|Biological agents; Immunotherapy; Monoclonal
antibodies; Autoimmune diseases; Cancers; Adverse effects;
|Many diseases have been observed to have an underlying
immune basis and include auto-immune diseases like systemic lupus
erythematosus (SLE), rheumatoid arthritis and systemic sclerosis .
Auto-reactive T cells are kept under control by the regulatory T cells
(Treg cells) and absence of co-stimulatory signalling leads to loss of Treg
cells . CD28 super-agonists have been observed to cause selective
expansion and activation of the regulatory T cells over conventional
T cells (Tconv cells) and so it was thought that this anti-CD28 antibody
represented a promising new treatment option for the large number of
human auto-immune diseases .
|The co-stimulatory signal
|Bretscher and Cohn in 1970 proposed the two signal model
of lymphocyte activation to enable understanding of self/non-self
discrimination, and this model suggested that T cell activation needs
two separate signals. The first is transmitted through the T cell receptor
(TCR) following binding of antigen, and the second co-stimulatory
signal occurs through the binding of a separate receptor (CD28)
on the T cell . It has been observed that antigenic stimulation of
the T cells without this co-stimulatory signal will result in a state of
unresponsiveness called anergy and the anergic T cells fail to produce
interleukin 2 (IL-2) and other cytokines required for T cell function and
regulation . CD28 is important for regulating immune responses and
maintaining peripheral tolerance. Binding of this receptor to its ligands
causes increased expression of CTLA-4 (Cytotoxic T lymphocyte
antigen-4), which has a negative effect on T cell activation. This
binding facilitates the differentiation of Th2 cells which have protective
functions in auto-immunity, as well as control of the regulatory T cells
|What is CD28?
|First cloned by Arrufo and Seed in 1987, and initially called TP44,
this is a protein that belongs to the IgSF (immunoglobulin superfamily)
group of receptors with a molecular weight of 44 KDa. It is
expressed by sub-populations of T cells and activated B lymphocytes
, as well as neutrophils and . It is expressed before activation of T
cells takes place.The ligands for this receptor are CD80/CD86 (B7.1 and
B7.2) co-stimulators which are generally found on antigen presenting cells (APCs), and also bind to the counter-stimulatory receptor CTLA-
4 that is expressed after T cell activation to produce a negative effect.
CTLA-4 has higher affinity for the ligands (B7.1 and B7.2) than CD28
and leads to blockade of further activation signal . B7.2 (CD86) is the
predominant co-stimulatory ligand for CD28 .
|Consequences of CD28-B7 Interaction
|CD28 stimulation causes transcriptional activation and posttranslational
stabilisation of mRNA leading to increased expression
of cytokines such as interleukin 2 (IL-2) and activation of protein
kinase AKT . It also prevents anergy and causes induction of the
anti-apoptotic factor BCL-XL, contributes to B cell differentiation,
antibody production, direction of T cell migration and the regulation
of chemokine receptors .
|The Role of regulatory T cells
|These are CD4+ CD25+ cells with regulatory function and known as
Tregs. They make up to about 5-15% of the peripheral CD4+ T cells. They
constitutively express the interleukin 2 receptor-α chain (CD25) and
CTLA-4. The development and survival of these cells in the thymus is
controlled by CD28, which maintains their function independent of
CTLA-4. This is important in the maintenance of tolerance, prevention
of auto-immunity, enhancement of T cell response to pathogens, and
prevention of transplant rejection . In addition to that, the CD28-
mediated up-regulation of the inhibitory molecule CTLA-4 prevents
potentially harmful inflammation by terminating normal immune
|Molecular properties of CD28 “super-agonist”
|The super-agonist anti-CD28 antibody has been observed to bind
to a lateral membrane –proximal loop of the molecule (C”D loop) and interaction of the antibody with the extracellular domain of human
CD28 leads to a linear complex formation. The conventional anti-
CD28 antibodies bind CD28 bivalently, but result in the formation of
tangled complexes. Complex formation of the CD28 causes aggregation
of stimulatory signalling components such as PI3 kinase and growth
factor receptor bound protein (Grb 2) leading to generation of strong
|Rationale for using anti-CD28 antibody
|Monoclonal antibodies and fusion proteins with specificity for the
T cell receptor (TCR) and CD28 have been found promising in the
treatment of auto-immune diseases . Both the anti-TCR monoclonal
antibodies and the “conventional” anti-CD-28 monoclonal antibodies
individually are unable to stimulate the T cells and need to be combined
to produce T cell activation and cytokine production. But the CD28
“super-agonists” are able to cause T cell activation without the need
for TCR stimulation, hence the name super-agonists. This is why it was
thought by many workers that these monoclonal antibodies will have a
place in the treatment of auto-immune diseases which are refractory to
the conventional treatments available to patients .
