Molecular characterization and functional activity of an IL-15 antagonist MutIL-15/Fc human fusion protein.

Fc fusion proteins are a new emerging class of molecules for immune-targeted delivery of therapeutic proteins. Biophysical and bioanalytical characterization is critical for clinical development and delivery of therapeutic proteins. Here we report molecular and functional characterization of a recombinant human fusion protein Mutant IL-15/Fc. MutIL-15/Fc has a molecular weight of ∼95 kDa as determined by multiangle laser light scattering with online size exclusion chromatography and migrated at a faster rate (lower retention time) in gel filtration column. The kinetics of binding of MutIL-15/Fc to Fcγ receptor is best fitted in a bivalent modal with K(D1) 5 µM and K(D2) 9 µM determined by surface plasmon resonance (BIAcore). N-Glycoprofiling analysis revealed extensive glycosylation of MutIL-15/Fc. The Fc and IL-15 components in the MutIL-15/Fc are detected using the dual mode ELISA. The HT-2 cell proliferation inhibition assay is qualified as a quantitative in vitro marker functional assay. Molecular state changes associated with forced stress analyzed by SEC-MALS resulted in changes in bioactivity and Fc:Fcγ receptor interaction affinity. These data provide a systematic approach to molecular and functional characterization of the MutIL-15/Fc to establish product consistency and stability monitoring during storage and under drug delivery conditions.

Analytical characterization of ch14.18: a mouse-human chimeric disialoganglioside-specific therapeutic antibody.

Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.

Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77.

PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.

Comparison of GD2 binding capture ELISA assays for anti-GD2-antibodies using GD2-coated plates and a GD2-expressing cell-based ELISA.

Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment.

MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

Evaluation of IRES-mediated, cell-type-specific cytotoxicity of poliovirus using a colorimetric cell proliferation assay.

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and cell-type specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor beta antibody (HuMikbeta(1)), and correlation with functional activity using an antigen-transfected murine cell line.

The HuMikbeta(1), a humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor beta-chain (CD122), inhibits the actions of the inflammatory cytokine IL-15, and may be useful for immunotherapy of an array of autoimmune disorders as well as diseases associated with the retrovirus human T-cell lymphotrophic virus 1 (HTLV-1). In order to facilitate the production of material for clinical investigation, we developed a cell-based ELISA (CbELISA) for measuring the binding activity, as a potential biological activity marker, of the HuMikbeta(1) monoclonal antibody to a transfected 32D mouse cell line (32Dbeta) expressing IL-2Rbeta antigen on the cell surface. There is specific binding of HuMikbeta(1) to the transfected cell line, titrating out in the concentration range of 1-1,000 ng/ml. Under identical conditions, there was no binding of HuMikbeta(1) to the parent cell line 32D. Satisfactory binding curves with HuMikbeta(1) were obtained with 32Dbeta cells grown between 3 and 19 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The binding was specific for Mikbeta antibodies recognizing the IL-2/IL-15 receptor beta subunit as demonstrated by binding of HuMikbeta(1), Mikbeta(2) and Mikbeta(3) antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32Dbeta cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMikbeta(1) binding. The assay could detect changes in binding activity of HuMikbeta(1) antibody under stressful conditions (heat and low pH) and the results paralleled the effect of stress on the physicochemical characteristics. More importantly, the binding activity shows an apparent correlation to inhibition of IL-15-induced proliferation of 32Dbeta cells with HuMikbeta(1). In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMikbeta(1) activity and could be used as a potency marker assay for monitoring the lot-lot consistency and functional stability of HuMikbeta(1) product.

Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Ralpha.

Interleukin-2 receptor alpha (IL-2Ralpha, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Ralpha peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Ralpha antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The assay shows reproducible dose-response curves in the concentration range of 10-1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.