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Background Aims: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies., Methods: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€)., Results: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices., Conclusions: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs., (Copyright © 2020 International Society for Cell and Gene Therapy. All rights reserved.)

EBV-CTL immunotherapy targets EBV antigens expressed by tumor cells in PTLD. Data on outcome of EBV-CTL in pSOT patients are limited. The aim of the study is to describe our experience with allogeneic, third-party EBV-CTL for the treatment of PTLD in pSOT patients in a single tertiary center. Retrospective review was performed of all pSOT patients who received EBV-CTL for PTLD. PTLD was diagnosed using World Health Organization histologic criteria. EBV-CTLs were derived from human leukocyte antigen-typed, EBV-seropositive third-party donors, and cryopreserved and maintained by an accredited national blood transfusion service. Ten patients received EBV-CTL for histologically proven PTLD from 1999 to 2016 following liver (n=5), combined intestinal/liver (n=4), and liver/kidney (n=1) transplantation. PTLD occurred at median age of 40 months (range: 12-144) and median post-transplant interval of 8 months (range: 2-107). Seven had monomorphic, two had polymorphic, and one had Hodgkin-type PTLD. All were of B-cell origin and EBV-positive on histology. EBV-CTL achieved an overall remission rate of 80% (8 of 10). Transient adverse effects included fever, tachycardia, and vomiting. None developed graft-versus-host disease or opportunistic infections. EBV-CTL is an effective treatment for PTLD in pSOT patients, with good remission rate and minimal toxicity., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes., (© 2014 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

We present the results of a multicenter clinical trial using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) generated from EBV-seropositive blood donors to treat patients with EBV-positive posttransplantation lymphoproliferative disease (PTLD) on the basis of the best HLA match and specific in vitro cytotoxicity. Thirty-three PTLD patients who had failed on conventional therapy were enrolled. No adverse effects of CTL infusions were observed and the response rate (complete or partial) in 33 patients was 64% at 5 weeks and 52% at 6 months. Fourteen patients achieved a complete remission, 3 showed a partial response, and 16 had no response at 6 months (5 died before completing treatment). At 5 weeks, there was a significant trend toward better responses with higher numbers of CD4(+) cells in infused CTL lines (P = .001) that were maintained at 6 months (P = .001). Patients receiving CTLs with closer HLA matching responded better at 6 months (P = .048). Female patients responded better than male patients, but the differences were not statistically significant. Our results show that allogeneic CTLs are a safe and rapid therapy for PTLD, bypassing the need to grow CTLs for individual patients. The response rate in this poor prognosis patient group is encouraging.

Adoptive immunotherapy using autologous Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes (auto-CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto-CTL remains constrained. Generation is time-consuming, and auto-CTL cannot be established in patients treated with the B-cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo-CTL) offers immediate accessibility. Allo-CTL has previously shown efficacy in "early" polyclonal- PTLD. We treated three patients with aggressive, advanced monoclonal-PTLD following solid-organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide-specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor-derived tumor targeted EBV-specific CTL; the other died of non-PTLD related pathology. In the third patient, autopsy demonstrated homing of allo-CTL at the tumor site. Larger prospective studies of EBV-specific allo-CTL in PTLD are warranted.

Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCRalpha beta+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

Background: Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) is a common, often fatal, complication of bone-marrow and solid-organ transplantation. Since tumour growth results from inadequate T-cell control of latent EBV, new immunotherapeutic approaches to treatment are being pioneered., Methods: In a phase 1/2 trial, eight patients with progressive PTLD unresponsive to conventional treatment were given one to six infusions of partly HLA-matched allogeneic EBV-specific cytotoxic T lymphocytes (CTLs) from a frozen bank of CTLs derived from healthy blood donors., Findings: Of the five patients who completed treatment, three had complete remission and two had no clinical response. One patient partly responded after two infusions. No graft-versus-host disease or allo-specific antibodies were detected, and graft function improved in three cases. Tumour responses were mainly seen in those with early, localised, polyclonal disease. EBV load in peripheral blood fell to undetectable levels in all patients who responded to treatment, but was more variable in those who did not., Interpretation: Treatment of EBV-associated PTLD with partly HLA-matched CTLs grown from unrelated donors is effective. Spontaneous remission is very unlikely to account for tumour regression in our patients; however, a larger, controlled trial is needed to assess this treatment further. The frozen bank of allogeneic CTLs is less prohibitively labour intensive and expensive for wide scale use than treatment with autologous CTLs. Such banks could be established to treat other infectious and neoplastic diseases in many patients.

