Method and Kit for Testing Immunomodulatory Potency of Immunoglobulin Compositions for Treatment of COVID-19
20230400453 · 2023-12-14
Inventors
Cpc classification
G01N33/54313
PHYSICS
International classification
Abstract
The present invention relates to the field of immunotherapeutics, in particular to a method for characterization and/or quality control of immunotherapeutics. It provides a method of testing potency of an immunoglobulin composition, e.g., plasma or a plasma-derived immunoglobulin composition such as an intravenous immunoglobulin composition (IVIG), as well as to use of a bead coated with an antigen and an antibody specifically bound to said antigen for testing po-tency of an immunoglobulin composition. Said immunoglobulin composition, or immunoglobulin test composition can be an IVIG, particularly and IgA- and/or IgM enriched (also sometimes re-ferred to as IVIG-AM). The potency is tested by the capability of the composition to inhibit an ef-fector function of an Fc-receptor expressing immune effector cell, such as a neutrophil, e.g., a HL60 cell, preferably, production of an inflammatory cytokine such as IL-8. The invention also relates to a method of preparing a standardized immunoglobulin composition, to a kit for carry-ing out the method, as well as a composition. The immunoglobulin compositions obtainable from said method may be used, e.g., in the treatment of inflammation, e.g., in the context of COVID-19 or pneumonia, e.g., severe community-acquired pneumonia.
Claims
1. A method for testing potency of an immunoglobulin test composition, the method comprising a) providing a bead coated with an antigen and an antibody specifically bound to said antigen, b) contacting said bead with said immunoglobulin test composition and with an immune effector cell expressing at least one Fc-Receptor (FcR), and c) determining an effector function of the immune effector cell.
2. (canceled)
3. The method of claim 1, wherein the antigen is a viral surface protein.
4. The method of claim 1, wherein said antibody specifically bound to said antigen is an antibody from a patient who has been infected with a pathogen expressing said antigen, or recombinant antibody, preferably, heat-inactivated plasma of a convalescent COVID-19 patient.
5. The method of claim 1, wherein the bead is selected from the group consisting of latex beads, agarose beads, glass beads and gold beads, preferably, latex beads.
6. The method of claim 1, further comprising preparing a bead coated with the antigen by incubating a bead with the antigen, wherein, preferably, the antigen is covalently linked to the bead, wherein, optionally, free binding sites on the bead are blocked after said incubation.
7. The method of claim 1, further comprising preparing the bead coated with an antigen and an antibody specifically bound to said antigen by incubating the bead coated with the antigen with antibodies to said antigen.
8. The method of claim 1, wherein the immunoglobulin test composition is a polyclonal immunoglobulin composition, wherein, optionally, the immunoglobulin test composition is plasma, e.g., from a convalescent patient who had a disease associated with the antigen.
9. The method of claim 1, wherein the immunoglobulin test composition comprises at least 30 g/L immunoglobulins and is derived from a plurality of human donors, and, optionally, comprises IgG, IgM and/or IgA, preferably, all three classes, e.g., the percentage of IgM and/or IgA being about 5-90% (w/total antibody w), respectively.
10. The method of claim 1, wherein the immunoglobulin test composition is derived from plasma or serum, optionally, plasma.
11. The method of claim 1, wherein the effector function is compared with the effector function of a control test carried out without contacting the bead coated with an antigen and an antibody specifically bound to said antigen with the immunoglobulin test composition to determine a change in effector function.
12. The method of claim 1, wherein the immune effector cell is selected from the group consisting of neutrophils, eosinophils, monocytes, macrophages, and dendritic cells, preferably, HL60 cells.
13. The method of claim 1, wherein the effector function is selected from the group consisting of cytokine production, phagocytosis of the beads, modulation of a surface marker, NETose, ROS release and degranulation, wherein, preferably cytokine secretion is determined.
14. The method of claim 1, wherein the effector function is production of IL-8, preferably, secretion of IL-8.
15. The method of claim 1, wherein the potency is immunomodulatory potency.
16. The method of claim 1, wherein the inhibition of the effector function by the immunoglobulin test composition is positively correlated to the potency of the immunoglobulin test composition, and, optionally, to efficiency of the immunoglobulin test composition in treatment of inflammation, optionally, in the context of COVID-19.
