Human AB Research Articles

Immunosuppressive human anti‐lymphocyte autoantibodies specific for the type 1 sphingosine 1‐phosphate receptor

Anti-lymphocyte antibodies (Abs) that suppress T-cell chemotactic and other responses to sphingosine 1-phosphate (S1P), but not to chemokines, were found in a lymphopenic patient with recurrent infections. Lymphocyte type 1 S1P receptor (S1P(1)) that transduces S1P chemotactic stimulation was recognized by patient Abs in Western blots of T cells, S1P(1) transfectants, and S1P(1)-hemagglutinin purified by monoclonal anti-hemagglutinin Ab absorption. The amino terminus of S1P(1), but not any extracellular loop, prevented anti-S1P(1) Ab suppression of S1P(1) signaling and T-cell chemotaxis to S1P. Human purified anti-S1P(1) Abs decreased mouse blood lymphocyte levels by a mean of 72%, suppressed mouse T-cell chemotaxis to S1P in vivo, and significantly reduced the severity of dextran sodium sulfate-induced colitis in mice. Human Abs to the amino terminus of S1P(1) suppress T-cell trafficking sufficiently to impair host defense and provide therapeutic immunosuppression.

Novel Autoantibodies against the Activated Coagulation Factor IX (FIXa) in the Antiphospholipid Syndrome That Interpose the FIXa Regulation by Antithrombin

We previously reported that some human antiphospholipid Abs (aPL) in patients with the antiphospholipid syndrome (APS) bind to the homologous enzymatic domains of thrombin and the activated coagulation factor X (FXa). Moreover, some of the reactive Abs are prothrombotic and interfere with inactivation of thrombin and FXa by antithrombin (AT). Considering the enzymatic domain of activated coagulation factor IX (FIXa) is homologous to those of thrombin and FXa, we hypothesized that some aPLs in APS bind to FIXa and hinder AT inactivation of FIXa. To test this hypothesis, we searched for IgG anti-FIXa Abs in APS patients. Once the concerned Abs were found, we studied the effects of the Ab on FIXa inactivation by AT. We found that 10 of 12 patient-derived monoclonal IgG aPLs bound to FIXa and that IgG anti-FIXa Abs in APS patients were significantly higher than those in normal controls (p < 0.0001). Using the mean + 3 SD of 30 normal controls as the cutoff, the IgG anti-FIXa Abs were present in 11 of 38 (28.9%) APS patients. Importantly, 4 of 10 FIXa-reactive monoclonal aPLs (including the B2 mAb generated against beta(2)-glycoprotein I significantly hindered AT inactivation of FIXa. More importantly, IgG from two positive plasma samples were found to interfere with AT inactivation of FIXa. In conclusion, IgG anti-FIXa Ab occurred in approximately 30% of APS patients and could interfere with AT inactivation of FIXa. Because FIXa is an upstream procoagulant factor, impaired AT regulation of FIXa might contribute more toward thrombosis than the dysregulation of the downstream FXa and thrombin.

Selection of CD271+ cells and human AB serum allows a Large expansion of mesenchymal stromal cells from human bone marrow

Mesenchymal stromal cells (MSC) are promising candidates for cell therapy and tissue engineering and may be used to treat acute graft-versus-host disease (GvHD). However, major obstacles for their clinical use are the required cell dose and the biosafety and potential immunogenicity of fetal bovine serum (FBS), which is a crucial supplement of all media currently used for the culture of MSC. In this study MSC were successfully expanded after selection of CD271 cells from human bone marrow (BM) mononuclear cells in medium supplemented with 10% pooled allogeneic human serum. We isolated MSC from 10 healthy donor BM by plastic adherence and immunomagnetic selection of the CD271(+) fraction and expanded MSC in medium supplemented with pooled human allogeneic serum and animal serum. We isolated a homogeneous multipotent population by CD271(+) selection with a proliferation rate that was higher than MSC isolated by plastic adherence, 6.8+/-1.57 compared with 2.07+/-1.40 logs. Similar to cells generated in animal serum medium, MSC from allogeneic human serum were positive for mesenchymal markers and negative for hematopoietic markers; moreover they expressed embryonic stem cell genes. A normal karyotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages and neurosphere-like structures were preserved throughout long-term culture. Expansion of MSC is both feasible and large with a CD271-selected population in medium supplemented with 10% pooled allogeneic human serum, without loss of multipotent differentiation capacity or karyotype alterations.

