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The storm has cleared: Lessons from the CD28 superagonist TGN1412 trial

Article

The storm has cleared: Lessons from the CD28 superagonist TGN1412 trial

Abstract

The life-threatening cytokine-release syndrome suffered by six volunteers in a Phase I clinical trial following administration of the CD28 superagonist antibody TGN1412 (developed by TeGenero) in March 2006 was completely unpredicted by the preclinical studies. Here, Thomas Hünig, main founder of TeGenero, describes the recent investigations into what went wrong and discusses the lessons learnt for future clinical trials.
The storm has cleared: lessons from
the CD28 superagonist TGN1412 trial
Thomas Hünig
The life-threatening cytokine-release syndrome suffered by six volunteers in a PhaseI
clinical trial following administration of the CD28 superagonist antibody TGN1412
(developed by TeGenero) in March 2006 was completely unpredicted by the preclinical
studies. Here, Thomas Hünig, main founder of TeGenero, describes the recent investigations
into what went wrong and discusses the lessons learnt for future clinical trials.
Thomas Hünig is at the
Institute for Virology and
Immunobiology,
Versbacher Strasse 7,
D-97078 Würzburg,
Germany.
e-mail: huenig@vim.uni-
wuerzburg.de
doi:10.1038/nri3192
Published online 10 April 2012
Corrected online 18 April 2012
When, on 13March 2006, six healthy young men received
an intravenous injection of TGN1412, a first-in-class
superagonist monoclonal antibody specific for the Tcell
co-stimulatory molecule CD28, a twofold storm arose.
First, all the volunteers experienced a life-threatening
systemic release of pro-inflammatory cytokines, termed
a cytokine-release syndrome (CRS), which they survived
with the help of expert care after being transferred to an
intensive care unit 16hours into the clinical trial1. In
addition, a shock wave went through the scientific com-
munity, the biotechnology industry and the regulatory
authorities, who asked why preclinical testing had failed
to warn of the impending catastrophe. Although the
European Medical Agency acted swiftly to change the
methodology for calculating the entry dose of first-in-
human biologicals (see below), it took 6years to under-
stand why the three sets of preclinical data (from rodents,
primates and human cells) used to support this PhaseI
clinical trial had failed to predict the cytokinestorm.
CD28 co-stimulates Tcell responses when it is engaged
by its ligands, CD80 and CD86, on ‘professional’ antigen-
presenting cells during antigen recognition. TGN1412
induces particularly strong CD28 signalling, allowing
Tcell activation without the need for simultaneous
strong Tcell receptor (TCR) engagement2. Nevertheless,
CD28 superagonists depend on weak or tonic TCR
signals, which they amplify3,4. TGN1412-mediated Tcell
activation can thus be viewed as a special type of
Tcell stimulation, in which most of the activating signal
is delivered through CD28 rather than through theTCR.
When CD28 superagonists were first discovered and
tested for invivo biological activity in rats, polyclonal Tcell
activation was observed, but the response was rapidly
dominated by an expanded population of hyperactive reg-
ulatoryT (TReg) cells5. Indeed, CD28 superagonists effec-
tively induced TReg cell activation and clinical improvement
in multiple models of autoimmune and inflammatory
diseases in both rats and mice6, and adverse effects, as
seen in the human volunteers, were absent. However,
recent work has shown that the administration of CD28
superagonists results in considerable levels of circulating
pro-inflammatory cytokines if mice are first depleted of
TReg cells, indicating that the TReg cell-mediated response is
crucial for preventing systemic inflammation in rodents7.
Why did this mechanism fail to protect the human
volunteers receiving TGN1412? It is now clear that CD4+
effector memoryT (TEM) cells — which are mostly found
in tissues and are poised for immediate cytokine release
— were the source of the cytokines interferon-γ (IFNγ),
tumour necrosis factor (TNF) and interleukin-2 (IL-2)
that mediated the CRS seen in the volunteers8,9. The accu-
mulation of TEM cells during the life of an individual is
driven by multiple exposures to infectious agents, which
does not occur in laboratory rodents housed under clean
conditions. In other words, in the presence of TGN1412,
the balance between TReg cell and TEM cell numbers is dis-
advantageous for humans compared with mice or rats. Of
note, however, studies in rats have shown that low doses
of a CD28 superagonist only expand TReg cell populations
and do not activate conventional CD4+ Tcells10, sug-
gesting that the very high dose of TGN1412 used in the
clinical trial (see below) aggravated theproblem.
