Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

Factor VIII-specific B cell responses in haemophilia A patients with inhibitors.

Development of inhibitory antibodies to factor VIII (FVIII) provides a major complication of replacement therapy in patients with haemophilia A. The risk of inhibitor formation is influenced by the underlying FVIII gene defect. Moreover, genetic determinants in the promoter region of IL-10 and TNFalpha have been linked to an increased risk of inhibitor development. Recent cohort-studies have provided evidence that the risk of inhibitor formation is linked to intensity of treatment. Eradication of FVIII inhibitors can be achieved by frequent infusion of high dosages of FVIII, so-called immune tolerance induction (ITI). Until now, the mechanisms involved in downmodulation of the immune response to FVIII during ITI have not been unraveled. Studies performed in an animal model for haemophilia A have suggested that elimination of FVIII-specific memory B cells by high dosages of FVIII contributes to the decline in FVIII inhibitor levels during ITI. Limited knowledge is available with respect to the development and persistence of FVIII-specific memory B cells in patients with haemophilia A. Two recent studies suggest that the frequency of peripheral FVIII-specific memory B cells in haemophilia A patients with inhibitors range from <0.01 to 0.40% of that of total IgG(+) B cells. No or very low frequencies of FVIII-specific memory B cells are observed in haemophilia A patients without inhibitors and in patients treated successfully by ITI. Possible implications of these findings are discussed in the context of currently available information on the role of antigen-specific memory B cells and long-living antibody producing plasma cells in humoral immunity.

Domain specificity of factor VIII inhibitors during immune tolerance induction in patients with haemophilia A.

INTRODUCTION: Frequent administration of high dosages factor VIII (FVIII), so-called immune tolerance induction (ITI), provides an efficient strategy to eradicate inhibitory antibodies in patients with haemophilia A. At present, our knowledge on the characteristics of inhibitory antibodies in patients undergoing ITI is limited. AIM: In this study we characterized the domain specificity of FVIII inhibitors in 11 haemophilia A patients during ITI. RESULTS: In three of six patients who were successfully tolerized, inhibitory antibodies were directed predominantly against the FVIII light chain. In two other patients within this group, a significant contribution of A2 antibodies was observed which did not change during treatment. In the sixth patient the relative contribution of A2 inhibitors declined which coincided with an increase in antilight chain antibodies. In four of five patients who failed ITI, A2 inhibitors were observed. In two patients the contribution of A2 inhibitors increased during treatment, while in two other patients the contribution of A2 inhibitor remained constant. The fifth patient had inhibitory antibodies predominantly directed against the FVIII light chain. CONCLUSION: Overall, our findings revealed changes in domain specificity of FVIII antibodies in five of 11 patients analysed. Remarkably, antibodies exclusively directed towards the light chain of FVIII were predominantly observed in patients who were successfully tolerized.

Opportunities and limitations of mouse models humanized for HLA class II antigens.

MHC class II molecules are essential for shaping the CD4+ T-cell repertoire in the thymus and for selecting antigenic peptides that are presented to CD4+ T cells in the periphery. A range of different mouse models humanized for HLA class II antigens have been developed to study the regulation of MHC-class II restricted immune responses. These mouse models have been used to identify immunodominant peptides that trigger diseases and to characterize the interactions of T-cell receptors with disease-associated peptides and MHC class II molecules. Peptides presented to CD4+ T cells in these mouse models were shown to be similar to peptides presented to CD4+ T cells in patients who carry the same MHC class II haplotype. Opportunities and limitations associated with these mouse models will be discussed and the potential application of these models for understanding the regulation of antibody responses against factor VIII in hemophilia A will be indicated.

Discordant antibody response in monozygotic twins with severe haemophilia A caused by intensive treatment.

Both genetic factors and environmental factors are suggested to play a role in the aetiology of inhibitor development in patients with severe haemophilia A. Monozygotic twins are ideal candidates to study the influence of environmental factors. We describe a pair of 3-year-old monozygotic twin brothers with severe haemophilia A. Prenatal diagnosis confirmed the presence of an intron 22 inversion. Other patient-related factors such as birth weight, vaccinations and the duration of breastfeeding were similar. At the age of 7 months, one boy suffered from a spontaneous subdural haematoma, which needed complete correction of haemostasis with continuous infusion of a third-generation recombinant factor VIII. A persistent high-titre inhibitor with severe clinical symptoms developed, that could only be eradicated with high-dose immune tolerance induction (ITI) for 36 months in combination with rituximab therapy. His twin brother first received treatment at 9 months of age with the same FVIII product. After treatment on nine exposure days, he developed a low-titre inhibitor at the age of 14.5 months. Unlike his brother, he was tolerized without difficulties with low-dose ITI within 2 months. The discordant antibody responses were underlined by dissimilar IgG1 and IgG4 levels in their plasma. The discordant immune response to FVIII in this pair of monozygotic twin brothers seemed to be related to intensity of treatment and severity of bleeds. This confirms that these environmental factors play an additional role in the development of inhibitors.