Monoclonal Antibodies

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at Nterminal Thr or Tyr residues. Simultaneous expression of core-2 ␤1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and Pselectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptorcounterreceptor pair.

The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes, and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules, in many cases, internalization to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic release of the therapeutic molecule is the membrane-barrier of the endocytic vesicles. Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides, and the chemotherapeutic bleomycin. PCI has also been shown to enhance the treatment effect of targeted therapeutic macromolecules. The present protocol describes PCI of an epidermal growth factor receptor (EGFR)-targeted protein toxin (Cetuximab-saporin) linked via streptavidin-biotin for screening of targeted toxins as well as PCI of nonviral polyplex-based gene therapy. Although describing in detail PCI of targeted protein toxins and DNA polyplexes, the methodology presented in these protocols are also applicable for PCI of other gene therapy vectors (e.g., viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), polymers, nanoparticles, and some chemotherapeutic agents.

This is a literature review about large granular lymphocyte leukemia (LGLL), a rare and misdiagnosed oncohematological disease, characterized by a clonal expansion of T-cells (T-LGLL) or NK-cells (NK-LGLL) in the bone marrow and/or peripheral blood. The clinical features of LGLL include cytopenias (anemia, neutropenia and thrombocytopenia), lymphocytosis (usually discrete), lymphadenopathy, hepatomegaly, splenomegaly, immune abnormalities and constitutional symptoms (fever, night sweats and weight loss). The diagnosis is based on the confirmation of the clonality of T-cells or NK-cells (polymerase chain reaction and Southern blot are the two methods most commonly used) and typical findings of the immunophenotypic analysis of peripheral blood lymphocytes (flow cytometry analyses for specific surface antigens). In contrast to the chronic and indolent course of T-LGLL, NK-LGLL has an acute presentation and poor clinical outcome. There are different current treatment options, depending on clinical presentation.

Localization of the interface between the candidate antibody and its antigen target, commonly known as epitope mapping, is a critical component of the development of therapeutic monoclonal antibodies. With the recent availability of commercial automated systems, hydrogen / deuterium eXchange (HDX) is rapidly becoming the tool for mapping epitopes preferred by researchers in both industry and academia. However, this approach has a significant drawback in that it can be confounded by 'allosteric' structural and dynamic changes that result from the interaction, but occur far from the point(s) of contact. Here, we introduce a 'kinetic' millisecond HDX workflow that suppresses allosteric effects in epitope mapping experiments. The approach employs a previously introduced microfluidic apparatus that enables millisecond HDX labeling times with on-chip pepsin digestion and electrospray ionization. The 'kinetic' workflow also differs from conventional HDX-based epitope mapping in that the antibody is introduced to the antigen at the onset of HDX labeling. Using myoglobin / anti-myoglobin as a model system, we demonstrate that at short 'kinetic' workflow labeling times (i.e., 200 ms), the HDX signal is already fully developed at the 'true' epitope, but is still largely below the significance threshold at allosteric sites. Identification of the 'true' epitope is supported by computational docking predictions and allostery modeling using the rigidity transmission allostery algorithm.

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two ''antibody mold structures": (1) X-ray structure of the free form of the Alzheimer's disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.

The CAMPATH-1 family of antibodies recognize an abundant glycoprotein expressed on virtually all human lymphocytes. All rat IgM and IgG antibodies of this specificity are lytic with human complement, but only IgG2b is active in antibody-dependent cell-mediated cytotoxicity (ADCC). We compared the ability of IgM, IgG2a, and IgG2b to deplete lymphocytes in vivo in two patients with prolymphocytic transformation of B-cell chronic lymphocytic leukemia (CLL). The IgM (CAMPATH-1M) produced transient depletion of blood lymphocytes with consumption of complement but had no effect on solid masses or bone marrow. Similar transient depletion of blood lymphocytes was noted with the IgG2a (YTH34.5). In contrast, the IgG2b (CAMPATH-1G) produced long-lasting depletion of lymphocytes from blood and marrow and improvement in splenomegaly but no detectable changes in complement levels. These differences probably reflect the importance of Fc receptor binding for effective clearance of target cells in ...

