OKT3 Research Articles

Monoclonal Antibodies and Antibody-drug Conjugates as Emerging Therapeutics for Breast Cancer Treatment.

When breast cells divide and multiply out of control, it is called breast cancer. Symptoms include lump formation in the breast, a change in the texture or color of the breast, or a discharge from the nipple. Local or systemic therapy is frequently used to treat breast cancer. Surgical and radiation procedures limited to the affected area are examples of local management. There has been significant worldwide progress in the development of monoclonal antibodies (mAbs) since 1986, when the first therapeutic mAb, Orthoclone OKT3, became commercially available. mAbs can resist the expansion of cancer cells by inducing the destruction of cellular membranes, blocking immune system inhibitors, and preventing the formation of new blood vessels. mAbs can also target growth factor receptors. Understanding the molecular pathways involved in tumor growth and its microenvironment is crucial for developing effective targeted cancer therapeutics. Due to their unique properties, mAbs have a wide range of clinical applications. Antibody-drug conjugates (ADCs) are drugs that improve the therapeutic index by combining an antigen-specific antibody with a payload. This review focuses on the therapeutic applications, mechanistic insights, characteristics, safety aspects, and adverse events of mAbs like trastuzumab, bevacizumab, pertuzumab, ertumaxomab, and atezolizumab in breast cancer treatment. The creation of novel technologies utilizing modified antibodies, such as fragments, conjugates, and multi-specific antibodies, must be a central focus of future studies. This review will help scientists working on developing mAbs to treat cancers more effectively.

Genetic code expansion in E. coli enables production of a functional ‘ready-to-click’ T cell receptor-specific scFv

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Emerging Antibodies in Cancer Therapy

Since the first monoclonal antibody, Orthoclone OKT3, was approved in 1986 for the treatment of acute allograft rejection in renal transplant recipients, the number of antibody‐based therapies has increased remarkably, with more than 130 monoclonal antibodies being currently available. In particular, due to their robust binding affinity and high specificity, various monoclonal antibodies have achieved success as complementary antitumor drugs. Moreover, therapeutic potency can be improved by engineering Y‐shaped binding sites of the antibodies to simultaneously recognize two distinct antigens; such molecules are called bispecific antibodies and function as a bridge, with one site binding to a tumor‐specific antigen and the other to immune cells to activate host immune responses. In addition, the development of trispecific antibodies has led to further enhancement of therapeutic specificity and effects. Indeed, a variety of structurally engineered antibodies have proven effective in tumor therapy. This article provides insights into the clinical translation of emerging antibody constructs, firstly describing conventional T cell‐redirecting bispecific antibodies and subsequently discussing the application of bispecific and trispecific antibody constructs based on innate immune cell recruitment. Finally, bispecific antibodies against mutant p53 and KRAS that have been actively studied in cancer treatment are introduced.

Monoclonal Antibodies for Protozoan Infections: A Future Reality or a Utopic Idea?

Following the SARS-CoV-2 pandemic, several clinical trials have been approved for the investigation of the possible use of mAbs, supporting the potential of this technology as a therapeutic approach for infectious diseases. The first monoclonal antibody (mAb), Muromonab CD3, was introduced for the prevention of kidney transplant rejection more than 30 years ago; since then more than 100 mAbs have been approved for therapeutic purposes. Nonetheless, only four mAbs are currently employed for infectious diseases: Palivizumab, for the prevention of respiratory syncytial virus (RSV) infections, Raxibacumab and Obiltoxaximab, for the prophylaxis and treatment against anthrax toxin and Bezlotoxumab, for the prevention of Clostridium difficile recurrence. Protozoan infections are often neglected diseases for which effective and safe chemotherapies are generally missing. In this context, drug resistance and drug toxicity are two crucial problems. The recent advances in bioinformatics, parasite genomics, and biochemistry methodologies are contributing to better understand parasite biology, which is essential to guide the development of new therapies. In this review, we present the efforts that are being made in the evaluation of mAbs for the prevention or treatment of leishmaniasis, Chagas disease, malaria, and toxoplasmosis. Particular emphasis will be placed on the potential strengths and weaknesses of biological treatments in the control of these protozoan diseases that are still affecting hundreds of thousands of people worldwide.