17. The method of claim 1, wherein the potency of the immunoglobulin test composition is compared to the potency of a standard immunoglobulin composition, and the ratio of the potency of the immunoglobulin test composition to the potency of the standard immunoglobulin composition is the relative potency, wherein the standard immunoglobulin composition preferably is a standard IgM and/or IgA containing immunoglobulin composition.
18. A method for preparing a standardized immunoglobulin composition comprising at least 30 g/L immunoglobulins derived from a plurality of donors, comprising i. pooling plasma or serum derived from a plurality of human donors to provide a pool; ii. isolating and concentrating immunoglobulins from the pool to produce an immunoglobulin test composition; iii. testing the potency of the immunoglobulin test composition of ii) by the method of claim 17, wherein said immunoglobulin test composition is discarded if the relative potency of said immunoglobulin test composition is not in a predetermined range; and iv. optionally, adapting the potency of the immunoglobulin test composition to a desired potency; and/or packaging an amount of the immunoglobulin test composition, e.g., an amount having a desired potency.
19. A kit for carrying out the method of claim 1, comprising the bead, the antigen and the antibody, wherein, optionally, the bead is coated with the antigen, or the bead is coated with an antigen and an antibody is specifically bound to said antigen, a standard immunoglobulin composition comprising at least 30 g/L immunoglobulins derived from a plurality of human donors, optionally, an IgM and/or IgA containing immunoglobulin composition, and, optionally, immune effector cells expressing FcR selected from the group comprising HL60 cells.
20. The method of claim 3, wherein the antigen is a SARS-CoV-2 surface protein.
21. The method of claim 20, wherein the antigen is a SARS-CoV-2 spike protein.
Description
DESCRIPTION OF THE FIGURES
[0233]
[0234]
[0235]
EXAMPLES
Example 1: Manufacture of an IgA and kW Containing Immunoglobulin Composition (IVIG-AM)
[0236] Human blood plasma for fractionation (2000 l) from more than 500 donors was used as starting material. The plasma was transferred to the pooling area and pooled.
[0237] A cryoprecipitation step was performed in order to separate coagulation factors such as Factor VIII, von Willebrand Factor, and Fibrinogen. In order to obtain the cryoprecipitate, the temperature of the plasma was adjusted under gentle stirring so that the temperature range was kept at 2±2° C. Under these conditions the cryoprecipitate remains undissolved in the thawed plasma. The cryoprecipitate was separated from the plasma by a continuously operating centrifuge such as a Westfalia separator.
[0238] From the supernatant of the cryoprecipitation step the Cohn fraction I/II/III was precipitated by ethanol precipitation as follows:
[0239] The temperature of the centrifugation supernatant remaining after separation of the cryoprecipitate was adjusted to 2±2° C. The protein solution was adjusted to pH 5.9. Subsequently, the temperature was lowered to −5° C. and ethanol was added to a final concentration of 20% by volume. Under constant slow stirring in a stainless steel vessel, Cohn Fraction I/II/III was precipitated. The Cohn Fraction I/II/III precipitate was separated from the supernatant by filtration with depth filter sheets under addition of filter aid such as Perlite or Diatomaceous Earth, using a filter press. The Cohn fraction I/II/III was recovered from the filter sheets. This Cohn fraction I/II/III precipitate comprised all immunoglobulins (IgG, IgA, IgM) in approximately the following percentages: 75% IgG, 13% IgM and 12% IgA.
[0240] 90 kg of the obtained Cohn fraction I/II/III precipitate were resuspended in 450 kg of 0.1 M sodium acetate puffer pH 4.8 and mixed for 60 minutes at 22° C. The pH of the suspension was adjusted to 4.8 with acetic acid.
[0241] In the following a treatment with octanoic acid was performed. The solution was treated by addition of 7.7 kg octanoic acid at room temperature. The octanoic acid was added slowly and the protein solution was further mixed for 60 minutes, using a vibrating mixer (Vibromixer®, Size 4, Graber+Pfenniger GmbH, Vibromixer adjusted to level 2-3).