Rapid diagnosis of Plasmodium falciparum malaria remains one of the main limitations to prompt treatment. Diagnosis based on clinical symptoms is decidedly unreliable, especially in areas of seasonal transmission like Zimbabwe. In view of this, the Plasmodium falciparum histidine-rich protein (HRP-II) antigen detection assay (ParaSight-F test) was tried at 10 health centres in 3 malaria endemicity zones of Zimbabwe, as a malaria diagnostic tool for primary health care. Parasitological evaluations were conducted using thick and thin film microscopy as gold standard, and ease of test operation and practicability to nurses were ascertained by questionnaire. The sensitivity of the test did not vary substantially by endemicity zone and was ]93%. Specificities were 85, 72 and 92% in the hyperendemic, mesoendemic and hypoendemic zones, respectively. Positive predictive values varied considerably with endemicity, the lowest being in the hypoendemic zone (56%). However, negative predictive values did not change significantly, with a mean of 94%. It was found that the ParaSight-F test reduced mistreatment for malaria, relative to clinical diagnosis, by up to 81%, especially in the hypoendemic region. Test acceptability evaluations were good.

T-cell growth factor (TCGF or interleukin-2) is an inducible glycoprotein hormone of molecular weight 15,000 (ref. 1) synthesized and secreted by T lymphocytes following activation with antigen or mitogen2,3. TCGF is required for proliferation and expansion of T cells following antigen encounter4,5 and to maintain them in long-term culture in vitro6-9. Full expression of the human immune response requires both the induction of TCGF synthesis and the formation of specific TCGF membrane receptors10,11. Monoclonal antibodies binding TCGF have been prepared12,13. In contrast, antibodies specific for the TCGF membrane receptor have not been identified, nor has the receptor been characterized. We have prepared a monoclonal antibody, termed anti-Tac14,15, which appears to bind to the human membrane receptor for TCGF. In support of this, we now demonstrate that anti-Tac suppresses TCGF induced proliferation of T cells and blocks binding of radiolabelled TCGF to cells from a cloned human continuous T-cell line. Also we have partially purified and characterized the putative TCGF receptor. This receptor is a glycoprotein with a molecular weight (Mr) of 47,000-53,000.

The prognosis of children with relapsed/refractory acute myeloid leukaemia (AML) is poor, and new therapies are needed. Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody linked to the antitumor antibiotic calicheamicin. We conducted an investigator-initiated phase II study with GO to assess its efficacy and safety, administering two dosages of 7AE5 mg/m 2 with a 14 d-interval. Thirty children who were refractory to re-induction at first relapse or suffered from second relapse of AML received a total of 64 infusions of GO. The response rate [complete remission (CR) and CR with insufficient platelet recovery] was 37%. Nine patients were subsequently transplanted (median time to transplant, 4 weeks, range 3-21 weeks), and three of these patients are currently in continuous CR with a median followup of >3 years, and can considered to be cured. This resulted in a statistically significant survival advantage for children who responded to GO versus those who did not [27% (standard error 13%) vs. 0%, respectively, P = 0AE001]. All other children died, mainly from progressive disease. The treatment was generally well tolerated by most patients. The frequency of transient transaminatis was low. All but one patient received defibrotide prophylaxis during the transplant procedure, and no cases of veno-occlusive disease were noted. This study showed a favourable safety/efficacy profile of single-agent GO in children with refractory first or second relapse of AML.

