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The use of chimeric antigen receptor (CAR)-T cell therapy for the treatment of hematologic malignancies has generated significant excitement over the last several years. From a transfusion medicine perspective, the implementation of CAR-T therapy as a potential mainstay treatment for not only hematologic but also solid-organ malignancies represents a significant opportunity for growth and expansion. In this review, we will describe the rationale for the development of genetically redirected T cells as a cancer therapeutic, the different elements that are required to engineer these cells, as well as an overview of the process by which patient cells are harvested and processed to create and subsequently validate CAR-T cells. Finally, we will briefly describe some of the toxicities and clinical efficacy of CAR-T cells in the setting of patients with advanced malignancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.

Background: The dog erythrocyte antigen (DEA) 1 blood group system remains poorly defined., Objectives: The purpose of the study was to determine the DEA 1 mode of inheritance and to characterize the DEA 1 antigen and alloantibodies., Animals: Canine research colony families, clinic canine patients, and DEA 1.2+ blood bank dogs were studied., Methods: Canine blood was typed by flow cytometry and immunochromatographic strips using anti-DEA 1 monoclonal antibodies. Gel column experiments with polyclonal and immunoblotting with monoclonal anti-DEA 1 antibodies were performed to analyze select samples. Cross-reactivity of human typing reagents against canine RBC and one monoclonal anti-DEA 1 antibody against human RBC panels was assessed., Results: Typing of 12 families comprising 144 dogs indicated an autosomal dominant inheritance with ≥ 4 alleles: DEA 1- (0) and DEA 1+ weak (1+), intermediate (2+), and strong (3+ and 4+). Samples from 6 dogs previously typed as DEA 1.2+ were typed as DEA 1+ or DEA 1- using monoclonal antibodies. Human typing reagents produced varied reactions in tube agglutination experiments against DEA 1+ and DEA 1- RBC. Polypeptide bands were not detected on immunoblots using a monoclonal anti-DEA 1 antibody, therefore the anti-DEA 1 antibody may be specific for conformational epitopes lost during processing., Conclusions: The autosomal dominant inheritance of DEA 1 with ≥ 4 alleles indicates a complex blood group system; the antigenicity of each DEA 1+ type will need to be determined. The biochemical nature of the DEA 1 antigen(s) appears different from human blood group systems tested., (© 2015 American Society for Veterinary Clinical Pathology.)

Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

For several decades, researchers have used erythrocytes for drug delivery of a wide variety of therapeutics in order to improve their pharmacokinetics, biodistribution, controlled release and pharmacodynamics. Approaches include encapsulation of drugs within erythrocytes, as well as coupling of drugs onto the red cell surface. This review focuses on the latter approach, and examines the delivery of red blood cell (RBC)-surface-bound anti-inflammatory, anti-thrombotic and anti-microbial agents, as well as RBC carriage of nanoparticles. Herein, we discuss the progress that has been made in surface loading approaches, and address in depth the issues relevant to surface loading of RBC, including intrinsic features of erythrocyte membranes, immune considerations, potential surface targets and techniques for the production of affinity ligands., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

ADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultralarge VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13(-/-)Plt(A13)), but not in wild-type and Adamts13(-/-), platelets. The expressed rADAMTS13 is released on stimulation with thrombin and collagen, but less with 2MesADP. Platelet-delivered rADAMTS13 is able to inhibit arterial thrombosis after vascular injury and prevent the onset and progression of Shigatoxin-2 or recombinant murine VWF-induced TTP syndrome in mice despite a lack of plasma ADAMTS13 activity resulting from the ADAMTS13 gene deletion or the antibody-mediated inhibition of plasma ADAMTS13 activity. These findings provide a proof of concept that platelet-delivered ADAMTS13 may be explored as a novel treatment of arterial thrombotic disorders, including hereditary and acquired TTP, in the presence of anti-ADAMTS13 autoantibodies., (© 2015 by The American Society of Hematology.)