[0242] A calcium phosphate treatment was performed in order to complete the octanoic acid reaction as follows:
[0243] Approximately 1.1 kg Ca 3 (PO 4) 2 were added and the protein solution was further mixed for more than 15 minutes and filtered over depth filter sheets. The filtrate was further processed. The obtained protein solution was subjected to ultrafiltration to a protein concentration of about 50 g/I. The protein solution was diafiltered against 0.02 M sodium acetate buffer pH 4.5 and afterwards adjusted to a protein concentration of about 40 g/I.
[0244] The protein solution was treated at pH 4.0 in order to inactivate viruses as follows: The pH was adjusted to pH 4.0 using 0.2 M HCl, and the resulting solution was incubated for 8 hours at 37° C. The resulting protein solution contains immunoglobulins with the following distribution: 90% IgG, 5% IgA, and 5% IgM.
[0245] The obtained protein solution was further processed by anionic exchange chromatography using a macroporous anion exchange resin in order to remove accompanying proteins and to obtain an IgG- and IgM-enriched immunoglobulin compositions. Per kilogram of the intermediate protein solution 0.00121 kg of tris(hydroxymethyl)aminomethane (Tris) were added and dissolved while stirring and the conductivity was adjusted to 6 mS/cm with solid NaCl. The protein solution was adjusted to pH 7.1 by adding 1 M NaOH. A macroporous anion exchange resin (POROS® 50 HQ anion exchange resin, Life Technologies, bed height of the column: 25 cm) was equilibrated with a 10 mM Tris buffer solution (pH 7.1, 50 mM NaCl, at a linear flow rate of 800 cm/h). The protein solution was loaded on the anion exchange resin with 40 g protein per liter of resin. The column was washed with the equilibration buffer (10 mM Tris, 50 mM NaCl, pH 7.1, at 800 cm/h).
[0246] An IgG-enriched immunoglobulin composition was obtained in the flow-through fraction and was further processed as described in Example 2 below.
[0247] An IgM-enriched fraction was eluted by increasing the conductivity as follows: 10 mM Tris buffer solution with 300 mM NaCl at pH 7.1 is used at 800 cm/h to elute the IgM-enriched fraction. The eluted fraction contained 58% IgG, 22% IgA and 20% IgM.
[0248] The protein solution was filtered through a Pall, Ultipor VF DV50 filter as a virus removal step. The filtrate was further processed by UVC light treatment at 254 nm, using a flowthrough UVivatech process device (Bayer Technology Services/Sartorius) at a UVC dose of 225 J/m.sup.2 for further virus inactivation. The flow velocity through the UVC reactor was calculated using the manufacturer's instructions. The irradiated protein solution was concentrated to a protein concentration of 50 g/I by ultrafiltration (and was subjected to diafiltration (using 0.3 M glycine buffer pH 4.5). The final product was filtered through a 0.2 μm filter and was stored at 2 to 8° C.
[0249] The obtained immunoglobulin composition had an IgM content of 22% by weight, an IgA content of 22% by weight and an IgG content of 56% by weight, based on the total immunoglobulin content, at an immunoglobulin concentration of 50 mg/ml. The ACA was 0.34 CH50/mg.
Example 2: Manufacture of a Purified IgG Containing Immunoglobulin Composition (IVIG)
[0250] The IgG-enriched immunoglobulin composition collected as the flow through fraction of the macroporous anion exchange chromatography (POROS® 50 HQ) in Example 1 was adjusted to pH 5.5 and to a conductivity of 22-26 mS/cm with sodium acetate buffer and NaCl and then was further purified by cation exchange chromatography in a flow-through mode on a cation exchange resin (POROS® 50 HS). The binding capacity of this resin is defined as 100-3000 g/I, and chromatography was carried out at a load of 3000 g/I and a flow-rate of 800 cm/h.
[0251] The cation exchange column was equilibrated with acetate buffer solution (pH 5.5, adjusted to 22-26 mS/cm with NaCl). The protein solution was loaded to the column and washed with acetate buffer (pH 5.5, adjusted to 22-26 mS/cm with NaCl). The flow through fraction and the wash are collected and further processed. The residual protein is eluted with 1.5 M NaCl.