A Highly Efficient Method to Expand CD3-CD56+ NK Cells from Cord Blood Segments

Background: The infusion of in-vitro expanded cord blood derived CD3−CD56+ NK cells could potentially be used to enhance graft-vs-tumor effects following cord blood transplantation. In order to infuse NK cells derived from the same cord unit used during allogeneic transplantation, we sought to develop a highly efficient culture method to expand large numbers of NK cells in vitro from < 1 ml of cord blood.Methods: Immunomagnetic beads were used to deplete CD3+ T-cells from thawed cord blood. CD3 depleted mononuclear cells (<0.1% CD56+) were co-cultured with either irradiated EBV-LCL feeder cells or 41BB transduced K562 cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 47 days.Results: Day 12 EBV-LCL expanded NK cell cultures contained up to 90% CD3−CD56+ NK cells with less than 0.5% CD3+CD56+ cells. Expanded cord blood derived CD56+NK cells had similar expression of CD16, NKG2D, LFA-1, perforin, and granzymes A and B and had similar cytotoxic function as NK cells expanded from adult PBMC. Surface expression of NK cell TRAIL increased dramatically with in vitro expansion. By day 12, TRAIL surface expression by FACS was at similar levels observed on expanded adult NK cells, although expression gradually declined with prolonged cell culture; on days 12, 20, and 34, 89%, 57% and 11% of cord derived NK cells expressed TRAIL respectively. NK cell cultures expanded with 41BB-transduced APCs had a similar phenotype and cytotoxic function against K562 cells and renal cell carcinoma (RCC) cells as EBV-LCL expanded cells. Furthermore, NK cell cytotoxicity against RCC tumor targets treated with 10 nM bortezomib for 18 hrs (bortezomib upregulates RCC surface expression of DR5) was higher than untreated RCC cells confirming the functional cytolytic activity of TRAIL expressed on cord blood derived NK cells. We next evaluated the feasibility of expanding CD56+ NK cells from thawed segments attached to the umbilical cord blood units. TNC numbers of each segment ranged from 3–9 × 106 cells. Unmanipulated thawed segments were co-cultured with irradiated EBV-LCL feeder cells as described above. On Day 12, 43% of viable cells were CD3+, 21.5% were CD3+CD56+ and 36% were CD3− CD56+. CD3−CD56+ NK cells from thawed segments increased 200 – 300 fold with in vitro expansion when maintained in culture for 2–3 weeks (Table). To enrich for pure NK cell populations, subsequent expansions were performed on CD3 depleted cells pooled from three thawed segments stimulated with EBV-LCL; cultures on day 12 contained a pure population of CD3−CD56+ NK cells with virtually an identical phenotypic and cytotoxicity profile as NK cells expanded from larger aliquots taken directly from the thawed cord blood unit. By day 33, CD3−CD56+ NK cells expanded by up to 30,000 fold; in one experiment, 391 ×106 CD3-CD56+ NK cells were expanded from only 13,000 NK cells obtained from a pool of three thawed cord segments.Conclusions: In vitro-expansion of a pure population of CD3−CD56+ cells derived from cord blood can be achieved using EBV-LCL or 41BB transduced feeder cells. Expanded cells have increased NKG2D and TRAIL expression and enhanced TRAIL-mediated tumor cytotoxicity. Even with very low starting numbers of TNCs, substantial numbers of CD3+, CD3+56+ and CD3−CD56+ cells can be expanded in vitro from thawed segments using EBV-LCL feeder cells in advance of thawing of the cord unit. These methods are being optimized to allow for clinical scale expansion of NK cells from the same cord unit used for hematopoietic cell transplantation.Cell numbers after expansion of individual segments with EBV – LCL feeder cellsDay 0 (×10e6)Day 16 (× 10e6)Fold expansionTNC3–678–11220–30CD3+0.42–0.9133–4750–70CD3+CD56-0.015–0.03216–24700–1000CD3-CD56+0.081–0.17528–40200–300Cell numbers after CD3+ depletion of pooled segments and expansion with EBV – LCL feeder cellsDay 0 (×10e6)Day 33 (× 10e6)Fold expansionTNC4.1640196CD3+00-CD3+CD56-00-CD3-CD56+0.013239129621

Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

Bone Marrow Mesenchymal Stem Cells Are Altered in B-Cell Chronic Lymphocytic Leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), CD5+CD19+ malignant cells home into the bone marrow (BM) and circulate in the blood. While CLL tumor cells are not susceptible to apoptosis in vivo, they die rapidly in vitro in the absence of specialized non-hematopoietic feeder cells, such as mesenchymal stem cells (MSC). Recent observations have suggested that there is a functional relationship between B cell clone and the stroma. We have thus compared BM-MSC obtained from B-CLL patients and healthy subjects. We first evaluated the influence of in vitro culture conditions on the number of BM-derived CFU-F and the proliferation of MSC and, in parallel, we quantified in unmanipulated normal and malignant BM samples the CD45negCD14negCD73pos cell subset that was previously shown to contain CFU-F (Veyrat-Masson et al., BJH, 2007). Changes in the level of 42 cytokines/chemokines, were then evaluated in MSC-conditioned media (4 CLL vs 4 normal BM-MSCs) using protein-array (RayBio Human Cytokine Antibody Array IIITM, Tebu-bio SA,). In addition, total RNA was extracted (Rneasy MiniKit, Qiagen,) from 9 expanded MSC at passage 1 (P1) in the presence of bFGF (5 untreated B-CLL BM-MSC: 2 Binet stage A, 2 stage B and 1 stage C; 4 normal BM-MSC) and then reverse transcribed (High Capacity cDNA RT Kit, Applied BioSystems). Quantitative PCR reactions, using dedicated microfluid cards screening 384 selected genes, were then performed (TLDAs, Applied Biosystem Courtaboeuf, France). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression level. Despite a 16-fold increase in total cell numbers tested, we found that most BM-MSC cultures from B-CLL patients failed under standard culture conditions (IMDM/10%FCS), in contrast with our experience with normal BM (69 % n = 13 vs <0.05 % n = 205 ; p <0.005). In agreement, CD45negCD14negCD73pos cells were under the threshold of detection in most of B-CLL BM samples (11/16). In productive cultures, we found more CFU-F from B-CLL BM formed by large, polygonal mesenchymal cells (58.1 ± 12.7 % vs 11.4 ± 3.6 % ; p = 0.008 ). These cells proliferated poorly and in most cases could not be further amplified. The use of normal human AB serum, CLL serum, or bFGF enabled us to detect CFU-F in most malignant samples and to amplify mesenchymal cells (19/21 (90 %)), but their frequency remained lower than in control BM. By using protein-array, we observed that MSC tended to release lower amounts of IL-6, IL-7, and MCP-1 and sometimes higher amounts of IL-8. The concentrations of these cytokines/chemokines in the MSC culture supernatant are under validation by ELISA. Finally, among the 384 genes tested by RT-qPCR, we identified 16 statistically up-regulated genes and 41 down-regulated genes (Mann Whitney U test, P< .05; and SAM permutation analysis, FDR<5%). Up-regulated genes included several growth and angiogenic factors as well as key players of the stroma - tumor cell crosstalk. Most down-regulated genes were involved in differentiation pathways.Conclusions: These results show that the BM-MSC from B-CLL patients were quantitatively and functionally altered and are dependent for their in vitro growth on circulating soluble factors or on growth factors like bFGF. Interestingly, from this small series, we observed 57 differentially expressed genes which could be involved in the B-CLL specific stromal cell alterations previously reported (dysregulation of cytokine secretion, angiogenesis, host-tumor relationships). These findings suggest the possible permissive role of MSC on B-cell clone progression and raise the question as to whether we are dealing with selection of a mesenchymal subset or with alteration of mesenchymal cells induced by malignant B-cells

Preclinical Development of Tissue-Engineered Vein Valves and Venous Substitutes using Re-Endothelialised Human Vein Matrix

