Different players generate positive responses in two in vitro cytokine assay formats with aqueous and immobilized TGN1412 analog.

To detect potential risk of severe cytokine release syndrome, in vitro assay formats with human cells have been developed. The two major testing platforms are a combination of whole blood with aqueous-phase test articles (whole blood cytokine assay, WBCA) and peripheral blood mononuclear cells with solid-phase articles (PBMC assay). Significant induction of cytokines was seen in both assays after treatment with a widely used control agent, TGN1412 or its analog CD28SA, but the WBCA cytokine profile differed from what was expected from clinical experience. In the WBCA, potential risk of CD28SA was detected by elevation of IL-8 whereas IL-2, a key cytokine after stimulation of CD28, was not induced in approximately 40% of donor samples. Therefore, further mechanistic understanding of the different responses in the in vitro assay was needed. In this study of donor samples treated with CD28SA, we compared the induction of cytokines and identified the cytokine-producing cells in the two assays. IL-2 was markedly elevated in all the donors in the PBMC assay but only in 1 of 3 donors in the WBCA. IL-8, the most sensitive biomarker in the WBCA, was produced by monocytes and granulocytes. T cells, the most relevant player in the PBMC assay with CD28SA, did not contribute to the positive response seen in two donors in the WBCA, which suggests that different players caused the positive cytokine responses to CD28SA in the two assays.

Rapid in vitro assessment of the immunogenicity potential of engineered antibody therapeutics through detection of CD4+ T cell interleukin-2 secretion.

Therapeutic antibodies sometimes elicit anti-drug antibodies (ADAs) that can affect efficacy and safety. Engineered antibodies that contain artificial amino acid sequences are potentially highly immunogenic, but this is currently difficult to predict. Therefore, it is important to efficiently assess immunogenicity during the development of complex antibody-based formats. Here, we present an in vitro peripheral blood mononuclear cell-based assay that can be used to assess immunogenicity potential within 3 days. This method involves examining the frequency and function of interleukin (IL)-2-secreting CD4+ T cells induced by therapeutic antibodies. IL-2-secreting CD4+ T cells seem to be functionally relevant to the immunogenic potential due to their proliferative activity and the expression of several cytokines. The rates of the donors responding to low and high immunogenic proteins, mAb1, and keyhole limpet hemocyanin were 1.3% and 93.5%, respectively. Seven antibodies with known rates of immunogenicity (etanercept, emicizumab, abciximab, romosozumab, blosozumab, humanized anti-human A33 antibody, and bococizumab) induced responses in 1.9%, 3.8%, 6.4%, 10.0%, 29.2%, 43.8%, and 89.5% of donors, respectively. These data are comparable with ADA incidences in clinical settings. Our results show that this assay can contribute to the swift assessment and mechanistic understanding of the immunogenicity of therapeutic antibodies.

In vitro human helper T-cell assay to screen antibody drug candidates for immunogenicity.

Monoclonal antibody (mAb) drugs offer a number of valuable treatments. Many newly developed mAb drugs include artificial modification of amino acid sequences from human origin, which may cause higher immunogenicity to induce anti-drug antibodies (ADA). If the immunogenicity of a new candidate can be understood in the nonclinical phase, clinical studies will be safer and the success rate of development improved. Empirically, in vitro immunogenicity assays with human cells have proved to be sufficiently sensitive to nonhuman proteins, but not to human/humanized mAb. To detect the weaker immunogenicity of human-based mAb, a more sensitive biomarker for in vitro assays is needed. The in vitro study here developed a proliferation assay (TH cell assay) using flow cytometry analysis that can detect a slight increase in proliferating TH cells. Samples from 218 donors treated with a low-immunogenic drug (etanercept) were measured to determine a positive threshold level. With this threshold, positive donor percentages among PBMC after treatment with higher-immunogenicity mAb drugs were noted, that is, 39.5% with humanized anti-human A33 antibody (hA33), 27.3% with abciximab, 25.9% with adalimumab, and 14.8% with infliximab. Biotherapeutics with low immunogenicity yielded values of 0% for basiliximab and 3.7% for etanercept. These data showed a good comparability with previously reported incidences of clinical ADA with the evaluated drugs. Calculations based on the data here showed that a TH cell assay with 40 donors could provide statistically significant differences when comparing low- (etanercept) versus highly immunogenic mAb (except for infliximab). Based on the outcomes here, for screening purposes, a practical cutoff point of 3/20 positives with 20 donors was proposed to alert immunogenicity of mAb drug candidates.

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity.

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method's high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains.

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.

Is an in vitro whole blood cytokine assay useful to detect the potential risk of severe infusion reaction of monoclonal antibody pharmaceuticals?

After the life-threatening cytokine release syndrome (CRS) occurred in the clinical study of the anti-CD28 monoclonal antibody (mAb) TGN1412, in vitro cytokine release assays using human blood cells have been proposed for non-clinical evaluation of the potential risk of CRS. Two basic assay formats are frequently used: human peripheral blood mononuclear cells (PBMC) with immobilized mAbs, and whole blood with aqueous mAbs. However, the suitability of the whole blood cytokine assay (WBCA) has been questioned, because an unrealistically large sample size would be required to detect the potential risk of CRS induced by TGN1412, which has low sensitivity. We performed a WBCA using peripheral blood obtained from 68 healthy volunteers to compare two high risk mAbs, the TGN1412 analogue anti-CD28 superagonistic mAb (CD28SA) and the FcγR-mediated alemutuzumab, with a low risk mAb, panitumumab. Based on the cytokine measurements in this study, the sample size required to detect a statistically significant increase in cytokines with 90% power and 5% significance was determined to be n = 9 for CD28SA and n = 5 for alemtuzumab. The most sensitive marker was IL-8. The results suggest that WBCA is a practical test design that can warn of the potential risk of FcγR-mediated alemtuzumab and T-cell activating CD28SA but, because there was apparently a lower response to CD28SA, it cannot be used as a risk-ranking tool. WBCA is suggested to be a helpful tool for identifying potential FcγR-mediated hazards, but further mechanistic understanding of the response to CD28SA is necessary before applying it to T cell-stimulating mAbs.

Impaired lymphopoiesis and altered B cell subpopulations in mice overexpressing Lnk adaptor protein.

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk(-/-) mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk(-/-) hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.