[0252] The resulting protein solution was further processed by a nanofiltration step, in order to remove potentially present virus. A Planova BioEx 20 nm filter (Asahi Kasei) was used as a virus filter. More than 50 kg of the protein solution were filtered over a 0.1 m 2 filter area at a protein concentration of 10 g/I. The maximum pressure was set according to the manufacturer's instructions.
[0253] The resulting protein solution was subjected to a concentration step to >100 g/L by ultrafiltration and diafiltered into formulation buffer (0.3 M Glycine pH 5.0). The resulting protein solution was filtered through a 0.2 μm filter in order to control sterility.
[0254] Obtained immunoglobulin compositions were analysed for their potency using the method of the invention, i.e., they were analysed as immunoglobulin test compositions in the method of the invention.
Example 3: Potency Assay
[0255] In brief, fluorescent latex beads were coated with an antigen, e.g., SARS-CoV-2 spike protein to generate antigen-coated beads. Antibodies to the antigen, e.g., anti SARS-CoV-2 spike protein antibodies (source: convalescent plasma donation or recombinant antibody) were added to generate a bead coated with an immune complex, here, a SARS-CoV-2 like immune complex. The beads coated with the immune complex were contacted with immune effector cells expressing at least one Fc, e.g., HL60 cells, in the presence or absence of an immunoglobulin test composition. The contact of the coated beads and resulting immune complex to the effector cells leads to induction of effector functions, e.g., inflammatory cytokine release or phagocytosis. For example, the coated beads incubated with specific antibodies to generate an immune complex were phagocytosed FcγRII and FcαRI dependently by the immune effector cells. The contact leads to induction of effector functions, e.g., inflammatory cytokine release, by the immune effector cell. This was reduced by addition of an immunoglobulin test composition such as an IVIG or IVIG-AM preparation, depending on the potency of said test composition. Immunomodulation caused by the immunoglobulins may be determined by monitoring, e.g., reduced pro-inflammatory cytokine release and bead uptake, preferably, IL-8 secretion.
[0256] Without intending to be bound by the theory, immunoglobulins added may e.g. inhibit immune complex uptake, target inhibitory cell signalling, modulate cell phenotype and neutralize cytokines.
[0257] Material and Methods
[0258] Differentiation of HL60 Cells
[0259] HL60 cells were cultivated in IMDM-Medium supplemented with 20% fetal bovine serum and 1% Penicillin (10.000 U/mL)/Streptomycin (10.000 μg/mL). After seeding, the cells were cultivated for 3-4 days at 37° C. in the incubator (5% CO.sub.2). For subculturing, the cells were count and seeded with a density of 2×10 5 cells/mL in fresh medium and a new flask. Up to 50 mL cell suspension was cultivated in a T-75 flask, up to 70 mL in a T-175 flask.
[0260] The assays were started, e.g., after passage 4-5 with a viability 95%. Cultivation may be maintained, e.g., until cells reach passage 25.
[0261] Differentiation of HL60 cells was induced by resuspending a cell pellet in complete culture medium supplemented with 1.3% (v/v) cell culture grade DMSO to reach a cell density of 6×10.sup.5 cells/mL. These cells were incubated for 3-5 days at 37° C. in the incubator to reach a neutrophil-like phenotype. Differentiation was monitored by flow cytometry.
[0262] Optionally, the HL-60 cells cultured in DMSO as described herein were further matured stimulated with LPS, e.g., 500 ng/mL LPS. The stimulation was for 6-48 h, preferably, at least over 24-48 hours.
[0263] Isolation of Primary Human Neutrophils
[0264] For the isolation of primary human neutrophils, fresh whole blood donations were used. The isolation was performed by using the MACSxpress® Whole Blood Neutrophil Isolation Kit (Miltenyi-Biotec, Bergisch Gladbach, DE). Isolation was performed according to manufacturer's instruction. In short: 8 mL of full blood donation was mixed with 4 mL of the Isolation Mix-cocktail and incubated for 5 min on a tube rotator, then the tube was transferred in the magnetic field of MACSxpress® Separator for 15 min. There the magnetic labeled cells were separated from the neutrophils and the red blood cells sediment to the bottom of the tube.
