Editor’s Note: This is part two of a three-part series on six men being hospitalized after taking part in TeGenero AG’s Phase I trial. Part one ran in Thursday’s issue. The series will conclude in Monday’s issue.

On March 15, six previously healthy volunteers were delivered to the intensive care unit in the UK after being dosed with the humanized antibody TGN1412, which was supposed to activate regulatory T cells.

Even in hindsight - supposedly 20/20 - it is not yet clear what caused the clinical trial to go so wrong.

From a scientific standpoint, other than contamination, there is a range of possibilities for what might have gone awry, some more likely than others. But "whatever it was, it was reproducible," Carl June, a professor at the University of Pennsylvania who is conducting Phase I trials that target the co-stimulation pathway, told BioWorld Today. "Six out of six. That makes an allergic syndrome, for example, very unlikely."

All experts who spoke to BioWorld Today noted that they had not seen the patients themselves, or their actual clinical data, and so it was impossible to say with certainty what they were affected with. But the most likely explanation was that TGN1412 massively stimulated all T cells instead of the regulatory subset it was supposed to affect preferentially.

Such stimulation would have led to the massive release of cytokines, which could explain the organ failure. That phenomenon’s technical name is "cytokine release syndrome." Its more dramatic stage name is "cytokine storm." It can occur as a side effect whenever antibodies to T cells or B cells, including such overall clinical successes as Genentech Inc. and Biogen Idec’s Rituxan (rituximab), are given.

"One way to rationalize the reports of swelling we see in the papers is massive T-cell activation," Joaquin Madrenas, professor of microbiology, immunology and medicine at the University of Western Ontario, told BioWorld Today.

While TeGenero scientists published peer-reviewed reports showing that TGN1412 preferentially affected regulatory T cells, Madrenas commented that "in vitro it was shown that [TGN1412] can activate regulatory T cells. But it can activate any T cell that expresses CD28. There was no priori reason to expect to see the selective activation of regulatory T cells."

If cytokine release syndrome is what happened, the next question is why there was no hint that it would occur in preclinical studies, including primate studies: The worst side effects there that TeGenero reported was a mild swelling of the lymph nodes in two monkeys - and that was with doses that were orders of magnitude higher than the Phase I volunteers received.

June said that what is known about species differences in the co-stimulation pathway could explain the results. While cautioning that co-stimulation research is "a rapidly evolving area," what is known to date suggests that the pathway is more active in humans than the so-called lower animals.

June said, "CD28 knockout mice have a normal life span, as long as you don’t stress them." In contrast, "studies that have looked in humans have never found someone with a nonfunctional CD28 receptor," which suggests that such a mutation would kill a human in embryonic development.

In primates, the problem seems to be a different one. Such testing "lacks a positive control," June said. Doing a primate test in and of itself "doesn’t necessarily mean they tested" a drug that has action in monkeys - it means they tested a drug.

In other words, small differences in species receptors make for big differences in how target and antibody interact, "perhaps more relevant to biologics than to small molecules."

For example, "you can inject OKT3" - Orthoclone OKT3, an anti-transplant rejection drug from Ortho Biotech Products LP and the first monoclonal antibody to be approved, in 1986 - "into a monkey and nothing happens," June said. "In a human, OKT3 can kill you."

The same phenomenon exists for co-stimulation. "My own antibody only works in about half of all primate species," June said. That antibody also targets CD28, though it is used ex vivo and T cells are reintroduced into patients only when cytokine secretion is at the desired level.

Nils Lonberg, senior vice president and scientific director of Princeton, N.J.-based Medarex Inc., said: "Clearly [TGN1412] must bind to the monkey CD28. But I wonder whether it binds in the same way as to the human CD28."

As part of the investigational new drug application for MDX-010, which is in clinical trials to treat different cancers, including pivotal trials for metastatic melanoma, Medarex and Bristol-Myers Squibb Co. submitted data to the FDA showing that the anti-CTLA-4 antibody bound to CTLA-4 in their primate models as it does in humans.

"I would assume [TeGenero was] as careful [as we were]," Lonberg said. "But I would want to see that data."

One factor that should not have made a difference is that TGN1412 was humanized. If anything, that should have made the data more predictive, since those parts of humanized antibodies that are not derived from humans are from mice - humanizing the antibody makes it more compatible to monkeys, not less.

MDX-010 is a full human antibody produced by transgenic mice, and Lonberg noted that in more than 600 patients who have been treated with anti-CTLA-4 to date, the company has never seen "anything like" the universal massive adverse reaction reported by TeGenero and Parexel. "The type of adverse events we see are very different," he said. "Our experience with the anti-CTLA-4 antibody has reinforced what was initially a bias, that a fully human antibody would be very low immunogenicity."