Objective The aim of the study was to evaluate the use of a decellularised scaffold and its re-endothelialisation in vitro in order to create human vascular substitutes containing venous valves. This research is clinically relevant particularly with regard to the development of venous (valve containing) transplants to replace a diseased femoral vein valve and/or obstructed veins. This technique may enable causal treatment of venous reflux and obstructions. Materials and methods Valve-bearing segments of human allogeneic great saphenous veins (GSVs) were decellularised using sodium deoxycholic acid (SD) and treated with DNase I. Human venous endothelial cells (ECs) were enzymatically harvested from the GSV, expanded up to the 3rd passage using FCS ( n = 20) or human AB serum (hABS; n = 8) supplemented media before used for re-seeding. In special bioreactors, 3D re-seeding of 28 decellularised GSV was performed with constant perfusion (A; n = 8), bidirectional perfusion (B; n = 8), bidirectional perfusion/reduced flow (C; n = 2), static conditions (D; n = 2), and bidirectional perfusion/reduced flow using hABS (E; n = 8) instead of FCS. Decellularised GSV, scaled-up EC and 3D-seeded tissue-engineered valve containing neo-veins underwent immunohistochemical and PCR characterisation. Results Intact collagen and elastin networks as well as complete acellularity were shown after GSV decellularisation. In EC culture, supplementation with hABS led to a significantly higher expression of vWF compared to FCS ( p = 0.025). Additional EC markers such as CD 31, FLK-1 and VE-Cadherin were not altered. EC re-seeding using hABS supplemented medium (E) led to a confluent monolayer of cells that were immunohistochemically positive for FLK-1, CD 31, vWF and VE-Cadherin and by means of PCR after RNA preparation in 7 of 8 cases but was unsuccessful if FCS was used (A–D). In A–D cells presented as conglomerates positive for CD 31 and VE-Cadherin, suggesting sufficient intercellular contact but not cell–matrix contact. Conclusions Treatment with SD and DNase enables complete decellularisation of human valve containing veins whereas 3D matrix components such as collagen and elastin remain preserved. The lumen of the scaffold including the valves can be successfully re-seeded with a human EC monolayer in a 3D bioreactor. There is substantial evidence that hABS and not FCS is essential for the completion of cell–matrix contacts in human veins.

Successful re-challenge with panitumumab in patients who developed hypersensitivity reactions to cetuximab: report of three cases and review of literature

Monoclonal antibodies (MAbs) targeting epidermal growth factor receptor (EGFR) are effective in treatment of metastatic colorectal cancer (mCRC). Cetuximab, a chimeric MAb targets EGFR. Even with premedication, cetuximab can cause a hypersensitivity reaction (HSR). In case of severe HSR, further therapy with cetuximab is contraindicated, thus preventing these patients from receiving potentially beneficial anti-EGFR therapy. Panitumumab is a fully human MAb also targets EGFR. To date, no human antihuman Ab have been detected, and unlike CET, HSR are infrequent, and no premedication is required. Safety of panitumumab in patients with a previous severe HSR with cetuximab is not fully known. We present three patients with GI cancers who tolerated panitumumab without HSR after experiencing severe HSR to cetuximab. Three patients were challenged with standard dose of panitumumab (6 mg/kg) after experiencing grade 3 HSR to standard dose of cetuximab under strict observation and no premedication. First patient, a 58-year-old male with mCRC developed grade 3 HSR during 8th dose of cetuximab. Second patient was a 58-year-old female with mCRC developed grade 3 HSR during 12th dose of cetuximab. Third patient was a 61-year-old male with pancreatic cancer who experienced grade 3 HSR during loading dose of cetuximab. Charts were reviewed to find history of prior allergy, including H1 blocker use, drug allergy, bee sting allergy, eczema, allergic reactive airways disease, or food allergy. All patients were Caucasians with an average age of 59 year with no history of prior allergy. No patient received any premedication. First patient received panitumumab for 2 months, second patient was treated for 6 months, and third patient who was rechallenged 1 week after HSR to cetuximab had a partial response following 6 months of therapy. HSR are serious complications associated with MAbs. Thanks to hybridoma technology that newer generations of MAbs contain less or no mouse-specific protein sequences, hence reducing the risk of HSR. Identification of individuals likely to develop severe and sometimes life-threatening HSR is challenging. Our report of three patients successfully treated with panitumumab after they had severe HSR to cetuximab warrant further investigation.