Pemphigus vulgaris (PV) is a prototypic tissue-specific autoantibody-mediated disease, in which anti-desmoglein 3 (Dsg3) IgG autoantibodies cause life-threatening blistering. We characterized the autoimmune B-cell response over 14 patient years in two patients with active and relapsing disease, then in one of these patients after long-term remission induced by multiple courses of rituximab (anti-CD20 antibody). Characterization of the anti-Dsg3 IgG(+) repertoire by antibody phage display (APD) and PCR indicated that six clonal lines persisted in patient 1 (PV3) over 5.5 years, with only one new clone detected. Six clonal lines persisted in patient 2 (PV1) for 4 years, of which five persisted for another 4.5 years without any new clones detected. However, after long-term clinical and serologic remission, ∼11 years after initial characterization, we could no longer detect any anti-Dsg3 clones in PV1 by APD. Similarly, in another PV patient, ∼4.5 years after a course of rituximab that induced long-term remission, anti-Dsg3 B-cell clones were undetectable. These data suggest that in PV a given set of non-tolerant B-cell lineages causes autoimmune diseases and that new sets do not frequently or continually escape tolerance. Therapy such as rituximab, aimed at eliminating these aberrant sets of lineages, may be effective for disease because new ones are unlikely to develop.

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The receptor for advanced glycation end products (RAGE) is a multiligand pattern recognition receptor implicated in multiple disease states. Although RAGE is expressed on systemic vascular endothelium, the expression and function of RAGE on lung endothelium has not been studied. Utilizing in vitro (human) and in vivo (mouse) models, we established the presence of RAGE on lung endothelium. Because RAGE ligands can induce the expression of RAGE and stored red blood cells express the RAGE ligand N(ε)-carboxymethyl lysine, we investigated whether red blood cell (RBC) transfusion would augment RAGE expression on endothelium utilizing a syngeneic model of RBC transfusion. RBC transfusion not only increased lung endothelial RAGE expression but enhanced lung inflammation and endothelial activation, since lung high mobility group box 1 and vascular cell adhesion molecule 1 expression was elevated following transfusion. These effects were mediated by RAGE, since endothelial activation was absent in RBC-transfused RAGE knockout mice. Thus, RAGE is inducibly expressed on lung endothelium, and one functional consequence of RBC transfusion is increased RAGE expression and endothelial activation.

Prevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia, antiphospholipid syndrome and thrombotic thrombocytopenic purpura, include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 in heparin-induced thrombocytopenia and β(2)-glycoprotein-I (β(2)GPI) in antiphospholipid syndrome. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In thrombotic thrombocytopenic purpura, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, that is, enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor. A corollary of these concepts is that disrupting platelet factor 4 and β(2)GPI conformation (or ultralarge multimer of von Willebrand factor oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.

Background: Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application., Results: Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3(550-558) and a truncated GPC3(368-548) fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC(50) ranging from 5.0-110.9 nM were identified., Conclusions: Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.

Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) were injected intravenously with AAV8 vector expressing green fluorescent protein. Pre-existing antibody titers in excess of 1:10 substantially diminished hepatocyte transduction that, in the absence of NAbs, was highly efficient. Vector-specific NAb diminished liver deposition of genomes and unexpectedly increased genome distribution to the spleen. The majority of animals showed high-level and stable sequestration of vector capsid protein by follicular dendritic cells of splenic germinal centers. These studies illustrate how natural immunity to a virus that is related to a vector can impact the efficacy and potential safety of in vivo gene therapy. We propose to use the in vitro transduction inhibition assay to evaluate research subjects before gene therapy and to preclude from systemic AAV8 trials those that have titers in excess of 1:10.