|It has been observed that CD28 super-agonist administration in vivo causes selective expansion and strong activation of the naturallyoccurring
CD4+CD25+CTLA-4+FOXP3+ Treg cells over conventional T
cells (Tconv cells), and absence of this signalling leads to loss of these
regulatory cells. Also, a functional and/or numerical deficit in Treg cells
compared to pathogenic auto-reactive cells leads to initiation of autoimmunity.
Therefore, it is believed that because Treg cells are the main
controllers of peripheral self tolerance, this new therapy which aims at
re-establishing the balance between auto-reactive T cells and Treg cells
could have important contributions in the treatment of auto-immune
|Furthermore, application of CD28 super-agonists was noted to
result in momentary increase in the percentage of Treg cells which was
dose-dependent. This preferential expansion of Treg cells over Tconv is
thought to result from generation of extremely strong or sustained
signal in Treg cells following super-agonistic anti-CD28 stimulation. This
is in addition to the production of BCL-XL which is an anti-apoptotic
factor that prevents cell death. Also, increased interleukin synthesis by
Tconv cells and its uptake by the Treg cells are thought to contribute to
the preferential expansion of the latter cells following super-agonistic
CD28 stimulation . Beyersdorf et al.  also suggested that Treg
cells may have selective advantage over Tconv cells because of active
suppression of the latter during super-agonistic CD28 stimulation by
activating the cell contact-dependent suppressor machinery which
only functions upon recognition of self antigens in vivo.
|Lastly, it has been observed that administration of CD28
super-agonist to animals with EAE (experimental auto-immune
encephalomyelitis) and experimental auto-immune neuritis (EAN)
which resemble multiple sclerosis and Gullain-Barre syndrome
respectively, prevented or reduced the clinical symptoms when given
before the onset of the disease. This monoclonal antibody also rapidly
stopped the disease progression and produced disease remission
even after the onset of symptoms . This evidence was supported by
adoptive transfer experiments which revealed that transfer of CD28
super-agonist-activated Treg cells protected recipient animals from
the clinical signs of EAE, and CD4+ CD25- T cells (Tconv) cells were
observed to lack any protective effect in spite of ten times greater dose
of administration .
|Because both the pharmaco-dynamics and toxicological profiles of
the CD28 super-agonist suggested that it was safe, it was thought to have
clinical relevance in patients with multiple sclerosis and rheumatoid
arthritis who are known to have deficits in Treg cell numbers and/or
function with the aim of curing them. The hope was that these patients
might benefit from a boost in their Treg cells following the super-agonist
administration to improve their clinical symptoms [1,2].
|What are biological therapies?
|These are monoclonal antibodies and fusion proteins which contain
specific recognition components that have antibody function. The
principle of biological therapy takes advantage of antigen specificity to
produce monoclonal antibodies to the desired antigens . Currently
used biological therapies in clinical practice include Adalimumab,
Etanercept, Infliximab, Rituximab, Ibritumomab tiuxetan,
Tositumomab and Iodinated Tositumomab (I-131), Basiliximab,
Daclizumab, Alemtuzumab, Cetuximab, Bevacizumab, Trastuzumab,
and Palivizumab .
|Sources of biological therapies
|Kohler and Milstein first developed hybridoma technology in early
1970s, and set the pace for the development of monoclonal antibody
therapeutics. This was followed later on by the development of
hybridomas by the fusion of B cells from immunised mice with murine
myeloma cell lines. The immortalised cell lines (clones) were then
selected according to the antibody of interest which is then produced
in large quantities. This came with problems because it was difficult
to produce human anti-mouse antibodies that can be used without
immunogenic complications .
|Genetic manipulations allowed the development of chimeric
antibodies which are obtained from a combination of murine
variable-region genetic material with human constant-region genes. A
combination of murine CDR genetic material with the remaining part
of the antibody obtained from humans results in humanised antibodies.
