Talk:Theralizumab/Archive 1

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MHRA interim report

I've added a section about the official inquiry that I thought could be updated as further reports come out. It's a bit rough and ready, but it is important. It could do with some perspectives and views from other sources. Great work wikipedians on this page!

Anthony Cox [Black Triangle http://www.blacktriangle.org] —Preceding unsigned comment added by 82.36.197.238 (talk • contribs)

--There was an allusion to cytotoxic properties of IgG4 in the recent Nature article. This would be news to me! -I went looking for information, and found this: http://rheumatology.oxfordjournals.org/cgi/content/full/44/5/573 among other things. Wow, something to learn every day. The technology to engineer human/humanized antibodies was in its bare infancy when I was prodding purified neutrophils to effect ADCC; I never tried it with an IgG4. (198.140.178.11)

--More news- (2 May 2006) -this is just out in the Journal of Immunology, from the Hunig lab: The Journal of Immunology, 2006, 176: 5725-5729.

Carl Zimmer article: http://loom.corante.com/archives/2006/05/02/wanted_hominids_for_clinical_drug_trials.php

Cutting Edge: Monovalency of CD28 Maintains the Antigen Dependence of T Cell Costimulatory Responses Kevin M. Dennehy, Fernando Elias, Gabrielle Zeder-Lutz, Xin Ding, Danièle Altschuh, Fred Lühder, and Thomas Hünig

CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement.

--This does not speak directly to TGN1412- but studies like this add to understanding of the biological neccessity for strict controls on signal transduction through CD28. These data were likely recorded months before the TGN1412 phase I trial was submitted for approval. What *else* did they know, I wonder? (198.140.178.11)

Cytokine storm, MoA, etc

Cytokine storm: The Wikipedia article Cytokine storm mentions that the drug OX40-Ig may be used to treat this condition. Does anyone know if this was used in the treatment of these patients? While OX40-Ig is in itself an experimental drug, in this extraordinary situation it might be worth trying it in the critically ill patients, if they did indeed suffer a cytokine storm. 12:59, 19 March 2006 (UTC)

I am deeply impressed by this Wikipedia article. I myself work in Drug Discovery for a pharmaceutical company that has a drug on the market that works by selective modulation of a signal involved in activating T-cells, and this Wikipedia article is the FIRST article that I've seen in any non-specialist publication with a decent description of this compound's intended MoA. Very well done, Wikipedians! —Preceding unsigned comment added by 64.105.73.7 (talk • contribs)

The whole approach of giving anti-cd28 is a bit naive. If someone believes CD28 is only stimulating T-cells they haven't bothered to do their science. AntiCD28 can be used to release Neurotoxins from Eosinophils which is definitively not what I would want to do. The reason that biology avoids regulation via a single pathway but uses "logical and gating" is to avoid disasters like this. User:Gerne1

The media are saying it's an anti inflammatory drug. All of the official sources say it's a monoclonal antibody. The article currently has both terms in but they appear to be contradictory. Secretlondon 18:35, 15 March 2006 (UTC)

They're not contradictory because the anti-body works as an anti-inflammatory.--Wikipediatastic 10:23, 16 March 2006 (UTC)

CD28 is the major co-stimulatory receptor on T-cells. This drug is a humanized monoclonal antibody which not only binds to CD28 but also activates this receptor, transmitting a very potent co-stimulatory signal to the T-cell. How this can be described as anti-inflammatory doesn't make any sense. Bizarre drug - not sure why anyone would want to do this - crudely put it massively activates every single T-cell in your body - not a good idea (evidently). Not sure what therapeutic potential it would have. It got orphan drug status in the EU so they must have some good data somewhere - wow. 16th March 13:10 GMT User:mxpule.

Fascinating - I have found the only academic reference to this anywhere. It was presented as a poster in the American Society of Haematology meeting in 2004. Here is the abstract for you (I don't know how to do nice formatting):

[2519] Superagonistic Anti-CD28 Antibody TGN1412 as a Potential Immunotherapeutic for the Treatment of B Cell Chronic Lymphocytic Leukemia. Session Type: Poster Session 732-II

Chia-Huey Lin, Thomas Kerkau, Christine Guntermann, Martin Trischler, Niklas Beyersdorf, Yvonne Scheuring, Hans-Peter Tony, Christian Kneitz, Martin Wilhelm, Peter Mueller, Thomas Huenig, Thomas Hanke. Non-clinical development, TeGenero AG, Wuerzburg, Germany; Institut for Virology und Immunbiology, University Wuerzburg, Wuerzburg, Germany; Medizinische Poliklinik, University of Wuerzburg, Wuerzburg, Germany

B cell chronic lymphocytic leukemia (B-CLL) is characterised by an accumulation of malignant B cells, and impaired humoral and cellular immune responses. Evasion strategies of leukemic cells appear to involve down-regulation of co-stimulatory molecules as well as increased resistance to apoptosis. Here we provide data supporting a novel concept to treat B-CLL with a humanized, superagonistic monoclonal antibody specific for CD28 (TGN1412). Superagonistic anti-CD28 antibodies have been shown to activate human T cells in vitro without requirement for engagement of the T cell antigen receptor (Luhder et al., J. Exp. Med. 2003. 197(8):955-66). Indicative of their activation, TGN1412-triggered T cells from healthy donors upregulate, among other activation markers, CD40L, that has been reported to promote anti-leukemic effects when ectopically expressed on B-CLL cells (Wierda et al., Blood. 2000. 96 (9): 2917-2924). In this report, the responses of PBMCs from B-CLL patients to soluble TGN1412 were examined. We show that in a dose-dependent fashion, polyclonal T cell activation was induced by TGN1412 including proliferation, cytokine production and induction of activation markers such as CD25, CD71, CD134 (Ox40), CTLA-4 (CD152) and CD154 (CD40L). Significantly, modulation of malignant B-CLL cells was also observed. MHC class II molecules (HLA-DR), CD95 and the co-stimulatory molecules CD80 and CD86, but not the proliferation marker Ki-67, were strongly up-regulated upon TGN1412 stimulation. These data suggested that improved antigen-presenting functions of B-CLL cells were induced by TGN1412. Accordingly, preliminary data indicate that B-CLL cells isolated from TGN1412 stimulated cultures induced enhanced proliferation of both allogeneic and autologous T cells, and importantly, TGN1412 activated T cells exhibited enhanced CTL-activity against B-CLL cells. In conclusion, our data suggest that TGN1412 induces polyclonal T cell expansion and activation as well as increased APC function of B-CLL cells. They imply that TGN1412 may have future therapeutic benefit for B-CLL patients. Abstract #2519 appears in Blood, Volume 104, issue 11, November 16, 2004 Keywords: T cell expansion|T cell costimulation|Non-Hodgkin's lymphoma

Not only was this agonistic but superagonist - not requiring even signal one to activate the T-cells. Basically this will fully activate every single T-cell in your body. Why someone would think this was a good idea is beyond me. User:mxpule

They thought that it would lead to a release of IL-10, this was supposed to mitigate the immune system rather than stimulate it. This is from their website (they have since pulled this information) "A pronounced T-cell activation and expansion mediated by CD28-SuperMAB® in animal models is accompanied by the expression of anti-inflammatory cytokines, like IL-10, rather than by the toxic cytokine storm of pro-inflammatory mediators induced by other agents that address the TCR complex. CD28-SuperMAB® over-proportionately expand regulatory T cells, a specialized T-cell subset that suppresses auto-aggressive T-cells present in the body and which has only recently been appreciated as important guardians of immune tolerance. Based on their functional potency in suppression of organ-specific as well as systemic autoimmune diseases, regulatory T-cells have been widely accepted as attractive targets for immunotherapeutic intervention. However, attempts to expand and activate this subset of CD4 T-cells in vivo and, ultimately, in autoimmune/inflammatory diseases have been hampered so far by the lack of therapeutic agents."-- Wikipediatastic 14:37, 16 March 2006 (UTC)

