Complement-dependent cytotoxicity method

Abstract

The present invention relates to a complement dependent cytotoxicity assay and a method for adding complement to cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 331,059, filed April 14, 2021, which is incorporated by reference in its entirety herein.

[0002] The present invention relates to methods of cytotoxicity, in particular to methods of complement dependent cytotoxicity and methods for reducing variability in complement-cytotoxicity assays. [Background technology]

[0003] The complement system is part of the immune system, composed of many different proteins that function to attack the cell membrane of pathogens. The classical complement cascade pathway can be initiated by the binding of complement (e.g., C1q) proteins to IgG antibodies. Complement-dependent cytotoxicity ("CDC") assays are used to measure complement cascade activation for many molecules, such as biologics. These assays can be used to evaluate the ability of products to activate the complement cascade to avoid unwanted safety concerns or they can be used to determine the mechanism of activation of the target pathway.

[0004] CDC assay methods are well known. See, for example, Duensing and Watson, Cold Spring Harb Protoc; 2018 Feb 1; 2018(2). The classical approach to CDC is to add serum containing components of the complement system to target cells to which the antibody being tested is bound and then determine cell membrane integrity or cell death through the use of dye-preloaded cells. Various sources of complement or tissue culture grade serum are commercially available from several manufacturers, including Cedarlane®, EMD Millipore, SigmaAldrich, and Bio-Rad.

[0005] When a CDC assay is used as part of an assay such as product release, control of variability is essential. However, the inventors of the present invention have found that CDC assays may exhibit variability and that a slow rate of complement addition contributed to this variability by causing nonspecific cytotoxicity. This nonspecific cytotoxicity before product addition results in undesirable assay variability. Although CDC assay methods are well known, Duensing and Watson (Cold Spring Harb Protoc; 2018 Feb 1; 2018(2)), for example, describe the CDC method but not the rate of complement addition. Wang et al. also describe a CDC assay, but to reduce antibody-independent nonspecific cytotoxicity, the assay includes an additional step of pre-adsorbing complement to target cells for 20 minutes (MAbs.2020 Jan-Dec;12(1):1690959).

[0006] The present invention seeks to solve the problem of non-specific cytotoxicity (and therefore method variability) by increasing the rate of addition of baby rabbit complement in the CDC assay. If complement is added to the cells at a faster rate, non-specific killing of target cells is reduced to acceptable levels. In addition, if complement is instead added to the assay plate prior to the addition of target cells, non-specific cytotoxicity is reduced. These improved methods result in less assay variability, leading to a more robust method. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Duensing and Watson,Cold Spring Harb Protoc;2018 Feb 1;2018(2) [Non-Patent Document 2] Wang et al.MAbs.2020 Jan-Dec;12(1):1690959 Summary of the Invention [Means for solving the problem]

[0008] The present invention provides a method for performing a cytotoxicity assay. In one embodiment, the present invention provides a method for performing a complement dependent cytotoxicity (CDC) assay. In one embodiment, the method includes adding complement to a mixture of target cells and test molecules, where the complement is added at a rate of at least about 250 μL / sec. In a specific embodiment, the complement is added at a rate of about 250 μL / sec. In a specific embodiment, the complement is added at a rate of about 300 μL / sec. In a specific embodiment, the complement is added at a rate of about 350 μL / sec. In a specific embodiment, the complement is added at a rate of at least about 400 μL / sec. In another specific embodiment, the complement is added at a rate of about 400 μL / sec. In another specific embodiment, the complement is added at a rate of about 450 μL / sec. In yet another specific embodiment, the complement is added at a rate of about 500 μL / sec.

[0009] In one embodiment, the method includes adding complement to the mixture of target cells and test molecules, wherein the complement is added at a rate of at least 250 μL / sec. In certain embodiments, the complement is added at a rate of between 250 μL / sec and 500 μL / sec. In certain embodiments, the complement is added at a rate of 250 μL / sec. In certain embodiments, the complement is added at a rate of 300 μL / sec. In certain embodiments, the complement is added at a rate of 350 μL / sec. In certain embodiments, the complement is added at a rate of at least 400 μL / sec. In another specific embodiment, the complement is added at a rate of 400 μL / sec. In another specific embodiment, the complement is added at a rate of 450 μL / sec. In yet another specific embodiment, the complement is added at a rate of 500 μL / sec.

[0010] In one embodiment, the complement is baby rabbit complement. In a specific embodiment, the complement is from Bio-Rad or Cedarlane®. In a specific embodiment, the complement is not from EMD.