Development of the first reference antibody panel for qualification and validation of cytokine release assay platforms – Report of an international collaborative study

Immunomodulatory therapeutics such as monoclonal antibodies (mAb) carry an inherent risk of undesired immune reactions. One such risk is cytokine release syndrome (CRS), a rapid systemic inflammatory response characterized by the secretion of pro-inflammatory cytokines from immune cells. It is crucial for patient safety to correctly identify potential risk of CRS prior to first-in-human dose administration. For this purpose, a variety of in vitro cytokine release assays (CRA) are routinely used as part of the preclinical safety assessment of novel therapeutic mAbs. One of the challenges for the development and comparison of CRA performance is the lack of availability of standard positive and negative control mAbs for use in assay qualification. To address this issue, the National Institute for Biological Standards and Control (NIBSC) developed a reference panel of lyophilised mAbs known to induce CRS in the clinic: human anti-CD52, mouse anti-CD3 and human superagonistic (SA) anti-CD28 mAb manufactured according to the respective published sequences of Campath-1H® (alemtuzumab, IgG1) , Orthoclone OKT-3® (muromonab, IgG2a) and TGN1412 (theralizumab, IgG4), as well as three isotype matched negative controls (human IgG1, mouse IgG2a and human IgG4, respectively).The relative capacity of these control mAbs to stimulate the release of IFN-γ, IL-2, TNF-α and IL-6 in vitro was evaluated in eleven laboratories in an international collaborative study mediated through the HESI Immuno-safety Technical Committee Cytokine Release Assay Working Group. Participants tested the NIBSC mAbs in a variety of CRA platforms established at each institution. This paper presents the results from the centralised cytokine quantification on all the plasma/supernatants corresponding to the stimulation of immune cells in the different CRA platforms by a single concentration of each mAb. Each positive control mAb induced significant cytokine release in most of the tested CRA platforms. There was a high inter-laboratory variability in the levels of cytokines produced, but similar patterns of response were observed across laboratories that replicated the cytokine release patterns previously published for the respective clinical therapeutic mAbs. Therefore, the positive and negative mAbs are suitable as a reference panel for the qualification and validation of CRAs, comparison of different CRA platforms (e.g. solid vs aqueous phase), and intra- and inter-laboratory comparison of CRA performance. Thus, the use of this panel of positive and negative control mAbs will increase the confidence in the robustness of a CRA platform to identify a potential CRS risk for novel immunomodulatory therapeutic candidates.

Variable Benefits of Antibody Induction by Kidney Allograft Type

BackgroundKidneys from acute renal failure (ARF), expanded criteria donors (ECD), and donation after cardiac death (DCD) donors are often discarded due to concerns for delayed graft function (DGF) and graft failure. Induction immunosuppression may be used to minimize these risks, but practices vary widely. Furthermore, little is known regarding national outcomes of transplant recipients receiving induction immunosuppression for receipt of high-risk kidneys. Materials and methodsUsing a center-level retrospective study, deceased donor transplants (115,485) from the Scientific Registry of Transplant Recipients from January 2003 to June 2016 were evaluated. Patients who received induction immunosuppression, including lymphocyte immune globulin, muromonab CD-3, IL-1 receptor antagonist, anti-thymocyte globulin, daclizumab, basiliximab, alemtuzumab, and rituximab, were included. Associations of center-level induction use with acute rejection in the first post-transplant year, graft failure, and patient mortality were evaluated using multivariable Cox and logistic regression. ResultsAmong all kidneys, increasing percentage of center-level induction was associated with lower risk of graft failure, acute rejection, and patient mortality. In recipients of ARF kidneys, the beneficial association of induction on graft failure and acute rejection was greater than in those that received non-ARF kidneys. Marginally greater benefit of induction was seen for acute rejection in ECD compared to standard criteria donor (SCD) recipients and for graft failure in DCD compared to donors after brain death (DBD). No benefit of induction was detected for patient and graft survival in ECD recipients, acute rejection in DCD recipients, and patient survival in DGF recipients. No difference in the benefit of induction was detected in any other comparisons. ConclusionsWhile seemingly beneficial for recipients of all kidneys, induction has more robust associations with lower graft failure and acute rejection probability for recipients of ARF kidneys. Given the lack of observed benefit for ECD recipients, induction policies should be carefully considered in these patients.

Monoclonal Antibodies: A Review.