While of course easy to say now, with my limited biochemical knowledge, I seem to agree that the rationale behind this drug seems srtange. If you look at the primary mechanism of action it would appear to be a strong immune stimulant - not an anti-inflammatory drugs. Not because of the fact that it is an antibody (after all, there are other monoclonal antibody drugs that DO suppress the immune system) but because it is superagonistic to CD28. It seems like too much of a risk to rely on this expected massive immune stimulation to go away due to subsequent events. At least, it doesn't seem very surprising that a drug like this could trigger the reactions described (even though the doctors that have spoken to the media appear very surprised). Apparently, animal models showed different behaviour but as has been shown many times before, that does not guarantee the same behaviour in human beings, especially not in relation to the immune system. For instance, HIV causes AIDS in humans whereas it appears to cause no such things in monkeys. I really hope that the company made every effort to test the drug in vitro on human blood / tissues before this trial.

BTW, in the patent application for the drug (http://www.freshpatents.com/Use-of-a-cd28-binding-pharmaceutical-substance-for-making-a-pharmaceutical-composition-with-dose-dependent-effect-dt20060112ptan20060009382.php) it is noted that "In the rat animal model, it could be shown that a novel monoclonal antibody (MAB) with specificity for the CD28 molecule--JJ316--efficiently activates T lymphocytes in vitro as well as in vivo without TCR stimulation (Tacke et al., 1997), i.e. acts "superagonistically". This antibody--in spite of its strong T cell-stimulatory properties--is very well tolerated in vivo, in contrast to all other known T cellactivating substances (Rodriguez-Palmero et al., 1999; Tacke et al., 1997). Maybe it was too good to be true (in humans at least). It is probably too early to tell as it could also be a dosing issue. BTW, this is pure speculation but from reading biochemistry texts it seems the immune system can choose between two types of responses, TH1 and TH2. This choice is made early in the process and is reinforced. It seems that only TH2 responses yield IL10. Could it be that humans respond with a TH1 reaction instead of TH2 in monkeys? Even between individual humans there can be differences in which response is chosen to a given disease due to genetic differences. Could someone with knowledge in this area comment on this? —This unsigned comment was added by Para82 (talk • contribs) .

OK, I've found another scientific reference to the parent antibody (before humanization). http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12707299 user:mxpule. Incidentally, the patent describes experimentation in non-human primates. I wonder how different human CD28 is from rhesus CD28. - just looked exactly the same.

Has anyone seen any media coverage discussing how this drug works (or was supposed to work) or references to the patent application? I think this is a "Wikipedia first" thing because I haven't found it anywhere. I really hope we can create a good article here that may generate more attention to the case. While I don't know if there is someone to blame yet, from the wording of the patent application etc. I can't help getting a feeling no-one really understood how the drug worked in detail, in particular why it exhibited immuno-surpressive effects rather than the opposite. While in vivo data demonstrating the effect of a drug in practice is of course important I think it would be reasonable to demand that the theoretical rationale for a drug's claimed effect is well-understood before it goes to market - particularly when it is opposite of what one would intuitively expect. Am I the only one feeling this way? Para82 21:26, 16 March 2006 (UTC)

I believe this is the most definitive public description anywhere - can we get it onto the front page of wikipedia? Does anyone know how to do that - I can't seem to edit that page. Mxpule 21:37, 16 March 2006 (UTC)

As an admin I can edit the main page but I'm just looking up the criteria. Secretlondon 21:41, 16 March 2006 (UTC)

I've added it to the main page In the news section. It may get swapped out again by someone else. It's pretty rare that science stories get in there and it may not be obvious why it belongs there. Secretlondon 21:53, 16 March 2006 (UTC)

Sorry, I'm not science literate. Could this article be dumbed down a bit for the non-science crowd.? After reading this I'm still not sure what this drug does, what diseases it was designed to fight, the implications etc. —Preceding unsigned comment added by 129.173.96.99 (talk • contribs)

Well we need both - we need a general para for the non-science literate. Secretlondon 22:33, 16 March 2006 (UTC)

I'm not an immunologist and I've only looked at a few scanty accounts for 15 minutes, but one thing glares at me: according to this, they tested an antibody against human CD28 in monkeys, then guessed the antibody would have roughly the same potency in the human? It would stand to reason that the antibody might bind the human protein that it was raised against much better than the related monkey protein. Now, they might have done an in vitro test to address this issue that I don't know about, but the whole thing is pretty creepy. They combine this uncertainty with the uncertainties of antibody treatments, the assumptions that the relative susceptibilities of each class of T cells will be the same in different species, and the inescapable fact that (even as they themselves admitted when they said that other T cell activating agents had problems) indiscriminately activating T cells is a gloriously bad idea. Even if the volunteers had come out perfectly fine I'd be left wondering if they'd suffer some kind of autoimmune problem later on in life because the wrong T cells had been activated. Now I'm not saying that dire diseases couldn't justify such risk - in patients - but if they truly just took the maximum safe monkey dose and divided by 500, that seems outrageous.Mike Serfas 23:35, 16 March 2006 (UTC)

A Reuters article from today [1] quotes an expert, Dr. David Glover, "I suspect the antibody was designed to work against human CD28 and because it was designed to work best in humans its performance in different animals may fall short of what you might have expected in humans". Just remember you heard it here first. ;) Mike Serfas 19:36, 19 March 2006 (UTC)

A Daily Mail article from March 20 [2] says: "Professor Trevor Smart, head of pharmacology at University College London, said that as well as any permanent damage to their organs, there could be long-term disruption to their immune systems."Mike Serfas 08:19, 22 March 2006 (UTC)

Rhesus monkey CD28 is 100% similar to human CD28 (primate/human genome data), hence I would have thought binding would have been similar - this is crucial. Monkeys are expensive animals to experiment on - in the patent they mention monkey (singular) rather than monkeys - so I don't know how many they tested. Data from rodents would be largely meaningless with this agent since their CD28 are significantly different to result in much less binding of this antibody. Hence going 1/500th of murine LD50 would have been a big mistake - I don't imagine these investigators would have made this mistake. I am thinking more and more that this must be a dosing error - they gave much more of the drug than intended. They would have needed to submit their data to the MHRA (equivalent to the FDA in the US), we should be able to get this under freedom of information act at some point.Mxpule 10:43, 17 March 2006 (UTC)

It could be a dosing error, but if that's the case I would have expected them to know these symptoms very well from the animal trials where they also give high doses of the drugs. However, a reason why they seem so puzzeled over the symptoms could be that they would face a great liability risk if they gave the impression they kind of expected that this could happen. Another thing to keep in mind is that the drug was humanized so that it wouldn't be rejected by the human immune system. Thus, it is not as surprising that it gives a different reaction in humans than in animals. As I understand the humanization process they replace part of the animal antibody with a part of the human body. Couldn't this mean that the resulting antibodies would be able to send a signal 1 to other human T-cells (in addition to also being able to signal 2 via binding to CD28) leading to an even stronger immune response. Perhaps that could explain why the same reaction wasn't seen in monkeys even if they have identical CD28. 14:40, 17 March 2006 (UTC)

I am an immunologist, and have studied the use of monoclonal antibodies, particularly in humans as therapeutics, for more than twenty-five years. I am by no means a T cell expert, but I do understand antibody structure and function, monoclonal antibody design, point mutations and their effects on binding specificity, and the basics of clinical immunology and inflammation. I also appreciate the differences between human and mouse immune systems, in particular their numerous disparities in divisions of labor in Th1 and Th2 cells.

