved cells 
For staining, 5x105 cells per sample tube (pelleted at 460 xg, 5 min) are resuspended in FACS staining buffer at 50 µL final volume containing the following antibodies: 
• 2.0 μL α-CD3-Pacific Blue (ID027) 
• 1.5 μL α-CD56-PE-Cy7 (ID031) 
• 3.0 μL α-CD4-FITC (ID043) 
• 1.5 μL α-CD8-APC (ID044)
• 1.5 μL α-CD25-FITC (ID047)
• 0.5 μL α-CD19-FITC (ID095)
• 5.0 μL α-CD69-PerCP (ID117)
• 0.125 μL eFluor 506
• 34.88 μL FACS staining buffer
A master mix is prepared if multiple samples are to be analyzed. This panel differs from the freshly-isolated PBMC, as cryopreserved PBMC may also be used for other  applications which require more detailed phenotyping. Thawed effectors are preferred to fresh cells, as  the cell populations of each donor are already well characterized. In addition, the EC50 value are  determined in a standard cytotoxicity assay3 after freezing and thawing.
s, for each assay, a specific donor can be chosen.  FACS analysis for phenotyping is performed only once for each donor (=PBMC-bank). Further aliquots for  use in assays are not analyzed again.
SECTION: Preparation
2.5. Cultivation of effector cells
PBMC
The isolated PBMC are kept light-protected in a closed sterile 50 mL tube at a cell density of  6 – 7x106 cells/mL in max. 30 mL assay medium at RT. The next day, the cells are centrifuged at 300 xg, 8 min, the supernatant removed with an aspiration pipette and the pellet resuspended in 1 mL assay medium by forward-reverse pipetting with a 1000 µL pipette. If the PBMC were  cultivated in multiple tubes, they can be pooled after this step. The volume is then topped up to 50 mL with assay medium and the cell count and viability are determined. The viability must be >80 %, below which the assay is not performed.

T cells 
Purified T cells are transferred into a cell culture flask with an appropriate pipette and the cell density is  adjusted to 5x106 cells/mL with assay medium. The flask is incubated overnight at 37 °C with 5 % CO2,  saturated humidity. The next day, T cells are transferred with a suitable pipette into a 50 mL tube. The  flask is rinsed with fresh assay medium, which is also transferred into the 50 mL tube and centrifuged . The resulting supernatant is removed with an aspiration pipette and the  pellet re-suspended in 1 mL assay medium with a 1000 µL pipette. The suspension is then topped up to 50 mL with assay medium, and the cell count and viability determined according. The viability must be >80 %, below which the assay is not performed.
SECTION: Preparation
3. Preparation of Target Cells
The target cells are to be in their exponential growth phase for the assay. The required cell number is  defined in the experimental plan and cells are expanded adequately prior to the assay day. The last  passage takes place one or two days before.     
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SECTION: Preparation
3.1. Harvest of target cells (for 175 cm2 cell culture flask) 
The culture medium is removed by aspiration and ~10 mL DPBS is then carefully added to the flask with an appropriate transfer pipette, avoiding disturbing the cell layer. The DPBS is distributed across the  adherent cell layer by gently rocking the flask by hand, followed by fluid aspiration. For cell detachment,  e.g. 4 mL Accutase® is added to the flask, gently handled as before and the cells placed in the incubator.  The detachment process is monitored by microscopy. As soon as the majority of cells is detached, e.g.  6 mL DPBS or assay medium is added with a 10 mL transfer pipette, the cells transferred into a 15 mL tube  and centrifuged at RT, 300 xg for 4 min. The resulting supernatant is discarded  and the pellet is re-suspended in 1 mL followed by cell counting. The viability must be >80 %, otherwise the assay is not performed.

Harvest of target cells (for 650 mL cell culture flask) 
The cell suspension is transferred into a 50 mL tube with an appropriate pipette and centrifuged at 300 xg,  4 min at RT. The resulting supernatant is discarded and the pellet is resuspended in 1 mL followed by cell counting. The viability must  be >80 %, otherwise the assay is not performed.