Over the last three decades, monoclonal antibodies (MAbs) have made a striking transformation from scientific tools to powerful human therapeutics. Muromonab CD3 a murine MAb was the first FDA approved therapeutic MAb for the prevention of kidney transplant rejection. Since its approval in 1986, there has been a decline in further application and approvals until the late 1990s when the first chimeric Mab, Rituximab was approved for the treatment of lowgrade B cell lymphoma in 1997. With the approval by licensing authorities of chimeric, followed by humanized and then fully human monoclonal antibodies, the rate of approval and monoclonal antibodies available in the market for the treatment of various diseases has increased dramatically. As of March 2017, FDA has approved approximately 60 therapeutic MAbs which are currently under evaluation in various phases of clinical trials. MAbs are approved for the treatment of diseases belonging to various systems like cardiovascular, respiratory, hematology, kidney, immunology and oncology. MAbs are approved for the treatment of orphan diseases or indications such as paroxysmal nocturnal hemoglobinuria as well as cancers and multiple sclerosis where hundreds of patients are treated and even diseases such as breast cancer, asthma and rheumatoid arthritis where millions are being treated. This review focuses briefly on types, molecular targets, mechanism of actions and therapeutic indications of FDA approved MAb products that are currently available in the market. With the advent of fully human MAbs, the efficacy and safety have improved in the treatment of various cardiovascular, cancer, respiratory, hematology, autoimmune diseases and infections. The introduction of biosimilars will increase the affordability and utilization of MAbs in the treatment of various diseases.

Revisiting the phenotypic and genetic profiling of anergic T cells mediating long-term transplant tolerance.

Herein our focus will be to revisit peripheral tolerance mechanisms and in particular 'active' or 'dominant' tolerance as originally defined and mediated by regulatory CD4FoxP3 T lymphocytes (Treg) and also T-cell anergy that appears as a major mainstay to support long-term allograft survival. It is at the same time interesting and rewarding that the tool that recently guided our efforts along this path is the in-vivo use of CD3 antibody, the first monoclonal introduced in the clinic (Orthoclone OKT3) about 35 years ago to treat and prevent rejection of renal allografts. Beyond their immunosuppressive activity, whenever administered judiciously, CD3 antibodies promote robust allograft tolerance through selective purging of alloreactive effectors, resetting Treg-mediated active tolerance and promoting a unique subset of anergic CD8 T cells. The new findings discussed open up new perspectives from both a fundamental and a clinical point of view. In basic research, concrete molecular signaling paths are now spotted to finely dissect the conditions that lead to the establishment and maintenance of robust T-lymphocyte anergy mediating allograft tolerance. In the clinic, this may rapidly translate into novel biomarkers to be used in parallel to the ones already available, to better adapt posttransplant immunotherapy and monitor for long-term allograft acceptance.

Clinical and pathological analysis of acute rejection following orthotopic liver transplantation.

Acute rejection is one of the most important factors for prognosis following liver transplantation. With the use of potent immunosuppressants, acute rejection does not always present typical manifestations. Moreover, other complications often occur concomitantly after liver transplantation, which makes early diagnosis of acute rejection more difficult. Acute rejection is best diagnosed by liver biopsy. Differentiation of clinical manifestations and pathological features plays an important role in achieving individualized immunosuppressive treatment and prolonging long term survival of patients given orthotopic liver transplants. From January 2004 to December 2006, 516 orthotopic liver transplantations were performed at the First Affiliated Hospital, Sun Yat-sen University. For patients who suffered acute rejection, clinical manifestations, histopathological features, diagnosis and anti-rejection treatment were summarized and analyzed. In 86 cases (16.7%), of the 516 recipients, 106 episodes of acute rejection occurred, which included 9 with histopathological borderline changes, 36 Banff I rejections, 48 Banff II and 13 Banff III. Among these, 36 were cured by adjusting the dose of immunosuppressant and 65 were reversed by methylprednisolone pulse treatment. Five were methylprednisolone resistant, 3 of whom were given OKT3 treatment and 2 underwent liver retransplantation. Due to potent immunosuppressive agents, acute rejection following an orthotopic liver transplantation lacks typical clinical manifestations and pathological features. Acute rejection is best diagnosed by liver biopsy. Designing rational individualized immunosuppressive regimen based on clinical and pathological features of acute rejection plays an important role in prolonging long term survival of patients.