Multi-organic failure in the context of autoimmune diseases is a multi-factorial condition where different pathways concur to produce a global system breakdown. Some of these pathways include the coagulation, fibrinolysis, kinin and complement cascades which in normal conditions work together to provide a comprehensive response to injury. In pathologic conditions these regulatory mechanisms are replaced by positive feed-back loops. The common response pattern is the activation of the immune system via endothelium activation. Furthermore, these different plasma-driven mechanisms may induce standardised endothelial cell responses of which the most relevant are the activation of p38, JNK, NF-kβ and IRF-3 pathways. In this paper we review the common points between these major pathways and how they become activated, contributing to a global clinical picture. We present two examples of apparently different clinical settings, caused by the same global dysfunction: the Macrophage Activation Syndrome and the iatrogenic "cytokine storm" triggered by the administration of anti-CD28 monoclonal antibody TGN1412 in a phase 1 trial.

The aim of this preliminary, observational study was to evaluate the value of ImmuKnow (IK), a new tool to measure the net state of immunefunction among renal transplant recipients, in correlation with clinical and laboratory data among unselected renal transplant recipients. Forty-nine recipients of mean age of 51 years were enrolled and followed for 1 year after transplantation. All subjects received the same immunosuppressive strategy with basiliximab induction and tacrolimus, mycophenolate mofetil and steroid maintenance therapy. Samples for IK were collected before transplantation as well as at 7, 14, 21 and 42 days and after 3, 6, and 12 months. There were 54 samples with IK Ͻ225 ng/mL, 201 samples with normal IK values, and 135 samples with Ͼ525 ng/mL. We divided recipients into 3 groups with respect to their basal IK values: Group 1 (Gr1; IK Ͻ225 ng/mL); Group 2 (Gr2; normal values of IK between 226 and 524 ng/mL); and Group 3 (Gr3; IK Ͼ525 ng/mL). At 1 year, we observed a significant difference among IK values at the start and the end of the study: Gr1 vs Gr2, P Ͻ .0001; Gr2 vs Gr3, P Ͻ .06 and Gr 1 vs Gr 3, P Ͻ .01). We observed reduced IK values to predict an increased risk of infection, particularly with cytomegalovirus (CMV) replication while higher IK value did not correlate with an increased risk of acute rejection episodes. Reduction of serum creatine levels occurred within 1 year in all groups (P Ͻ .005), but there was a significant difference between Gr 2 versus Grs 1 and 3 (P Ͻ .0001 and P Ͻ .0005, respectively). There findings suggested that more stable IK values were associated with clinical quiescence and laboratory stability. In conclusion, our preliminary analysis showed a beneficial capacity of this assay to represent the global depression of the immune system. We noted that reduced IK values, as a sign of excessive immunosuppressive therapy, were associated with an increased risk of infection. We did not confirm the predictive value of higher IK values for an increased risk of an acute rejection episode.

Background: Several of the currently used anticancer drugs may variably affect thyroid function, with impairment ranging from modified total but not free concentration of thyroid hormones to overt thyroid disease. Summary: Cytotoxic agents seem to alter thyroid function in a relatively small proportion of adult patients. Anticancer hormone drugs may mainly alter serum levels of thyroid hormone-binding proteins without clinically relevant thyroid dysfunction. Old immunomodulating drugs, such as interferon-a and interleukin-2, are known to induce variably high incidence of autoimmune thyroid dysfunction. Newer immune checkpoint inhibitors, such as anti-CTLA4 monoclonal antibodies, are responsible for a relatively low incidence of thyroiditis and may induce secondary hypothyroidism resulting from hypophysitis. Central hypothyroidism is a well-recognized side effect of bexarotene. Despite their inherent selectivity, tyrosine kinase inhibitors may cause high rates of thyroid dysfunction. Notably, thyroid toxicity seems to be restricted to tyrosine kinase inhibitors targeting key kinase-receptors in angiogenic pathways, but not other kinase-receptors (e.g., epidermal growth factor receptors family or c-KIT). In addition, a number of these agents may also increase the levothyroxine requirement in thyroidectomized patients. Conclusions: The pathophysiology of thyroid toxicity induced by many anticancer agents is not fully clarified and for others it remains speculative. Thyroid dysfunction induced by anticancer agents is generally manageable and dose reduction or discontinuation of these agents is not required. The prognostic relevance of thyroid autoimmunity, overt and subclinical hypothyroidism induced by anticancer drugs, the value of thyroid hormone replacement in individuals with abnormal thyrotropin following anticancer systemic therapy, and the correct timing of replacement therapy in cancer patients need to be defined more accurately in well-powered prospective clinical trials.

