Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

Hamilton, Douglas A.; Goodwin, Joseph; Clarke, Mary Beth; Moulin, Gary C. du; Liu, Victor; Caplan, Barry; Babbitt, Bruce

doi:10.1002/bit.260430805

Cited by 4 publications

(1 citation statement)

References 8 publications

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“…The assay is based on the ability of activated, not resting, T cells to proliferate in response to the low dose (1 ng/ml) of PMA, the protein kinase C activator (3,4). When this assay was used to evaluate the effect of RBC on the levels of proliferative response of the PBMC in the secondary culture, there were no statistical differences of proliferation of the ex vivo-activated T cells between PBMC with and without RBC addition.…”

Section: Compositions Of Cd3mentioning

confidence: 99%

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Goodwin¹,

Zhang²,

Babbitt³

et al. 2002

Clin Vaccine Immunol

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Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4 ؉ and CD8 ؉ T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1␤ (IL-1␤) produced by PBMC upon stimulation by OKT3 were increased (P < 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.Lymphocytes are among the most extensively studied cells of the hematopoietic system because of their central role in the generation of immune responses. Information provided from the study of T lymphocytes is important not only in understanding the basic concepts of immune function but also in enabling the development of lymphocyte-based adoptive immune therapies. Lymphocytes can be collected from the peripheral blood, lymphoid tissues, and certain internal organs. In most cases, lymphocytes are initially isolated from the peripheral blood compartment and purified by Ficoll density gradient centrifugation. However, regardless of the method used to isolate T cells or peripheral blood mononuclear cells (PBMC), there always exists a low level of contaminating red blood cells (RBC). In addition, when PBMC are isolated on a large scale, as with most ex vivo adoptive immunotherapy approaches, the level of contaminating RBC increases even further. It has been shown previously that lymphocytes in whole blood stimulated with mitogen produce more interleukin-2 (IL-2) than Ficoll-Hypaque-purified lymphocytes in culture (5). What remains unknown is the effect various levels of contaminating RBC have on the ability of well-characterized Tcell stimulants to activate lymphocytes under normal cell culture conditions.A unique form of outpatient adoptive immunotherapy referred to as autolymphocyte therapy (ALT) for the treatment of patients with metastatic renal cell carcinoma has been developed (6, 6a). Patients are infused monthly with ϳ10 9 T lymphocytes activated ex vivo in a conditioned medium containing a mixture of OKT3 (mouse monoclonal anti-CD3 antibody) and a broad panel of autologous cytokines. The cytokine mixture is generated by stimulation of patient PBMC ex vivo with 25 ng of OKT3/ml for 3 days during the first cycle of the therapy (8). During the secondary cycles, i.e., monthly, of therapy, patient PBMC are cultured with the autologous cytokine mixture from the first cycle of therapy for 5 days and then infused back into the patient.For this procedure, lymphoapheresis is performed for each cycle to collect large numbers of PBMC. The resulting apheresis cell products (ACP) are highly enriched in white blood cells and contain various amounts of RBC, platelets, ...

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Section: Compositions Of Cd3mentioning

confidence: 99%

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Goodwin¹,

Zhang²,

Babbitt³

et al. 2002

Clin Vaccine Immunol

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Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Moulin

Pitkin

Shen

et al. 1994

Cell Culture Engineering IV

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Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Moulin¹,

Pitkin²,

Shen³

et al. 1994

Cytotechnology

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Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congenital and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA. Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company's ex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activated ex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody. We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 in fusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations.(ABSTRACT TRUNCATED AT 250 WORDS)

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Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

Cited by 4 publications

References 8 publications

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

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