Erika Gustafsson

Immunoglobulin-Secreting Cells of Maternal Origin Can Be Detected in B Cell-Deficient Mice

Abstract It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offsprings immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (μ−/−) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to μ+/− mothers. In some of these serum IgG-positive μ−/− mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, μ−/− offspring born to μ+/− dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these μ−/−GFP−/− offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in μ−/− mice that were born to μ−/− dams and transferred to a lactating μ+/+ foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.

Locally Delivered CD40 Agonist Antibody Accumulates in Secondary Lymphoid Organs and Eradicates Experimental Disseminated Bladder Cancer

Comparing the efficacy and biodistribution of local and systemic delivery of anti-CD40 agonistic antibodies, Sandin and colleagues show that local low-dose antibody therapy is effective against disseminated bladder cancer with reduced toxic side effects. Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic. Cancer Immunol Res; 2(1); 80–90. ©2013 AACR.

Maternal antibodies protect immunoglobulin deficient neonatal mice from mouse hepatitis virus (MHV)-associated wasting syndrome

PROBLEM: Neonatal mice nursed by dams lacking immunoglobulins (Igs) may often suffer from lethal runting if raised under conventional conditions. The present study was performed in order to clarify a) the cause of the wasting syndrome and b) the protective role of antigen‐specific milk antibodies.

Purification of truncated and mutated chemotaxis inhibitory protein of Staphylococcus aureus—an anti-inflammatory protein

The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2. The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.

Neonatal ingestion of IgG-containing milk increases the survival of adoptively transferred B-lineage cells in B cell-deficient mice

This study shows that neonatal ingestion of immunoglobulin-containing milk increases the survival of adoptively transferred B-lineage cells in non-irradiated B cell-deficient (mu-/-) mice. Neonatal mu-/- mice were either transferred to lactating mu+/+ foster dams, allowing them to suckle IgG-containing milk, or were kept with their mu-) dams, without IgG in the milk. After adoptive transfer of spleen cells as adults, serum-IgG levels, numbers of plasma cells, T and B cells in spleen and bone marrow were determined. The results showed that the mice which had received milk-IgG had significantly higher levels of serum-IgG and splenic B cells, as well as a higher number of Ig-secreting cells in spleen and bone marrow. This indicates that the mice might have been tolerised against IgG as neonates, if allowed to ingest Ig-containing milk. There is, however, a possibility that B cells in the milk may also contribute to the observed tolerising effect. In summary, the results suggest that cross-fostering could be a suitable method to facilitate the long-term reconstitution of B- and plasma cell numbers in non-irradiated B cell-deficient mice.

Human choriocarcinoma-derived JEG-3 cells transfected with murine MHC class II Aq expression vectors present antigen to Aq-restricted murine T-cell hybridoma

MHC class II molecules are not normally expressed on the cell surface of placental cells. This absence of class II molecules is assumed to be of importance for mammalian reproduction, since such expression is likely to increase the risk of harmful anti-placental immune responses. The present study was aimed to clarify whether post-transcriptional events prohibit proper cell surface expression of MHC class II molecules in cell lines of placental origin. The murine trophoblast cell line SM9-1 as well as the human choriocarcinoma-derived cell line JEG-3 were transiently co-transfected with MHC class II Aq a and b genes under the control of viral promoter systems. The transfected cells were stained for surface expression of MHC class II and assayed for antigen presentation in vitro. Only a small proportion of the transfected murine SM9-1 cells showed detectable class II cell surface expression, which made functional studies of this cell line difficult. The transfected JEG-3 cells, however, showed a high proportion of cells with distinct surface expression of murine class II Aq molecules and the antigen presentation assays revealed T cell activation upon addition of processed antigen, but not with unprocessed antigen. These results show that ectopic MHC class II gene transcription can result in cell surface expression of immunohistochemically detectable MHC class II on cells of placental origin. The fact that murine class II molecules could be expressed in a functional manner on human JEG-3 cells also strongly suggests that proper accessory gene activities are not essential for obtaining surface expression.

ADC-1013, an agonistic CD40 antibody optimized for local immunotherapy of cancer

Local administration of immune activating antibodies may increase the efficacy and reduce the immune-related adverse events associated with systemic immunotherapy of cancer. Here we report the development of a fully human agonistic CD40 antibody (IgG1), ADC-1013, which has been optimized for local immunotherapy by increasing potency and tumor retention. ADC-1013 activates CD40 receptors on antigen-presenting cells such as dendritic cells, resulting in up-regulation of the co-stimulatory molecules CD80 and CD86, and induction of IL-12. In addition, ADC-1013 induces direct tumor killing of CD40+ tumors, e.g. via antibody-dependent cellular cytotoxicity (ADCC). The anti-tumor effects of ADC-1013 were first assessed in a bladder cancer model (EJ) in immunodeficient NSG mice. Significant anti-tumor responses were demonstrated, and further augmented in mice repopulated with human moDCs/T cells. To study the anti-tumor effects related to the immune activating properties of ADC-1013 in more detail, a human CD40 positive transgenic mouse (hCD40tg) in C57/BL-6 background was used. This transgenic mouse strain has an intact immune system and fully functional dendritic cells that are activated upon ADC-1013 treatment. Furthermore, the dendritic cells obtained from this strain are able to induce antigen specific T cell activation in vitro upon stimulation with ADC-1013. Importantly, treatment with ADC-1013 in a syngeneic bladder cancer (MB49) model, which is hCD40 negative, demonstrated that ADC-1013 induce significant tumor protection and long term immunity independent of direct tumor targeting. In addition, the anti-tumor immunity was shown to be T-cell dependent. To our knowledge, ADC-1013 represents the first immunomodulatory antibody optimized for local immunotherapy of cancer. It is currently in late pre-clinical development and will enter clinical trials in 2014.