No, they didn't use OX40-Ig- that's a compound designed for use in mice, and the humanized equivalent is not available, as far as I know...

They say in the end it all boils down to sex or money. My sense is that someone (at TeGenero? –at Parexel?) was in a hurry to save money, or paid someone well to hurry for them. This horror has received almost no press in the USA: has Parexel silenced the media here? (I notice that their upper management is largely European.) Only a very few points of scientific importance as regards the TGN1412 disaster have yet been addressed. Start small…

A receptor/signal transduction molecule called CD28 is present on all T lymphocytes (T cells). Overall, the structure of CD28 is indeed well conserved across species- but TeGenero performed research, posted on their website and published in peer-reviewed scientific literature, on the fine crystal structure of the specific few amino acids to which JJ316 and TGN1412 bind- pointing out the requirements for valine(s) in different positions on the immunoglobulin-like region (the C’-D loop) where these agonistic antibodies bind, not to the “second-signal” determinant engaged by the natural ligands for CD28 (molecules called B7-1 and B7-2). Accompanying this is some information as regards CD28 amino acid differences among the species, which is already acknowledged in the present Wikipedia discussion.

The mouse IgG1 monoclonal (JJ316) antibody that was first engineered and tested in their rat model for arthritis differed significantly from the final form "humanized" and manufactured for use in trials in humans. In the patent for TGN1412 are references to phage-display engineered monoclonal antibodies described only by their DSM accession numbers and short pieces of their amino-acid sequences; the full identity of these antibodies is kept secret, stated at the DSMZ repository website as protected by the Budapest Treaty. Whether TGN1412 is identical or similar to either 9D7G3H11 or 5.11A1C2H3 is not revealed. Of particular importance to its pharmacokinetics (how long the antibody stays in the body, and where it goes/how it is cleared) is the antibody subclass of IgG: a humanized monoclonal might be expected to be either IgG1 or IgG4. Human and mouse IgG1 molecules clear similarly, form complexes and clear through the liver. IgG4 (no mouse equivalent) does not form complexes, is in essence functionally monovalent and persists in the circulation much longer.

Under the radar is the possible role of LPS (bacterial endotoxin): it is a common contaminant of biological drugs, and its content is severely restricted in preparations for humans because of its extreme toxicity; described in peer-reviewed literature is the amplification of this toxicity by ligands of the CD28 system. Notably, rodents are extraordinarily insensitive to LPS toxicity. The LD50 of LPS in mice is 250-500 micrograms per 20-gram mouse; the LD50 of LPS in humans is only 1-2 micrograms per 70-kg person. The GMP limit on endotoxin (LPS) contamination in a biological product intended for injection in humans is 1 nanogram of LPS per milligram of (antibody) and assumes no drug/LPS interaction.

Segue to dosing: the best I can figure is that TeGenero got approval to use 1/500th of the primate dose in their Phase I human trial. Dosing is ordinarily calculated on a weight:weight basis, and expressed in milligrams (or micrograms) of drug per kilogram of recipient. Average mouse 20 grams Average rat 200-400 grams Average rhesus macaque 10 kilograms Average human 70 kilograms

From a careful read of their TGN1412 patent application, they used a “low dose” for suppression of inflammation in monkeys of 1 mg/kg, which might well be a 10 mg dose per animal. For any other garden-variety antagonistic or tissue-antigen binding monoclonal antibody drug, a human dose of 1 mg/kg is indeed considered “low.” What really concerned me about their determination of TGN1412 toxicity in monkeys is that is was done “in vitro” and they had “no negative clinical observations” to report. Hm. “In vitro” does NOT mean they shot the antibody into the monkey; re-reading some of their statements further on in the patent application suggests that they took some blood from the animal, purified the lymphocytes, incubated these cells in culture with some TGN1412 antibody, and later re-infused the cells back into the donor animal. I’d not be surprised in the least to find activated T cells homing to lymph nodes- in the neck (cervical) or elsewhere.

Do the arithmetic: 1/500th of 1 mg/kg is 2 ug/kg; if you assume a big 100 kg man, that’s a 200 ug dose of antibody. The molecular weight of IgG is generally taken to be 150,000- at 6x10e23 molecules per mole and 150,000 grams per mole, that’s something around 10e15 molecules of IgG in a 200 ug dose. Standard clinical medicine estimates a 70 kg human to have a 5L blood volume. I took the liberty of expanding this to 6L in a 100 kg human; working with ordinary hematology tables for normal white blood cell counts, as well as my own experience in purifying human peripheral T cells, there are 10e10 T cells in circulation. That 200 ug dose of TGN1412, at 10e15 molecules, is more than enough to hit every T cell in the body ten times over. The time it takes to disseminate an IV bolus throughout the circulation is about five minutes, and the time it takes for an antibody of reasonable affinity to bind a reasonably available target is about fifteen minutes at body temperature- voila, about the amount of time elapsed from administration of TGN1412 to onset of inflammatory symptoms.

There are reports, including this one (Blood, 15 June 2004, Vol. 103, No. 12, pp. 4594-4601: CD28 disruption exacerbates inflammation in Tgf- 1-/- mice: in vivo suppression by CD4+CD25+ regulatory T cells independent of autocrine TGF- 1 , by Mizuko Mamura, WoonKyu Lee, Timothy J. Sullivan, Angelina Felici, Anastasia L. Sowers, James P. Allison, and John J. Letterio) -that speak to involvement of the CD28 family of receptors in inflammation.

This one (Infection and Immunity, December 2000, p. 6962-6969, Vol. 68, No. 12: Dose-Dependent Activation of Lymphocytes in Endotoxin-Induced Airway Inflammation, by Roland Larsson,1 David Rocksén,1 Bo Lilliehöök,1 Åsa Jonsson,1 and Anders Bucht1,2,*) -speaks to the involvement of the CD28 receptor family in responses to bacterial endotoxin.

Also described, in another paper (The Journal of Pathology Volume 199, Issue 3 , Pages 335 – 344: Early up-regulation of chemokine expression in fulminant hepatic failure, by Ludger Leifeld 1 *, Franz-Ludwig Dumoulin 1, Ingvill Purr 1, Katrin Janberg 1, Christian Trautwein 2, Martin Wolff 3, Michael Peter Manns 2, Tilman Sauerbruch 1, Ulrich Spengler 1 . 1Department of Internal Medicine I, University of Bonn, Germany 2Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Germany, and 3Department of Surgery, University of Bonn, Germany) -is the fulminant liver failure elicited in humans by systemic stimulation/upregulation of the CD28 system and concomitant wanton antigen presentation in this organ. If this doesn’t make the thought of a turbocharged CD28 system curl your hair, I don’t know what will.

Other publications in peer-reviewed journals (sorry, I didn’t keep all the refs handy) discuss the perplexingly opposite effects of CD28 ligation in healthy people vs those observed in RA and MS patients, and in localized lesions as compared to systemic exposure. Try searching on “cd28” and “inflammation” and dig for your subset(s) of interest-?

RA and MS are largely diseases of women, whilst the initial testing of this intended therapeutic antibody was undertaken in healthy men. Sigh, again.