[0265] The untouched neutrophils were pipetted in to a clean tube and centrifuged for 5 min at 350×g. Red blood cells were lysed by resuspending the cell pellet in 10 mL red-blood-cells lysis solution for 10 min. The reaction was stopped by addition of 25 mL D-PBS. After centrifugation the cells were resuspended in RPMI-1640 medium and the cell number were determined. The purity of the cells and the success of the isolation was controlled by flow cytometry. The isolated neutrophils were cultured in RPMI-1640 supplemented with 10% FBS and 1% Penicillin (10.000 U/mL)/Streptomycin (10.000 μg/mL) or were directly used in cell-based assays.
[0266] The assay of the invention can be carried out with primary neutrophils.
[0267] Preparation of Antigen-Coated Beads and Immune Complexes Thereof
[0268] For the preparation of antigen-coated beads fluorescent latex beads were coated with recombinant antigen, e.g., virus protein such as spike protein from SARS-CoV-2. Coating of carboxylate-modified polystyrene yellow fluorescent latex beads (L4655-1ML Sigma-Aldrich) was done by same day as experiments. First beads were washed twice with a buffer comprising 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES) and 1.3 mM N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in Aqua dest. pH 6.1. MES/EDAC-Buffer has to be prepared freshly because of the low stability of EDAC.
[0269] The bead suspension was transferred into a 15 mL falcon tube and centrifuged (4.700×g for 20 min). Supernatant was discarded and beads were resuspend in MES/EDAC buffer. This wash step was performed twice. To completely dissolve the beads, a short incubation in ultrasonic cleaner was done for 1:30 min. Coating of latex beads was performed by adding 5 μg/mL antigen, e.g., reSARS-CoV-2 full-length spike protein (SPN-052H4, Acro Biosystems) to 2×10.sup.8 beads/mL in 1 mL MES/EDAC-Buffer. The mixture was incubated for 2 h at 37° C. with 500 rpm in a heat block in the dark.
[0270] To block remaining binding sites, the double volume of 2% (w/v) BSA/PBS was added to the bead/spike-protein mixture after incubation. After a centrifugation at 4,700×g, 15 min, the pellet was resuspended in 2% (w/v) BSA/PBS and centrifuged again. Finally the pellet was resuspended in 0.1% (w/v) BSA/PBS to 1×10.sup.8 beads/mL.
[0271] Heat inactivation of plasma was done by incubation for 30 min at 56° C., followed centrifugation at 4,000×g for 10 min. Precipitate was discarded and supernatant used for preparation of the immune complexes.
[0272] Preparation of the immune complex with SARS-CoV-2 virus-like particles was done by adding either 5 μg/mL specific anti-SARS-CoV-2 chimeric monoclonal antibody (40150-D001-50, SinoBiological) or 400 μg/mL heat inactivated convalescent COVID-19 plasma (Deutsches Rotes Kreuz) to the coated and blocked beads. The beads were incubated for 45 min at 37° C. with the antibody or the plasma.
[0273] As controls, antibodies (negative for SARS-CoV-2) or BSA were mixed with blocked beads and incubated as described above.
[0274] The binding of antibodies to the antigen-coated beads is essential. IgG, IgA and IgM could be detected on the surface of beads prepared as described herein by flow cytometry (data not shown).
[0275] Potency Assay
[0276] HL60 cells were prepared as described above.
[0277] 1*10.sup.8 latex beads were coated with SARS-CoV-2 spike protein, as described above, and immune complexes formed by incubation with 5 μg/mL anti-SARS CoV-2 IgG or, preferably, 400 μg/mL convalescent heat-inactivated plasma, e.g., for 45 min at 37° C. The antigen-coated beads bound by antibodies (beads coated with immune complexes) were washed with D-PBS. After resuspension in 100 μL IMDM without FBS, the beads were added to the HL-60 cells and incubated. Directly after addition of the cells, an immunoglobulin test composition, e.g., IVIG or IVIG-AM at 0.005-15 g/L (or a buffer control) was added. Remaining volume (to the highest concentration) was filled up with formulation buffer.