Endosialin/TEM1 is predominantly expressed on neovasculature, thus ideally suited for diagnostic, targeted imaging and therapy of cancer. To isolate TEM1-specific affinity reagents, we thought to screen a recombinant antibody (scFv) library derived from the repertoire of a patient with thrombotic thrombocytopenic purpura (TTP), as autoimmune disorders may produce self-reactive specificities. The yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage in the novel vector pAGA2 for yeast-display expression. The TTP yeast-display library (10⁹ members) was screened by magnetic and flow sorting with human TEM1 recombinant protein. A pool of yeast-display scFv able to detect 2nM of TEM1 was obtained and transformed into yeast-secreted scFv by homologous recombination using the novel p416 BCCP vector for yeast secretion of biotinylated scFv. Anti-TEM1 yeast-secreted scFv were independently validated in vitro by flow cytometry analysis and ELISA assays, then in vivo biotinylated in N-termini to produce biobodies. Biobody-78 bound specifically to Endosialin/TEM1-expressing ovarian tumor in vivo, with functional stability over 48 h. Our results suggest that our novel paired display-secretory yeast libraries can serve as an ideal platform for the rapid isolation of high-affinity reagents, and that anti-TEM1 biobody-78 can be used for in vitro assays including flow cytometry analysis, as well as in vivo for targeted imaging and therapy of cancer., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Single chain variable region fragments (scFvs) are composed of an immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain joined by a flexible serine-glycine linker. They represent the smallest antibody fragments that maintain antigen specificity and they hold significant potential for therapeutic antigen targeting in vivo. Here we report on the design and validation of a series of degenerate primers that amplify the recombined variable regions of canine Ig heavy and light chain genes from lymphocyte cDNA. We show that these VH and VL amplicons can be randomly combined by a flexible linker using splicing by overlap extension PCR to form scFv constructs that can be expressed on the surface of M13 bacteriophage. To demonstrate that scFvs with specificity for previously encountered antigens are contained within these scFv phage display libraries we used simple panning procedures to isolate canine parvovirus (CPV) specific scFvs from a library made from the splenocytes of a dog immunized against CPV. These studies reveal the feasibility of this approach for generating diverse canine scFv libraries and pave the way toward future studies to isolate canine antigen-specific scFv of interest that may be tested as targeting agents for the treatment of infectious, inflammatory and neoplastic diseases in the dog., (2010 Elsevier B.V. All rights reserved.)

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.

Pemphigus is a life-threatening autoimmune disease in which antibodies specific for desmogleins (Dsgs) cause loss of keratinocyte cell adhesion and blisters. In order to understand how antibodies cause pathogenicity and whether there are commonalities among antibodies in different patients that could ultimately be used to target specific therapy against these antibodies, we characterized Dsg-specific mAbs cloned by phage display from 3 patients with pemphigus vulgaris and 2 with pemphigus foliaceus. Variable heavy chain gene usage was restricted, but similar genes were used for both pathogenic and nonpathogenic mAbs. However, the heavy chain complementarity-determining region 3 (H-CDR3) of most pathogenic, but not nonpathogenic, mAbs shared an amino acid consensus sequence. Randomization of the H-CDR3 and site-directed mutagenesis indicated that changes in this sequence could block pathogenicity but not necessarily binding. In addition, for 2 antibodies with longer H-CDR3s, a tryptophan was critical for pathogenicity but not binding, a result that is consistent with blocking the tryptophan acceptor site that is thought to be necessary for Dsg-mediated adhesion. These studies indicate that H-CDR3 is critical for pathogenicity of a human autoantibody, that a small region (even 1 amino acid) can mediate pathogenicity, and that pathogenicity can be uncoupled from binding in these antibodies.

Background: Recent evidence suggests that storage-induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form advanced glycation end products (AGEs) after increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE N-(carboxymethyl)lysine (N(ε) -CML) during standard blood banking conditions., Study Design and Methods: Purified, fresh RBCs from volunteers were compared to stored RBCs (35-42 days old) obtained from the blood bank. N(ε) -CML formation was quantified using a competitive enzyme-linked immunosorbent assay. The receptor for advanced glycation end products (RAGE) was detected in human pulmonary microvascular endothelial cells (HMVEC-L) by real-time polymerase chain reaction, Western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester-based assays., Results: Stored RBCs showed increased surface N(ε) -CML formation when compared with fresh RBCs. HMVEC-L showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both human umbilical vein endothelial cells and HMVEC-L. RBC-induced endothelial ROS generation was attenuated in the presence of soluble RAGE or RAGE blocking antibody., Conclusions: The formation of the AGE N(ε) -CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE-RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage., (© 2010 American Association of Blood Banks.)

