Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4 + effector memory T‐cells

May combine two claims.

Subsequent analysis of the TGN1412 used at the trial showed that it was formulated correctly, complied with its product specification and was free from pro-inflammatory contaminants.

“Subsequent analysis of the TGN1412 used at the trial showed that it was formulated correctly, complied with its product specification and was free from pro-inflammatory contaminants (Duff, 2006;Stebbings et al., 2007).”

• cites Duff, 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Stebbings et al., 2007 sourcePartly supported

  The introductory section (page not available) says TGN1412 used in the trial tested negative for endotoxin and describes subsequent analysis at NIBSC. The passages available from our copy do not establish that the trial material was correctly formulated, met its product specification, or was free of all pro-inflammatory contaminants; a negative endotoxin test supports only the narrower contamination claim. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

TGN1412 was called a superagonist because it could activate T-cells without prior engagement of the T-cell receptor complex.

“TGN1412 was called a 'superagonist' because it could activate T-cells without the need for prior engagement of the T-cell receptor complex (Tacke et al., 1997;Lühder et al., 2003).”

• cites Tacke et al., 1997 sourceNot checked — no full text of the source
• cites Lühder et al., 2003 sourcePartly supported

  The Introduction defines anti-CD28 superagonists as antibodies that fully activate resting T cells without TCR signaling, and the Figure 4 caption describes human T-cell stimulation without TCR triggering. Our copy does not identify TGN1412, so it supports the reason for the label but not the claim about that specific antibody. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

PBMCs from cynomolgus macaques were not stimulated in vitro by immobilized TGN1412, in agreement with the failure of in vivo administration at any dose tested to evoke any adverse response.

“Significantly, PBMCs from cynomolgus macaques were not stimulated in vitro by immobilized TGN1412 in agreement with the failure of in vivo administration at any dose tested to evoke any adverse response (Duff, 2006;Stebbings et al., 2007).”

• cites Duff, 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Stebbings et al., 2007 sourcePartly supported

  The Lymphocyte proliferation section reports very little proliferation of cynomolgus macaque lymphocytes with immobilized TGN1412, and the Discussion says all tested in vivo doses were well tolerated. But the Activation and intracellular cytokine staining section reports significant up-regulation of an activation marker in macaque T cells. Thus, saying the cells were not stimulated at all overstates the in vitro finding. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Cynomolgus and rhesus macaque CD4+ effector memory T-cells do not express CD28, so they cannot be stimulated by CD28 superagonists including TGN1412.

“This is because we and others (Pitcher et al., 2002;Moniuszko et al., 2004) have shown that cynomolgus and rhesus macaque CD4 + effector memory T-cells do not express CD28; therefore, they cannot be stimulated by CD28 superagonists including TGN1412.”

• Also stated in: § Figure 6 · plain

  “In contrast, both cynomolgus and rhesus effector memory T-cells (CCR7 -CD28 -) do not express CD28.”

• cites Pitcher et al., 2002 sourceNot checked — no full text of the source
• cites Moniuszko et al., 2004 sourcePartly supported

  The Results (page unavailable) describe effector memory T cells in Indian rhesus macaques as CD28-low or CD28-negative, supporting the CD28-expression point only in qualified form. The cited paper does not establish that all CD4+ effector memory T cells in both rhesus and cynomolgus macaques lack CD28, or test whether they can be stimulated by CD28 superagonists such as TGN1412. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

In the authors' in vitro assays, Mabthera and Campath-1H did not induce lymphoproliferation or IL-2 release, which are cardinal features of TGN1412 stimulation.

“When we investigated other therapeutic mAbs, known to be capable of causing adverse reactions, Mabthera and Campath-1H (Wing et al., 1995;Chung, 2008), using our new in vitro procedures, we did not detect lymphoproliferative responses or IL-2 release, the cardinal features of TGN1412 stimulation (Stebbings et al., 2007).”

• cites Stebbings et al., 2007 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Peripheral blood contains only 2-5% of all lymphocytes, while lungs and gastrointestinal mucosa harbour up to 90% of the body's lymphocytes.

“Moreover, peripheral blood contains just 2-5% of all lymphocytes, while the lungs and gastrointestinal mucosa harbour up to 90% of the body's lymphocytes (Cerf-Bensussan and Guy-Grand, 1991;Smit-McBride et al., 1998).”

• cites Cerf-Bensussan and Guy-Grand, 1991 sourceNot checked — no full text of the source
• cites Smit-McBride et al., 1998 sourcePartly supported

  The Abstract states that peripheral blood contains 2–5% of total lymphocytes and that the gastrointestinal tract harbors over 90%. It does not mention the lungs, and its figure is “over 90%,” rather than “up to 90%.” (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Central memory CD4+ T-cells are characterized by strong lymphoproliferative responses, which TGN1412 stimulates from human but not macaque PBMCs.