[0011] The invention also provides a method for performing a CDC assay comprising adding target cells and a test molecule to a mixture comprising complement and medium. In one embodiment, the target cells are added to the mixture of complement and medium, followed by the addition of the test molecule. In one embodiment, the target cells and the test molecule are added simultaneously with the mixture of complement and medium. In one embodiment, the complement is baby rabbit complement. In one embodiment, the complement is human complement. In certain embodiments, the complement is from Bio-rad, Cedarlane®, or EMD.

[0012] The present invention also provides a method for adding complement to a target cell, comprising adding complement to the target cell at a rate of at least about 250 μL / sec. In one embodiment, complement is added to the target cell at a rate of at least about 400 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 250 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 300 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 350 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 400 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 450 μL / sec. In one embodiment, complement is added to the target cell at a rate of about 500 μL / sec. In one embodiment, complement is added to the target cell at a rate of at least about 500 μL / sec.

[0013] In certain embodiments, complement is added at a rate of 250 μL / sec to 500 μL / sec. In one embodiment, complement is added to the target cells at a rate of at least about 400 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 250 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 300 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 350 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 400 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 450 μL / sec. In one embodiment, complement is added to the target cells at a rate of about 500 μL / sec. In one embodiment, complement is added to the target cells at a rate of at least about 500 μL / sec.

[0014] In one embodiment, the complement is baby rabbit complement. In one embodiment, the complement is Bio-rad or Cedarlane. In one embodiment, the complement is not from EMD. In one embodiment, the complement is added to target cells in an assay to determine cell death. In one embodiment, the assay is a complement dependent cytotoxicity assay. In one embodiment, the complement is baby rabbit complement. In certain embodiments, the complement is Bio-rad or Cedarlane®. In certain embodiments, the complement is not from EMD.

[0015] In one embodiment, the test molecule is an IgG1 or IgG3 molecule.

[0016] The invention also provides a method for performing a CDC assay (Assay 1), where if unwanted non-specific cell killing is observed in Assay 1, the CDC assay is subsequently performed in a manner that includes an increased rate of complement addition (Assay 2). The invention also provides a method for performing a CDC assay (Assay 1), where if unwanted non-specific cell killing is observed, the CDC assay is subsequently performed in a manner that includes adding target cells and a test molecule to a mixture that includes complement and medium (Assay 2). In one embodiment, non-specific cell killing is reduced in Assay 2 compared to non-specific cell killing in Assay 1. [Brief description of the drawings]

[0017] [Figure 1A] FIG. 1 illustrates the effect of the rate of addition of various materials of baby rabbit complement (BRC) on the ratio to control. "Control (medium)" refers to wells with cells and medium. "Cells + complement" refers to wells with cells, medium and complement. The rate of complement (or medium only) is represented in the legend and is a manual addition of 10 μL / sec, 50 μL / sec, 100 μL / sec, 200 μL / sec, 400 μL / sec or 429 μL / sec. * indicates a statistically significant p-value <0.0001 and "ns" indicates not statistically significant. [Figure 1B] FIG. 1 illustrates the effect of the rate of addition of various materials of baby rabbit complement (BRC) on the ratio to control. "Control (medium)" refers to wells with cells and medium. "Cells + complement" refers to wells with cells, medium and complement. The rate of complement (or medium only) is represented in the legend and is a manual addition of 10 μL / sec, 50 μL / sec, 100 μL / sec, 200 μL / sec, 400 μL / sec or 429 μL / sec. * indicates a statistically significant p-value <0.0001 and "ns" indicates not statistically significant. [Figure 1C]FIG. 1 illustrates the effect of the rate of addition of various materials of baby rabbit complement (BRC) on the ratio to control. "Control (medium)" refers to wells with cells and medium. "Cells + complement" refers to wells with cells, medium and complement. The rate of complement (or medium only) is represented in the legend and is a manual addition of 10 μL / sec, 50 μL / sec, 100 μL / sec, 200 μL / sec, 400 μL / sec or 429 μL / sec. * indicates a statistically significant p-value <0.0001 and "ns" indicates not statistically significant. [Figure 1D] FIG. 1 illustrates the effect of the rate of addition of various materials of baby rabbit complement (BRC) on the ratio to control. "Control (medium)" refers to wells with cells and medium. "Cells + complement" refers to wells with cells, medium and complement. The rate of complement (or medium only) is represented in the legend and is a manual addition of 10 μL / sec, 50 μL / sec, 100 μL / sec, 200 μL / sec, 400 μL / sec or 429 μL / sec. * indicates a statistically significant p-value <0.0001 and "ns" indicates not statistically significant. [Diagram 2] FIG. 2 shows luminescence demonstrating increased non-specific killing of WIL2-S cells when cells are added prior to the addition of BRC compared to when BRC is added prior to the addition of cells (compare “cells+BRC” with “BRC+cells”). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention provides improved methods for reducing variability in cytotoxicity assays, for example by reducing the amount of non-specific cell killing observed in the cytotoxicity assay, In particular, the present invention provides complement dependent cytotoxicity methods in which non-specific cell killing is reduced.