Note: in December 2005, Orencia (CTLA4-Ig) which is described as a first-in-class antagonist of CD28 costimulation was approved by the FDA for treatment of rheumatoid arthritis in humans. CTLA4 is a member of the CD28 family of molecules. —Preceding unsigned comment added by 198.140.178.11 (talk • contribs) ````

Can someone wikify this comment? --Chl 13:43, 22 March 2006 (UTC)

Here is the FDA page with the patient information sheet for Orencia: http://www.fda.gov/CDER/drug/InfoSheets/patient/abataceptPIS.htm (sorry, I'm pretty much HTML-illiterate or I would be happy to link and/or annotate all this stuff) -notice the side effects and contra-indications, remember this drug is an ANTAGONIST of CD28 activation, not a superagonist.

Posted at the Research Diagnostics website (a vendor of antibodies for R&D use) is a product description sheet for JJ316- they sell it:

DATA SHEET: Mouse anti-Rat CD28 clone JJ316 Catalog#: RDI-RTCD28-JJ316 $625.00/vial Package Size: 500ug purified in 0.5ml PBS 1mg/ml (no carrier, no preservative) sterile filtered, **endotoxin <0.01ng/ug antibody** at time of bottling. Species: mouse IgG1k CLONE: JJ316 Source: purified Activity: reacts with rat CD28 which is expressed on virtually all T cells bearing AlphaB T cell receptors (TCR), on most DG TCR-bearing T cells and on a subset of NK cells. In the thymus, CD28 expression is regulated during the maturation of AB TCR-bearing T cells. CD28 is a co- timulatory receptor required for activation of T cells, its ligands include CD80 (B7-1) and CD86 (B7-2). Soluble Clone JJ316 enhances proliferative responses and IL-2 production by T cells activated by anti-T cell receptor Mab. At hugh concentrations, cross linked clone JJ316 mab stimulates resting CD4+ and CD8+ T cells to proliferate, secrete IL-2, express activation markers, and gain CTL activity in the absence of signalling through TCR. **In VIVO administration of mab JJ316 results in blastogenic transformation of peripheral CD4+ cells and dramatic lymphocytosis.**

Applications: include... -in vitro AND IN VIVO T cell stimulation and in vitro co- stimulation of T cell activation via TCR. ... References: J. Immunol 154:5121-5127; Eur J Immunol 27:239-247; Eur J Immunol. 25:328-332; Immunity 2:555-559.

It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product. ... All of this is pretty standard information for ANY antibody sold for research purposes. Remember, this is NOT TGN1412, but its research-grade predecessor.

... This is one of the references provided on the data sheet for JJ316:

Eur J Immunol. 1997 Jan;27(1):239-47.

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.

Tacke M, Hanke G, Hanke T, Hunig T.

Institute for Virology and Immunobiology, University of Wurzburg, Germany.

JJ316 and JJ319 are rat CD28-specific monoclonal antibodies (mAb) of the gamma1 kappa isotype with identical co-stimulatory potency. At a concentration 100-1000-fold higher than that required for co-stimulation, JJ316, but not JJ319 induces massive proliferation of all T cell subsets in vitro without T cell receptor (TCR) triggering. "Direct" stimulation by JJ316 is fully blocked by JJ319, indicating that it is not due to cross-reactivity of JJ316 with the TCR complex or other activating receptors. JJ316 binds much more slowly to primary T cells than JJ319, whereas both antibodies bind with similar kinetics to CD28-transfected L-929 cells, suggesting that JJ316 binding to T cells requires redistribution or a conformational change of CD28. In vivo, JJ316 but not JJ319 induces rapid and transient proliferation of most CD4 T cells and, indirectly, of B cells. These data show that TCR engagement is not an absolute prerequisite either in vitro or in vivo for the induction of T cell proliferation through CD28 and suggest that mAb JJ316 is able to stimulate resting T cells directly by recruiting CD28 molecules from an inactive to an active form. ... -and now I am even more confused and at a complete and utter loss as to WHY an antibody like JJ316 was ever administered to humans. 198.140.178.11

Media etc.

How much is the media playing up the "we have no clue how to treat this" "we're totally in the dark" angle of the doctors in this case? It would seem that if they know that this isn't a case of overdosing then they have general reason to believe that the drug really did 'hyperstimulate' the immune system to attack it's own body. Knowing that, wouldn't it be reasonable to turn to immunosuppressants such as Cyclosporine as a next course of action? Not knowing much about how clinical trials for pharmaceuticals like these go, I have to say it is rather shocking that they are allowed to tinker so freely with something as horrendously complex as the immune system. You would think that one would be more careful about not conjuring demons which one doesn't know how to cage. scary.--Deglr6328 04:11, 17 March 2006 (UTC)

Correction to article

..then delete this comment. I just don't have time to be "bold". original: "Criticism has been raised that 6 participants were given the drug in such a short time, which is against the recommendations of some standard literature."

correct: "Criticism has been raised that 6 participants were given the drug simultaneosly or in VERY short time, which is against anything even healthy brain and no SOME literature. I've read it in some serious newspaper yesterday that it is against all the odds to give such EXPERIMENTAL NON-CHEMICAL(I mean genetical here, e.g. not similar to 99.999% of the common drugs) DRUG to 6 patient at once. No some "standart literature" are we fuc"£ing joking here?

And also: "The initial dose, he said, was one five-hundredth of that which the animal studies indicated was the maximum safe amount.[11]." This is FUC£"ING lie, just delete it. I've read 4 newspapers on this subject yesterday(Times, FT, Guardian and ES) and I can't recall some of them said it. Maybe some Tabloid but I REALLY doubt it was 1/500 of the maximum animal dose. This is satan's first line of defence to say it. —This unsigned comment was added by 80.2.38.195 (talk • contribs) 10:15, 17 March 2006 (UTC).

It was on the Channel 4 news last night. It was a spokesman from the MHRA (I think). It wasn't a lie. That is standard procedure. They build up the doses over time. --Wikipediatastic 10:46, 17 March 2006 (UTC)

The statement about "one five-hundredth" on C4 news is unfortunately not verifiable online. The C4 web site has not been updated to include this interview. If the edit had been done by an anon, then it would probably have to be deleted. Since it was done by a logged-in user, with whom I have corresponded, I would like to assume good faith and leave it for now. Obviously, once better information is available, this section is likely to be rewritten. Colin°^Talk 11:34, 17 March 2006 (UTC)

No, it's not verifiable online. But that isn't a requirement, else we wouldnt be able to cite any book not released into the public domain. GeeJo _{(t) (c)} • 16:08, 17 March 2006 (UTC)

Lots of references on Wikipedia are not easily verifiable. They are, however, still verifiable. However, "I saw/heard on the news last night" is not verifiable unless someone has subsequently transcribed the interview and made it publicly available, or has otherwise written about it.

It is amusing that one primary source for this scientific article is The Sun. Colin°^Talk 17:07, 17 March 2006 (UTC)

Daily Mail.co.uk Article Okay its online now. I have read the 500 number elsewhere. The original question remains however: 500th of what. --ParticleBry 16:34, 17 March 2006

I would advise, as the original poster did, against the use of tabloids as sources of information. --Oldak Quill 22:33, 17 March 2006 (UTC)

A search on Google News for "CD28" and "500" turns up over thirty news articles citing that 1/500th of a safe animal dose was used. Admittedly, none of these are primary literature. However, the number seems believable because it has apparently been a common practice to use this dosage, although unfortunately not advisable in this situation. Mike Serfas 19:46, 19 March 2006 (UTC)

The fact that thirty newspapers or online news services all repeat the same thing does not make it more true, or more likely that any of them have checked the facts with a primary source. Agencies such as Reuters supply a considerable amount of the text that goes into a newspaper article. In addition, they all work off the same terse press releases by the hospital or drugs company. Most importantly, The Sun has "exclusive" access to one of the volunteers (who got the placebo). For this info, the Sun is a primary source and if The Telegraph or The Guardian repeats it, it doesn't make it more scientifically accurate. Pretty much all our info about the way the volunteers were given the drug and the immediate aftermath comes from one non-scientific, non-medical witness, being paid by a tabloid to give a sensational story. Draw your own conclusions as to how much weight we should be giving to such a source in an encyclopedia. Colin°^Talk 20:46, 19 March 2006 (UTC)

Conjecture

Any conjecture in this article must be made by a third party otherwise it is Original Research. We can report facts (such as the difference between rodent and primate biology, etc) but postulating as to the cause of error must be attributable to some other source, preferrably with a citation/reference to online material that can be verified.

