Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs.

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy.

Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML).

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures.

HuCAL PLATINUM, a synthetic Fab library optimized for sequence diversity and superior performance in mammalian expression systems.

This article describes the design of HuCAL (human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production.

In vitro affinity maturation of HuCAL antibodies: complementarity determining region exchange and RapMAT technology.

Monoclonal antibodies gain ever-increasing importance in the treatment of human diseases across a broad range of indications. Diverse technologies currently exist, which are used to generate recombinant therapeutic antibodies that are basically indistinguishable from naturally occurring human immunoglobulins. We describe how human combinatorial antibody libraries are used together with unique optimization techniques to produce such therapeutically relevant proteins, for instance in the areas of oncology and inflammation.

Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface.

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm.

The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies.

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family.

Crystallization and molecular-replacement solution of a diagnostic fluorescent dye in complex with a specific Fab fragment.

Tetrasulfocyanine (TSC) has been described as a fluorescent probe for tumour imaging. The complex of TSC and the Fab antibody fragment MOR03268 has been crystallized in three different crystal forms. MOR03268 was identified from the HuCAL GOLD library and further optimized to bind TSC with high affinity (Kd = 0.

Tetracycline-dependent gene regulation: combinations of transregulators yield a variety of expression windows.

In addition to the originally described Tet transactivator tTA, several variants including transrepressors (tTRs) and reverse transactivators (rtTAs) have been constructed, which we employ here to establish a set of HeLa cell lines carrying different combinations of chromosomally integrated Tet transregulators. We first compare the regulatory properties of these lines using transient transfection of a luciferase reporter gene. Cell lines carrying rtTA-S2 or rtTA-M2 show reduced activity in the absence of dox and higher activation levels in its presence compared to an rtTA line.

Tetracycline-inducible systems for Drosophila.

Since their inception, tetracycline (Tet)-inducible systems have become the method of choice for transgenic research. The Tet-Off systems have a number of advantages, including robust target induction using a relatively benign effector molecule. However, use of the Tet-On system has been fraught with difficulties, including high background expression in the absence of effector molecules and inconsistent gene induction.

Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity.

Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter.

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity.

Even though renal cell carcinomas (RCC) are thought to be immunogenic, many tumors express variations in surface molecules and intracellular proteins that hinder induction of optimal antitumor responses. Interferon gamma (IFNgamma) stimulation can correct some of these deficiencies. Therefore, we introduced the complementary DNA (cDNA) encoding human IFNgamma into a well-characterized RCC line that has been selected for development of an allogeneic tumor cell vaccine for treatment of patients with metastatic disease.

The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system.

The tc-responsive TetR protein allows the investigation of various transcriptional activators in respective fusion proteins. We have fused eight well-known human activator domains to the C-terminus of TetR and determined the properties of the resulting transactivators using a tetracycline-responsive promoter in three human cell lines (HeLa, BJAB, and Jurkat). Several-hundred-fold activation was exclusively obtained with the acidic p65 domain from NF-kappaB and with VP16, which served as a positive control.

Tetracycline-inducible expression systems with reduced basal activity in mammalian cells.

We describe a modification of the tetracycline-inducible eukaryotic gene expression system with decreased basal levels of expression in HeLa cells. It employs the tetracycline-inducible transactivator and a tetracycline-regulated repressor fusion acting on the same promoter. To avoid heterodimerization or competition for the same DNA site, each was provided with different DNA recognition and/or protein dimerization specificities.

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts.

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer.

Hyperexpression of transporter in antigen processing-1 (TAP-1) in thyroid glands affected by autoimmunity: a contributing factor to the breach of tolerance to thyroid antigens?

According to the 'aberrant HLA expression' hypothesis, endocrine autoimmunity is driven by presentation of self antigens by target cells over-expressing HLA molecules. In autoimmune thyroid diseases (AITD), thyroid follicular cells (thyrocytes) over-express HLA class I and HLA class II molecules. Since efficient presentation of endogenous peptides via class I requires transporters that translocate endogenous peptides from the cytoplasm to the endoplasmic reticulum, i.

Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast.

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae.

Interferon-gamma rescues HLA class Ia cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins.

Human placental trophoblast cells that constitute the materno-fetal interface during pregnancy escape maternal alloimmune attack. The different trophoblast cell subpopulations have developed efficient regulatory mechanisms to prevent expression of beta2-microglobulin-associated HLA class Ia molecules at their cell surface. We previously reported the presence of HLA class Ia messages in villous cytotrophoblast cells and in the syncytiotrophoblast differentiated in vitro purified from term placenta.

The activity of the gluconate-H+ symporter of Schizosaccharomyces pombe cells is down-regulated by D-glucose and exogenous cAMP.

Schizosaccharomyces pombe cells take up D-gluconate, as an alternative carbon source for growth, during glucose starvation or when cultured on glycerol-containing medium. Gluconate uptake is not detectable while cells are growing logarithmically on glucose. The addition of D-glucose as well as its non-metabolizable analogues to glycerol-grown cells causes an immediate loss of gluconate transport within 1 min.