[0278] After incubation, e.g., for 1 h at 37° C., the supernatant was analysed for cytokine secretion, and the cells were analysed by flow cytometry.
[0279] Measurement of Cytokine Release
[0280] For measurement of the cytokine-release, cell culture supernatant from the co-incubation of the beads coated with the immune complex and the immune effector cells, e.g., the HL-60 cells, may be analysed by different techniques, e.g., cytokine arrays or multiplex assays may be performed. Quantification of selected cytokines was done with commercial human cytokine ELISA-Kits.
[0281] For example, quantification of IL-8 levels in cell culture supernatants was performed with an Abcam human IL-8 ELISA Kit, based on a classical sandwich ELISA. The assay was performed according to manufacturer's instructions. If needed, the supernatant was diluted before the analysis.
[0282] Results
[0283] The uptake of fluorescent beads and IL-8 secretion by HL-60 cells incubated with different amounts of immunoglobulin test compositions, IVIG and IVIG-AM is shown in
[0284] The release of IL-8 into cell culture supernatant was decreased by addition of both immunoglobulin test preparations. IVIG-AM reduces IL-8 level significant more than classical IVIG preparation. Therefore, IVIG-AM demonstrates stronger immunomodulatory properties then IVIG preparation in this assay when IL-8 secretion is analysed.
[0285] By replacing chimeric anti-SARS-CoV-2 IgG (
[0286]
[0287] It was shown, via FcR Blocking experiments, that the convalescent plasma immune complex uptake and IL-8 secretion is FcαRI dependent. Thus, IgA species in convalescent plasma induce inflammation and this in turn could be modulated by the IgA component of immunoglobulin test compositions. Therefore the assay of the present invention shows an increased immunomodulatory potency of IVIG-AM compared to IVIG, which is in line with possible benefits for immunomodulatory treatment of COVID-19 patients with IVIG-AM over standard IVIG.
[0288] In summary, the data demonstrate the establishment of a new assay capable of determining the immunomodulatory potency of immunoglobulin preparations in a system modelling immune stimulation by a virus such as SARS-CoV-2. The analysis of cytokine secretion is believed to more closely mirror the physiologic relevance, as it is able to demonstrate the improved immunomodulatory potency of IVIG-AM. An improved immunomodulatory potency of IVIG-AM has also been shown in vivo, e.g., IgM-concentrate was developed and effectively tested for severe community acquired pneumonia (sCAP) (Welte et al., 2018), which has symptoms similar to COVID-19. Therefore IVIG, in particular, IVIG-AM could be an ideal treatment for severe COVID-19 patients. First clinical studies already show promising results in treatment of COVID-19 patients with IVIG preparations (Cao et al. 2020. Open Forum Infect. Dis. 7.; Xie et al. 2020. J. Infect. S0163-4453(20)30172-9).
1.1 Example 4: Addition of IVIG and IVIG-AM Preparation Reduces Inflammation
[0289] More aspects of immunomodulation in the method of the invention, as described herein, were investigated by the addition of various concentrations of immunoglobulin test compositions. IgG containing IVIG (IgG Next Generation, Biotest AG), as well as IgG, IgA and IgM containing IVIG-AM (trimodulin, Biotest AG) were compared. Used lots were tested negative for anti-SARS-CoV-2 neutralizing antibodies. IL-8, IL-10, MCP-1, and IL-1ra release were measured as markers of inflammation.
[0290] Addition of IVIG or IVIG-AM to HL60 cells significantly and equally decreased bead uptake of SARS-CoV-2-like particles opsonized with COVID-19 plasma (data of this experiment not shown). The corresponding cytokine release is also affected: IL-10, MCP-1 and IL-8 level are reduced by IVIG and significantly more by IVIG-AM addition (
[0291] Thus, HL60 cells are particularly advantageous effector cells, if potency of IgA- and/or IgM-containing immunoglobulin test compositions is to be analysed. In this case, also, the effector function determined can be the release of different pro- or anti-inflammatory cytokines such as IL8, IL-10, MCP-1 and/or IL-1ra. For example, as shown in