Production and characterization of pigs transgenic for human hemeoxygenase‐I by somatic nuclear transfer

Introduction: The hyperacute rejection after porcine‐to‐human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors (done in this study) or by knocking out the gene for α1,3‐galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase‐I (hHO‐1) is known to have anti‐apoptotic and cell protective properties. Thus, the expression of hHO‐1 on porcine endothelial cells could have beneficial effects in a xenotransplantation setting. Here, we describe the generation and characterization of pigs transgenic for hHO‐1.Methods: Fibroblasts were obtained by an ear punch from a CD55 transgenic female pig and were cultured in vitro (Kues WA et al. 2005 Biol Reprod 72: 1020–1028). Cells reaching confluency of 70 to 80%, were detached with EDTA/trypsin and subsequently transfected by electroporation at 450 V/350 μF with a vector coding for hHO‐I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 μg/ml) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone was selected for somatic nuclear transfer.In total, 205 reconstructed embryos were transferred to two synchronized peripuberal German Landrace gilts which gave birth to nine live piglets, all with normal birth weights. The expression of the transgenes (CD55 and hHO‐1) was determined by flow cytometry and Western blot in endothelial cells and peripheral blood lymphocytes. The expression pattern of the transgenes in different organs was shown by immunostaining, RT‐PCR and Northern blot. Organ survival and XAC following the contact of human blood with porcine endothelium, were determined by using an ex‐vivo perfusion circuit based on low‐dose heparin mediated anticoagulation. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled human AB blood.Results: PCR and Southern blot analyses revealed that all of the offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for further characterization. In these animals, the expression of CD55 and hHO‐1 in peripheral blood lymphocytes and cultured endothelial cells was shown by flow cytometry and Western blot. In addition, albeit all animals were cloned from the same cell clone, variations in the expression pattern and levels could be observed in immunostaining, RT‐PCR and Northern Blot. The animals showed only weak expression of hHO‐1 in most of the xenorelevant organs like heart, kidney and liver. Average survival of wildtype pig kidneys during organ perfusion with human blood and addition of heparin was 42 ± 26 min. Application of soluble complement inhibition (C1‐inhibitor) prolonged organ survival to 126 ± 72 min. XAC was observed with significantly elevated concentrations of d‐Dimer and thrombin antithrombin complex (TAT) combined with consumption of fibrinogen and antithrombin. The histological analyses revealed numerous microthrombi. In contrast, the perfusion of the CD55/hHO1 transgenic porcine kidneys was feasible for more than 240 min in all perfusion experiments (with and without C1‐inh). Although elevated levels of d‐Dimer and TAT were measured, no significant consumption of fibrinogen and antithrombin occurred. In addition, no microthrombi were detectable histologically.Conclusion: These results show that hHO‐1 in combination with CD55 can be expressed in transgenic pigs. Somatic cell nuclear transfer is a powerful tool for the generation of transgenic animals for xenotransplantation. These results are encouraging and warrant further studies on endothelial cell activation and the biological function of hemeoxygenase‐I in the context of xenotransplantation.This study was funded by the Deutsche Forschungsgemeinschaft Ni 256/ 22‐1, ‐2, ‐3.

Management of loss of response to anti-TNF drugs: Change the dose or change the drug?