SECTION: Preparation
3.2. Antigen expression check 
Staining of target cells (S1 material) can be performed under non-sterile conditions outside the biosafety cabinet. 5x105 cells are added to each 5 mL FACS sample tube, centrifuged at 460 xg, 5 min. and the supernatant decanted. 50 µL staining buffer including all antibodies and live/dead dye is  added to the cell pellet with an appropriate pipette or dispenser. All samples are vortexed for 3 s at level  4 (moderate speed) and incubated in the dark at 2 – 8 °C for 30 min.

Sample list for staining with unconjugated primary antibodies:
 
 Sample list for staining with fluorochrome-conjugated primary antibodies:
 
Examples for frequently used antibodies for staining of tumor antigens: 
 
For the washing step, 2 mL DPBS is added to all samples, centrifuged at 460 xg for 5 min. and the DPBS supernatant is decanted. If a secondary antibody is required, add 50 µL staining  buffer incl. secondary antibody. The tubes are vortexed for 3 s on level 4 (moderate speed) and incubated  in the dark at 2 – 8 °C for 30 min. After this, 2 mL DPBS again is added to all samples, centrifuged at 460 xg for 5 min. and the is decanted. The tubes are vortexed for 3 s on level 4 (moderate speed) and the washing  procedure is repeated once more.
Finally, 1x BD™ fixative solution, e.g. 100 µL, is added to each tube and vortexed (3 s, level 4 at moderate  speed). The samples can be stored sealed for max. three days at 2 – 8 °C, protected from light. If the  primary antibody is conjugated to a fluorescent dye, the washing step is also performed twice.  The FACS acquisition is performed. Data analysis is done by FlowJo.
SECTION: Preparation
3.3. Adjustment of target cells
1x104 targets cells/96-well and 2x103 target cells/384-well plates, respectively, are needed. 40 µL/10 µL  target-cell suspension are prepared. The total volume is calculated according to the total count of assay  wells on all assay plates.  Analyzing mouse-derived samples i.e. plasma/serum (5 µL per well) requires the total volume of cell  suspension to be adapted appropriately. 
Examples for cell counts and extra volumes for multiple assay plates:
 
Examples for cell counts per well with adapted volumes for the use of purified proteins, CCSN or  plasma/serum:
  
Volumes for 384-wells are not changed using mouse plasma/serum samples. The adjusted target cells are kept at RT until further use. 
For plating, the effector and target cells are  mixed and seeded within 30 min after target cell preparation. 
Note: cell line PA-1-SC12 loses viability quickly outside the incubator, thus best processed quickly.

SECTION: Assay Setup
4. Plating of effector and target cells
The cytotoxicity assay is performed in a 96- or 384-well luminescence plate. With unknown test material, a  transparent luminescence plate can be used or an additional clear cell-culture plate for microscopy  analysis is prepared in the same manner to be able to observe cytotoxic effects (T-cell clustering,  proliferation or target-cell lysis). 
Usually, two time points are measured per assay (24 h and 48 h) i.e. each  plate needs to be pipetted twice. If further relevant time points e.g. 72 h or 96 h more plates need to be  prepared accordingly.

The adjusted cell suspensions with targets and effectors are first pooled in a volume ratio of 1:1 in an  appropriate tube. The total volume results from the total well count. An adequate extra volume needs to  be calculated (e.g. 10 mL suspension per 96-well plate/3 mL per 384-well plate). Target cells are adjusted  to 2.5x105 cells/mL for 96-well plates, and 2x105 cells/mL for 384-well plates, respectively. The effector-target mixed cell suspension is plated as follows: 
96-well format = 80 µL/well; 
384-well format =  10 µL/well. 
For analyzing mouse plasma/serum, the volume is reduced accordingly. 
Example: 5 μL mouse plasma →  75 μL effector-target mixed cell suspension consisting of 37.5 μL effectors and 37.5 μL targets. The suspension is homogenized by inverting the tube multiple times and transferring the contents to a  single-use, sterile reagent reservoir. Distribute cells promptly on the plates, preferably with an electric  Eppendorf Xplorer®, manual multichannel pipette or Integra Voyager and the appropriate filter tips. The  operating volume is set, and 100 µL or 25 µL DPBS or cell suspension is dispensed into empty wells in a  96-well or 384-well plate, respectively. An hour of incubation time before proceeding is recommended.  For 384-well plates, an Integra Voyager pipette is used. 
Note: 384-well plates are additionally sealed with a semipermeable membrane and centrifuged quickly at  max. 300 xg by pressing the “short” button.


