Irene Michalk

The histone demethylase UTX regulates stem cell migration and hematopoiesis.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Redirection of T cells with a first fully humanized bispecific CD33–CD3 antibody efficiently eliminates AML blasts without harming hematopoietic stem cells

Redirection of T cells with a first fully humanized bispecific CD33–CD3 antibody efficiently eliminates AML blasts without harming hematopoietic stem cells

Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3–anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.

Retargeting of human regulatory T cells by single-chain bispecific antibodies

Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8+ and CD4+ effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8+ and CD4+ effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4+ T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection.

Costimulation improves the killing capability of T cells redirected to tumor cells expressing low levels of CD33: description of a novel modular targeting system

Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.

A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.

Bispecific antibody releasing-mesenchymal stromal cell machinery for retargeting T cells towards acute myeloid leukemia blasts

Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future.

Characterization of a Novel Single-Chain Bispecific Antibody for Retargeting of T Cells to Tumor Cells via the TCR Co-Receptor CD8

There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

TCR/CD3 activation and co-stimulation combined in one T cell retargeting system improve anti-tumor immunity

We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.

Gel drying methods.

For some instances, protein gels need to be dried after SDS-PAGE, for example, if autoradiography should be performed from radioactive-labeled proteins after their separation on SDS-polyacrylamide gels. Another reason may be to simply store the gel in the laboratory book. Aside from expensive commercial solutions, especially for storage of the dried gel in the lab book, the simple and cheap drying protocol here presented may be sufficient.