Usefulness of Rabbit Anti-thymocyte Globulin in Patients With Giant Cell Myocarditis

Giant cell myocarditis (GCM) is an aggressive inflammatory myocardial disease. Immunosuppression is an effective treatment for some cases. However, the duration of action of agents such as muromonab CD3 is short and others such as the calcineurin inhibitors may lead to renal failure. Here we describe the outcome of a novel approach to treatment using rabbit anti-thymocyte globulin (RATG). A retrospective analysis of 6 patients treated with RATG for GCM was performed. Diagnosis was confirmed by endomyocardial biopsy, and RATG was administered with a high dose of corticosteroids. None of the patients had cytokine release syndrome or leukopenia, and 5 had thrombocytopenia (2 of them severe). Only 1 had a serious bleeding event that occurred after implantation of mechanical circulatory support. None developed impaired renal function after the treatment. Five were successfully discharged home with an increase in global left ventricular ejection fraction of 29%. Four are currently alive without recurrent disease, 1 of them after heart transplantation, with a mean follow-up of 970days (423 to 1,875days), left ventricular ejection fraction of 53%, and all in current New York Heart Association Classification class ≤II. Only 1 case had GCM recurrence. There were 2 deaths: one because of intracranial bleeding after mechanical circulatory support implantation and the other caused by primary graft dysfunction. In conclusion, patients with GCM can be successfully immunosuppressed with RATG and corticosteroids, thereby avoiding renal impairment. Early thrombocytopenia is the main adverse event. Larger cohorts of patients are necessary to compare the different immunosuppressant strategies available for GCM in a randomized fashion.

Comparative Analysis of T-Cell Depletion Method for Clinical Immunotherapy—Anti–Hepatitis C Effects of Natural Killer Cells Via Interferon-γ Production

Liver transplantation (LT) is a life-saving treatment for liver cirrhosis patients with hepatocellular carcinoma (HCC). However, 10%–20% HCC recurrence rate after LT is due to the immunosuppression inducing tumor growth. We recently reported a novel immunotherapy with donor liver natural killer (NK) cells to prevent HCC and hepatitis C virus (HCV) recurrence after LT. In this cell processing procedure, Muromonab-CD3 (Orthoclone OKT3, an anti-CD3 antibody) was added to the culture medium to deplete CD3+ T cells to prevent graft-versus-host disease. However, the manufacture of OKT3 was discontinued in 2010, when other treatments with similar efficacy and fewer side effects became available. In this study, we examined alternative reagents for T-cell depletion-MACS GMP CD3 pure (GMP CD3), antithymocyte globulin, and alemtuzumab-for NK cell immunotherapy in the allogeneic setting. We observed that GMP CD3 showed exactly the same effects on liver mononuclear cells as OKT3, including activation of NK cells and depletion of T cells. Interestingly, binding of T-cell depletion antibodies to NK cells led to an anti-HCV effect via interferon-γ production. These results with the use of in vitro culture systems suggested that antibodies which produce T-cell depletion affected NK cell function.

Remarkable stability of clinical grade reagents for gene-engineered T-lymphocyte production

Gene-engineered T-cell immunotherapy has emerged as a promising and potentially curative treatment for patients with numerous cancers, including renal cell carcinoma, metastatic melanoma, synovial cell sarcoma, colon cancer, neuroblastoma and multiple types of leukemia (1), and holds promise for many additional cancers and lifethreatening diseases, including HIV/AIDS. A major limitation to the widespread implementation of gene-engineered T-cell immunotherapy is the cost of obtaining current Good Manufacturing Practices (cGMP) clinical-grade reagents with which to prepare transduced cells. In this issue of Cytotherapy, Lamers et al. show the potential for long-term cold storage and continued use of multiple clinical-grade immunotherapy reagents, in some cases up to 14 years after their indicated expiration dates (2). In their article, the authors methodically evaluate the sterility and function of the T cell-stimulating anti-CD3 monoclonal antibody Orthoclone OKT3 (Janssen Biotech, Inc., Horsham, PA, USA), the widely used transduction reagent RetroNectin (TAKARA BIO INC., Shiga, Japan) and previously produced and qualified clinical-grade retroviral vector supernatant encoding a chimeric antigen receptor for the carbonic anhydrase IX protein (3). These results suggest that the shelf-lives of many required gene therapy transduction reagents can be significantly extended, provided that they are prepared as single-use aliquots according to cGMP standards and stored at 80 C. Although the needs for patient safety and qualityassured reagents are paramount, the prescribed dates of usage for reagents often fall within a short window,

Adenovirus in Solid Organ Transplantation

Abbreviations: CDC, Centers for Disease Control and Prevention; CMV, cytomegalovirus; CTL, cytotoxic T lymphocyte; DNA, deoxyribonucleic acid; EBV, Epstein–Barr virus; FDA, Food and Drug Administration; GALT, gut associated lymphoid tissue; GVHD, graft-versus-host disease; HICPAC, Healthcare Infection Control Practices Advisory Committee; HLA, human leukocyte antigen; HSCT, hematopoietic stem cell transplantation; OKT3, Orthoclone OKT3, muromonab CD3; PCR, polymerase chain reaction.

Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells

The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

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With availability of specific and sensitive HLA antibody assays, the importance of donor specific antibodies (DSA) in liver transplant has grown. Patients undergoing 2nd liver transplant are at high risk of DSA. We present contrasting outcomes in 2 patients. We retrospectively reviewed the DSA in 2 liver recipients and compared them to the clinical outcomes. Pt #1: A 28 year old woman with PBC underwent a living donor liver transplant in 2006. In the next 4 years she received her 2nd, 3rd and 4th liver transplants due to graft failures. Plasmapheresis and IVIG were used at the 3rd & 4th transplants due to high PRA. A positive B cell flow crossmatch was noted at the last transplant. DSA monitoring was initiated after the 4th transplant. Elevated levels of class II antibodies to DR4, DR53 and DQ8 persisted. Patient had recurrence of jaundice after 6 months and showed short lived response to thymoglobulin, plasmapheresis, IVIG and Rituximab with rebound of DSA within a month of antibody therapy. Coincidentally, donors 2, 3 and 4 all had DR4, DR53 and DQ8. The patient underwent Bortezomib therapy as liver biopsy continued to show mixed portal infiltrate & bile duct injury. Pt #2: A 25 yr old woman with autoimmune hepatitis underwent liver transplant in 2006. Graft function deteriorated following steroid non-responsive rejection treated with Thymoglobulin & OK T3. Retransplant was done in early 2011. B cell crossmatch was positive and patient received thymoglobulin, plasmapheresis and IVIG. DSA assay showed the presence of anti-A2, -A11, -DR53, and -DQ8. During a follow up of 11 months, graft function was excellent despite the persistence of high titers of Class II antibodies. In contrast, level of class I antibodies remain under cutoff. Sensitization status needs serious consideration in retransplantation of liver. Avoidance of donors with unacceptable antigens may be crucial in some liver retransplant recipients. A positive B cell crossmatch may portend a poor outcome.

Clinicopathologic Analysis of Acute Vascular Rejection Cases After Renal Transplantation

IntroductionHistopathologic change of acute vascular rejection (AVR) is characterized by intimal arteritis and transmural arteritis. In this report, we discuss the clinicopathologic analysis of AVR cases after renal transplantation. PatientsAVR was diagnosed in 28 renal transplant recipients followed up in our institute between January 2003 and November 2010. ResultsAmong 28 cases of AVR, 18 were mild (v1 in Banff 07 classification), 8 were moderate (v2), and 2 were severe (v3). Interstitial inflammation was present in 25 biopsy specimens. Moderate to severe tubulitis (t2–t3) was present in 10 biopsy specimens and transplant glomerulitis in 17; peritubular capillaritis was in 25 of the 28 biopsy specimens. C4d deposition in peritubular capillaries was observed in 11/28 cases. By using assays with plastic beads coated with human leukocyte antigen (HLA) in the 28 cases, we detected circulating anti-HLA alloantibody in 18 patients, among which 11/28 were donor-specific. Acute antibody-mediated rejection was diagnosed in 6 cases. Among AVR cases, 19/28 displayed steroid-resistant rejection (SRR) requiring greater anti-rejection therapy (ART), including muromonab CD3 injection, gusperimus injections, plasmapheresis, intravenous immune globulin, and/or rituximab injections. Twenty of 28 patients recovered renal allograft function after ART, and 26/28 grafts are functioning. Among the 2 cases of graft loss, only 1 patient lost his graft due to AVR. ConclusionsIn some cases, AVR might be provoked by anti-donor antibodies. The prognosis of the graft exhibiting AVR was relatively good using available immunosuppression.