SECTION: Assay Setup
5. Dilution of test material
Test material (bsAb - purified, in CCSN or plasma/serum) is diluted in its storage buffer, Mock4 CCSN or  assay medium, respectively. If available, the same volume of mock CCSN is added to the Lmin and Lmax control wells. Dilution rows are always prepared freshly by preparing a serial dilution starting with the  highest and down to the lowest concentration.  
Generally, for the first dilution at least 5 µL of the test item is used to reduce pipetting errors and falsified  concentrations. If the protein concentration is known and high enough, a concentration as high as  5000 ng/mL may be used as the starting point at the investigator’s discretion for each experiment. Depending on the dilution volumes different 96-well plates or reaction cups are chosen to dilute the test  items. 
Up to 2 mL total volume per dilution step a plate can be used:
  
Prior to use, all solutions need to be at RT to ensure that the assay requirements for temperature are  complied. For optimal mixing of proteins in diluent, the sample is mixed multiple times with an  appropriate pipette (e.g. 1 mL protein dilution is mixed with a 1000 µL pipette adjusted to 500 µL). It is  also possible to use a suitable pipette i.e. Eppendorf Xplorer® with the function “pipette and mix”. If only one concentration of test item is required, the dilution can be prepared in a suitable reaction tube.  Prepared dilutions are stored at RT and used within 30 min; otherwise, storage is at 2 – 8 °C. Dilutions can  be stored at RT for 2 h max., since proteins may bind to the surface and mask the actual concentration.  If available, a standard reference is prepared in the protein concentration range (e.g. 10-point, 5-step  dilution from 5000 – 0.00256 ng/mL). This serves as positive control and intra-assay comparison between  different plates. If no reference is available e.g. for new target proteins, no intra-assay comparison can be done.



SECTION: Assay Setup
6. Addition of test material
The pre-diluted test items in multi-well plates can be added to the assay plate(s) using an electric  Eppendorf Xplorer® Multichannel Pipette, manual Eppendorf multichannel or Integra Voyager. If the test  item is prepared in a single tube, a manual or electric dispenser (e.g. Multipette Xstream® with a 1 mL  Combitip advanced® plus) is used. 20 µL (96-well) or 5 µL (384-well) of test item and equal amounts of buffer or mock medium is added to each corresponding Lmin and Lmax wells.  If only low amounts of test material are available with no extra volume to consider, 20 µL or 5 µL test item  is then added with an appropriate single channel pipette. The tips have to be changed after the test item  addition to each well/row/column. For the analysis of mouse plasma/serum, 75 µL/20 µL E:T cell mixture is added. During in vivo cytotox  planning the sample concentration is determined by the investigator. Mouse serum/plasma can be used  as percentage from 1 – 5 % per well or as defined concentration after Gyros ELISA quantification. If using  defined mouse sample concentrations, the reference protein dilutions are used correspondingly. Subsequently diluted or undiluted mouse test item (1 – 5 µL, according experiment plan) is added. IF less  than 5 µL serum/plasma are used, they are diluted with assay medium up to 5 µL. The pre-dilutions with can be prepared in 96-well round bottom plates and be directly transferred with an appropriate multi-channel pipette.
Tabular overview about the components’ volumes per well:
  
Moreover, negative control serum/plasma is added correspondingly to the reference protein dilutions, as  well as the protein buffer is added to the mouse samples. 
Example for 96-well: 
Control: 3 µL reference protein  
           + 5 µL Mock serum/plasma/CCSN 
           + 17 µL assay medium 
           + 75 µL cells 
= 100 µL total volume 
Samples: 5 µL serum/plasma/CCSN  
             + 3 µL storage buffer 
             + 17 µL assay medium 
             + 75 µL cells 
= 100 µL total volume 

Note: 384-well plates are additionally sealed with a semipermeable membrane and centrifuged quickly  at max. 300 xg by pressing the “short” button. 