[0019] Cytotoxicity assay refers to an assay that determines cell death. As used herein, "cell death" may be used interchangeably with "cell death" or "cytolysis." "Complement-dependent cytotoxicity" refers to cell death observed in a CDC assay. In a CDC assay, when a target cell, a test molecule, and complement interact, a membrane attack complex is formed on the surface of the target cell, and the target cell then undergoes cytolysis. The CDC assay determines the amount of cytolysis.

[0020] As used herein, "complement dependent cytotoxicity assay" or "CDC assay" refers to an assay used to measure the killing of target cells mixed with complement and a test molecule, such as in medium in a cell culture plate. In some embodiments described herein, the CDC assay is performed without the addition of a test molecule to determine the amount of non-specific cell killing. Non-specific cell killing refers to cell death in the absence of a test molecule.

[0021] CDC assay can be performed by methods widely known by those skilled in the art, including modified methods. For example, CDC assay can be performed by adding target-expressing cells in the medium of a plate, and then adding complement (such as baby rabbit complement) and a test molecule that binds to the target. For example, if the test molecule is an antibody with Fc, the antibody binds to the target expressed on the cell, and the Fc portion of the antibody binds to complement. The bound complement initiates the complement cascade, resulting in the death of the target-expressing cell. The present invention provides an improved method of cytotoxicity assay by adding complement at a faster rate. Such a faster rate of complement addition results in a decrease in non-specific cell killing, increasing the precision and accuracy of the method.

[0022] The CDC assay of the present invention can also be carried out by a method that includes adding target cells and test molecules to a mixture that includes complement and medium. In such a method, complement and medium are added to the plate first, and then target cells and test molecules are added to the plate. In this particular method, the rate of complement addition is not important to reduce non-specific cell killing.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0024] As used herein, "plate" is used interchangeably with "wells" of a plate, flasks or other devices suitable for cell culture.

[0025] CDC assays may be performed for times and temperatures suitable for the target cell type and test molecule.

[0026] As used herein, the term "about" when used in reference to a specific recited numerical value means that the value may vary by up to 10% from the recited value.

[0027] CDC assays may be performed according to known methods and improved methods described herein. With respect to CDC assays, "target cells" refers to cells used in CDC assays that express an antigen to which a test molecule can bind. Examples of target cells include WIL2-S, A431, CHO-M7 and BT474. "Test molecule" refers to a molecule that can bind to a target antigen and a complement protein (e.g., C1q) expressed on a target cell. Examples of types of molecules that can be test molecules include antibodies and heterodimeric molecules.

[0028] As used herein, an "antibody" is an immunoglobulin molecule that contains two HCs and two LCs interconnected by disulfide bonds. The amino-terminal portion of each LC and HC contains a variable region of about 100-120 amino acids that is primarily responsible for recognizing an antigen through the CDRs contained therein. The CDRs are interspersed with more conserved regions called framework regions ("FRs"). Each LCVR and HCVR is composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with antigens. Thus, the functional ability of an antibody to bind a particular antigen is largely influenced by the amino acid residues within the six CDRs. The general consensus is that CDC is typically high in IgG1 or IgG3 molecules and low in IgG2 and IgG4 molecules.

[0029] As used herein, a heterodimeric molecule can bind to two or more different antigens or two or more different epitopes on the same antigen. Heterodimeric molecules may be used interchangeably with multispecific antibodies. A non-limiting example of a heterodimeric molecule is a bispecific antibody. Examples of bispecific antibody formats can be found, for example, in Labrijn et al., Nat. Rev. Drug Disc.; 18, 585-608 (2019).