Also, this talk page is for discussion regarding this article, and how to improve it. It is not a forum for general discussion/speculation about this drug and the clinical trial. Colin°^Talk 11:34, 17 March 2006 (UTC)

That rule only applies to the article itself. Talk pages can and do contain whatever people want to talk about regarding the subject. With a story like this one, especially since it's still ongoing, it is only natural that this page will be full of conjecture like so many others. --Deglr6328 13:39, 17 March 2006 (UTC)

See What may talk pages be used for?. Whilst talk pages have relaxed some rules (e.g. you can express a POV), they are not a substitute for an Internet forum or blog. This is just a Guideline, not a Rule. I'm not trying to stop any discussion here, just to keep it focussed on the article. Colin°^Talk 14:40, 17 March 2006 (UTC)

Only academic reference?

The article presently states something like "this is the only academic reference" in connection with an abstract. What about the articles returned from Google Scholar: [3], e.g., [4] Sorry Dude #1 isn't academic (i.e) peer reviewed #2 is about the antibody before it got humanized.Mxpule 15:38, 17 March 2006 (UTC) If you can find a peer reviewed paper or abstract about the humanized antibody that would be fab.

I thought it was pretty obvious that the first Google Scholar entry wasn't academic. Also note that the abstract of the second writes "superagonistic anti-CD28 antibodies offer a promising novel treatment option for human autoimmune diseases ..."- fnielsen 00:18, 27 March 2006 (UTC)

Since when is LD50 "safe"???

"The critical point here is whether investigators gave 1/500th of the murinae LD50 or a safe dose in primates."

LD50 is the dose that has a 50% chance of having death as a side effect. LD50 is an unsafe dose. Don't talk about it like it's a safe dose. It isn't, the whole point of labelling it such is that it isn't.

Er did you actually read the sentence? What it is trying to say is we don't know whether it is 1/500th of the murinae LD50 which would be a very serious mistake OR 1/500th of the safe dose in primates. I believe the original quote is something like 1/500th of the maximal dose in animals. We cannot conclude from this quote what exactly they mean by 1/500th of the maximal dose in animals, it could be the LD50, it could be the safe dose... A number of people including me find it unlikely that anyone would be stupid enough to give 1/500th of the LD50 in murines as the starting dose given that the people administring this trial were dumb enough to give it to 6 people in a short space of time, who knows? Nil Einne 17:10, 17 March 2006 (UTC)

Multicentre trial

This article links to multicentre trial. Was this intended to be a multicentre trial and were there other centres going to test the drug in a few days or hours time? If so, I would guess the people recruited in the other centres must be thanking their lucky stars that they weren't the first Nil Einne 17:10, 17 March 2006 (UTC)

Time frame

Does anyone have any idea what the time frame is like? This article supports what I think many of us not knowledgable in clinical testing thought, that it is not standard practice to give the drug to 8 different people (although 2 received the placebo of course) in such a short space of time but what kind of time frame are we talking about. Did they administer doses within minutes? Hours? How long is it normal to wait? Nil Einne 17:16, 17 March 2006 (UTC)

Criticism has been raised that six participants were given the drug in such a short time, which is against the recommendations of standard literature

Could anyone more medical-literate find a citation to support the "recommendations of standard literature"? - Eric 17:03, 18 March 2006 (UTC)

Diagram

I think what this article needs is a diagram on the mechanism of action - even if it is just a molecule binding to a T Cell. Do we know enough about the mechanism to be able to pull it off? Secretlondon 21:09, 17 March 2006 (UTC)

Agreed. 4.232.195.49 01:19, 18 March 2006 (UTC)

A partial diagram can be found here: http://www.tegenero.com/research__development/technology/index.php —Preceding unsigned comment added by 64.105.73.7 (talk • contribs)

MOST importantly...

What sort of compo are they looking at if they make it? Presumably they took part in these trials to earn a bit of extra dosh. They should be able to sue after this. Probably never need to work again. ;-).

Sounds like some of these subjects will be lucky to make it out of ICU alive, probably won't be able to ever work again. Mxpule 11:20, 18 March 2006 (UTC)

I've been wondering about this too and it's something that should be added somewhere IMHO. Since this is the UK were talking about not the US and given that the people taking part in the trial must have signed some agreement severely restricting their ability to claim compensation, I would guess it'll depends a LOT on whether anyone was actually neglegent or not and how neglegent they were. From the story as it stands, it appears they may have been at least partially neglegent if they did violate the supposed agreed protocol of 2 hours between treatments. Does anyone know if it's standard practice to insure test paticipants against ongoing medical complications/costs from negative reactions? Nil Einne 13:18, 20 March 2006 (UTC)

Mistake comparing CD28

Sorry I made a mistake, I thought drug tested in Pan troglodytes not Macaca mulatta. The Macaque's CD28 is slightly different - a few amino acids. You can look at the links below - the sequence starts "MLRLLLALNLLP...." with each letter representing a different amino acid. If there are some protein chemists out there they might tell us if the small differences could effect where the antibody binds. This is probably explains what happened. http://feb2006.archive.ensembl.org/Macaca_mulatta/protview?transcript=ENSMMUT00000008414;db=core http://feb2006.archive.ensembl.org/Homo_sapiens/protview?transcript=ENST00000340642;db=core http://feb2006.archive.ensembl.org/Mus_musculus/protview?transcript=ENSMUST00000027165;db=core

Mxpule 14:47, 18 March 2006 (UTC)

At the "user" level for antibodies (trying to stain where a protein is located in a tissue, rather than really trying to understand the immune process) the (oversimplified) generalization often given is that a monoclonal antibody binds a small piece (epitope) of a protein about six amino acids in length - and changing any of them may greatly reduce binding. I know from sad experience that just because a butterfly protein has many stretches of more than six amino acids homologous to fruit flies doesn't mean that a monoclonal raised against a fruit fly protein that beautifully lights up the distribution of a protein in a fruit fly will give any signal at all when used in immunohistochemistry on a butterfly. It is true that the monkey CD28 is extremely similar to human (unless there's some difference in the preferred splice forms...) but any difference at all would be something to worry about when considering what dosage is safe.—Preceding unsigned comment added by Mike Serfas (talk • contribs)

Which makes me wonder, I'm not a medical research but surely you would carry out binding affinity tests and the like to compare the binding affinity of their drug to human CD28 and their animal model CD28? I can't remember much from what little I learnt about T-cell binding but surely something could be done, perhaps with cell culture or purified proteins? Of course, it may not simply be a case of binding affinity, it could have different levels of activation with equal binding affinity and the cascade is probably too complex to study in isolation (I guess it's probably not that well understood even in normal circumstances) but isn't it possible to try to determine the amount of activation from cell culture? Obviously this may not necessarily provide much useful information but it sounds like something that should have been tried first. I would guess some of this may have been done but perhaps hasn't been published yet. Nil Einne 12:39, 20 March 2006 (UTC)

