ABBV-184: A Novel Survivin-specific TCR/CD3 Bispecific T-cell Engager is Active against Both Solid Tumor and Hematologic Malignancies.

CD3 bispecific T-cell engagers (TCE), comprised of a tumor-targeting domain linked to a CD3 binding domain, function by bridging target-positive tumors and CD3-expressing effector T cells enabling redirected T cell-mediated killing of tumor cells. Although the majority of CD3 bispecific molecules in clinical development incorporate tumor-targeting antibody-based binding domains, many tumor-associated antigens derive from intracellular proteins and are not accessible to targeting via antibody. Intracellular proteins processed into short peptide fragments and presented on the cell surface by MHC proteins are recognized by T-cell receptors (TCR) on the surface of T cells. Here we describe the generation and preclinical evaluation of ABBV-184, a novel TCR/anti-CD3 bispecific composed of a highly selective soluble TCR that binds a peptide derived from the oncogene survivin (BIRC5) bound to the class I MHC allele human leukocyte antigen (HLA)-A*02:01 expressed on tumor cells, linked to a specific binder to the CD3 receptor on T cells. ABBV-184 drives an optimal distance between T cell and target cell thereby enabling sensitive recognition of low-density peptide/MHC targets. Consistent with the expression profile of survivin across a broad range of both hematologic and solid tumors, treatment of acute myeloid leukemia (AML) and non-small cell lung cancer (NSCLC) cell lines with ABBV-184 results in T-cell activation, proliferation, and potent redirected cytotoxicity of HLA-A2-positive target cell lines, both in vitro and in vivo, including patient-derived AML samples. These results indicate that ABBV-184 is an attractive clinical candidate for the treatment of patients with AML and NSCLC.

Author Correction: Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assessment of Proteus mirabilis Antigen Immunological Complexes by Atomic Force Microscopy.

Atomic force microscopy (AFM) is being increasingly used to directly measure protein interactions in nearly physiological environments. Here, protocols for atomic force microscopy (AFM) for visualization of antigen-antibody complexes are presented. The technique is used to demonstrate complexes formed by rheumatoid arthritis patient antibodies with lipopolysaccharide (LPS) isolated from P. mirabilis (O3) strain S1959 and a synthetic antigen (LPS epitope of 6 N-alpha-(D-galacturonoyl)-L-lysine residues).

Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus.

The three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells. We observe high frequencies of homologous chromosome X colocalization (or coalescence), typically associated with initiation of X-chromosome inactivation, occurring in XX cells outside of early embryogenesis. Moreover, during chromosome X coalescence significant changes in Xist, H3K27me3, and X-linked gene expression occur, suggesting the potential exchange of gene regulatory information between the active and inactive X chromosomes. We also observe significant differences in chromosome X coalescence in disease-implicated lymphocytes isolated from systemic lupus erythematosus (SLE) patients compared to healthy controls. These results demonstrate that X chromosomes can functionally interact outside of embryogenesis when X inactivation is initiated and suggest a potential gene regulatory mechanism aberration underlying the increased frequency of autoimmunity in XX individuals.

Novel tool in rheumatoid arthritis diagnosis-The usage of urease flap region peptidomimetics.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non-RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms. Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test.

A Phase I Clinical Trial with Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants.

There is considerable interest in therapeutic transfer of regulatory T cells (Tregs) for controlling aberrant immune responses. Initial clinical trials have shown the safety of Tregs in hematopoietic stem cell transplant recipients and subjects with juvenile diabetes. Our hypothesis is that infusion(s) of Tregs may induce transplant tolerance thus avoiding long-term use of toxic immunosuppressive agents that cause increased morbidity/mortality. Towards testing our hypothesis, we conducted a phase I dose escalation safety trial infusing billions of ex vivo expanded recipient polyclonal Tregs into living donor kidney transplant recipients. Despite variability in recipient's renal disease, our expansion protocol produced Tregs which met all release criteria, expressing >98% CD4+CD25+ with <1% CD8+ and CD19+ contamination. Our product displayed >80% FOXP3 expression with stable demethylation in the FOXP3 promoter. Functionally, expanded Tregs potently suppressed allogeneic responses and induced the generation of new Tregs in the recipient's allo-responders in vitro. Within recipients, expanded Tregs amplified circulating Treg levels in a sustained manner. Clinically, all doses of Treg therapy tested were safe with no adverse infusion related side effects, infections or rejection events up to two years post-transplant. This study provides the necessary safety data to advance Treg cell therapy to phase II efficacy trials.

Generation and Characterization of Alloantigen-Specific Regulatory T Cells For Clinical Transplant Tolerance.