Introduction 2 2. Pigment cells are a distinct mesodermal lineage 3 3. Pigment cell precursors transition to mesenchyme, migrate, and re-insert in epithelium 5 4. Sp1 and NCAM 8 5. Pigment cells and archenteron formation 11 6. Localized maternal factors in the egg lead to short range signals that cause differentiation of pigment cells 12 7. A network of interacting genes controls pigment cell differentiation 16 8. Detailed descriptions of lineage specific genes provide insights into the enigmatic functions of pigment cells 18 9. Pigment cells as immunocytes 22 10. Conclusions 24 Acknowledgments 28 References 28

Non-human primate models of human disease have an important role in the translation of a new scientific finding in lower species into an effective treatment. In this study, we tested a new therapeutic antibody against the IL-7 receptor α chain (CD127), which in a C57BL/6 mouse model of experimental autoimmune encephalomyelitis (EAE) ameliorates disease, demonstrating an important pathogenic function of IL-7. We observed that while the treatment was effective in 100 % of the mice, it was only partially effective in the EAE model in common marmosets (Callithrix jacchus), a smallbodied Neotropical primate. EAE was induced in seven female marmoset twins and treatment with the anti-CD127 mAb or PBS as control was started 21 days after immunization followed by weekly intravenous administration. The anti-CD127 mAb caused functional blockade of IL-7 signaling through its receptor as shown by reduced phosphorylation of STAT5 in lymphocytes upon stimulation with IL-7. Groupwise analysis showed no significant effects on the clinical course and neuropathology. However, paired twin analysis revealed a delayed disease onset in three twins, which were high responders to the immunization. In addition, we observed markedly opposite effects of the antibody on pathological changes in the spinal cord in high versus low responder twins. In conclusion, promising clinical effect of CD127 blockade observed in a standard inbred/SPF mouse EAE model could only be partially replicated in an outbred/non-SPF non-human primate EAE model. Only in high responders to the immunization we found a positive response to the treatment. The mechanism underpinning this dichotomous response will be discussed.

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Combotox is a 1:1 mixture of RFB4-dgA and HD37-dgA which are immunotoxins that target the CD22 and CD19 antigens, respectively. Combotox has different toxicities and targets than chemotherapy and is, thus, a new candidate for the treatment of patients with relapsed ALL. Preclinical data have demonstrated which Combotox is effective in killing pre-BALL cell lines and cells from patients with pre-B ALL. Methods: We designed and conducted a Phase 1 dose-escalation study using Combotox in children with refractory or relapsed B-lineage-ALL. Seventeen patients aged 1 to 16 years were enrolled in this multi-institution study. They were treated at 4-dose levels: 2 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , and 6 mg/m 2. Results: The maximum tolerated dose was 5 mg/m 2 and graft versus host disease defined the maximum tolerated dose. Three patients experienced complete remission. Six additional patients experienced a decrease of >95% in their peripheral blood blast counts, and 1 patient experienced a decrease of 75%. Conclusions: Combotox can be safely administered to children with refractory leukemia. It has clinically important anticancer activity as a single agent. The recommended dose for future studies is 5 mg/m 2 /dose.

