Biomarkers in chronic graft-versus-host disease: quo vadis?

Biomarkers are increasingly used for diagnosis and treatment of transplant-related complications including the first biomarker-driven interventional trials of acute graft-versus-host disease (GvHD). In contrast, the development of biomarkers of chronic GvHD (cGvHD) has lagged behind due to a broader variety of manifestations, overlap with acute GvHD, a greater variation in time to onset and maximum severity, and lack of sufficient patient numbers within prospective trials. An international workshop organized by a North-American and European consortium was held in Marseille in March 2017 with the goal to discuss strategies for future biomarker development to guide cGvHD therapy. As a result of this meeting, two areas were prioritized: the development of prognostic biomarkers for subsequent onset of moderate/severe cGvHD, and in parallel, the development of qualified clinical-grade assays for biomarker quantification. The most promising prognostic serum biomarkers are CXCL9, ST2, matrix metalloproteinase-3, osteopontin, CXCL10, CXCL11, and CD163. Urine-proteomics and cellular subsets (CD4+ T-cell subsets, NK cell subsets, and CD19+CD21low B cells) represent additional potential prognostic biomarkers of cGvHD. A joint effort is required to verify the results of numerous exploratory trials before any of the potential candidates is ready for validation and subsequent clinical application.

B-cell activating factor (BAFF) plasma level at the time of chronic GvHD diagnosis is a potential predictor of non-relapse mortality.

Biological markers for risk stratification of chronic GvHD (cGvHD) could improve the care of patients undergoing allogeneic hematopoietic stem cell transplantation. Increased plasma levels of B-cell activating factor (BAFF), chemokine (C-X-C motif) ligand 9 (CXCL9) and elafin have been associated with the diagnosis, but not with outcome in patients with cGvHD. We evaluated the association between levels of these soluble proteins, measured by ELISA at the time of cGvHD diagnosis and before the initiation of therapy, with non-relapse-mortality (NRM). Based on the log-transformed values, factor levels were divided into tertiles defined respectively as low, intermediate, and high levels. On univariable analysis, BAFF levels were significantly associated with NRM, whereas CXCL9 and elafin levels were not. Both low (⩽2.3 ng/mL, hazard ratio (HR)=5.8, P=0.03) and high (>5.7 ng/mL, HR=5.4, P=0.03) BAFF levels were associated with a significantly higher NRM compared with intermediate BAFF level. The significant effect of high or low BAFF levels persisted in multivariable analysis. A subset of cGvHD patients had persistently low BAFF levels. In conclusion, our data show that BAFF levels at the time of cGvHD diagnosis are associated with NRM, and also are potentially useful for risk stratification. These results warrant confirmation in larger studies.

Effectiveness of etoposide chemomobilization in lymphoma patients undergoing auto-SCT.

The effectiveness of stem cell mobilization with G-CSF in lymphoma patients is suboptimal. We reviewed our institutional experience using chemomobilization with etoposide (VP-16; 375 mg/m(2) on days +1 and +2) and G-CSF (5 µg/kg twice daily from day +3 through the final day of collection) in 159 patients with lymphoma. This approach resulted in successful mobilization (>2 × 10(6) CD34+ cells collected) in 94% of patients (83% within 4 apheresis sessions). Fifty-seven percent of patients yielded at least 5 × 10(6) cells in 2 days and were defined as good mobilizers. The regimen was safe with a low rate of rehospitalization. Average costs were $14 923 for good mobilizers and $27 044 for poor mobilizers (P<0.05). Using our data, we performed a 'break-even' analysis that demonstrated that adding two doses of Plerixafor to predicted poor mobilizers at the time of first CD34+ cell count would achieve cost neutrality if the frequency of good mobilizers were to increase by 21%, while the frequency of good mobilizers would need to increase by 25% if three doses of Plerixafor were used. We conclude that chemomobilization with etoposide and G-CSF in patients with lymphoma is effective, with future opportunities for cost-neutral improvement using novel agents.

Suspected case of venous air embolism in an infant undergoing hip arthrogram.

An otherwise well ten-week-old girl underwent an air contrast hip arthrogram and application of a hip spica for a developmentally dislocated hip. The child displayed signs consistent with venous air embolism after injection of 5 ml of air into the hip joint. These signs included a decrease in arterial haemoglobin oxygen saturation as measured by pulse oximetry, decreased end-tidal carbon dioxide level and tachycardia. The signs initially resolved, but the patient deteriorated with injection of a further 5 ml of air. The patient responded to cessation of injection and resuscitative measures. The infant remained well postoperatively. The need for the use of air to confirm intra-articular placement of the needle prior to injection of contrast during a hip arthrogram is questioned.

Recombinant polyclonal antibody libraries.

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Generation of a polyclonal fab phage display library to the human breast carcinoma cell line BT-20.

We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.

A bidirectional phage display vector for the selection and mass transfer of polyclonal antibody libraries.

An approach to the creation of antigen-specific polyclonal libraries of intact antibodies is presented. A polyclonal library of Fab antibody fragments would be expressed using a phage display vector, and selected for reactivity with an antigen or group of antigens. For conversion into a sublibrary of intact polyclonal antibodies, the selected heavy (H) and light (L) chain variable (V) region gene combinations would be transferred in mass, as linked pairs, to a eukaryotic expression vector which provides immunoglobulin (Ig) constant (C) region genes. To enable this selection and transfer, a bidirectional phage display vector was generated, in which the V region gene pairs are linked head to head in opposite transcriptional orientations. The functionality of this vector was demonstrated by the selection, transfer and expression of linked V region gene pairs derived from an A/J mouse that had been immunized with p-azophenylarsonate (Ars)-coupled keyhole limpet hemocyanin (KLH). As expected, the expressed IgG2b anti-Ars antibodies with selected V region gene pairs were shown to have V region sequences and Ars-binding characteristics similar to those of anti-Ars hybridoma antibodies. The technology presented here has potential for many diagnostic and therapeutic applications. These include the generation of polyclonal antibody libraries against multiple epitopes on infectious agents or cancer cells, and of polyclonal libraries encoding chimeric molecules composed of antibody V regions and T cell receptor C regions.

Generation of a polyclonal Fab phage display library to the protozoan parasite Cryptosporidium parvum.

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.

A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries.

Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.

Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells.

MRL/1pr mice demonstrate anatomic specificity in their development of vasculitis including the small- and medium-sized muscular arteries of the mesentery. To define the functional role of endothelium in vasculitis, we have cloned endothelial cells derived from inflamed small- and medium-sized arteries. Primary cells were derived by enzymatic dispersement and endothelial cells were selected by utilizing a combination of specific culture conditions. Cloned endothelium were developed utilizing limiting dilution cultures supplemented by endothelial cell growth factor. The cloned endothelial cells express many structural features of mature endothelial cells including Factor VIII-RA, non-muscle-specific actin, and Weibel-Palade bodies. Functionally, the clones express functional receptors for the scavenger pathway for LDL metabolism. The cells do not express Class I MHC antigens; however, IFN-beta and IFN-gamma stimulate Class I MHC expression after 24 h, which induces lysis of virus-infected cloned endothelium by Class I-restricted virus-primed T cells. In direct contrast to site-identical vascular smooth muscle cells (VSMCs), endothelial cells do not spontaneously express Class II MHC antigens, nor do they secrete biologically relevant levels of IL-1 unless triggered by lipopolysaccharide. The availability of site-specific cloned endothelium along with cloned VSMCs from autoimmune mice should resolve major experimental controversies involving the pathophysiology of inflammatory vascular disease.