On the question of possible species differences in the reactivity of the superagonist anti-CD28 mAb, TGN1412, the following data should be taken into consideration: The amino acid (aa) sequence differences between Macaca mulatta monkeys and man is only about 4 % (i.e. 9 of 220 aa) (1,2). However one of these substitutions (a glutamic acid for a glycine at the 83rd position in the protein sequence) occurs in what has been crystallographically determined to be a point of contact between CD28 and the active site of the antibody in question (3).The exchange of uncharged glycine with the larger negatively-charged glutamic acid is likely to have a marked effect on the strength of antibody-antigen interaction. That is to say, TGN1412’s binding affinity for Macaca CD28 (and thereby its potency as an immunoreactive drug) maybe several orders of magnitude lower than that for human CD28. Direct measurement of the binding affinities of TGN 1412 for both human and Macaca CD28 would appear to be off critical importance in assessing the relative potency of TGN1412 in the two species. 1) http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=5453611 2) http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=13650008 3) Evans-EJ et al. Nature Immunology 6 :271 (2005).38.119.107.81 09:21, 24 March 2006 (UTC)

front page news?

While this is an interesting article it doesn't really seem to me to be front page news; other media don't seem to be covering it intensively. Phr 23:15, 18 March 2006 (UTC)

I disagree. Novel treatments based on monoclonal antibodies have been proposed for many diseases and a number of them have been released. There are prospects for remarkably effective treatments and of course large sums of money are involved. It also touches on the issues of animal experimentation, a highly political issue in some countries, and can be used as evidence by both sides of that issue as support for their position. ("Animal testing didnt detect this, so we shouldnt test on animals" vs "This will be more common if we dont test on animals"). The implications arising from this case in relation to drug testing of newer drugs are profound, and the complexity of the issue has resulted in many reports of it being overly simplistic (as commenetd on above), something wikipedia can correct since esoteric subjects like this are one of the wiki's strong points compared to most mainstream news sources, thanks to the expertise of members of the wiki community. bignoter 05:16, 19 March 2006 (UTC)

I don't think the front page of wikipedia is an appropriate mechanism for telling media outlets what the news should be. Wikinews is a better choice and was created for that purpose. Phr 05:44, 19 March 2006 (UTC)

The UK media have been covering it intensely. The question is - why have the in the news section - to tell people what's in the news (they can get this anywhere) or to link to good articles on current topics. We still have the best article on this subject - much better than the UK media despite it having been on their front pages. This is an encyclopedia article - not a current affairs article. Discussion on "in the news" is probably best placed at Template talk:In the news. Secretlondon 08:28, 19 March 2006 (UTC)

I think it is okay it is on the front page as it is still developing and is one the front page of many media outlets, especially in the UK. As others have pointed out it could have profound implications for medicine too and I think it is strange that it is covered so little in, for instance, the Danish news media since a lot of my friends, colleagues etc. seem to be talking about how these are doing. The Wikipedia article currently seems to have all the credible information about the drug in one place whereas most other news stories have only bits and pieces and many have serious errors and misunderstandings. This article is also a good example of Wikipedias advantages. It seems it was the first on the web to go into detail on the mechanism of action by collecting info from the company, the scientific literature, sequence databases and from the patent applications. In the case of a complex subject as this it is also nice to have direct links to the related articles on the immune system, cytokine storms etc. This serves as a good example of Wikipedias potential. On the other hand, many other news stories are emerging and not all stories can be on the front page, so of course it can't be on the front page indefinitely.

Oops

From the Times:

EXPERTS knew that drugs similar to the one that nearly killed six men at a London hospital last week could have had dangerous side effects.[5]

Nomen Nescio 00:41, 19 March 2006 (UTC)

Link this to "serious adverse event"

Does anyone think this article should be linked to the Serious adverse event article? The article is a stub, but this article seems to be a pretty good example of a serious adverse event. 02:55, 20 March 2006 (UTC) —Preceding unsigned comment added by 69.86.35.181 (talk • contribs)

Yes. Rich Farmbrough 14:00 20 March 2006 (UTC).

Update

Can someone update the article please. http://news.bbc.co.uk/1/hi/england/london/4823992.stm for example. I don't have time right now. Rich Farmbrough 14:00 20 March 2006 (UTC).

It was done earlier. Last para. Secretlondon 05:00, 21 March 2006 (UTC)

Fee higher than average?

The article links to a Daily Mail article claiming that the fee (£2000) was many times higher than the "few hundred quid" normally offered. This isn't the case - I'll try and find some sources. The Daily Mail really isn't a very good source to be honest. Secretlondon 09:00, 23 March 2006 (UTC)

Yes, please try to clear this, we've had the same issue on the German article. Paparodo 11:06, 23 March 2006 (UTC)

Making a career out of drug tests is the best I can find at the moment. Secretlondon 18:09, 26 March 2006 (UTC)

I would expect it depends significantly on how long you are required. Nil Einne 16:00, 1 April 2006 (UTC)

Of course. My comment is that the Daily Mail are trying to make a story out of the fee to imply that it was known this drug was more dangerous than average. Secretlondon 16:09, 1 April 2006 (UTC)

I am deeply impressed by the learned arguments herein and I shall enjoy plowing through them during the short summer nights to come. Have you heard of MDX-010? ( This the drug refered to in "From the London Times 4 papragraphs above.) I googled TGN1412+MDX010 and I put out a google alert for TGN1412+MDX 010 and have come up with a load of shocking truths.

Isn't it misleading to call these MAB's drugs? True they come in tablet form but don't they really belong with other bits of genetic engineering like that laboratory created bladder the other day? I mean if they had told the volunteers that they were going to give them body cells which were 95% human and 5% mouse and would replicate in their bodies like any other cell and would possibly change their genetic structure permanently and that change wou;ld be passed on to their children then.......you get my drift

"Fees higher than average" above. the daily mail was right. The normal is rate is £100 for every day spent in the clinic and £30 per follow up visit. Apparently it was all done in a great hurry. At least some of them turned up on the day expecting to take part in a different trial but were told that was cancelled and were offered the MAB trial. The rest as they say is history. —Preceding unsigned comment added by 87.74.227.217 (talk • contribs)

I have no idea what you are talking about as I expect with other people on this board. The drug in particular was a drug. It is not a cell, nor would it replicate in the human body, nor would it change anyone's genetic structure.

Finally, no ever that we know of has attempted an experiment that could potentially modify the human germ line. This has never, ever been approved and is almost definitely not going to be approved any time soon. All gene therapy experiments most specifically ensure the germ line will not be affected. Believe me, if any drug affected the germ line, this would be front page news throughout the world and would not have gone away so fast.

I have no idea whether your fee claim is correct although I doubt it. In any case, given then clear complete lack of credibility of your other information, I think we can safely say the whatever sources you rely on are highly circumspect and your ability to understand and evalute information is also highly circumspect. Nil Einne 11:23, 6 April 2006 (UTC)

Looking through the material published by the MHRA it would appear that the test subjects would have been required to attend the hospital on at least more than 15 days (it does not appear unlikely that 20 visits would have been required), in which case the fee paid would have been standard for a trial of this length.

