The molecule was genetically engineered by transfer of the complementarity determining regions CDRs from heavy and light chain variable region sequences of a monoclonal mouse anti-humanC28 [sic] antibody 5. Humanised variable regions were subsequently recombined with a human gene coding for the IgG4 gamma chain and with a human gene coding for a human kappa chain, respectively.
Naive T-cells normally require both signal 1 the antigen receptor and signal 2 co-stimulation to become fully activated. Studies of monoclonal antibodies specific for mouse, rat or human CD28 identified a so-called "superagonistic" antibodies that could stimulate T-cells without concurrent antigen-receptor stimulation. Whether this activity represents merely a "stronger" activity or a different activity is uncertain. Two antibodies specific for human CD28 were identified. The more active of the two, TGN originally called 5.
However, in vitro and in vivo data from animal studies later suggested that administration would lead to preferential activation of regulatory T cells , leading to a net effect of T cell downregulation.
On its website, the company writes: "A pronounced T-cell activation and expansion mediated by CDSuperMAB in animal models is accompanied by the expression of anti-inflammatory cytokines, like IL, rather than by the toxic cytokine storm of pro-inflammatory mediators induced by other agents that address the TCR complex. A new explanation for the trial mishap was suggested by the findings of a recent paper in Clinical immunology.
Pillai et al. However, eventually most of these cells downregulate their regulatory capabilities and become effector cells. Other cells activated by CD28 ligation in humans are eosinophil granulocytes. To function as an agonist, it has been suggested that TGN needs to be a whole antibody including the constant Fc region. According to a report by TeGenero the F ab 2 is not able to generate the required stimulation.
However, cell opsonisation by antibody leads normally to phagocytosis of the labelled cells as for example seen in the case of HIV. Mark's Hospital, London, on 13 March Good Clincal Practice GCP prohibits payments being made to Phase I trial volunteers on the basis of risk, and specifies that payments must be based upon the amount of time given up and the number of invasive procedures e.
All six of the trial subjects who received the drug were male, aged 19 to 34 median He took his shirt off, complaining that he felt like he was burning. Shortly after, the remaining participants who received the actual drug also became ill, vomiting and complaining of severe pain. The first patient was transferred to the Northwick Park hospital's intensive care unit 12 hours after infusion, with the others following within the next 4 hours. This led to his description as being similar to the "Elephant Man".
All of the men were reported to have experienced cytokine release syndrome resulting in angioedema , swelling of skin and mucous membranes, akin to the effects of the complement cascade in severe allergic reaction.
The patients were treated with corticosteroids to reduce inflammation, and plasma-exchange to attempt to remove TGN from their circulation. The treating doctors confirmed in August that all six men had suffered from a cytokine storm , and that, paradoxically, the men's white blood cells had vanished almost completely several hours after administration of TGN According to a press release from 5 July on the North West London Hospitals NHS Trust website, where the men were treated, the patients continued to improve and "five of them went home within a month of the incident, while one patient remained in hospital until 26 June, when he also went home.
An article by The Sunday Times on July 30 reported lawyers' claims that the long-term damage to the patients may be worse than originally thought. Medical assessment by immunologist Professor Richard Powell were said to have revealed that the blood of the patients contained a low number of regulatory T-cells, below one percent compared to three to five percent for healthy male adults - although the clinical significance of any such finding are unknown. Powell also reportedly claimed that one of the patients has "definite early signs that a lymphoid malignancy is developing".
Some of the men involved in the trial are said to have been told that they face "a lifetime of contracting cancers and all the various auto-immune diseases from lupus to MS , from rheumatoid arthritis to ME.
TGN had not previously been given to humans; however, the trial was preceded by animal testing, including in non-human primates. In the TGN trial protocol, there was no mention of projected plasma concentration of TGN, CD28 receptor occupancy as well as the possible pharmacological effects of the FIH dose in immunocompetent humans. Additional concern with the administration of a novel biological agent is the dosing interval between subjects.
The unexpected event must also be expected in preparations for FIH trial of novel biological agents. Moreover, it would have been much appropriate if a hospital premise was selected for the trial site other than a privately leased unit by Parexal which was inadequately resourced for the diagnosis and treatment of affected volunteers.
This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and build upon your work non-commercially. Withdrawal Guidlines. Publication Ethics. Withdrawal Policies Publication Ethics. Home PPIJ A decade after the tgn disaster what have we learnt about safety predicting methods for new biological agents. Commentary Volume 4 Issue 7.
Author declares that there is no conflict of interest. Final Report. Clinical trial protocol: A phase-1, single-centre, double-blind, randomised, placebo-controlled, single escalating dose study, to assess the safety, pharmacokinetics, pharmacodynamics and immunogenicity of TGN administered intravenously to healthy volunteers. Attarwala H. J Young Pharm. Toxicol Pathol. Loss of Siglec expression on T lymphocytes during human evolution. Weis JH. Allergy test might have avoided drug-trial disaster.
It was found that one category of these antibodies was capable of activating T cells irrespective of signal received from T-cell receptor. They were named as CD28 superagonists. These antibodies did not differ in antibody class or the binding avidity for the CD28 receptor but differed in the epitope-binding site. From these studies, TGN, a genetically engineered humanized anti-CD28 antibody was produced by transferring complement-determining regions from variable regions of heavy and light chains of monoclonal anti-mouse CD28 antibody 5.
