Exploring Issues With TGN1412 Biology Essay

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TGN1412, a Monoclonal antibody drug produced by TeGenero, German pharmaceutical company to solve devastating illnesses like leukaemia, and auto immune diseases like rheumatoid arthritis. The Phase I clinical trial of the superagonist antibody caused a life threatening "cytokine storm" in all the six healthy trial volunteers which signalled the failure of pre-clinical testing safety. The volunteers suffered from severe panic attacks, shooting pains, breathing problems and many more violent diseases.

The product TGN1412 is an immunoglobin protein which was produced by mice in response to the transgenic antigens that were artificially inserted so as to get the antibody that could act as a drug for treatment of few diseases.

The Background of the working of the drug specifies that TGN1412 will act upon the human immune cells and tier them up to their maximum potential so as to get rid of the auto immune diseases (the case where immune system is over activated and causes harm to itself). But the unexpected results from the first phase of trial raised many serious questions upon the procedures followed and the pre-clinical tests in the first place.

Medicines and Healthcare Products Regulatory Agency, MHRA approved the drug and also stated that PAREXEL, the company which supervised the drug trial, followed the agreed protocol for the trial which was approved.

But where the company TeGenero stated the drug to be based on a totally different production basis of an antibody of mouse, there questions have been raised about the humanised form of drug, which was supposedly not done, thus causing the drug to be immunologically rejected by humans. Also the statements given by the company explaining the safe pre-clinical tests on the non-human primates was not justified as genetic engineering to make the drug suitable for human-primates was not done, thus the pre clinical tests on animals such as mice and rats were not trustworthy to proceed for human trial.

Though the volunteers started recovering after two weeks of trial and their families were even paid for the troubles they faced but the whole event raised many serious questions on the approval of the trial by MHRA in the first place which caused a total collapse of the science and ethics.



TGN1412 (also known as CD28-SuperMAB) was thought to be a landmark discovery and was expected to solve cancer-related immunodeficiency such as B cell chronic lymphocytic leukaemia and auto immune/ inflammatory diseases like rheumatoid arthritis.

TeGenero, a German Pharmaceutical research company's most advanced product TGN1412, a monoclonal antibody, first to reach human testing. The first trial (phase I) was conducted out in a private unit in London's Northwick Park & St. Marks Hospital on 13th March 2006 caused the six paid healthy volunteers to be immediately hospitalized in intensive care units who were suffering from inflammation and multiple organ failure. All of the volunteers were reported to have experienced swelling of skin and mucous membranes (cytokine release syndrome) and all the white blood cells were almostly vanished after several hours of trial of the drug. Also one of the volunteer was supposed to have developed cancer. [1]

According to a press release from North West London Hospitals NHS Trust website on 5th July 2006, the condition of the patients improved continuously and five of them were sent home while the one patient remained in the hospital until 26th June 2006 when he was also sent home, but it was also suggested by Head Of Pharmacology, Trevor Smart at University College London that these men may never fully recover, and may suffer long-term disruption to their immune systems.

Figure 1. Hand fingers of one of the volunteers turned black and started shedding off. [11]


Description of the Drug.

TGN1412, a monoclonal antibody (MAB) is an immunoglobin protein made by the descendants of a single antibody producing cell as described by TeGenero on its website. [2]. TGN1412 was said to be an immunomodulatory humanized agonist anti-CD28 which binds to the CD28 marker cells present on the surface of the T lymphocytes, causing

more T-cells to be created. (Updated Statement by TeGenero, 24th March 2006)

TGN1412, a MAB type drug is very different from the conventional synthetically prepared drugs. It can't be produced chemically but had to be genetically engineered and "Humanised" as this MAB is based on an antibody produced in mouse, thus it had to be genetically engineered so as to make it immunologically accepted by humans. [3]

MAB drugs are typically produced by making the mice to produce antibodies in response to the protein antigen (genetically prepared) which is repeatedly injected into it together with the transgenic cells (continuously producing the protein). The spleen cells of the killed mice are then isolated and fused with lymphoma (cancer) cells to create 'hybridoma' cells, clones of which give permanent cell lines which grows and secretes the aimed, protein antibody or the monoclonal antibody continuously. Then for these mice monoclonal antibodies to be humanised, another part of genetic engineering had to be done so as not to be rejected by human immune system. [4]

Figure 2. A fully human Monoclonal Antibody.

(A genetically engineered Monoclonal Antibody Humira (drug), produced by Abbot Laboratories to treat moderate to severe rheumatoid arthritis in April, 2003 was totally successful and is being used now widely).



Body is protected from diseases and external infections by the immune system but mistakenly if the immune system attacks itself, then the person becomes sick and get

auto-immune diseases which could be scientifically said as an overactive immune response of the body against substances and tissues present normally in its own body.