But what about primates? TGN1412 had been given
to cynomolgus macaques at up to 500 times the dose
given to the healthy volunteers. Contrary to claims made
in the wake of the disaster, macaque and human CD28
have identical extracellular domains and bind TGN1412
with the same affinity11. However, a surprisingly sim-
ple explanation for the absence of a TGN1412-induced
CRS in macaques has recently been found: macaque
CD4+ Tcells, but not human CD4+ Tcells, lose CD28
expression during their differentiation into TEM cells8.
Unfortunately, this was a detail that had gone unnoticed
for many years of primatetesting.
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Finally, the failure of assays using human periph-
eral blood mononuclear cells (PBMCs) to predict
the TGN1412-mediated CRS required an explanation.
TGN1412 does not elicit cytokine or proliferative
responses when added in a soluble form to PBMC cul-
tures. By contrast, the therapeutic monoclonal antibody
OKT3, which is specific for the TCR component CD3
and has been in clinical use for decades to treat transplant
rejection, induces cytokine production from PBMCs and
is a notorious CRS inducer12. Based on a serendipitous
observation, we have now found that if the Tcells are first
cultured at a high cell density, soluble TGN1412 can sub-
sequently activate these Tcells as potently as OKT3 (REF.9).
We suggest that cellular contacts during the high-density
pre-culture of PBMCs restore the tonic TCR signal that
TGN1412 needs as a ‘substrate’ for amplification and that
Tcells lose during their transient sojourn in the blood (see
Supplementary informationS1 (figure)). Indeed, mouse
CD4+ Tcells leaving lymphoid tissues and entering the
circulation have previously been shown to lose this sub-
threshold signal, which is strictly dependent on cell–cell
contacts and involves the scanning of MHC molecules13.
Pre-culturing PBMCs under tissue-like conditions
before using them in standard activation assays allowed
us to study the response to soluble TGN1412 in detail9.
We found that a TGN1412 concentration of 1 μg per ml
of bloodthe level predicted to result from the bolus
application of TGN1412 at a dose of 0.1 mg per kg of
body weight during the London trial14 — was not only
close to saturation with regard to receptor occupancy15,
but also way beyond the amount required for maximum
cytokine release9. In fact, small amounts of TNF can be
detected at a 50-fold lower concentration of TGN1412,
and if TGN1412 had been administered using the current
‘minimum anticipated biological effect level’ (MABEL)-
based calculations for first-in-human applications, the
entry dose would have been at least 200-fold lower.
Furthermore, we showed using this novel invitro assay
that the TGN1412-induced release of pro-inflammatory
cytokines is as sensitive to inhibition by the cortico steroid
dexamethasone as the release induced by OKT3 (for
which glucocorticoids are an effective means to prevent
the CRS). Thus, building on the clinical experience with
OKT3, it seems likely that corticosteroid co-medication,
and appropriate dosing, could provide appropriate
management of the side-effects ofTGN1412.
As expected, the pre-conditioning assay to mimic
tissue-like conditions also increases Tcell responses to
other activating antibodies and to antigens. Beyond help-
ing us to understand what went wrong in the TGN1412
trial, the introduction of the pre-conditioning assay will,
hopefully, improve the relevance of preclinical research
using Tcells from the blood for numerous therapeutic
antibodies.