Clinicopathological analysis of acute vascular rejection cases after renal transplantation

Histopathological change of acute vascular rejection (AVR) is characterized by intimal arteritis and transmural arteritis. In this report, we discuss the clinicopathological analysis of AVR cases after renal transplantation (RTX). AVR was diagnosed in 17 patients from 17 renal transplant patients followed in our institute between January 2003 and September 2008. We retrospectively reviewed these 17 patients. Among 17 cases of AVR, 10 cases were mild (v1 in Banff 07 classification), five were moderate (v2), and two were severe (v3). Interstitial inflammation (i1-i3) was present in all 17 biopsies. Moderate to severe tubulitis (t2-t3) was present in seven biopsies, and transplant glomerulitis (g1-g3) was present in 11, peritubular capillaritis (ptc1-ptc3) was in 15 of 17 biopsies. C4d deposition in peritubular capillary (PTC) was observed in 6 of 17 cases. By assaying with plastic beads coated with anti-human leukocyte antigen (HLA) antigen performed in 17 cases, the circulating ant-HLA alloantibody was detected in 10 patients, of which 5/10 were donor-specific antibodies (DSA). Acute antibody-mediated rejection (AAMR) was diagnosed in three cases. Many of v1 cases, steroid pulse therapy (SP) were effective. In v2 and v3 cases, six of seven were steroid-resistant rejection and were need more anti-rejection therapy (ART), such as muromonab CD3 (OKT3) injection, gusperimus (DSG) injection, plasmapheresis, intravenous immune globulin, and injection of rituximab. Ten of 17 patients recovered their renal allograft functions by ART, and 16 of 17 patients' grafts are functioning. Deterioration of renal allografts' function after biopsies was seen in seven patients with one of them lost their graft. In some cases, AVR might be provoked by anti-donor antibodies. The prognosis of the graft exhibiting AVR was relatively good in present immunosuppression and ART.

Muromonab‐CD3 therapy for refractory rejections after liver transplantation: a single‐center experience during two decades in Japan

Refractory rejections still occur in the liver transplantation (LT) field. The aim of this study was to investigate significant factors for the introduction of therapy with muromonab-CD3 (MCD3) after LT. A total of 1415 LT patients were retrospectively evaluated, and 11 of the recipients received MCD3 therapy because of steroid-resistant rejections. The clinical factors before LT and before MCD3 therapy were investigated. The recipients were retrospectively divided into two groups based on responses to MCD3 therapy, including their clinical courses after MCD3 therapy and their outcomes. The MCD3 therapy had positive effects in LT recipients with the following four factors: low score of model for end-stage liver disease or pediatric end-stage liver disease; earlier time point of the first incidence of rejection; more frequent steroid pulse therapy (SPT) within 2 weeks after LT; and the expression of CD3 in the peripheral blood before MCD3 introduction. Optimal induction of MCD3 triggered recovery from refractory rejections, especially in LT recipients in a stable condition, but not in those in a critical or compromised condition.

Successful Living-Donor Liver Transplantation for Chronic Hepatic Graft-Versus-Host Disease After Bone Marrow Transplantation for Chronic Granulomatous Disease