Using matrices based upon Affi-Gel and Affi-Prep, we have examined conditions during the immobilization of antibodies (immunoglobulin G, IgG) that influence the performance of immunosorbents. Such conditions include: coupling pH, coupling kinetics, antibody density on the immunosorbent and the activation chemistries utilized for the immobilization process. These studies have shown that the capacity for antigen does not increase with increased antibody coupling efficiency. Presumably, increased coupling times or efficiencies lead to multi-site attachment of the antibody to the matrix, thereby causing inactivation. Immunosorbents containing low densities of IgG were found to have greater capacity for antigen on a per mole IgG basis. This suggests steric crowding of antigen at high antibody density. Finally, immunosorbents prepared through IgG carbohydrate linkages (oriented coupling) show dramatic increases in antigen capacity over those prepared by stochastic (random) coupling through IgG primary amino groups. A combination of low IgG density and oriented coupling of the IgG via the carbohydrate moiety may represent the best strategy for the preparation of immunosorbents.

Objective: To describe the clinical characteristics, histopathologic features, and outcomes of patients in whom vasculitis developed in association with use of tumor necrosis factor-␣ (TNF-␣) inhibitors. Patients and Methods: This is a retrospective review of patients evaluated at Mayo Clinic, Rochester, Minnesota, from January 1, 1998, through March 31, 2011, with a diagnosis of vasculitis induced by anti-TNF-␣ therapy. Results: Of 8 patients with vasculitis associated with anti-TNF-␣ therapy (mean age, 48.5 years), 6 (75%) were female. Four (50%) had rheumatoid arthritis, 1 (13%) had Crohn disease, and 3 (38%) had ulcerative colitis. Five (63%) were treated with infliximab, 2 (25%) with etanercept, and 1 (13%) with adalimumab. The mean duration of treatment before development of vasculitis was 34.5 months. The skin was the predominant organ affected (5 patients [63%]), with the most common cutaneous lesion being palpable purpura (4 of 5 [80%]). Two organs involved in systemic vasculitis were the peripheral nervous system (4 patients [50%]) and kidney (1 patient [13%]). All cases of vasculitis were histopathologically confirmed. Seven of 8 patients improved with discontinuation of therapy (mean time to resolution, 6.9 months) and adjuvant treatment (all 8 received prednisone; another agent was also used in 7); rechallenge with anti-TNF-␣ therapy was not attempted in any patient. At last follow-up, no patients had experienced a recurrence of vasculitis after therapy discontinuation. Conclusion: Cutaneous small-vessel vasculitis was the most common finding, but systemic vasculitis, including peripheral nerve and renal vasculitis, was also frequently observed.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Two rat IgGza antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220+ but not B220 murine spleen cells, and immunoprecipitate a 45-kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up-regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti-IgM-induced growth arrest. The other monoclonal antibody (designated 4Fll) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti-IgM antibodies or the 1ClO agonistic antLCD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of lCl0 antibody to rescue B lymphoma cells from anti-IgM-induced growth arrest. The lCl0 and 4Fll antibodies were unable to cross-block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.

The selection and characterization of a set of mouse mAbs against high-risk human papillomavirus (HPV) E7 oncoprotein and the development of protocols for immunocytochemistry (ICC) are described here. A large number of antibodies raised towards HPV16 and 18 E7 were tested for high-risk specificity by ELISA using a panel of HPV E7 proteins. Antibodies detecting low-risk E7 were discarded, resulting in 38 high-risk HPV E7-specific antibodies. The corresponding epitopes were mapped using overlapping HPV E7 fragments displayed on phage particles. Functionality in ICC against formalin-fixed cervical cancer cell lines was demonstrated for ten mAbs; their high-risk specificity was confirmed by Western blot analysis and ICC on transiently transformed cells expressing high-or low-risk HPV E7. These mAbs were specific for one or several of the high-risk strains HPV16, 18, 31, 35 and 45. Specific E7 staining of liquid-based cytology (LBC) samples was demonstrated for seven mAbs and optimized protocols were established. The E716-41 and E718-79 mAbs demonstrated particularly strong and specific staining of cells stored in LBC fluid for at least 6 months. It is proposed that the high-risk HPV E7 staining protocols established in this study may have the potential to be included in a complementary test for the detection and identification of malignantly transformed cells, in for example atypical squamous cells of undetermined significance samples.