The dosage frequency issue

However, it appears the MHRA approved a protocol involving the doses being given within 2 hours whereas one of the placebo-receiving participants has explained the doses were given with 2-minute intervals

Has anyone seen the issue of dosage frequency raised in any newspaper or whatever discussing the story? We've discussed it earlier and although it did seem a bit bizarre to me, the fact that I haven't seen any discussion/mention of it in any media discussion and the fact that the report doesn't discuss it makes me wonder whether this isn't really an issue. Perhaps they we're planning to administer the actual primary doses within 2 hours of each other however the initial 1/500 dose was going to be administered at abouy the same time. I guess since this was such a minute dose, the expectation was that absolutely nothing would happen and even if something did happen, they weren't in their wildest dreams expecting anything this severe... Nil Einne 11:36, 6 April 2006 (UTC)

From the statement released by the MHRA, and a followup article released last night (please see http://www.the-scientist.com/news/display/23275/) by The Scientist, the eight men were dosed at ten-minute intervals and the first of them were already sickening before the last of them were administered their injections. Here is a link to the press release on the MHRA page: http://www.mhra.gov.uk/home/idcplg?IdcService=SS_GET_PAGE&useSecondary=true&ssDocName=CON2023515&ssTargetNodeId=389 and at the bottom are links to a number of pdf files, including the clinical trial protocol. 198.140.178.11 15:30, 7 April 2006 (UTC)

Thanks. The interim report and other documents have lots of information. The interim report says the 6 volunteers experienced a life threatening incident of "cytokine release syndrome" (page 2). Clinical trial protocol 6.3.2 (page 33 in the pdf file) says a "cytokine burst" or anaphylactic reactions theoretically could occur within the first few hours. My reading is that the trial documentation covered the possibilty of the reaction occuring. (I'm presuming that cytokine burst and cytokine release syndrome are the same). The documents contain lots of info about the trial protocol, dosing, and previous studies. We should be able to add lots to the article from this. Secretlondon 16:31, 7 April 2006 (UTC)

The New York Times recently reported that there was a 10 minute interval between dosages and that the first man was expereriencing ill effects before the last man was injected. http://www.nytimes.com/2006/04/08/world/europe/08britain.html Cshay 04:20, 10 April 2006 (UTC)

Immunological activity of TGN1412 at low doses was known. According to the IB and the IMPD available on the MHRA website, rats receiving an equivalent monoclonal had immunological effects at 0.3mg/kg - but no no observed effect level was stated. The primates in the 28 day study showed T cell activation and increases in some cytokines at 5mg/kg. Again, it is not known at what dose this effect ceases. Unfortunately, most of the in vitro activity data were deleted. These can give some clue as to what tissue/plasma concentrations might be active. However, it is clear that immunological changes would reasonably have been expected in volunteers at the starting dose of 0.1mg/kg. In calculating a safe starting dose TeGenero disregarded the immunological effects and MHRA endorsed this. Their reasoning seems to have been that the T cell and cytokine changes were not in themselves adverse and no cytokine release syndrome had been detected in the primates. Note, the primate study was not robust - two animals were killed because of diarrhoea and it is likely many others were affected but less so. Was it reasonable to disregard the immunological changes likely to be present when calculating the starting dose? If I had been doing this assessment (and I have done dozens)I would have wanted a lot more information than was apparent from the released documentation before recommending such a high dose to volunteers - we know all too well the serious adverse reactions that can accompany T cell and cytokine stimulation and TeGenero appreciated this. And why they started with 6 volunteers apparently to be dosed within a total of 2 hours defeats me. Sandy Wilson 14:18, 10 April 2006 (UTC)

Immunological changes at trial doses were expected

Firstly, my apologies, I initially placed this in the wrong position. Immunological activity of TGN1412 at low doses was known. According to the IB and the IMPD available on the MHRA website, rats receiving an equivalent monoclonal had immunological effects at 0.3mg/kg - but no no observed effect level was stated. The primates in the 28 day study showed T cell activation and increases in some cytokines at 5mg/kg. Again, it is not known at what dose this effect ceases. Unfortunately, most of the in vitro activity data were deleted. These can give some clue as to what tissue/plasma concentrations might be active. However, it is clear that immunological changes would reasonably have been expected in volunteers at the starting dose of 0.1mg/kg. In calculating a safe starting dose TeGenero disregarded the immunological effects and MHRA endorsed this. Their reasoning seems to have been that the T cell and cytokine changes were not in themselves adverse and no cytokine release syndrome had been detected in the primates. Note, the primate study was not robust - two animals were killed because of diarrhoea and it is likely many others were affected but less so.

Was it reasonable to disregard the immunological changes likely to be present when calculating the starting dose? If I had been doing this assessment (and I have done dozens)I would have wanted a lot more information than was apparent from the released documentation before recommending such a high dose to volunteers - we know all too well the serious adverse reactions that can accompany T cell and cytokine stimulation and TeGenero appreciated this. And why they started with 6 volunteers apparently to be dosed within a total of 2 hours defeats me. Sandy Wilson 14:31, 10 April 2006 (UTC)

The MHRA released, albeit incompletely, the TeGenero TGN1412 investigator's brochure and Parexel's clinical trial study design. TeGenero's data showed that JJ316 (mouse IgG1 anti-rat CD28) apparently induced expansion of rat Treg cells without also stirring up a cytokine storm. They then engineered a mouse anti-human CD28 equivalent (5.11) which was then further modified by grafting the antigen-binding domains into human IgG1 and IgG4 frameworks.

TeGenero states flat out that rhesus and human CD28 are identical in the TGN1412 binding region. Although they say that they did indeed shoot antibody into monkeys (cyno, not rhesus) for a tox study, as high as 50 mg/kg on a weekly basis for a month, what these monkeys were given was NOT TGN1412 but TGN1112. TGN1412 is a humanized IgG4; TGN1112 is its humanized IgG1 equivalent. The affinity (Kd by Biacore binding) of TGN1412 is given as 1.88 x 10e-9 M, decent high-affinity binding. (A *similar* value for TGN1112 might be expected because the antigen-binding site is identical, but the electrical & configuration changes upon ligation of the molecules are likely to be different.) Their cyno data indicate a mean serum half-life of eight days for TGN1112; onset of T-cell expansion at day 3, induction of IL-5 and IL-6 (but not TNF-alpha or IFN-gamma), and a delayed expansion of B cells at around day 20. In humans, CD28 is reported also to be expressed on plasma cells. A "significant number" of these animals developed antibodies to TGN1112. In rhesus monkeys, TGN1412 has a much longer serum half-life than TGN1112 does in cyno monkeys.

Eerie in hindsight is mention of a relatively small dose of antibody rapidly saturating the CD28 on "virtually all" circulating T cells; also binding to T cells in lungs and gut epithelium, tissue reactivity on cells in the CNS (astrocytes?) and in the cervix, and noticeably less effect of T cell expansion in female cyno monkeys than in males. Not addressed is the fate of TGN1112 (or TGN1412) after ligation of CD28: does it remain on the cell surface? -is it internalized? cleaved? -is CD28 recycled to the cell surface? It is reported that ligation of CD28 upregulates expression of itself and of its ligands (CD80, CD86) within a few hours. This is consistent with the kinetics of fulminant liver failure observed after abrupt perturbation (and upregulation of expression) of markers in the CD28 family, reported in the 1999 Journal of Pathology reference mentioned in another part of this discussion.

TeGenero justified use of TGN1112 in the tox study because it "worked better" for T cell expansion in monkeys than TGN1412; they addressed the possibility of IgG1-mediated damage by ADCC and by recruitment of inflammatory neutrophils (via IL-8 chemoattraction). Neutrophils express Fc gamma RI and are plenty capable of ADCC in fairly short order- if monkey neutrophils bound TGN1112 by Fc gamma RI, they might have targeted and killed the T cells ligated by the antibody’s other end and cleared out the whole business before a cytokine storm ever developed its first cloud. No mention of ADCC is made in the rat studies, although JJ316 is an IgG1 and perfectly capable of this function. Rodents do not have IL-8 and mediate inflammatory chemoattraction differently.