[0030] The antibody or heterodimeric molecule may bind complement via the Fc region of the antibody or heterodimeric molecule. The Fc region is primarily responsible for binding to cell receptors and / or complement. The Fc region is therefore involved in antibody effector functions and initiation of the complement cascade.

[0031] As used herein, "complement" refers to proteins circulating in the blood that can become activated in a reaction, such as an immune reaction. Examples of complement proteins include C1, C2, C3, C4, C5, C6, C7, C8, and C9. Complement can be human complement, rabbit complement, or another type of complement, which can be purchased or made by known methods. Complement can be derived from one of a variety of species, such as rabbit, baby rabbit, human, goat, llama, or guinea pig. In a preferred embodiment, complement is tissue culture grade complement, which is bacteria-free, mycoplasma-free, virus-free, and low endotoxin. Human complement refers to complement proteins obtained from human serum. Rabbit complement refers to complement proteins obtained from rabbit serum. In some embodiments, rabbit complement is obtained from baby rabbit serum. Complement can be purchased from a manufacturer or other commercially available material. Complement can be lyophilized and then reconstituted in solution or complement can be prepared in solution, such as a frozen solution in a vial.

[0032] In a CDC assay, complement can be added manually or by automated equipment, such as a Tecan Freedom EVO® automated liquid handling system, programmed to add complement at a desired rate according to the manufacturer's instructions. In addition, electronic pipettes can be set to add complement at a desired rate, and such methods can be determined from the associated user manual.

[0033] Cell death (also referred to as cell death or cell injury) in CDC assay can be measured by methods known to those skilled in the art, including, but not limited to, CellTiter-Glo®, detecting the release of cellular components during cell death, such as GAPDH, through fluorescence or luminescence quantification, staining and detecting dead cells, adding Alamar Blue, which is converted to a fluorescent molecule only in living cells, and measuring radioactive compounds released from cells during cell death. Tetrazolium compounds can also be used to detect live cells. Examples of tetrazolium compounds include MTT, MTS, XTT, and WST-1. EXAMPLES

[0034] Example 1: Rate of complement addition in a CDC assay To determine whether the rate of complement addition affects non-specific cell killing and variability, a liquid handling program script was written for the Tecan Freedom EVO® automated liquid handling system. The script added 50 μL of assay medium and 50 μL of cells (from two cell banks (Cell Bank A and Cell Bank B) derived from WIL2S cells) at a net concentration of 1.6 μL. e Three 96-well assay plates were then added at 5 cells / mL. 50 μL of BRC or Assay Media was then programmed to add one column at a time to each of the three plates at five different speeds. Each speed was programmed into the script via the Dispense Speed ​​parameter of the Liquid Class. Five Liquid Classes were created to dispense at the following speeds: 10 μL / sec, 50 μL / sec, 100 μL / sec, 200 μL / sec or 400 μL / sec.

[0035] Assay medium or baby rabbit complement ("BRC") was also added manually to each plate row using a Rainin XLS™ multichannel electronic pipette set at a dispense speed of 9 (out of 10). Plates were incubated for 45-75 minutes at 37°C and 5% CO2. After a 5-10 minute cool down, 50 μL of CellTiter-Glo® (Promega) reagent was added to all wells of the plate. After a 15-30 minute incubation, plates were read on an Envision® plate reader to measure luminescence. The difference in signal between wells containing cells and medium and wells containing cells, complement, and medium is a measure of nonspecific killing of cells by complement. A higher ratio indicates more nonspecific killing of cells. The results are shown in Table 1.

[0036] [Table 1]

[0037] These data demonstrate that the rate of addition of BRC is an important factor for nonspecific killing and that faster rates of complement addition result in decreased nonspecific cell killing.

[0038] Example 2: Complement Sources in CDC Assays Other materials from BRC were also tested to determine whether different addition rates would affect non-specific cell killing. Different lots of Cederlane BRC (cat#CL3441-S100) used in Example 1, two lots from Bio-rad (cat#C12CA), and one lot of BRC from EMD Millipore (cat#234400) were tested. The test was essentially performed as described in Example 1, except that four plates were prepared instead of three plates, and different lots of complement from the four lots were used on each plate. The Cederlane lot was diluted (1:16.5), while the other three lots were diluted 1:12 based on previously suitable concentrations. The results are shown in Figure 1.

[0039] These data, similar to those in Example 1, demonstrate that the rate of addition of other lots of complement and materials (the Cederlane lot and the two Bio-rad lots) affected the rate of cell viability of WIL2-S cells. In particular, nonspecific cell killing decreased when complement was added at a faster rate. However, the rate of addition was not a factor for the EMD Millipore lot of rabbit complement at the dilution tested (1:12).