Optimization of a screening hit from uHTS led to the discovery of TGR5 agonist 32, which was shown to have activity in a rodent model for diabetes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Elevated Lipoprotein (a) (Lp(a)) levels are associated with atherosclerosis and are independent risk factors for coronary artery disease and stroke [Ariyo et al., N Engl J Med 2003;349:2108–2115; Price et al., Atherosclerosis 2001;157:241–249]. Low-density lipoprotein (LDL)-apheresis is the most effective therapy for reducing Lp(a) levels [Parker, Chem Phys Lipids 1994;67–68:331–338; Stefanutti et al., Transfus Apher Sci 2010;42:21–26]. Dextran sulfate-cellulose adsorption (Liposorber®) removes both LDL and Lp(a) particles with minimal effect on high-density lipoprotein levels. During the procedure, high levels of bradykinin are generated as the kallikrein-kinin system is activated by contact with the negatively charged dextran-sulfate cellulose [Krieter et al., Artif Organs 2005;29:47–52]. Bradykinin is a potent vasodilator and a substrate of the angiotension converting enzyme (ACE). ACE inhibitors are contraindicated for apheresis procedures because these drugs prevent bradykinin degradation, which causes anaphylatoid reactions characterized by hypotension, bradycardia, dyspnea, and flushing [Owen and Brecher, Transfusion 1994;34:891–894]. Turmeric is a yellow spice that is used as an herbal remedy to treat a myriad of conditions ranging from abdominal pain to pulmonary infections. Scientific investigations of the ethnomedicinal properties of curcumin, the major derivative of turmeric, suggest that this compound has anti-inflammatory, antioxidant, and antineoplastic properties [Lobo et al., J Pharm Pharmacol 2009;61:13–21]. We report a case of a patient undergoing Liposorber® therapy for treatment of hyperLp(a)lipidemia who had three episodes of anaphylactoid-like reactions after starting therapy with the spice turmeric.

In pemphigus foliaceus (PF), autoantibodies against desmoglein 1 (Dsg1) cause blisters. Using Ab phage display, we have cloned mAbs from a PF patient. These mAbs, like those from a previous patient, were directed against mature Dsg1 (matDsg1) on the cell surface of keratinocytes and precursor Dsg1 (preDsg1) in the cytoplasm. To determine whether individuals without pemphigus have B cell tolerance to Dsg1, we cloned mAbs from two patients with thrombotic thrombocytopenic purpura and a healthy person. We found mAbs against preDsg1, but not matDsg1. All but 1 of the 23 anti-preDsg1 mAbs from PF patients and those without PF used the VH3-09 (or closely related VH3-20) H chain gene, whereas no PF anti-matDsg1 used these genes. V(H) cDNA encoding anti-preDsg1 had significantly fewer somatic mutations than did anti-matDsg1 cDNA, consistent with chronic Ag-driven hypermutation of the latter compared with the former. These data indicate that individuals without PF do not have B cell tolerance to preDsg1 and that loss of tolerance to matDsg1 is not due to epitope shifting of anti-preDsg1 B cells (because of different V(H) gene usage). However, presentation of peptides from Dsg1 by preDsg1-specific B cells may be one step in developing autoimmunity in PF.

Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.

Therapeutic options for patients with multiple myeloma whose disease has relapsed after a prior auto-SCT include novel biologic therapies, traditional chemotherapy or a second transplant, with no clear standard of care. Few published studies address the safety and efficacy of a second auto-SCT for relapsed disease. We reviewed the Abramson Cancer Center experience with salvage auto-SCT for relapsed multiple myeloma. Forty-one patients had received a salvage auto-SCT at our institution; the median time between transplants was 37 months (range 3-91). The overall response rate in assessable patients was 55%, and treatment-related mortality was 7%. With a median follow-up time of 15 months, the median PFS was 8.5 months and the median overall survival (OS) was 20.7 months. In a multivariate analysis of OS, independent prognostic factors were >or=5 prior lines of therapy and time to progression after initial auto-SCT of

Pemphigus foliaceus (PF) is a blistering disease caused by autoantibodies to desmoglein 1 (Dsg1) that cause loss of epidermal cell adhesion. To better understand PF pathophysiology, we used phage display to isolate anti-Dsg1 mAbs as single-chain variable fragments (scFvs) from a PF patient. Initial panning of the library isolated only non-pathogenic scFvs. We then used these scFvs to block non-pathogenic epitopes and were able to isolate two unique scFvs, each of which caused typical PF blisters in mice or human epidermis models, showing that a single mAb can disrupt Dsg1 function to cause disease. Both pathogenic scFvs bound conformational epitopes in the N terminus of Dsg1. Other PF sera showed a major antibody response against the same or nearby epitopes defined by these pathogenic scFvs. Finally, we showed restriction of the heavy-chain gene usage of all anti-Dsg1 clones to only five genes, which determined their immunological properties despite promiscuous light-chain gene usage. These mAbs will be useful for studying Dsg1 function and mechanisms of blister formation in PF and for developing targeted therapies and tools to monitor disease activity.