Donor-specific CD4+CD127-CD25+FOXP3+ regulatory T cells (AgTregs) have the potential to induce clinical transplant tolerance; however, their expansion ex vivo remains challenging. We optimized a novel expansion protocol to stimulate donor-specific Tregs using soluble 4-trimer CD40 ligand (CD40L)-activated donor B cells that expressed mature antigen-presenting cell markers. This avoided the use of CD40L-expressing stimulator cells that might otherwise result in potential cellular contamination. Purified allogeneic "recipient" CD4+CD25+ Tregs were stimulated on days 0 and 7 with expanded "donor" B cells in the presence of IL-2, TGFß and sirolimus (SRL). Tregs were further amplified by polyclonal stimulation with anti-CD3/CD28 beads on day 14 without SRL, and harvested on day 21, with extrapolated fold expansion into the thousands. The expanded AgTregs maintained expression of classical Treg markers including demethylation of the Treg-specific demethylated region (CNS2) and also displayed constricted TcR repertoire. We observed AgTregs more potently inhibited MLR than polyclonally expanded Tregs and generated new Tregs in autologous responder cells (a measure of infectious tolerance). Thus, an optimized and more clinically applicable protocol for the expansion of donor-specific Tregs has been developed.

Cross-Reactivity of Polyclonal Antibodies against Canavalia ensiformis (Jack Bean) Urease and Helicobacter pylori Urease Subunit A Fragments.

Overlapping decapeptide fragments of H. pylori urease subunit A (UreA) were synthesized and tested with polyclonal antibodies against Canavalia ensiformis (Jack bean) urease. The linear epitopes of UreA identified using the dot blot method were then examined using epitope mapping. For this purpose, series of overlapping fragments of UreA, frameshifted ± four amino acid residues were synthesized. Most of the UreA epitopes which reacted with the Jack bean urease polyclonal antibodies had been recognized in previous studies by monoclonal antibodies against H. pylori urease. Fragments 11 - 24, 21 - 33, and 31 - 42 were able to interact with the Jack bean urease antibodies, giving stable immunological complexes. However, the lack of recognition by these antibodies of all the components in the peptide map strongly suggests that a non-continuous (nonlinear) epitope is located on the N-terminal domain of UreA.

[analysis of adherence and compliance to methotrexate therapy in rheumatoid arthritis patients].

Rheumatoid arthritis is chronic inflammatory disease leading to disability and increasing the risk of premature death. Early treatment is crucial for remission and maintaining it for a maximal time. Adherence of the patient to the therapeutic recommendations is necessary. Although this issue is important, its role in achieving of therapeutic success is not fully noted by patients as well as medical stuff. Patients with RA are often non-adherent to rheumatologist's recommendations and non-compliance to the therapy regimen. The causes of this phenomenon are numerous and lie in the patient's attitude to therapy or are patient-independent. To effectively increase the level of adherence and compliance, the detailed medical care, including education of the patient, as well as adaptation of the drug form, by concentrating the active compound, is essential.

Characterization of Microbial Communities in Acidified, Sulfur Containing Soils.

Over a period of three years, microbial communities in acidified soil with high sulfur content were analyzed. In soil water extracts ureolytic, proteolytic, oxidoreductive, and lipolytic activity were detected. The presented results indicate that the enzymatic activity of soil microbial communities varied considerably over time. Isolated 26 (80%) bacterial strains belonged to genus Bacillus sp. and were identified by cultivation and 16S rRNA methods. The commercially available procedures for bacterial DNA isolation from acidified soil failed, therefore a new, specific DNA isolation method was established. Ureolytic activity, detected in soil extracts as well as in isolated Bacillus sp. strains may be considered as a tool for the bioremediation of acidified soils with high sulfate content.

Use of Fourier-transform infrared spectroscopy in the diagnosis of rheumatoid arthritis: a pilot study.

Rheumatoid arthritis is an autoimmune inflammatory disease leading to joint cartilage, bone degradation and limitation of mobility. Diagnosis of RA is difficult and complex. There are also no effective methods for clear discrimination between RA patients and non-RA individuals. In this work we use IR spectroscopy to differentiate RA patients and blood donors' sera. We found differences between investigated sera (RA and non-RA) in range of 3000-2800 and 1800-800 cm-1 (W1-W5 regions). Based on mathematical analysis we developed a K-NN model characterized by 85 % of sensitivity and 100 % of specificity. Also we found that, wavenumber 1424 cm-1, comprising in W3 region, was the most effective in human sera distinguishing. We conclude that IR spectroscopy may serve as a fast and easy method useful in RA serology.

Hoxa10 null animals exhibit reduced platelet biogenesis.

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions in vitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.

Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants.

Nineteen subjects have more than 18 months' follow-up in a phase IIb tolerance protocol in HLA-mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4 and CD8 T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.

The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.