[0040] In a separate experiment, the rate of addition of EMD Millipore rabbit complement serum did not appear to have an effect on WIL2-S or CHO-M7 cells at dilutions of 1:2, 1:15 and 1:30 relative to the control ratio.

[0041] Example 3: Order of Addition of BRCs To determine whether adding BRC to the plate before or after the cells would affect non-specific cell killing and assay variability, preparations of product dilutions, cells (WIL2-S) and BRC (purchased from Cederlane) were prepared. For this assay, product dilutions were added, followed by BRC, then cells. Less than about 1 minute elapsed between the addition of each component, and BRC was added at a rate of 105 μL / sec. Plates were incubated at 37° C. with 5% CO2 for 60 minutes. Cell killing was determined using Cell Titer Glo. The cell viability ratio after these procedures was 0.94, indicating a relatively low level of non-specific cell killing when BRC was added to the plate before the cells were added.

[0042] Without being bound by theory, these data suggest that adding cells to the plate first, followed by BRCs, results in non-specific cell killing if a gradient of BRCs is created, which can be reduced by adding complement at a faster rate (e.g., 250 μL / sec to about 500 μL / sec) or by adding BRCs to the plate first, followed by the cells.

[0043] In another experiment, BRC was added to the plate before or after the addition of cells to analyze non-specific cell killing. In a 24-well plate, WIL2-S cells were added first, followed by BRC (Cederlane). In another 24-well plate, BRC was added first, followed by cells. Non-specific killing of WIL2-S cells was calculated by comparing the ratio of the mean of treated wells to the mean of control wells containing cells and medium only. The closer the ratio is to 1, the less non-specific killing there is in the assay. As shown in Table 2 and Figure 1, there is less non-specific killing of WIL2-S cells when BRC is added first. Therefore, adding BRC before adding cells provides more reliable assay results.

[0044] [Table 2]

Claims

1. A method for performing a complement-dependent cell-mediated cytotoxicity (CDC) assay, comprising adding complement to a mixture of target cells and a test molecule, wherein the complement is added at a rate of at least 250 μL / second.

2. The method according to claim 1, wherein the complement is added at a rate of at least about 400 μL / second.

3. The method according to claim 1 or claim 2, wherein the complement is added at a rate of approximately 400 μL / second.

4. The method according to claim 1 or claim 2, wherein the complement is added at a rate of approximately 450 μL / second.

5. The method according to claim 1 or claim 2, wherein the complement is added at a rate of approximately 500 μL / second.

6. The method according to claim 1 or claim 2, wherein the complement is added at a rate of at least about 500 μL / second.

7. The method according to claim 1 or claim 2, wherein the complement is a juvenile rabbit complement.

8. The method according to claim 1 or claim 2, wherein the complement is of Bio-rad or Cedarlane.

9. The method according to claim 1 or claim 2, wherein the complement is not of EMD.

10. A method for performing a CDC assay, comprising adding target cells and a test molecule to a mixture comprising complement and culture medium.

11. The method according to claim 10, wherein the complement is a juvenile rabbit complement.

12. A method comprising adding complement to target cells, wherein the method comprises adding complement to the target cells at a rate of at least 250 μL / second.

13. The method according to claim 12, wherein the complement is added to the target cells at a rate of at least about 400 μL / second.

14. The method according to claim 12 or claim 13, wherein the complement is added to the target cells at a rate of approximately 400 μL / second.

15. The method according to claim 12 or claim 13, wherein the complement is added to the target cells at a rate of approximately 450 μL / second.

16. The method according to claim 12 or 13, wherein the complement is added to the target cells at a rate of at least about 500 μL / second.

17. The method according to claim 12 or claim 13, wherein the complement is a juvenile rabbit complement.

18. The method according to claim 12 or claim 13, wherein the complement is of Bio-rad or Cedarlane.

19. The method according to claim 12 or 13, wherein the complement is added to target cells in an assay to determine cell death.

20. The method according to claim 19, wherein the assay is a complement-dependent cytotoxicity assay.

21. The method according to claim 1 or claim 2, wherein the test molecule is an antibody.

22. The method according to claim 21, wherein the test molecule is an IgG1 antibody or an IgG3 antibody.

23. The method according to claim 1 or claim 2, wherein the test molecule is a heterodimer molecule.

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