Today there are around 40 known auto-immune diseases which had affected 5 - 7% of the total population. The most common is rheumatoid arthritis, which was thought to be cured by the company TeGenero's product TGN1412, a superagonist. [2]

The Antigen Presenting Cells, APC binds an antigen to itself upon its entering into the body and presents them on their surface attached to MHC molecules which then carries the antigen molecule to the immune cells of the body (either T-cells or B-cells).

Figure 3. Interaction of antigen presenting cells with T-cells.

(The APC interacts with the T-cells via two bonds. The first link is formed between the MHC and T-cell receptor, TCR molecules of APC and T-cells respectively whereas the second link is formed between the costimulatory molecules CD80/86 and CD28). [5]


The Antigen Presenting Cells upon presenting the antigen to the T-cells, the receptor - ligand interaction occurs via an antigenic peptide bond (the receptor is present on the surface of the T-cells, known as TCR whereas the antigen on the MHC molecules acts as the ligand). Also few amino acid side chains or links have to be completed between T-cells and the Antigen Presenting cells. These links are basically composed of the costimulatory molecules CD80/CD86 on the surface of Antigen Presenting Cells and CD28 on the surface of T-cells which binds to each other for the full signaling and activation of T-cells so that it can proliferate and produce an effective response to attack and eliminate the foreign pathogen from which the antigen was derived and to provide immunity to the body. (Refer fig. 3) [5]

Figure 4. Crystal Structure of Human CD28.

(The structure of human CD28 in complex with the Fab fragment of a mitogenic antibody was resolved by T-cell biology group at the University of Oxford in collaboration with TeGenero). [6]


Mechanism of Action.

The Antigen - receptor complex acts as the primary messenger for T-cells activation and the complex upon entering into the cell causes a cascade of steps to occur which activates number of various kinds of protein complexes and secondary messenger (like Ca2+ or cyclic AMP) inside the cell. Then finally an activated protein molecule (totally different from the receptor - ligand complex) enters the nucleus, attaches itself to DNA and alters the gene expression. This alteration either causes the cell to respond to the dangers caused by the antigen in the body and to cure them or the cell (like phagocytic cells) eats up the antigen itself to protect the body. This gene expression basically leads to the secretion of cytokines from the T-cells which spur the growth of more T-cells. Some T-cells become cytotoxic and track the other cells infected with the antigen, also some T-cells become helper cells and secrets more cytokines. (Refer fig.5).

Figure 5. T-cell Activation and release of Cytokines.

(The T-cells get activated upon encountering with its cognate antigen present on the MHC molecule on the surface of Antigen Presenting Cells or on the surface of an infected cell or a phagocyte). [7]

But surprisingly, in the case of autoimmune diseases, the molecule CD28 on the surface of T-cells has already been activated even without the presence of any foreign antigen in the body and thus as a result the T-cells start attacking and destroying up its up body cells thus causing diseases that are named as Autoimmune Diseases.


Unexpected Results.

The main thought behind TGN1412, a monoclonal antibody drug was that this drug would act as an super agonist, that will massively enhance the working of immune cells (or T-cells) when it will get attached to CD28 and will activate it too much that the T-cells would exhaust and get switched off and thus preventing autoimmune diseases.

Whatever happened in the first drug trial of this monoclonal drug was completely unexpected as the super stimulation of the immune system (T-cells), backfired and led to the violent cytokine storm precipitating the cascade and the release of endogenous molecules causing various problematic situations in other cells of all the trial volunteers. The volunteer who was thought to have developed cancer had to get his foot fingers chopped off as they were turning black and dry continuously. Also one of the patient was then described being similar as Elephant-Man as his head ballooned up and grew in size three times its original (Refer fig.6). Two of the patients started fainting, vomiting and writhing around their beds, they started complaining about fever and severe headache. One patient's breathing went disordered and he went through a terrible panic attack, started convulsing and was getting shooting pains in his back. The drug completely acted as a trouble for the vital organs of all the 6 healthy volunteers. [8]

A statement from TeGenero stated that they were of course saddened by the unforeseen side effects that resulted from the drug and they also said that they will continue to work to establish exactly what happened and will make possible ongoing efforts to make the future trials as safe as they can be.

Figure 6. One of the trial volunteer became ballooned head as a side effect of the drug. [11]


Question at Clinical Trials.

This finding and trial increased the challenge to anyone involved in developing such treatments to improve and find new ways to reduce the risk of future clinical trials.

But whether the standards of trial were maintained during the drug trial conducted for TGN1412? Whether prior to the trial it was adequately been tested on human blood in vitro? Also, was the drug humanized before being trialed on human primates as the drug was based on an antibody produced in mouse ?

Thus numerous medical, ethical and scientific questions were raised at the possible dangers associated with the medical or pharmaceutical industries.

Answer statements from the concerned organizations!