Monoclonal antibodies (MAbs) were generated against Leishmania major promastigote lipophosphoglycan (LPG) to use as tools in defining functional epitopes of this major cell surface glycoconjugate. Epitope mapping of four MAbs, designated 4A2-A2, 2Gll-A3, 5E6-D10 and 5E10-F2, revealed that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(/31-3)]Gal(/3 1-4)Man ot 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(/31-2)Gal(/31-3)]Gal(/31-4)Manc~l-, P4a, with less affinity than P3, while 2Gll-A3 recognised P4a with greater affinity than for P3. The L. major metacyclic-specific antibody 3F12 only recognised repeat units terminating with arabinose residues. In particular, 3F12 recognised P4a, which is upregulated in metacyclic LPG compared to the procyclic form of the molecule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG. The epitopes of 4A2-A2, 2G1 l-A3, 5E6-D10 and 5E10-F2 were found on the cell surface and in the flagellar pocket of both procyclic and metacyclic V121 promastigotes, but were only detected at very low levels on amastigotes. The repeat unit P3 is able to inhibit attachment of procyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit attachment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishmaniasis recognised purified P3. The data suggests that while P3 is an immunogen in the natural course of infection of the human host, P3 plays no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.

Murine monoclonal antibodies, HB88A, B29A and DH59B separately identify the CD2 T lymphocyte molecule, a unique pan-B lymphocyte surface marker and a pan-granulocyte/monocyte surface molecule, respectively, in the horse. Specificity was shown by two-color immunofluorescent flow cytometry and immunofluorescent microscopy. MAb HB88A reacted with a 52 kDa pan-T lymphocyte molecule present on 75 % ± 7 of peripheral blood lymphocytes (PBL) (n = 15 horses). It also reacted with lymphocytes restricted to T lymphocyte dependent areas of lymph node and spleen. Specificiry of mAb HB88A to CD2 was demonstrated by its reactivity to COS 7 cells which expressed a transfected 1.5 kb equine lymphocyte c-DNA clone having 77.5 % overall sequence homology with human CD2 c-DNA. MAb B29A reacted with a pan-B lymphocyte specific cell surface complex, 143, 72, 50, 40, 27 and 14.5 kDa, present on 19% ± 7 of PBL (n = 15 horses). This complex has not been described in the horse or other species. MAb DH59B reacted with a 96 kDa pan-granulocyte/monocyte specific surface protein and identified macrophages and Kupffer cells in equine tissue sections. Together these mAbs can be used to identify and quantitate the major constituents of equine leukocytes. Abbreviations: PBL = peripheral blood lymphocytes; FACS = fluorescent-activated cell sorting; PBS = phosphate-buffered saline; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cells; r-XIgM = fluorescein-labeled polyclonal rabbit anti-equine IgM antibody; BSA = bovine serum albumin; PBS-BSA = PBS with 0.5 % BSA and 0.02 % sodium azide; PAGE = polyacrylamide gel electrophoresis mAbs define equine leukocytes . 49