The advent of biological therapy has dramatically changed our concept of treating refractory inflammatory bowel disease (IBD). Chimeric and more humanized anti-TNF Abs have shown to be highly efficacious in these illnesses, but all issues have not been resolved. Indeed, still 20–30% of patients with refractory Crohn's disease1–6 and 30–40%7 of those with refractory ulcerative colitis do not respond to anti-TNF treatment. Moreover, the long-term use of anti-TNF monoclonal antibodies is associated with immunogenicity, which interferes with efficacy, and with the risk of infectious complications. Secondary loss of response to monoclonal antibodies is a reality and clinicians should be prepared to optimize therapy. A rapidly declining response to a given drug in patients responding to the first doses is usually called tachyphylaxis. Underlying reasons for a rapid loss of response can be very diverse. First, the human body usually reacts to external activators of endogenous proteins by decreasing the expression of the target protein or by internalizing membrane bound receptors. Second, alternative pathways can be recruited to restore the bioactivity targeted by a given drug. Third, the bioavailability and/or pharmacokinetics of therapeutic compounds are highly variable among individuals and liable to dramatic changes over time. The last mechanism can also induce a gradual loss of response, which is more commonly observed with anti-TNF agents. We will focus on loss of response with the use of anti-TNF antibodies in IBD and suggest strategies to optimize therapy in these patients. Several strategies have been followed in drug development to improve the efficacy and tolerability of biological agents. Progress in protein engineering has resulted in the elimination of immunogenic non-human peptide sequences from anti-human antibodies, a technique called humanization.8 Third generation, humanized antibodies (± 95% human) and fourth generation, fully (100%) human antibodies, are usually associated with less …

Features of Rituximab‐Induced Killing of B Lymphocytes

Features of Rituximab‐Induced Killing of B Lymphocytes

The Potency of Erythropoietin-Mimic Antibodies Correlates Inversely with Affinity

Preclinical animal studies have shown that Ab12.6, an agonistic human Ab targeting the erythropoietin receptor (EPOR), exhibits several potential dosing and safety features that make it an attractive clinical candidate for the treatment of anemia. Ab12.6 was derived by yeast display affinity maturation of parental Ab12, a strategy initially intended to improve off-rate and affinity for EPOR, thereby enhancing erythropoietic activity. Analysis of full-length IgGs derived from yeast clones identified sequences within Ab12 CDRH2 that independently influenced both affinity and potency. The Ab12.6 derivative displayed improved in vitro potency and in vivo efficacy, although its binding affinity to the EPOR was lower than that of the parent Ab12. Additional Ab12 derivatives also exhibited an inverse correlation between affinity and potency. These results suggest that for this class of agonistic Abs, faster off-rates may permit continuous receptor stimulation and more efficient erythropoiesis.

Induction of an anti-Fab, anti-DNA and anti-RNA polymerase I autoantibody response net work in rabbits immunized with SLE anti-DNA antibody

SUMMARYSystemic lupus erythematosus (SLE)-like anti-IgG Fab autoantibodies (autoAb) were induced in rabbits by immunization with either human, mouse or rabbit anti-DNA Ab. In direct-binding radioimmunoassay (RIA), affinity-purified anti-normal rabbit (NR) Fab autoAb cross-reacted with normal mouse (NM) Fab, ssDNA (but not dsDNA), poly(dA,dT), and RNA polymerase I (RPI). Affinity-purified anti-NM IgG Ab isolated from the same antisera cross-reacted with NR Fab, ssDNA and RPI. In inhibition RIA, soluble NR Fab inhibited anti-NR Fab binding to NR Fab and ssDNA, but enhanced binding to RPI. In contrast, ssDNA or RPI inhibitors had no effect upon autoAb binding to NR Fab. Anti-DNA, anti-RPI and anti-RPI 190 kD subunit autoAb, induced by immunization with lupus mouse anti-DNA Ab, also reacted with NM Fab, but were idiotypically specific for lupus mouse anti-DNA Fab. Further, rabbit anti-DNA and anti-RPI IgG autoAb, induced by immunization with rabbit anti-DNA IgG, were each idiotypically specific for homologous and autologous rabbit anti-ssDNA Fab. Together, these data provide evidence that anti-DNA, anti-RPI and anti-Fab autoAb are linked in a complex, multiple-specific and perhaps regulatory, immune response idiotype network in SLE.

Detection of auto-anti-idiotypic antibodies toLol p I(rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy

Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

Combined Pancreatic Islet-Lung Transplantation With Islet Percutaneous Portal Embolization in Cystic Fibrosis

Combined Pancreatic Islet-Lung Transplantation With Islet Percutaneous Portal Embolization in Cystic Fibrosis

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

BackgroundComplementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal FindingsCDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/SignificanceThe high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.