The report from TeGenero stated that highest standards were observed in developing this drug and the animal study results showed that there were no drug related deaths in the test. Also they stated that the volunteers were given one five-hundredth of the dose that was given in animal studies and that value was considered as maximum safe starting level for humans. [2]

The question that was raised on the PAREXEL, that whether they maintained the whole range of current drug testing regulations and the bioethics of the trials or not?- this question was answered in an interim report produced by MHRA which cleared that PAREXEL ran the complete trial according to the agreed protocol, with the exact amount of dosage given to the trial volunteers. [4]

In a provisional report by The Medicines and Healthcare Products Regulating Agency, MHRA which gave approval for the trial said that the drug was correctly engineered and was not even found contaminated and also concluded that the cause of the adverse effects in the trial volunteers was most likely due to an unpredictable biological action, which wasn't ever seen in the pre-clinical tests conducted. [3]


Public Views, Criticism & Controversy.

MHRA, Medicines and Healthcare Products Regulatory Agency, suggested that an unpredictable biological action of the drug in human is the most likely cause of adverse reaction in the volunteers. But they also questioned TeGenero preclinical standards that whether before the trial the drug was humanized or was just tested upon non-human primates?

Authors of The Times, Thomas Hanke and Thomas Hunig, stated that although mouse and rat CD28 share 93% sequence identity in the extracellular part of the molecule, but human CD28 is only 65% identical to the mouse sequence. Thus it was not fully safe to rely on the preclinical trials conducted on mouse and to conduct a trial on humans then.

A report in The Journal of Immunology [9], suggests that the dose of the drug TGN1412 given to the trial volunteers was near to the maximum immunostimulatory dose, which can easily stimulate the quick release of cytokines and can destroy the body's own lymphocytes and also it stated that TGN1412 is not a superagonist in nonhuman primates.

Dr. Mae-Wan Ho and Prof. Joe Cummins, members of Institute of Science in Society, ISIS stated that the drug was an unconventional member of a new class of drugs and it was known to have caused side effects and even deaths, yet was approved for clinical trial on human, solely based on unpublished animal tests. [4]

A former executive of the company TeGenero, who asked to remain anonymous, stated that the company should not have trialled so many persons at onec when there was no prior human test experience. [4]

Family members of the volunteers questioned at the death of a dog during the pre-clinical tests, but the company clearly denied at TGN1412 being ever tested on dogs. [4]

Two monkeys in the pre-clinical trials experienced transient increase in the size of their lymph nodes, but TeGenero denied that too by stating that side effect was not drug related. [3]


One of the member of a science organization on March 26, 2006 spoke in the favour of the drug and the company using the company's own statement itself: Statement given by Te Genero's Application - There is 100% homology between the structures of CD28 in the rhesus (Macacca mulatta) and cynomologous (Macacca fascicularis) monkeys, mice and humans.

The person stated that in monkeys, CD28 is also expressed by the mast cells and if is true in humans too then binding of the antibody drug to T-cells have nothing to do with this devastating response as this would be caused by the antibody binding to CD28 on the mast cells and creating an allergic response within minutes. [5]

Another member of the same organization explained that if CD28 on the mast cell would have been affected by this antibody, then this reaction should have been seen in mice too which was not seen because the statement published by TeGenero regarding the 100% homology between human, monkeys and mice was not the true case and then that member also posted the original alignment in a published sequence of CD28 cells of monkeys, mice and humans.

Alignment of Macacca mulatta, Macacca nemestrina and Homo sapiens CD28 protein sequences :-


Important residues in the protein sequences of CD28 and related molecules in humans and mice :


Report from a medical company questioned the trial phases rules of testing the drugs on healthy volunteers rather than patients as the concerned risks are high and the benefits are non-existent. [10]

Questions were raised on the company TeGenero at the "Informed Consent Form", which is given to anyone who participates in any type of a drug trial to make them informed about the dangers associated but these six trial volunteers were just given ten minutes to read the document just half an hour before the trial begun.


What Went Wrong?

Although TeGenero couldn't find the actual fault and the main reason behind the drug responding so wrong, but a conclusive answer that came up after the whole study was the on hand danger of the action of the drug, TGN1412 on the most important and reactive system of the body, The Immune System.

All of the licensed MAB drugs work on the principle of acting and suppressing the immune responses, whereas TGN1412 working was a bit complex and was not well understood as it was designed to stimulate or activate the immune response.

As known by the scientific process of signal amplification, that a very small stimulus may produce a dramatic or a large effect because at each step of amplification cascade, the signalling intermediate persists long enough to stimulate the production of large number of molecules for the next step ( for e.g. A single epinephrine ligand can elicit the production of hundreds of millions of glucose molecules). Thus assumption of only a small effect being produced by a low dosage (of one five-hundredth of that given in animal studies) can't be anyhow explained scientifically and should not have been considered as a safe starting dosing level for humans.

Figure 7. Foot fingers of a trial volunteer that turned black due to the drug's side effect showing exactly-What Went Wrong? [13]