SECTION: Incubation
7. Assay plates ready for analysis are placed in an incubator set at 37 °C with 5 % CO2 and saturated humidity and removed only at the designated time points e.g. 24 h, 48 h etc. for reading on a luminometer.
SECTION: Luminescence Measurement
8. All following steps are performed under non-sterile conditions. The Lmax-defined wells (maximal lysis) get 10 µL (96-well) or 5 µL (384-well) of 20 % Triton™ X-100 lysis  solution with an electric Xplorer® pipette, or a suitable 8-channel manual pipette or Integra Voyager,  adjusted to dispense 10 µL/5 µL. For maximal effects the wells are mixed by forward-reverse pipetting.  The “pipette and mix” mode can be used on Xplorer®. The plate is placed in the incubator for further  10 min. During the Triton™X-100 incubation time, luciferin solution is prepared in a light-protected  15 mL tube (amber or aluminum foil-wrapped). The solution is transferred by decanting into a reagent  reservoir and the residual solution added by pipetting. Quickly afterwards 20 µL luciferin solution per 96- well and 12.5 µL per 384-well are distributed onto the assay plates, respectively, preferably with an  electric Xplorer® Multichannel pipette (mode “dispense”) or Integra Voyager. Start adding luciferin into column 1, rows A – H and proceed uniformly with an 8-channel manual pipette. All steps need to be  proceeded with quickly. The finished plate is incubated immediately in an incubator for 30 min, protected from light. Handle a  maximum of four 96-well plates or two 384-well plates at the same time. 
The measurement must be performed 5 min after the 30-min incubation time ends e.g. with an Infinite  M200 Pro or F200 Pro luminescence reader with Tecan i-control software. 
                                                                                                                                                                                                                                 
SECTION: Preparation
1.1. Stock Solutions

BD Monolight™ Luciferin 
Final concentration: 12 mg/mL in water, pH 6.0 – 6.3 
Due to possible racemization of D-luciferin to L-luciferin at pH above 6.3, this salt dissolution process  needs to be performed quickly.  250 mg BD Monolight™ D-luciferin potassium salt is dissolved in 20 mL Aqua B. Braun at RT, protected  from light. Therefore, 10 mL water is pipetted with a 10 mL transfer pipette into the BD™ glass bottle. The  luciferin salt is dissolved by forward-reverse pipetting several times. With the same pipette, the dissolved  luciferin is transferred into an amber or aluminum-wrapped 50 mL tube. Using a fresh 10 mL pipette, the  glass bottle is rinsed with 10 mL water which is then transferred into the 50 mL tube. This step is repeated  one to two times for collecting all luciferin salt in the original bottle, after which the remaining solution is  collected with a 100 µL pipette. The 50 mL tube is then closed and inverted several times, followed by pH  adjustment to 6.0 – 6.3 using a pH electrode, for which approx. 2 µL of 6 M HCl solution is needed. After pH adjustment, the final volume of the luciferin stock solution of 20.833 mL is reached by topping  up with water using a 1000 µL pipette. The stock solution is aspirated with a 20 mL syringe and filtered  through a syringe filter (0.2 µm) into a fresh 50 mL tube. Finally, aliquots of 1 mL are prepared in 1.5 mL  amber reaction tubes and stored at −20 ± 5 °C. 
Storage: −20 ± 5 °C, amber 1.5 mL reaction tubes.
Shelf life: 12 months.