Lastly, fully human monoclonal antibodies have their origin completely
from human beings . Humanised antibodies are superior because
they have better interaction with human Fc receptors and effector cells,
which gives a greater effector function and less immunogenic reaction.
This prolongs the therapeutic half-life of the drug and reduces the side
effects especially following subsequent exposures .
|The function of the monoclonal antibody can be determined by
selecting a particular Fc component. This can be coupled with a specific
molecule to form a fusion protein that has both the antibody effector
function and any other desired effects. Finally, monoclonal antibodies
can be changed and used for immunotherapy by labelling them with
toxins specific for cancer cells without affecting the normal cells .
|Problems of biological therapies
|Biological therapies are associated with a lot of problems which
include regulatory/licensing issues, documentation of the trials
(recording and reporting), funding, side effects and complications
(short- and long-term), ethical considerations, media attention and
publicity, selection of the immune component to be modified, time
considerations, risk-benefit analysis and when to use the therapy .
I shall therefore attempt to briefly highlight each of the issues involved.
|Regulatory and Licensing Issues
|Biological therapies are potentially harmful agents and so there
is need for involvement of licensing and regulatory agencies like the UK Medicines and Healthcare Products Regulatory Agency (MHRA),
and independent research ethics board (REB) right at the beginning of
conception of the idea through to the pre-clinical and clinical studies
. This will help to create checks and balances and highlight any
potential hazardous effects of the agent before it is tried on humans.
This protocol has been observed in the case of TGN1412 because three
independent bodies reviewed the drug before it was tried in the six
volunteers. It is also important for drug investigators and sponsors to
provide accurate, complete and unbiased information to the regulatory
bodies and ensure transparency throughout the development of
new drugs, in addition to the need for compulsory registration and
disclosure of the trials and protocols to the regulatory agencies and
|Problems of Funding
|It is important to note that the process of drug development is very
expensive and capital intensive , hence the need for research grants
and financial support from governments, international agencies and
pharmaceutical companies, which will help to convert the idea into a
finished product ready for use. In addition to that, even the trials (both
pre-clinical and clinical) are capital intensive and add to the cost of
drug manufacturing. Lastly, use of the drug is also expensive because
it has to be procured by the governments or sometimes the individuals
have to pay for the drug according to its availability, this may be
worsened by the added treatment of side effects and complications. In
addition to all that, the cost of not treating the patient also needs to be
considered as it may be more expensive than treating the patient with
the therapy in question.
|Side Effects of the Drugs
|Safety issues in drug trials are of utmost importance, because of
the possible side effects which can be short- or long-term. Any system
can be affected depending on the type of monoclonal antibody used
and its intended target(s). Immunosuppression can lead to fungal and
opportunistic infections, involvement of the gastro-intestinal system
may lead to nausea, vomiting, and diarrhoea, while affectation of the
respiratory system will lead to difficulty in breathing, chest pain and
even respiratory as well as cardiovascular failure. In addition to that
shock and hypovolaemia may result in acute renal failure which may
present in decreased or complete lack of urine formation .
|Occasionally, multiple organ failure can occur with affectation
of virtually all the systems at once as can be seen in the case of trial
with TGN1412 . Within 90 minutes of receiving this monoclonal
antibody, all the six volunteers developed a systemic inflammatory
response syndrome as evidenced by headache, myalgias, nausea,
diarrhoea, erythema, vasodilatation and hypotension. There was also
recurrent fever, respiratory difficulty, cardiovascular shock, renal failure,
hospital-acquired infections, peripheral ischaemia, disseminated intravascular
coagulation and generalised skin desquamation .
|Long-term side effects and complications are also to be expected
in some cases and these can include chronic low blood cell count,
anaemia, cancers, autoimmune diseases, decreased life expectancy
and resurgence of infections like tuberculosis in patients that received
anti-TNFα therapy . This is because TNF-α is required for the
formation of localised tuberculous granuloma, which helps to localise
the infection in one place and so may remain dormant for a long time.