What the humans got was a completely different molecule- alluded-to in the Nature report by way of mention of differences in antibody tails. IgG4 has a truncated Fc that does not bind Fc gamma RI- no immune complex formation, no ADCC- and it’s functionally monovalent, so no “complexing” by way of crosslinking multiple antibodies with antigen-in-common, for accelerated clearance. Ligation of CD28 is reported to up-regulate expression of CD28, CD80, and CD86 (this is stated in the TeGenero documents also), and it happens fairly quickly- within a few hours.

Mice aren't humans, or rabbits. Monkeys aren't humans. Rhesus aren't cynomolgus. CD28 is under different controls for expression, and distributed on diffeent cell types, in the model animal species chosen. JJ316 isn't TGN1412; IgG1 is not IgG4; transfected CHO cells are not hybridomas. And although the study design appeared well written, somehow "subjects are to report to the hospital at 7:00 am" was construed to mean that ALL members of that first cohort were to appear SIMULTANEOUSLY for the very first dose-?

I’d better stop now. ````(198.140.178.11)

The IMPD or the IB (available on the MHRA website)describe the primate studies and state the compound administered in the pilot and 28 day cyno studies was TGN1412. See page 36 and pg 40 respectively of the IMPD. An earlier study in rhesus used both TGN1112 and TGN1412. TeGenero also carried out rat studies using JJ316, a mouse anti-rat CD28. Having said that, it is clear that immunological effects could reasonably have been expected in the volunteers at 0.1 mg/kg. I have been able to find very little information on the value of animals (via the web and immunology friends and focusing particularly on primates)in predicting CRS etc in man. Your interesting paragraphs on the different species/antibody effects enlarge on the difficulty in extrapolation between species. Which makes disregarding the immunological effects for calculating the safety factors even more inexcusable. Sandy Wilson

- My turn to make apologies- I did indeed print out and read in their entireties the four documents posted on MHRA's website, and they were quickly marked up in yellow highlighter and littered with notes to myself in the margins- but I'm not seasoned in wading through this kind of document, and evidently blew it! -it's a good thing that you have such experience, and thank you for catching me up on my mistake so quickly. I think I homed in on the PK figures, and knowing that IgG4 normally persists far longer in circulation than IgG1, flipped the other parameters in my head.

I'm more accustomed to reading research journals, and have skimmed through half a dozen of the reports on JJ316 (and JJ319) findings in rats- I noted the inclusion of JJ316 data (second sentence in my first paragraph, above) and I can certainly understand mention of JJ316 as the "founding-father" molecule for TGN1412 as far as host target specificity, but perplexed at TGN1112- particularly its being taken as far as primate studies, when that expense with an antibody clearly inappropriate for human use would be unjustified (in my mind, at least). The basic immune-complexing and ADCC studies could have very easily been carried out with human peripheral blood cells and serum immunoglobulins. Neutrophils are pretty touchy, but I know from direct experience that they can be purified from whole blood and used as effector cells in fairly standard ADCC assays, with both mouse and human antibodies, to explore killing of target cells bearing (or not) appropriate target molecules. They could have looked at this with and without IL-8, also, if they were worried about recruiting or generally activating inflammatory cells. (It's all moot with an IgG4, which is borne out with their ADCC data.)

Mention of TGN1112 as a stepping-stone between JJ316 (mouse IgG1 anti-rat CD28), to TGN1112 (humanized 5.11 IgG1 anti-human CD28), to TGN1412 (humanized 5.11 IgG4 anti-human CD28) makes a kind of sequential-logic sense, but AFAIK humanized IgG1 monoclonals are carried into human use only when their bivalent antigen-binding and/or functional properties of their Fc moiety are desirable. (If you're interested, you might look up humanized IgG1 antibodies to TRAIL-R1 and TRAIL-R2. I've done in vitro studies with some of these antibodies, and it's fascinating. A few have made it into early clinical trials.)

Non-human primates for preclinical studies: what I *do* know is that rhesus are way more expensive than cyno monkeys, and to the best of my knowledge considered the closest approximation of the human for overall immune function- although I do not know the details. For flow cytometry studies, commercially available reagents for CD antigens are offered for human markers- and a select few are described specifically as having ability to bind the homologue in various primate species (cyno, rhesus, baboon...) a little background is NIAID's ongoing study of HIV/SIV in baboons and other primates, where these reagents would be needed. Perhaps TeGenero's tox study was done in cyno monkeys both for cost containment (cyno monkeys would require less antibody- they're smaller than rhesus- and be cheaper overall to use, as well as easier to get- and the appropriate fluorescent marker antibodies were available-?

Totally inexcusable, to my mind, is the apparently cavalier assumption that ligation of CD28 invokes similar sequelae in rodents and in humans. Rodents and humans divvy up the jobs done by their Th1 and Th2 cells very differently- it all gets done, but if it matters by WHOM, a lot of parallels disappear. What might be expected of a human Th2 cell when its CD28 is tweaked would be very different from what a mouse Th2 cell would do. No mention is made of the expression of CD28 being under completely different kinds of genetic control; no mention is made of its appearance on significantly different families of cells. In mice (and rats also, I think) NK cells bear CD28, but in humans NK cells do NOT bear CD28. Mouse mast cells bear CD28, human mast cells do not. Human plasma cells bear CD28 (these are mature, antibody-secreting B cells), but not mouse plasma cells.

More disturbing is the apparent assumption that immunological responses to such ligation would be similar in normal individuals and in the kind of immune-compromised patients they intended to treat with TGN1412: normal folks typically have few CD28(null) cells (cells bearing CD28 but neither CD4 nor CD8), but a diagnostic indicator of impending or active autoimmune diseases such as RA and MS is accumulation of such CD28(null) cells- and what I don't know is what they do when their CD28 is ligated, and how it differes from such ligation in normals. Patients with autoimmune disease are *already* in a very different state of function from that of normal healthy individuals- it just stuns me that this antibody was approved for use in normal humans at all.

When I read the Nature report that mentioned functional differences in antibody "tails" it was almost comforting to know that this difference had been noted and was under scrutiny- but the media take on the subject seems to be backwards, I'm reading statements that the antibody given to the human volunteers was somehow cytotoxic, and the one used in the primates was not- and I think it's the other way around! -the humans got the IgG4 which cannot form immune complexes either through Fc binding or by binding multiple antigenic determinants, nor can it mediate ADCC. For this I have pictures... not of TGN1412, but of human IgG1 and IgG4 with the same antigenic specificity offered their (soluble, not cell-bound) antigen in vivo and followed for biodistribution, clearance, and immune complexes... bound, crosslinked IgG1/antigen complexes go straight to the liver within an hour, while IgG4/antigen remains in the bloodstream for days. it was a poster at the Clinical Immunology Society conference, in 1989.

The issue of calling ALL of the volunteers in at one time for the first dose of TGN1412, injecting them at very short intervals, AND continuing even when at least one or two of the first recipients were already in trouble- how that made sense to anyone is far beyond my comprehension.

(198.140.178.11)

Criticism and controversy

I don't wish to split hairs, but if the transient swelling of the lymph nodes is the consequence of the increased T cell production which is the drug's objective, then this swelling is not, strictly speaking, a "side effect" (as it is referred to in this section) but evidence of the drug's mode of action. Any objection to my changing this? Rrburke 04:29, 11 April 2006 (UTC)

I certainly have no objection: a "side effect" after all is a major effect that's undesirable, n'est-ce pas? (aka 198.140.178.11) ````