Recent developments in molecular immunology have facilitated the expression of immune repertoires in the form of immunoglobulin fragments on the surface of filamentous bacteriophage. Such approaches, known as "phage display", have provided powerful tools for producing monoclonal antibodies for research, clinical, and therapeutic applications. Our laboratory has combined these techniques with novel selection methods to isolate extraordinarily large arrays of human antibodies that can be used for developing new diagnostic reagents and methods for their use, as well as reagents that may serve as leads for the design of novel therapeutic molecules. In particular, application of phage display to the study of antibody-mediated autoimmune disease, notably idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and pemphigus have facilitated comprehensive genetic and serologic analyses of pathogenic autoantibodies thereby offering new insights into disease pathophysiology and approaches for their treatment.

High-dose melphalan with autologous stem cell support improves survival as part of initial therapy for myeloma. Previous studies of pre-transplant induction regimens have compared paraprotein response rates but not long-term outcomes after transplant. We reviewed the records of all patients with multiple myeloma who received an autologous stem cell transplant at the University of Pennsylvania Medical Center. We compared outcomes for 69 patients who received high-dose melphalan conditioning after January 1, 2003 as part of initial therapy for myeloma, including 41 patients who received anthracycline-based induction (VAD or DVD) and 28 patients who received thalidomide and dexamethasone induction. Baseline characteristics in these two groups were not different, though potentially clinically important differences were apparent in assignment to post-transplant consolidation and maintenance therapy. Despite similar response rates during induction therapy, thalidomide and dexamethasone induction was associated with better progression-free survival (hazard ratio 0.18, P = 0.011) after transplant. This effect persisted in multivariable regression models including baseline characteristics and post-transplant treatment. Overall survival was not different between the two groups. These results suggest that the use of thalidomide during induction therapy may lead to improved long-term outcomes after transplant. Future trials comparing induction therapies should examine progression-free and overall survival after transplant to confirm this benefit.

Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against cell surface adhesion proteins desmoglein (Dsg) 3 and Dsg1. Previous studies using phage display to clone Dsg-reactive monoclonal antibodies from a PV patient demonstrated that a limited number of antibody variable region genes encode the autoantibody repertoire, with different genes for pathogenic and non-pathogenic mAbs. Here, we investigated the feasibility of specific autoantibody targeting in pemphigus. We produced rabbit anti-idiotypic antibodies against two pathogenic and two non-pathogenic PV mAbs. Antisera inhibited binding of the immunizing mAb to Dsgs by ELISA as well as pathogenicity against cultured human keratinocytes. Antisera also inhibited other mAbs using the same variable region heavy chain (V(H)) genes, despite different light chains or somatic mutations. Additionally, peptide phage display identified peptide sequences that bound PV mAbs in a V(H)-specific manner. To evaluate the therapeutic potential of V(H) gene-targeted reagents, preimmune sera and antisera were used to adsorb pathogenic antibodies from PV sera. Pooled antisera significantly reduced pathogenic activity from the original PV patient's serum and bound pathogenic antibodies from two other PV sera, suggesting shared autoantibody V(H) gene usage among PV patients. Together, these data suggest novel V(H) gene-targeted approaches toward PV treatment.