Nimotuzumab (h-R3) is a humanized anti-epidermal growth factor receptor monoclonal antibody (mAb) registered for treating head and neck tumours. The present study was designed to evaluate the systemic and skin toxicity of chronic intravenous administration of the h-R3 in a relevant species demonstrated by comparing the h-R3 binding affinity constants (Kd) in microsomal placental fractions from Homo sapiens and Cercopithecus aethiops monkeys using an EGF-Receptor radioligand competition assay. The Kd obtained for Nimotuzumab were 9.1 x 10 -8 M for monkeys and 4.5 x 10 -8 M for humans. Monkeys (n = 18) were distributed into three groups with three animals of each sex in each group. Group I received saline; group II received 2.85 mg/kg of h-R3; and group III received 28.57 mg/kg of the h-R3, which represent 1 and 10 times the human dose, and they were weekly intravenously treated during 26 weeks. During the study there were no deaths. Electroneurophysiological, sanguine chemistry and haematological results did not evidence alterations. Areas of haematomas, probably related with the administration procedure, were observed at the administration zones of all animals. The electrocardiography study showed at the end of the study a slight increase in the cardiac frequency of four treated animals without other signs. Unexpectedly, skin biopsies and a detailed clinical inspection of the animals did not detect the presence of cutaneous rash or any other skin toxicity sign reported for the majority of the anti-EGF-R monoclonal antibodies. It is concluded that doses up to 28.5 mg/kg of h-R3, intravenously administered during 26 weeks to Cercopithecus aethiops monkeys, do not produce considerable toxic effects. © 2 0 0 8 L A N D E S B I O S C I E N C E .

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1,335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulenceassociated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1negative strains differed significantly (P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P≤0.0007).

The occurrence of legionnaires' disease has been described previously in passengers of cruise ships, but determination of the source has been rare. A 67-year-old, male cigarette smoker with heart disease contracted legionnaires' disease during a cruise in September 1995 and died 9 days after disembarking. Legionella pneumophila serogroup 1 was isolated from the patient's sputum and the ship's water supply. Samples from the air-conditioning system were negative. L. pneumophila serogroup 1 isolates from the water supply matched the patient's isolate, by both monoclonal antibody subtyping and genomic fingerprinting. None of 116 crew members had significant antibody titers to L. pneumophila serogroup 1. One clinically suspected case of legionnaires' disease and one confirmed case were subsequently diagnosed among passengers cruising on the same ship in November 1995 and October 1996, respectively. This is the first documented evidence of the involvement of a water supply system in the transmission of legionella infection on ships. These cases were identified because of the presence of a unique international system of surveillance and collaboration between public health authorities.

Objective. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for treatment of B-cell malignancies. Natural killer (NK) cells, through the activating receptor FcgRIIIa (CD16), play a major role in rituximab-mediated ADCC. We have studied the in vitro effect of NK stimulators, such as interleukin-15 (IL-15) and CpG oligodeoxynucleotides A-Class (CpG ODN A), in the enhancement of rituximabmediated ADCC against B-cell lymphoma. Methods. Peripheral blood mononuclear cells (PBMC), purified NK cells, or NK-depleted PBMC from healthy donors, were activated with IL-15 or CpG ODN A, and cocultured with B-lymphoma cells in the presence of rituximab to evaluate the enhancement of the cytotoxicity.

Denosumab, a fully human monoclonal antibody to RANKL, decreases bone remodeling, increases bone density, and reduces fracture risk. This study evaluates the time course and determinants of bone turnover marker (BTM) response during denosumab treatment, the percentage of denosumab-treated women with BTMs below the premenopausal reference interval, and the correlations between changes in BTMs and bone mineral density (BMD). The BTM substudy of the Fracture REduction Evaulation of Denosumab in Osteoporosis every 6 Months (FREEDOM) Trial included 160 women randomized to subcutaneous denosumab (60 mg) or placebo injections every 6 months for 3 years. Biochemical markers of bone resorption (serum C-telopeptide of type I collagen [CTX] and tartrateresistant acid phosphatise [TRACP-5b]) and bone formation (serum procollagen type I N-terminal propeptide [PINP] and bone alkaline phosphatase [BALP]) were measured at baseline and at 1, 6, 12, 24, and 36 months. Decreases in CTX were more rapid and greater than decreases in PINP and BALP. One month after injection, CTX levels in all denosumab-treated subjects decreased to levels below the premenopausal reference interval. CTX values at the end of the dosing period were influenced by baseline CTX values and the dosing interval. The percentage of subjects with CTX below the premenopausal reference interval before each subsequent injection decreased from 79% to 51% during the study. CTX and PINP remained below the premenopausal reference interval at all time points in 46% and 31% denosumab-treated subjects, respectively. With denosumab, but not placebo, there were significant correlations between CTX reduction and BMD increase (r ¼ À0.24 to À0.44). The BTM response pattern with denosumab is unique and should be appreciated by physicians to monitor this treatment effectively. ß