A 10-year-old boy diagnosed postnatally with chronic granulomatous disease (CGD) underwent human leukocyte antigen-identical bone marrow transplantation (BMT) from an unrelated donor. After engraftment, he developed an erythematous rash, diarrhea, hepatomegaly, and elevated liver enzyme levels and was treated for acute graft-versus-host disease (GVHD) by increased immunosuppressive therapy with tacrolimus and corticosteroids. Although the gut and skin GVHD improved, the hepatic GVHD persisted and was confirmed as chronic hepatic GVHD on liver biopsy. Owing to the persistence of hyperbilirubinemia, elevated liver enzyme levels, thrombocytopenia, and coagulopathy, the chronic GVHD was managed with low-dose corticosteroids. After transplantation, the patient showed complete engraftment of the transplanted bone marrow (99%), and bone marrow biopsy and other investigations showed no evidence of the relapse of CGD. At 3 years after BMT, liver function deteriorated, and the boy underwent living-donor liver transplantation (LDLT) from his mother, a heterozygous donor. The left-lobe graft weighed 510 g, and the graft-to-recipient weight ratio was 1.6%. Both the portal vein and hepatic artery of the graft were flushed with muromonab-CD3 (Orthoclone OKT3) contained in a histidine-tryptophan-ketoglutarate solution to wash out and eliminate donor lymphocytes. Histopathological examination of the explanted liver revealed the presence of ductopenia with foam-cell arteriopathy, which was consistent with chronic hepatic GVHD. Postoperative immunosuppression consisted of low-dose tacrolimus and corticosteroids to prevent GVHD. Acute cellular rejection occurred during the postoperative course, but severity decreased after treatment. The patient was discharged on postoperative day 60 without complications, and has done well on 12 months follow-up with normal liver function. Graft-versus-host disease, a major cause of liver dysfunction after BMT, occurs in 60% to 80% of patients receiving marrow from a partially human leukocyte antigen-matched family donor. Given the host immunosuppression related to BMT, the risk of passenger lymphocyte-associated GVHD in the present case was an additional complication which required additional measures to avoid allogeneic reactions arising from passenger lymphocytes in the graft. To reduce the number of donor-derived passenger lymphocytes, muromonab-CD3 was used for graft perfusion just after harvesting. OX40 (CD134), a member of the tumor necrosis factor receptor superfamily, is expressed on activated T cells, including CD4(+)CD25(+) T regulatory (Treg) cells. We monitored CD4 OX40/CD4 CD25++ ratio as a marker of GVHD before and after LDLT because CD4 OX40/CD4 CD25++ ratio was recently reported to be a reliable indicator of the onset of chronic GVHD donor (1). In the present case, CD4 OX40/CD4 CD25++ ratio remained stable before and after LDLT (Fig. 1), and no evidence of GVHD was obtained.FIGURE 1.: Change in CD4 OX40/CD4 CD25++ ratio after transplantation.Although many cases of liver transplantation for chronic liver GVHD after BMT have been reported, few have involved LDLT, especially the use of liver tissue from the same donor (2–11). Bone marrow transplantation is commonly indicated in leukemia patients. In the case of benign diseases such as CGD, when BMT is successful, the possibility of recurrence of original disease is low. On this basis, liver transplantation is a suitable approach to the treatment of chronic liver GVHD after BMT. Our case was a LDLT to CGD patient after BMT. The liver donor was different to the BMT donor, and heterozygous to the CGD. At least with regard to CGD, no problems derived from the use of a heterozygous donor have occurred. Satoshi Yokoyama Mureo Kasahara Akinari Fukuda Shuichi Sato Department of Transplant Surgery National Center for Child Health and Development Tokyo, Japan Tetsuya Mori Department of Pediatric Oncology National Center for Child Health and Development Tokyo, Japan Atsuko Nakagawa Department of Pathology National Center for Child Health and Development Tokyo, Japan Akira Matsui Department of Transplant Surgery National Center for Child Health and Development Tokyo, Japan

Severe acute‐hybrid rejection occurring nine months after kidney transplantation: a report of rescue by orchestration of antirejection therapies

Abstract: Although a majority of acute rejection (AR) in non‐sensitized recipients is T‐cell‐mediated by primed T cells, recent studies have shown that antibody‐mediated acute rejection occurs in 20–30% of AR, and that it is often refractory to conventional antirejection therapy; possibly leading to graft loss. We report a case of severe acute‐hybrid rejection consisting of both features in a non‐sensitized kidney recipient, which was rescued by the orchestration of antirejection therapies. A 33‐yr‐old Japanese male, with advanced‐stage chronic kidney disease with an unknown etiology, underwent a HLA 3/6 mismatch and ABO‐compatible living‐related kidney transplantation preemptively. He had an excellent clinical course, except for initial cytomegalovirus infection, with good graft function [serum creatinine (sCr) 1.1 mg/dL]. Nine months later, his creatinine abruptly increased to 2.1 mg/dL, when graft biopsy revealed acute T cell‐mediated rejection (ATMR) grade IA, and simultaneous acute antibody‐mediated rejection (AAMR) grade I. Antirejeciton therapy, comprising methyl‐prednisolone pulse and 15‐deoxyspergualin, and second line rituximab and plasmapheresis, was ineffective. Moreover, histologically and clinically, the rejection status deteriorated (ATMR grade III and AAMR grade III, max sCr 4.0 mg/dL). Next, we administered muromonab CD3 and basiliximab, which could eradicate the complicated severe AR without opportunistic infection, even under the strong immunosuppression. The present case implies that high‐grade combined rejection can respond to anti‐CD 20 and anti‐CD25 mAbs, without serious complication; however, post‐operative, thorough appropriate monitoring of immunosuppression is important because its effects are limited.