We conducted a retrospective analysis of 50 lymphoma patients (Hodgkin's disease and non-Hodgkin's lymphoma) who had an 18F-fluoro-deoxyglucose positron emission tomography (FDG-PET) scan after at least two cycles of salvage chemotherapy and before autologous stem cell transplantation (ASCT) at our institution. The patients were categorized into FDG-PET negative (N = 32) and positive (N = 18) groups. The median follow-up after ASCT was 19 months (range: 3-59). In the FDG-PET-negative group, the median progression-free survival (PFS) was 19 months (range: 2-59) with 15 (54%) patients without progression at 12 months after ASCT. The median overall survival (OS) for this group was not reached. In the FDG-PET-positive group, the median PFS was 5 months (range: 1-19) with only one (7%) patient without progression at 12 months after ASCT. The median OS was 19 months (range: 1-34). In the FDG-PET-negative group, chemotherapy-resistant patients by CT-based criteria had a comparable outcome to those with chemotherapy-sensitive disease. A positive FDG-PET scan after salvage chemotherapy and prior ASCT indicates an extremely poor chance of durable response after ASCT.

Nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis (NFD/NSF) is a disorder occurring exclusively in patients with renal disease. Until recently, it has been considered a fibrosing disorder essentially confined to the skin and underlying superficial soft tissue. Recent reports, however, have described patients with involvement of other organ systems, suggesting that this disorder is actually a systemic disease with preferential cutaneous manifestations. We describe a patient with end-stage renal disease with diagnosed NFD/NSF who subsequently developed respiratory failure leading to his death. Autopsy findings showed NFD/NSF involving the skin of all extremities, as well as diffuse diaphragm involvement.

Although follicular lymphoma (FL) is generally responsive to conventional-dose chemotherapy, improved survival in patients with this disease has been difficult to demonstrate. High-dose chemo/radiotherapy followed by autologous stem-cell transplantation (ASCT) can improve response rates, although its effects on survival remain controversial. Between 1990 and 2003, we transplanted 49 patients with low-grade FL at our institution. Twenty-two patients (45%) had undergone histologic transformation at the time of ASCT. In all, 44 patients (90%) had relapsed disease and five patients (10%) were resistant to chemotherapy at the time of transplantation. After ASCT, 30 patients (61%) were in complete remission (CR). The median overall survival (OS) has not been reached, while the median event-free survival (EFS) is 2.4 years. At a median follow-up of 5.5 years (longest 12.4 years), a plateau has been reached with 56% of patients remaining alive, and 35% event-free. ASCT was well tolerated except for two (4%) treatment-related deaths. In multivariable analysis, CR after ASCT and age less than 60 years are the best predictors of EFS and OS. ASCT is thus a safe therapeutic approach in FL, resulting in long-term EFS and OS for some patients, even with transformed disease.

Current technologies used in blood banks for blood group antigen typing and other pretransfusion tests are extraordinarily labor-intensive, prone to human error, and an order of magnitude more expensive per test than those in other clinical laboratories. With a growing shortage of skilled medical technologists, dwindling supplies of human plasma-derived phenotyping reagents, and an inherent difficulty in fully automating agglutination-based methodologies, the ability to perform rapid and accurate pretransfusion testing in a cost-effective manner has become a significant challenge. This paper reviews the latest advances in the use of phage display technologies to address these issues through the development of inexpensive and renewable blood bank testing reagents that are amenable to high-throughput, automatable assay systems.

Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.

The Rh blood group proteins are erythrocyte proteins important in neonatal and transfusion medicine. Recent studies have shed new light on the possible biological function of Rh proteins as members of a conserved family of proteins involved in ammonium transport. The erythrocyte Rh-associated glycoprotein (RhAG) mediates uptake of ammonium when expressed in Xenopus laevis oocytes, and functional studies indicate that RhAG might function as an NH(4)(+)-H(+)-exchanger. To further delineate the functional properties of RhAG, in this study we have expressed RhAG in both a Saccharomyces cerevisiae ammonium-transport mutant (mep1Delta mep2Delta mep3Delta) and a wild-type strain. RhAG was able to complement the transport mutant, with complementation strictly pH-dependent, requiring pH 6.2-6.5. RhAG also conferred resistance to methylamine (MA), a toxic analog of ammonium, and expression in wild-type cells revealed that resistance was correlated with efflux of MA. RhAG-mediated resistance was pH-dependent, being optimal at acid pH. The opposite pH dependence of ammonium complementation (uptake) and MA resistance (efflux) is consistent with bidirectional movement of substrate counter to the direction of the proton gradient. This report clarifies and expands previous observations of RhAG-mediated transport in yeast and supports the hypothesis that ammonium transport is coupled to the H(+) gradient and that RhAG functions as a NH(4)(+)/H(+) exchanger.