A mouse antibody used in humans may have toxicity problems related to immunogenicity and problems related to effective functioning of antibody. To avoid these problems, the above humanized antibody TGN was constructed. These assays showed specificity of TGN for CD28 receptor and that TGN did not cross react with other closely related molecular targets such as Cytotoxic T-lymphocyte-antigen-4 and inducible co-stimulator. In vitro studies for cross reactivity of TGN with CD28 expressed on T cells of rodents and non-human primates revealed that TGN had low-binding affinity for rodent CD 28 whereas the same was high in case of T cells from for CD 28 to T cells derived from cynomolgus monkey and rhesus monkey.
When incubated with different subsets of T cells obtained from healthy donars, only TGN but not conventional CD28 antibody was able to cause rapid proliferation of T cells in the absence of stimuli from T-cell receptor. These results showed that TGN had superagonistic activity for T cells obtained from healthy donars and that they could specifically react with CD28 receptor having sequence homology with human CD28 receptor.
Prior to use of TGN different antibody variants were used for preclinical studies. All these studies demonstrated that these superagonist are safe and efficacious Investigation brochure, These encouraging results demonstrated high possibility for the use of this superagonist for the treatment of different T-cell deficiency syndromes like auto-immune diseases and B-cell lymphoma.
To further evaluate its efficacy, humanized antibody as described above was engineered from 5. Selection of proper non-human primate model was an important issue for testing further safety and efficacy of this antibody.
On the basis of this hypothesis, it was decided that results obtained from pharmacokinetic and pharmacodynamic studies in these closely related species would most closely predict fate of drug response when tested in humans. A repeat dose study for toxicokinetic evaluation of TGN was conducted.
Plasma half-life of TGN was found to be 8 h which was as expected for a large protein molecule like an antibody. Despite four increasing repeated doses of TGN resulting in four plasma peaks concentrations of TGN, only one peak for increase in T-cell number was observed.
This was because extent of expansion of T cells by TGN is highly dependent on availability of T cells and saturation kinetics of CD28 co-stimulator receptor. After these studies, toxicological studies using rhesus and cynomolgus monkeys were conducted. A repeat dose pilot study was conducted in cynomolgus and rhesus monkey.
In addition, no signs of toxicity were observed in any of the physiological systems including cardiovascular system, respiratory system, or central nervous system. In addition, there was no signal from any of the animals treated with any dose of superagonist indicating symptoms of anaphylactic shock or development of autoimmune disease, or systemic immune suppression. In addition to these studies, tissue cross-reactivity studies were performed where distribution of lymphocytes was observed by lymphocyte staining.
These studies revealed a consistent tissue staining in lymphoid tissue as expected demonstrating target-tissue specificity of CD28 superagonist.
In addition, studies for immunogenicity of TGN were performed on primate model. Anti-TGN antibody titers were observed in all animals, which were thought to be as a consequence of the humanized antibody being used in primate model.
After getting approval from regulatory authorities, phase 1 trials were conducted. The main aim was to establish safe human dose which can be further be used for subsequent drug trials. For this purpose, it was decided to conduct the trials on healthy human volunteers because disease free subjects have comparable CD28 receptors as in case of rhematioid arthritis or B-cell lymphoma. Also, immunological safety was expected to be more in healthy subjects compared to those with pre-existing disease.
In addition, healthy subjects would not only exclude effects of other medications administered to diseased patients, but also exclude the effects of functional activation or dysfunctionalization of T cells as a result of prior diseased condition. Since TGN showed specificity toward CD28 receptor expressed on human and non-human primate T cells, safe dose calculated from preclinical studies in non-human primate model was considered of suitable relevance for calculation of first in human dose.
Various tests for expected pharmacological activity of TGN and unexpected toxicological effects of TGN were conducted in non-human primates cynomolgus and rhesus monkeys. After collection of this large amount of preclinical data, when TGN was administered to six healthy human volunteers in phase 1 clinical trial conducted by Paraxel for TeGenero at Northwick hospital in London, UK, minutes after the first infusion of humanized CD28 superagonist TGN, all patients started suffering from severe adverse reaction resulting from rapid release of cytokines by activated T cells.
Adapted from Dayan and Wraith[ 8 ]. They did not find any flaw in trial procedure or in manufacture of drug. They mentioned that the severe reactions were as a result of unexpected biological effect of the drug. This raises doubts on whether trial met the criteria on scientific validity of preclinical data. Toward this end, expert scientific group under Professor Gordon Duff was formed which further investigated the biological and ethical concerns which may have resulted in the disastrous aftermath.
Despite of knowing these facts infusion of TGN given to all six volunteers within a short span of time was a serious concern in conduct of the trial. Moreover, when the last volunteer was to be infused, the first volunteer had already started showing adverse effects. Despite of this observation, sixth volunteer was still infused with the drug.
This was important because CD28 is also expressed by the cells responsible for allergy and the fact that the adverse reactions were immediate, relates to the release of preformed cytokines in granules of allergy-mediating immune cells. Inclusion of an allergy test in preclinical studies might have predicted the massive cytokine release. In an another clinical trial conducted by National Institute of Health for the drug Fialuridine, a thymidine analog having antiviral activity against Hepatitis B virus showed adverse reactions in phase 2 clinical trials leading to death of five human volunteers due to severe hepatic toxicity and lactic acidosis.
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