The infectivity of 15 cryopreserved Theileria spp. sporozoite stabilates was assessed semi-quantitatively by titration using naive peripheral blood mononuclear cells (PBM) in vitro in multi-well plates. Using the method described, the effective dilution, which would result in 50% of replicate wells infected (ED50), was calculated. The ED50 for 11 Theileria annulata stabilates in bovine PBM ranged from 10(-2.6) to 10(-4.2) dilutions of 1 tick equivalent (t.e.) ml(-1), one stabilate of Theileria parva 10(-2.2)t.e.ml(-1); and three Theileria lestoquardi stabilates in ovine PBM, from 10(-1.5) to 10(-1.8)t.e.ml(-1). Two of the T. annulata stabilates had been used individually to infect groups of calves: stabilate 52 produced more severe disease responses than stabilate 67, as measured by prepatent period, parasitosis, parasitaemia and death or recovery. This corresponded with the sixfold difference found in vitro between the ED50's of these two stabilates. This method is useful not only to measure the infection potential of the sporozoite stabilates but also as an in vitro model for chemotherapeutic and immunological studies of the early stages of theileriosis.

Background: Adoptive immunotherapy with autologous and donor-derived cytotoxic T lymphocytes (CTL) has recently been used to treat Epstein Barr virus (EBV)-positive posttransplant lymphoproliferative disease (PTLD)., Methods and Results: We report complete regression of EBV-positive PTLD in an 18-month-old small bowel and liver transplant recipient after one infusion of partially human leukocyte antigen (HLA)-matched EBV-specific CTL grown ex vivo from an EBV seropositive unrelated blood donor. No infusion-related toxicity or evidence of graft-versus-host disease was observed. The tumor showed signs of regression within 1 week and EBV load in peripheral blood dropped to undetectable levels. Limiting dilution analyses (LDA) detected no EBV-specific CTL precursor (CTLp) cells before the infusion, and high numbers of CTLp at 4 hr and 24 hr post-CTL infusion. There was a reversal of the CD4/8 ratio in peripheral blood and an increase in HLA-DR positive CD8 cells. The patient has been in complete remission for 24 months., Conclusion: If this success is repeated in more PTLD patients, then stored CTL could be used for antiviral and antitumor therapies in immunocompromised patients.

An in vitro method for testing activity of buparvaquone in serum on the infection and development of Theileria in its bovine host mononuclear cells is described and results compared with the effect exhibited in vivo. Serum samples were collected over a time course from calves in a clinical trial of 5 mg kg(-1) buparvaquone prophylaxis on Theileria annulata or T. parva experimental infection. To evaluate drug levels and persistence in each animal for a period of 14 days and its effect on the early infection stages, the sera were tested on established macroschizont infected cell lines and against the in vitro infection and development process of the sporozoite and trophozoite stages of the two Theileria species. Results from the in vitro assays show that buparvaquone in serum can completely prevent the establishment of Theileria infection during the first 48 h after administration at 5 mg kg(-1). After seven days, levels are sufficient to delay the establishment of infection. The drug is more effective in the prevention of the de novo development of the parasite in cells than against established macroschizont infected cell culture. At low concentrations, it is more effective against T. parva than against T. annulata. Drug effect peaks during the first 24 h but residual effect persists for 14 days, particularly against T. parva infection. These novel findings demonstrate how high doses of buparvaquone could over-protect calves if used in the 'infection-and-treatment' method of immunisation when drug is administered prophylactically at the same time as infection with live sporozoites. It is suggested that in certain high Theileria risk situations there may be potential for the immunoprophylactic use of buparvaquone without simultaneous infection. The in vitro assay itself has been shown to be of value as a model for Theileria establishment in cattle.

A clinical trial testing the prophylactic effect of a 5 mg kg-1 dose of buparvaquone on either Theileria annulata or Theileria parva experimental infections of calves demonstrated its efficacy for periods of at least seven days. The drug given 1 h or seven days before 50% lethal T. annulata sporozoite infection protected all eight calves, but prophylaxis was insufficient after 14 days to protect two out of four calves from severe reaction. When immunity was challenged by a lethal second parasite dose a month after the first, all these calves were immune. In the T. parva trial, calves given drug 1 h or seven days before a 25% lethal infection underwent minimal reaction, but some were over-protected and were susceptible to a similar challenge sporozoite dose. Although drug levels remaining 14 days after prophylaxis protected these calves from the mild challenge, some parameters measured were within the range of the 'no drug' control group. These results indicated the effectiveness of a single 5 mg kg-1 dose of buparvaquone for more than seven days but also the potential risk of its use in the infection and treatment method of immunisation. It is suggested that there may be circumstances where simple field prophylactic treatment with buparvaquone may be beneficial.