Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells.

Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability. CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation.
A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 10(6) vs 1502 ± 1066 × 10(6); P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 10(6) transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients.
Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes.

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

BACKGROUND
Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation.
OBJECTIVE
Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA).
METHODS
The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA).
RESULTS
During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed.
CONCLUSIONS
The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise.

BACKGROUND
The safety and clinical efficacy of adoptive transfer of prospectively isolated antigen-specific T cells are well established. Several competing selection methods are available, one of which is based on immunomagnetic enrichment of T cells secreting IFNγ after incubation with the relevant antigen. The proprietary, GMP-conforming selection technology, called ‘cytokine capture system’ (CCS) is established in many laboratories for the CliniMACS Plus system. It is robust and efficient, but labour-intensive and incompatible with a single-shift working schedule. An automatic immunomagnetic cell processing system, CliniMACS Prodigy (‘Prodigy’), including a protocol for fully automatic CCS execution was recently released.
METHODS
Feasibility of clinical-scale CMV-specific T-cell selection using Prodigy was evaluated using leukoapheresis products from five healthy CMV sero-positive volunteers. Clinical reagents and consumables were used throughout.
RESULTS
The process required no operator input beyond set-up and QC-sample collection, that is, feasibility was given. An IFNγ-secreting target T-cell population was detectable after stimulation, and >2 log-scale relative depletion of not CMV-reactive T cells in the target population was achieved. Purity, that is the frequency of CMV-reactive T cells among all CD3(+) cells ranged between 64 and 93%.
CONCLUSIONS
The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

Detection and identification of platelet-associated alloantibodies by a solid-phase modified antigen capture enzyme-linked immunosorbent assay method and its correlation to platelet refractoriness in multiplatelet concentrate-transfused patients.

Platelets express a variety of polymorphic glycoproteins (GPs), such as GPIIb/IIIa, GPib/IX, GPla/Ila, GPIV, and class I human leukocyte antigen. In the platelet transfusion setting, alloimmunization involves the production of antibodies against these glycoproteins. Patients transfused with multiple units of platelet concentrates for longer periods are the main individuals with platelet alloimmunization.
This study was performed to detect the development of platelet antibodies in patients who are transfused with multiple units of leukodepleted platelet concentrates, such as those with hemato-oncologic diseases and bone marrow failure syndromes. The method used was solid phase modified antigen capture enzyme-linked immunosorbent assay. Platelet refractoriness was assessed by measuring the corrected count increment at 1 and 24 hours after transfusion.

BSA Antigen Concentrate

F031G Cygnus Technologies 1 ml 219 EUR

Insulin Antigen Concentrate

F043H Cygnus Technologies 100 ul 373 EUR

HSA Antigen Concentrate

F057H Cygnus Technologies 1 ml 296 EUR

S.cerevisiae Antigen Concentrate

F138H Cygnus Technologies 500 ul 411 EUR

P.pastoris Antigen Concentrate

F143Z Cygnus Technologies 0.5 ml 873 EUR

P.fluorescens Antigen Concentrate

F453W Cygnus Technologies 1 ml 2683 EUR

P.fluorescens Antigen Concentrate

F453X Cygnus Technologies 1 ml 411 EUR

PER.C6 Antigen Concentrate

F533H Cygnus Technologies 200 ul 642 EUR

MDCK Antigen Concentrate

F803X Cygnus Technologies 1 ml 527 EUR

MDCK Antigen Concentrate

F804 Cygnus Technologies 1 mL 989 EUR

HeLa Antigen Concentrate

F813X Cygnus Technologies 1 ml 527 EUR

CAPÐ’ Antigen Concentrate

F823W Cygnus Technologies 1 ml 1605 EUR

CAPÐ’ Antigen Concentrate

F823X Cygnus Technologies 400 ul 604 EUR

SF9 Antigen Concentrate

F843X Cygnus Technologies 200 ul 488 EUR

CHO Lysate Antigen Concentrate

F018H Cygnus Technologies 200 ul 604 EUR

CHO Lysate Antigen Concentrate

F018R Cygnus Technologies 1 ml 6225 EUR

hTransferrin Reference Antigen Concentrate

F037RAC Cygnus Technologies 1 ml 411 EUR

Protein A Antigen Concentrate

F044 Cygnus Technologies 200 ul 257 EUR

Bovine IgG Antigen Concentrate

F073G Cygnus Technologies 200 ul 257 EUR

Bovine IgG Antigen Concentrate

F073H Cygnus Technologies 1 ml 835 EUR

Bovine Transferrin Antigen Concentrate

F121G Cygnus Technologies 300 ul 373 EUR

Pichia pastoris Antigen Concentrate

F143X Cygnus Technologies 200 ul 873 EUR

SP2/0 Antigen Concentrate

F186C Cygnus Technologies 200 ul 565 EUR

NS/0 Antigen Concentrate

F227C Cygnus Technologies 1 ml 642 EUR

A549 HCP Antigen Concentrate

F233G Cygnus Technologies 200 ul 450 EUR

Protein A Antigen Concentrate

F403H Cygnus Technologies 200 ul 257 EUR

E.coli HCP Antigen Concentrate

F413H Cygnus Technologies 200 ul 488 EUR

L.lactis HCP Antigen Concentrate

F493G Cygnus Technologies 200 ul 488 EUR

Detection and Identification of Platelet-Associated Alloantibodies by a Solid-Phase Modified Antigen Capture Elisa (MACE) Technique and Its Correlation to Platelet Refractoriness in Multi platelet Concentrate Transfused Patients.

  • Platelets express glycoproteins (IIb/IIIa, Ib/IX, Ia/IIa, IV, and HLA-1) that are polymorphic and can become targets for antibody responses. Patients at threat are those who received multiple platelet transfusions. Modified antigen capture elisa (MACE) is a qualitative solid phase Elisa designed to detect IgG antibodies against platelet specific antigens. The study has been carried out over a period of 2 years. A total of 100 patients were selected, who had been transfused with at least 15 units of platelet concentrate.
  • All patients were having either hematological malignancies or bone marrow failure syndromes. Platelet antibodies were identified using MACE-1&2. Data was analysed statistically, using odds ratio (OR) with 95 % confidence interval. 39 % of the patients were found to be alloimmunized against platelet antigens, of which eleven showed refractoriness. Six patients (54.5 %) with HLA-1, two patients (9.5 %) with GPIb/IX, two patients (40 %) with both HLA-1 and GPIIb/IIIa, and one patient with GPIIb/IIIa antibodies showed refractoriness.
  • Production of HLA-1 antibody and the development of refractoriness was found to be significant with OR 14.05 and P value 0.0025. MACE-1&2 enabled specific detection and identification of platelet antibodies, which in turn correlated well with the development of refractoriness in multi transfused patients. GPIb/IX was detected as the commonest antibody in our patient population, which is in variance with Europian studies where it is GPIa/IIIa (HPA-1a/5b). This technique should be utilised in patients who are at an increased risk of developing alloimmunisation due to repeated platelet transfusions.

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