Idiopathic thrombocytopenic purpura (ITP) is a common immune disorder caused by platelet-reactive autoantibodies. Antibody-coated platelets are cleared more rapidly from the circulation, often in the spleen, than they can be replaced by compensatory stimulation of platelet production in the bone marrow. In some patients, platelet production is depressed as well. ITP in adults does not generally remit spontaneously, and most patients require treatment to prevent bleeding at one time or another. Therapy with corticosteroids, danazol, intravenous immune globulin, anti-D antibody, and several other agents inhibits clearance of the antibody-coated platelets but is rarely curative. Most patients will sustain a hemostatic response after splenectomy, although relapses may occur at any time. Patients may be more responsive to these same modalities after splenectomy, but treatment with an immunosuppressant that inhibits T- and B-cell function and cooperation, including azathioprine, cyclophosphamide, cyclosporine, mycophenolate mofetil, or anti-CD20, may be required. Antiviral therapy is useful in patients with HIV or hepatitis C infection, but no consensus has been reached as to the efficacy of antibiotics to eradicate Helicobacter pylori. Promising results have been seen in several patients treated with a modified thrombopoietin. It may be possible to design therapeutics that exploit the apparent restricted immunoglobulin gene usage by antiplatelet antibodies, perhaps in the form of engineered anti-idiotypic antibodies or other compounds that specifically target autoantibody-producing B cells. Rationale therapy awaits a more thorough understanding of autoantibody production.

Although idiopathic thrombocytopenic purpura (ITP) is the most common autoimmune hematologic disorder, little is known about the associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig) gene usage, clonality, and antigen specificity. Phage display libraries were constructed from splenocytes from 2 patients with chronic ITP, and competitive cell-surface selection was used to isolate several dozen unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in both patients were associated almost exclusively with rearrangements of a single Ig heavy-chain variable-region gene (V(H)3-30), despite an apparent diversity of antigen specificities. Comparative analysis of platelet-reactive Fab Ig gene rearrangements from each patient suggested that they evolved from a restricted number of B-cell clones through somatic mutation with high replacement-to-silent mutation ratios. Although V(H)3-30-encoded heavy chains were found with light chains encoded by several different Ig genes, molecular repairing experiments showed exquisite restriction on the specific heavy- and light-chain pairings that permitted platelet reactivity. Together, these data suggest that the development of platelet-reactive antibodies associated with ITP is driven by an encounter with diverse platelet antigens through the clonal expansion of B cells using genetically restricted and highly specific combinations of heavy- and light-chain gene products. The extraordinarily high usage of the V(H)3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.

With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Background: ABO incompatibility is a common cause for mild hemolysis in the newborn, ranging from 1 in 30 to 1 in 150 births. Fortunately, hemolysis requiring transfusion is rare and restricted to blood group O mothers, because blood group A and B individuals make poor IgG anti-B and anti-A responses. No human IgG ABO antibody sequences have been reported, in part because of the difficulty in obtaining human IgG hybridomas. Phage-display technology may be able to circumvent these difficulties, but its application to carbohydrate antigens is poorly studied., Study Design and Methods: A human IgG1 phage-display Fab library was constructed from splenocytes derived from a nonhyperimmunized blood group O person, and panned against group B RBCs., Results: After five rounds of panning, essentially all phage bound to group B RBCs. Nucleotide sequence analysis of a single monoclonal IgG1lambda phage, FB5.7, revealed a highly mutated VH4 family heavy chain, and a nearly germline VL7 family lambda light chain. The Fab agglutinated group B, but not group A, random-donor RBCs. However, group B ELISA reactivity could be inhibited by soluble B-trisaccharide, soluble A-trisaccharide, galactose, and N-acetyl galactosamine. Similarly, galactose and N-acetyl galactosamine were able to inhibit group B RBC agglutination., Conclusion: FB5.7 is the first human IgG ABO MoAb described. Alhough it behaves serologically like a group B-specific antibody, it demonstrates interaction with both the A and B epitopes. Phage-display technology can be used to better define the relationship between antibody genotype and phenotype in anti-carbohydrate responses in nonhyperimmunized hosts, and thus to improve our understanding of the composition of the antibody repertoire.