To cite this article: Ulrichts H, Harsfalvi J, Bene L, Matko J, Vermylen J, Ajzenberg N, Baruch D, Deckmyn H, Tornai I. A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations. J Thromb Haemost 2004; 2: 1622-28.

Root canal samples, taken from periapical tissue exudates during routine root canal treatment procedures, were processed for identification of tumor necrosis factor using a mouse anti-human monoclonal antibody and enzyme-linked immunosorbent assay. Detectable levels of tumor necrosis factor were identified in periapical tissue exudates in chronic apical periodontitis.

Ultraviolet irradiation of skin induces various DNA photolesions. Here we demonstrate that irradiation of human buttock skin with 300 nm UVR in situ induces thymine dimers and 6-4 photoproducts. Irradiation with 260 nm immediately followed by UVA (320 nm) induces the Dewar photoisomers of the 6-4 lesions. All three lesions can be detected in methanol-fixed paraffin sections using specific monoclonal antibodies.

The addition of cetuximab (CTX) to the combination of cisplatin and 5-fluorouracil increases the overall survival (OS) in recurrent/metastatic head and neck squamous cell carcinoma. Only a few patients are eligible for this treatment because of its toxicity. The combination of CTX and paclitaxel (TXL) could be included in sequential treatment strategies. Patients were treated with CTX (400/ 250 mg/m 2 ) and TXL (60-80 mg/m 2 ) weekly until disease progression or unacceptable toxicity. Efficacy and safety outcomes were determined retrospectively. A total of 42 patients were included in this analysis. The overall response rate was 38% [95% confidence interval (CI); 23-53%]. The disease control rate with TXL and CTX combination was 74%. Seven (17%) patients progressed before the first evaluation. The median progression-free survival was 3.9 months [95% CI; 3.1-4.7 months] and the median OS was 7.6 months [95% CI; 5.3-9.9 months]. Neurotoxicity and skin rash were the most frequent grade Z 2 toxicities, reported in 17 and 12% of patients, respectively. Previous chemotherapy seems to be associated with a lower response rate and progressionfree survival but not with the OS. The combination of CTX and TXL was an active and well-tolerated treatment in this series of patients with a poor prognosis and who were mostly symptomatic.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-s...

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several hu-manized monoclonal antibodies that are required at large ...

Vial ''Fogging'' is a phenomenon observed after lyophilization due to drug product creeping upwards along the inner vial surface. After the freeze-drying process, a haze of dried powder is visible inside the drug product vial, making it barely acceptable for commercial distribution from a cosmetic point of view. Development studies were performed to identify the root cause for fogging during manufacturing of a lyophilized monoclonal antibody drug product. The results of the studies indicate that drug product creeping occurs during the filling process, leading to vial fogging after lyophilization. Glass quality/ inner surface, glass conversion/vial processing (vial ''history'') and formulation excipients, e.g., surfactants (three different surfactants were tested), all affect glass fogging to a certain degree. Results showed that the main factor to control fogging is primarily the inner vial surface hydrophilicity/hydrophobicity. While Duran vials were not capable of reliably improving the level of fogging, hydrophobic containers provided reliable means to improve the cosmetic appearance due to reduction in fogging. Varying vial depyrogenation treatment conditions did not lead to satisfying results in removal of the fogging effect. Processing conditions of the vial after filling with drug product had a strong impact on reducing but not eliminating fogging.