|These are very important issues to be considered so as to be sure the subject/patient is given his/her right to know and make judgements
before any clinical trials can be done on them. This can be complicated
by the need to reward or pay the subjects for their participation which
many believe can cloud or bias their judgements in taking a reasonable
decision to partake in a drug trial . But Emanuel and Miller 
caution that although the role of money cannot be over-emphasised,
bad things can happen in ethical research, but money does not make
them unethical, unscrupulous or malignant. They further noted
that a research is ethical from the beginning, good outcomes do not
necessarily make it ethical, and a bad outcome does not make an ethical
research unethical. Among the many ethical issues to be considered
include the need to conduct research in a particular disease area and
whether it is justified. Others are the need for scientific validity which
require that pre-clinical studies are conducted using cell cultures and
laboratory animals before clinical trials are carried out, fair subject
selection should be done without targeting vulnerable and marginalised
communities, taking informed consent and respecting the enrolled
participants as well as granting them the right to withdraw from any
trials at any time [1,9].
|It is important to make a careful assessment in order to weigh the
risks of any drug use against the benefits so as to avoid mistakes and
end up with litigation. In the case of the TGN1412 phase 1 clinical trial,
healthy male adult volunteers were used even though the drug was
meant for patients with compromised immune systems, and therefore
some felt that real patients who stood to benefit from the drug should
have been co-opted into the trial . But Emanuel and Miller 
suggested that even though the volunteers had no chance of benefiting
directly from the trials (medically), the medical knowledge to be gained
could be used to justify the risk of the trials.
|Although it is important for drug developers to be open and
transparent during drug research, negative media attention and bad
publicity need to be discouraged, in order to prevent fear and panic
in the public which can lead to refusal or rejection of new therapies.
It is important for health personnel to educate themselves and their
patients on the benefits and/or hazards of new therapies and how they
work. This will ensure patient’s trust and compliance with medication,
an important consideration in their treatment to ensure good outcomes
|Anti-CD28 monoclonal super-agonist and other biological agents
have definite roles in the treatment of auto-immune diseases and even
cancers, because of their ability to enhance regulatory T cell function,
but there is need to carry out careful and meticulous pre-clinical trials
before trying them on humans under close observation, given to one
person at a time to prevent disastrous outcomes. Proper and strict
regulation of research on humans, as well as use of these biological
agents should be enforced and monitored by the authorities to ensure
maintenance of transparency and good standards.
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- Beyersdorf N, Hanke T, Kerkau T, Hünig T (2005) Superagonistic anti-CD28 antibodies: potent activators of regulatory T cells for the therapy of autoimmune diseases. Ann Rheum Dis64: 91-95.
- Mehrishi JN, Szabó M, Bakács T (2007) Some aspects of the recombinantly expressed humanised superagonist anti-CD28 mAb, TGN1412 trial catastrophe lessons to safeguard mAbs and vaccine trials. Vaccine 25: 3517-3523.
- Green JM (2000) The B7/CD28/CTLA4 T-cell activation pathway. Implications for inflammatory lung disease. Am J Respir Cell Mol Biol 22: 261-264.
- Male D, Brostoff J, Roth D, Roitt I (2012) Immunology. (8thedn) Elsevier Limited, Canada.
- Bour-Jordan H, Blueston JA (2002) CD28 function: a balance of costimulatory and regulatory signals. J Clin Immunol 22: 1-7.
- Delves PJ, Martin SJ, Burton DR, Roitt IM (2006) Roitt's Essential Immunology. (11thedn) Blackwell Publishing, Oxford 176-180.
- Lin CH, Hunig T (2006) Efficient expansion of Regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur J Immunol 33: 626-638.
- Emanuel EJ, Miller FG (2006) Money and distorted ethical judgments about research: ethical assessment of the TeGenero TGN1412 trial. American J Bioeth 7: 76-81.
- Goodyear M (2006) Learning from the TGN1412 trial. BMJ 332: 677-678.
- Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, et al. (2006) Cytokine storm in a phase 1 trial of the anti-CD28 Monoclonal Antibody TGN1412. N Engl J Med 355: 1018-1028.