Antibodies to PF4/heparin can be demonstrated in almost all patients with heparin-induced thrombocytopenia/thrombosis (HIT/HITT) and in some persons exposed to heparin who do not have clinical manifestations. The role of anti-PF4/heparin antibodies in the pathogenesis of HIT/HITT has been difficult to establish because the antibodies found in serum are generally polyclonal and polyspecific. To circumvent this problem, we developed a murine monoclonal antibody (mAb) to human (h) PF4/heparin complexes. A monoclonal IgG(2bkappa )antibody (designated KKO) was identified that bound specifically to hPF4/heparin complexes. Maximal binding of KKO to hPF4/heparin complexes occurred at similar molar ratios of PF4:heparin observed for HIT/HITT antibodies. KKO also bound to hPF4 in association with other glycosaminoglycans. Platelet activation by KKO required heparin and was abrogated by blockade of FcgammaRIIA. In the presence of PF4, KKO bound to endothelial cells, but not to CHO cells lacking heparan sulfate proteoglycans. Variants of PF4 complexed to heparin were recognized equally well by KKO and HIT/HITT sera. KKO competes for binding with a subset of HIT/HITT antibodies that are relatively spared by mutations in the 3rd domain of PF4. The nucleotide and predicted amino acid sequences of KKO and RTO, a murine anti-hPF4 mAb that does not require heparin for binding, revealed no obvious relationship in either the heavy- or the light-chain immunoglobulin variable regions. These studies suggest that KKO recapitulates the antigenic and functional specificity of a subset of HIT/HITT antibodies and may, therefore, provide insight into the pathogenesis of thrombocytopenia and thrombosis in affected persons. (Blood. 2000;95:1533-1540)

The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.

Understanding anti-Rh(D) antibodies on a molecular level would facilitate the genetic analysis of the human immune response to Rh(D), lead to the design of therapeutically useful reagents that modulate antibody binding, and provide relevant information regarding the structural organization of Rh(D) epitopes. Previously, we described a Fab/phage display-based method for producing a large array of anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single alloimmunized donor. In the current study, we present a detailed analysis of 83 randomly selected clones. Sequence analysis showed the presence of 28 unique gamma1 heavy chain and 41 unique light chain gene segments. These paired to produce 53 unique Fabs that had specificity for at least half of the major Rh(D) epitopes. Surprisingly, despite this diversity, only 4 closely related heavy chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21). Similarly, nearly all Vkappa light chains (15/18) were derived from one germline gene (DPK9). lambda light chains showed a more diverse VL gene usage, but all (23/23) used the identical Jlambda2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In particular, 2 such clones differed by only 3 residues, which resulted in a change from epD2 to epD3 specificity. These results suggest a model in which footprints of anti-Rh(D) antibodies are essentially identical to one another, and Rh(D) epitopes, as classically defined by panels of Rh(D) variant cells, are not discrete entities. Furthermore, these data imply that the epitope specificity of an anti-Rh(D) antibody can change during the course of somatic mutation. From a clinical perspective, this process, which we term epitope migration, has significance for the design of agents that modulate antibody production and for the creation of mimetics that block antibody binding in the settings of transfusion reactions and hemolytic disease of the newborn.

A major goal of current immunologic research is to develop specific therapeutic strategies by which the enormous diversity in immune response can be enhanced, attenuated, or eliminated, depending on the particular disease process. For nearly a century, the human immune response to red blood cell antigens has served as a paradigm for understanding the pathophysiology of autoimmune disorders and alloimmune reactions to foreign cells and tissues. Recent developments in molecular biology have facilitated the expression of immune repertoires in the form of immunoglobulin Fab fragments on the surface of filamentous bacteriophage. Such approaches have provided powerful means for producing monoclonal antibodies for research, clinical, and therapeutic applications. Our laboratory has combined these techniques with novel cell-surface selection methods to isolate extraordinarily large arrays of human antibodies to the clinically relevant red blood cell Rh(D) antigen. Our results have provided a comprehensive genetic and serologic analysis of anit-Rh(D) antibodies within single alloimmunized individuals thereby offering new insights into the development of human immune repertoires.