The key lesson from the disastrous TGN1412 trial
is not to confuse ‘absence of evidence’ with ‘evidence of
absence’ in preclinical trials. The three test systems used
— an analogous rodent model, a primate model using
TGN1412 itself, and conventional human PBMC cultures
— all failed to provide evidence for the toxic potential of
the antibody for distinct and unrelated reasons. It was an
inherent deficit of the ‘no observed adverse effect level’
(NOAEL)-based calculations of first-in-human doses that
if the animal model failed to respond for some unfore-
seen reason (in this case, the lack of CD28 expression by
macaque CD4+ TEM cells), the requested dose escalation
leads to a very high tolerated level (here, 50 mg per kg),
from which the human starting dose was calculated based
on a prescribed adjustment factor plus a safety margin. By
contrast, the MABEL method does not rely on an adverse
invivo effect, but uses receptor occupancy assays (when
feasible) and invitro surrogate response markers (such as
cytokine release or the upregulation of activation mark-
ers) to arrive at an invitro dose at which the drug may just
begin to work, and then adds a safety margin to the calcu-
lated dose required to obtain corresponding plasma levels
invivo. By teaching us the superiority of the MABEL-
based approach over the NOAEL-based approach for
entering innovative and highly active biopharma ceuticals
into humans, the disastrous outcome of the London trial
of 2006 may have made a positive contribution to the
future development of immunomodulatorydrugs.
1. Suntharalingam, G. etal. Cytokine storm in a phase1 trial of the
anti‑CD28 monoclonal antibody TGN1412. N.Engl. J.Med. 355,
1018–1028 (2006).
2. Tacke, M., Hanke, G., Hanke, T. & Hunig, T. CD28‑mediated
induction of proliferation in resting Tcells invitro and invivo
without engagement of the Tcell receptor: evidence for functionally
distinct forms of CD28. Eur. J.Immunol. 27, 239–247 (1997).
3. Dennehy, K.M. etal. Mitogenic CD28 signals require the exchange
factor Vav1 to enhance TCR signaling at the SLP‑76–Vav–Itk
signalosome. J.Immunol. 178, 1363–1371 (2007).
4. Levin, S.E., Zhang, C., Kadlecek, T.A., Shokat, K.M. & Weiss, A.
Inhibition of ZAP‑70 kinase activity via an analog‑sensitive allele
blocks Tcell receptor and CD28 superagonist signaling. J.Biol.
Chem. 283, 15419–15430 (2008).
5. Lin, C.‑H. & Hunig, T. Efficient expansion of regulatory T‑cells invitro
and invivo with a CD28 superagonist. Eur. J.Immunol. 33,
626–638 (2003).
6. Hunig, T. Manipulation of regulatory T‑cell number and function with
CD28‑specific monoclonal antibodies. Adv. Immunol. 95, 111–148
(2007).
7. Gogishvili, T. et al. Rapid regulatory T‑cell response prevents
cytokine storm in CD28 superagonist treated mice. PLoS ONE 4,
e4643 (2009).
8. Eastwood, D. etal. Monoclonal antibody TGN1412 trial failure
explained by species differences in CD28 expression on CD4+
effector memory T‑cells. Br. J.Pharmacol. 161, 512–526 (2010).
9. Römer, P.S. etal. Preculture of PBMC at high cell density increases
sensitivity of T‑cell responses, revealing cytokine release by CD28
superagonist TGN1412. Blood 118 , 6772–6782 (2011).
10. Beyersdorf, N. etal. Selective targeting of regulatory Tcells with
CD28 superagonists allows effective therapy of experimental
autoimmune encephalomyelitis. J.Exp. Med. 202, 445–455 (2005).
11. Hanke, T. Lessons from TGN1412. Lancet 368, 1569–1570;
author reply 1570 (2006).
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and γ‑interferon in serum after injection of OKT3 monoclonal
antibody in kidney transplant recipients. Transplantation 47,
606–608 (1989).
13. Stefanova, I., Dorfman, J.R. & Germain, R.N. Self‑recognition
promotes the foreign antigen sensitivity of naive T lymphocytes.
Nature 420, 429–434 (2002).
14. Duff, G.W. et al. Expert Scientific Group on Phase One Clinical
Trials: Final Report (Stationary Office, Norwich, UK, 2006).
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Acknowledgements
Thanks go to my dedicated laboratory team, in particular P. Römer and
S. Berr, and to TheraMAB for providing TGN1412. The author is supported
by the Deutsche Forschungsgemeinschaft through CRC52.
Competing interests statement
The author declares competing financial interests: see Web version fordetails.
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