Lysozyme (1,4-ß-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur.

Defining the alloreactive T cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture.

The cellular immune response is the most important mediator of allograft rejection and is a major barrier to transplant tolerance. Delineation of the depth and breadth of the alloreactive T cell repertoire and subsequent application of the technology to the clinic may improve patient outcomes. As a first step toward this, we have used MLR and high-throughput sequencing to characterize the alloreactive T cell repertoire in healthy adults at baseline and 3 months later. Our results demonstrate that thousands of T cell clones proliferate in MLR, and that the alloreactive repertoire is dominated by relatively high-abundance T cell clones. This clonal make up is consistently reproducible across replicates and across a span of three months. These results indicate that our technology is sensitive and that the alloreactive TCR repertoire is broad and stable over time. We anticipate that application of this approach to track donor-reactive clones may positively impact clinical management of transplant patients.

Administration of nicotinamide does not increase platelet levels in mice.

Elucidating ways to enhance megakaryopoiesis in vivo would have therapeutic applications for thrombocytopenia and transfusion medicine. Nicotinamide has been shown to enhance endomitosis in megakaryocytes cultured in vitro, suggesting that it may be beneficial for the production of platelets in culture. We hypothesized that regular injections of nicotinamide in mice would also increase platelets in vivo. However, we found that platelet counts were reduced by about 25% with daily injections of nicotinamide. Altering the schedule, duration, or nicotinamide dose did not improve platelet production. Consistent with lower platelet levels, nicotinamide also tended to decrease megakaryocyte frequency in sternum and spleen sections, as well as colony formation in vitro by bone marrow progenitor cells. However, there was no effect on the fraction or ploidy of CD41(+) cells harvested from bone marrow. Together, our results suggest that, although nicotinamide increases polyploidization of megakaryocytes in culture, it does not have translatable effects in vivo.

Detection of antibodies against synthetic peptides mimicking ureases fragments in sera of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic disease with an autoimmunological background. RA is mostly characterized by systemic inflammation and injuries of synovial joints. There is a hypothesis that bacterial infections may be connected with development of the disease. It has been suggested that molecular mimicry between bacterial and human antigens may be one of possible mechanisms of RA development. One of potential antigens involved in this mechanism is urease - enzyme with high structural conservatism, occurring in pathogenic and commensal bacteria. We found that the level of antibodies against peptide mimicking urease "flap" region is significantly higher in sera from patients with rheumatoid arthritis in comparison with volunteer blood donor sera. We also observed that antibodies present in RA sera may bind not only specific peptide antigens but also peptides with a slightly different structure.

Bacterial urease and its role in long-lasting human diseases.

Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymatic activity, urease has a toxic effect on human cells. The presence of ureolytic activity is an important marker of a number of bacterial infections. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as molecular mimicry, leads to the appearance of autoantibodies, which take part in host molecules destruction. Detection of antibodies- binding motives (epitopes) in bacterial proteins is a complex process. However, organic chemistry tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serologic investigations. In this review, we present a synthetic report on a molecular organization of bacterial ureases - genetic as well as structural. We characterize methods used in detecting urease and ureolytic activity, including techniques applied in disease diagnostic processes and in chemical synthesis of urease epitopes. The review also provides a summary of knowledge about a toxic effect of bacterial ureases on human body and about occurrence of anti-urease antibodies in long-lasting diseases.

Interferon consensus sequence binding protein (ICSBP) decreases beta-catenin activity in myeloid cells by repressing GAS2 transcription.

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and beta-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases beta-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases beta-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased beta-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in beta-catenin activity in Bcr/abl-positive (Bcr/abl(+)) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences beta-catenin activity in myeloid leukemia.

Are anti-Helicobacter pylori urease antibodies involved in atherosclerotic diseases?

OBJECTIVES: The ureB subunit of urease is a major target recognized by the antibodies of Helicobacter pylori-infected patients. The minimal epitope was determined to be an 8-mer peptide (H-SIKEDVQF-OH). DESIGN AND METHODS: The aim of this study was to discover whether this synthetic 8-mer peptide (BK-61A) directly recognizes the anti-ureB subunit antibodies of H. pylori-infected and atherosclerotic patients. To achieve a better presentation of the epitopes to antibodies, a new isocyanuric linker was designed and used for to immobilize the peptides on a cellulose support. RESULTS: In this study a new peptide synthesis method is presented. Anti-ureB antibodies were evaluated by the dot blot technique in 26 H. pylori-infected donors and the sera of 20 H. pylori-infected patients with atherosclerosis using the 8-mer peptide. CONCLUSIONS: The results reveal that the BK-61A peptide could be used for diagnosing the presence of anti-ureB antibodies that may be involved in the initiation of atherosclerosis.