Fixative Solution for FACS
Final concentration: 1x in water 
To prepare the FACS fixative solution, one part BD™ Stabilizing Fixative is diluted with two parts water in  volume. 100 µL fixative is used per sample. The required total volume, tube size and pipettes are based  on the total sample count. The solution is thoroughly mixed by manual inversion or vortexing prior to use.
Storage: 2 – 8 °C in polystyrene tubes.
Shelf life: 1 day.

Antibody staining solution (FAC staining buffer)
Final concentration: 5 % FBS, 5 mM EDTA in 1xDPBS 30 mL from a 500 mL DPBS bottle is discarded. Afterwards 25 mL FBS using a 25 mL transfer pipette and  5 mL 0.5 M EDTA using a 5 mL transfer pipette are added. The flask is tightly closed and manually inverted  multiple times to obtain a homogenous solution. 
Storage: 2 – 8 °C 
Shelf life: 3 months.

Assay medium                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
Final concentration: 10 % FBS, 0.5 % P/S in RPMI 1640 medium
Under sterile conditions, 52.5 mL RPMI 1640 medium is removed from the medium flask using a 50 mL  transfer pipette. 50 mL heat-inactivated FBS and 2.5 mL P/S are transferred into the medium flask using a 25 mL/50 mL transfer pipette or a  5 mL transfer pipette, respectively. The flask is tightly closed and inverted several times to obtain a  homogenous solution. A sterility check is then performed according to SOP-020-009 “Ansetzen von  Medien und Zusätzen für die Zellkultur“ and documented in FOR-020-009A.  The heat inactivation of FBS destroys components of the complement system a part of the IgG effector  function hence not heat inactivated assay medium has to be used for the investigation of the cytotoxic  potential of IgG antibodies. 
Storage: 2 – 8 °C 
Shelf life: 1 month.

Triton™ X-100 lysis solution (for Lmax)
Final concentration: 20 % (v/v) in water.
10 mL Triton™ X-100 are transferred with a dispenser pipette into a 100 mL glass bottle, topped up with  40 mL ultra-pure water and mixed by vortexing.
Storage: RT
Shelf life: 12 months.

Luciferin solution
Final concentration: 3 mg/mL luciferin, 150 mM HEPES buffer To prepare a master mix for the working solution, the following components are sequentially mixed into  a brown or aluminum-wrapped tube of appropriate size (usually 15 mL). The final volume is calculated  according to the number of sample-containing wells. Sufficient extra volume also needs to be calculated. 
Example for a 96-well plate incl. extra volume, if 20 µL luciferin solution per well is used: 
• 1440 µL Aqua B. Braun 
• 360 µL 1 M HEPES buffer 
• 600 µL Luciferin stock solution 
The working solution is prepared at RT and is ready to use.
Long-term storage is not possible.


SECTION: Preparation
1.2. Sample Material
Samples for investigation can be chromatographically-purified bispecific antibodies (bsAb) in buffer or  antibody-containing CCSN from producer cell lines (e.g. HEK-293, CHO-K1, K-562 cells) or mouse serum and plasma. 
To determine the concentration Gyros ELISA, ForteBio BLItz, Nano Drop or Western Blot can  be performed. Sample material needs to be stored at 2 – 8 °C.   
SECTION: Preparation
1.3. 
SECTION: Preparation
2.6. Adjustment of effector cells 

Effector cells are re-suspended in assay medium for the desired E:T ratio. Effector-cell suspension volumes  of 40 µL and 10 µL are required for 96-well plates and 384-well plates, respectively, as shown in the table  below. When analyzing samples from mouse i.e. plasma/serum (5 µL), the total cell suspension volume  needs to be adapted appropriately for 96-well plates only. For 384-well plates the volumes and cell counts  are not changed, the mouse samples are just added on top to the wells. The following table shows the counts for effector cells depending on the common E:T ratios:

 
Different E:T ratios or volumes can be used equally. The adjusted effector cell suspension is stored in a  50 mL tube upon use at the same day at RT.
2. 
2. 
SECTION: Workflow
9.  Workflow for luciferase-based cytotoxic assay protocol