Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412

Abstract

Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-␥, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs. (Blood. 2011;118(26):6772-6782)

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

“…Differential activation of T cells by superagonistic mAbs may therefore encompass different conformational forms of CD28 and/or differing proportions of monovalent binding versus divalent ligation and lattice formation, which give rise to differential signaling properties. This is consistent with the expression of CD28 by virtually all CD4 + T cells and about half of CD8 + T cells but preferential superagonistic activation of only a proportion of cells correlating with specific subset categories such as effector memory cells (11,13), demonstrating that not all CD28 molecules are signalingcompetent through superagonistic stimulation.…”

“…Nevertheless, in the first-in-man phase I trial of TGN1412, all six volunteers receiving the Ab suffered systemic inflammatory response syndrome and life-threatening multiorgan failure (4). The reason(s) for the failure of preclinical testing to predict the severity of the reactions in humans has been the subject of speculation and investigation (5)(6)(7)(8)(9)(10)(11)(12)(13). Suggested contributing factors have included a role for differences between species in Fcmediated interactions with FcgRs in bringing about cross-linking of CD28 and thereby triggering T cell activation and associated cytokine release and proliferation of PBMCs (8,9), although experimental data on the possible influence of Fc on the immunomodulatory activity of TGN1412 has to date been lacking.…”

Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors

“…Although initially proposed to be related solely to CD28 expression on a particular subset of T cells and independent of hFcgR expression, it is now evident that hFcgR and in particular hFcgRIIB is critical for this response, at least in predictive in vitro assays (53,54) and our own observations (55). However, what is not yet resolved is where the hFcgR interaction occurs in vivo to cause the resulting, rapid, cytokine storm.…”

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

“…For example, CAR-T cells undergo rapid proliferation (up to 10 4 -fold expansion) upon encountering antigen-positive cells in the patient, which has resulted in serious cases of tumor lysis syndrome (TLS) and fatal cytokine release syndrome (CRS) (6)(7)(8). Further complications may be caused by the persistent on-target activity of CAR-T cells.…”