Selection, diversity and evolutionary patterns of the MHC class II DAB in free-ranging Neotropical marsupials

BackgroundResearch on the genetic architecture and diversity of the MHC has focused mainly on eutherian mammals, birds and fish. So far, studies on model marsupials used in laboratory investigations indicated very little or even no variation in MHC class II genes. However, natural levels of diversity and selection are unknown in marsupials as studies on wild populations are virtually absent. We used two endemic South American mouse opossums, Gracilinanus microtarsus and Marmosops incanus, to investigate characteristic features of MHC selection. This study is the first investigation of MHC selection in free-ranging Neotropical marsupials. In addition, the evolutionary history of MHC lineages within the group of marsupials was examined.ResultsG. microtarsus showed extensive levels of MHC diversity within and among individuals as 47 MHC-DAB alleles and high levels of sequence divergence were detected at a minimum of four loci. Positively selected codon sites were identified, of which most were congruent with human antigen binding sites. The diversity in M. incanus was rather low with only eight observed alleles at presumably two loci. However, these alleles also revealed high sequence divergence. Again, positive selection was identified on specific codon sites, all congruent with human ABS and with positively selected sites observed in G. microtarsus. In a phylogenetic comparison alleles of M. incanus interspersed widely within alleles of G. microtarsus with four alleles being present in both species.ConclusionOur investigations revealed extensive MHC class II polymorphism in a natural marsupial population, contrary to previous assumptions. Furthermore, our study confirms for the first time in marsupials the presence of three characteristic features common at MHC loci of eutherian mammals, birds and fish: large allelic sequence divergence, positive selection on specific sites and trans-specific polymorphism.

Successful re-challenge with panitumumab (PAN) in patients (pts) with GI cancers who developed hypersensitivity reactions (HSR) to cetuximab (CET)

20744 Background: Monoclonal antibodies (Mabs) targeting epidermal growth factor receptor (EGFR) are effective in treatment of metastatic colorectal cancer (mCRC). CET, a human-murine chimeric Mab targets EGFR. Even with premedication, CET can cause an HSR in select pts. In case of severe HSR, further therapy with CET is contraindicated, thus preventing these pts from receiving potentially beneficial anti-EGFR therapy. PAN is a fully human Mab also targets EGFR. To date, no human antihuman Ab have been detected, and unlike CET, HSR are infrequent, and no premedication is required. Safety of PAN in pts with a previous severe HSR with CET is not fully known. We present 3 pts with GI cancers who tolerated PAN without HSR after experiencing severe HSR to CET. Methods: 3 pts were challenged with standard dose of PAN (6mg/kg) after experiencing grade (g) 3 HSR to standard dose of CET and monitored very closely. 1st pt, a 58-yr-old M with mCRC developed g3 HSR during 8th dose of CET. 2nd pt, a 58-yr-old F with mCRC develpoed g3 HSR during 12th dose of CET. 3rd pt, a 61-yr-old M with pancreatic cancer experienced g3 HSR during loading dose of CET. Charts were reviewed to find prior allergy, including H1 blocker use, drug allergy, bee sting allergy, eczema, allergic reactive airways disease, or food allergy. Results: All pts were Caucasians with average age of 59 yr and no history of prior allergy. No pt received any premedication. 1st pt received PAN for 2 mths, 2nd pt was treated for 6 mths, and 3rd pt who was rechallenged 1 week after HSR to CET had a partial response after 6 mths. Conclusions: HSR are serious complications associated with modern Abs. In order to reduce incidence and severity of such reactions, newer generations of Mabs contain less or no mouse-specific protein sequences. Identification of individuals likely to develop severe and sometimes life-threatening HSR is challenging. Our report of 3 pts successfully treated with PAN after they had severe HSR to CET warrant further investigation. Summary of safety and response to re-challenge with PAN Case Age/Sex Diagnosis CET Dose Grade of HSR Time Interval Between CET and PAN PAN Dose Tolerance Duration of PAN Therapy 1 58/M CRC 250mg/kg G3 1 month 6mg/kg No HSR 2 months 2 58/F CRC 250mg/kg G3 3 months 6mg/kg No HSR 6 months 3 61/M Pancreatic cancer 400mg/kg G3 1 week 6mg/kg No HSR 6 months M: male, F: female No significant financial relationships to disclose.

Activation of T-Cells from Allergic Patients and Volunteers by p-Phenylenediamine and Bandrowski's Base

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.