“Central memory CD4 + T-cells are characterized by strong lymphoproliferative responses (Sallusto et al., 1999;2004), which TGN1412 stimulates from human, but not macaque PBMCs (Stebbings et al., 2007).”

• cites Sallusto et al., 1999 sourceNot checked — no full text of the source
• cites [2004]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Stebbings et al., 2007 sourceSupported

May combine two claims.

Naive and central memory CD4+ T-cell subsets have limited cytokine expression potential, whereas the effector memory subset has considerable cytokine production capability, with IL-2 as a major component.

“This notion is also consistent with the known capability of CD4 + T-cell subsets to produce cytokines (i.e.naive and central memory CD4 + T-cell subsets both have limited potential for cytokine expression, whereas the effector memory subset has a considerable capability for cytokine production, with IL-2 being a major component of this) (Sallusto et al., 1999;Amyes et al., 2005).”

• cites Sallusto et al., 1999 sourceNot checked — no full text of the source
• cites Amyes et al., 2005 sourceSupported

May combine two claims.

Human CD4+ effector memory T-cells express CD28, whereas cynomolgus and rhesus macaque CD4+ effector memory T-cells do not.

“All human CD4 + T-cell memory subsets (naive, central and effector) expressed high levels of CD28 with the effector memory subset defined by a lack of CCR7 expression (Sallusto et al., 1999;2004; Figure 6A).”

• Also stated in: § CD28 is expressed by human, but not macaque, CD4 + effector memory T-cells · plain

  “However, we found that the CD4 + effector memory T-cell subsets of both cynomolgus and rhesus macaques did not express CD28, as previously reported for rhesus macaques (Pitcher et al., 2002;Moniuszko et al., 2004; Figure 6A).”

• cites Pitcher et al., 2002 sourceNot checked — no full text of the source
• cites Moniuszko et al., 2004 sourcePartly supported

  The Results (page unavailable) characterize rhesus macaque CD4+ effector memory T cells as CD28-low or CD28-negative, supporting only the rhesus portion in qualified form. The copy does not establish CD28 expression in human effector memory T cells or the claimed CD28 status of cynomolgus CD4+ effector memory T cells; it also does not say all rhesus cells lack CD28. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

It has been previously published that ANC28.1 induces greater or similar in vitro cytokine release than TGN1412, which contrasts with the authors' finding that TGN1412 is far more potent than ANC28.1.

“Moreover, it has been previously published that ANC28.1 induces greater or similar in vitro cytokine release than TGN1412 (Waibler et al., 2008), which is in contrast to our findings here that TGN1412 is far more potent than ANC28.1.”

• cites Waibler et al., 2008 sourceSupported

May combine two claims.

Immobilized TGN1412 primed macaque CD4+ T-cells, and they underwent marked lymphoproliferation only when exogenous IL-2 was added.

“However, we previously reported that immobilized TGN1412 primed macaque CD4 + T-cells and that they underwent marked lymphoproliferation only when exogenous IL-2 was added (Stebbings et al., 2007).”

• cites Stebbings et al., 2007 sourcePartly supported

  The Activation and intracellular cytokine staining section reports that immobilized TGN1412 activated macaque CD4+ T cells without inducing proliferation. The Lymphocyte proliferation section reports proliferation after recombinant IL-2 was added, but says that response was only about 10% of the human response or the macaque mitogen response; calling it “marked” overstates the finding. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

The mechanism of cytokine release by TGN1412 differs from that of other therapeutic mAbs that can cause adverse reactions, because those other mAbs stimulate cytokine release primarily from natural killer cells.

“This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells.”

May combine two claims.

CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so those cells cannot be stimulated by TGN1412.

“In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412.”

May combine two claims.

Other therapeutic mAbs tested stimulated cytokine release only from CD4- lymphocytes, with high TNF-a, very low IFN-g and no IL-2.

“In contrast, other therapeutic mAbs that we tested only stimulated CD4 -lymphocyte cytokine release that comprised high levels of TNF-a, but very low levels of IFN-g and no IL-2.”

May combine two claims.

Initial data showed that human CD4+CD45RO+ memory T-cells were the predominant source of cytokines after TGN1412 stimulation, but this did not explain why in vitro IFN-g levels were low relative to TNF-a.

“Initial data showed that human CD4 + CD45RO + memory T-cells were the predominant source of cytokines following TGN1412 stimulation, but this did not explain why in vitro IFN-g levels were low when compared with TNF-a levels.”

May combine two claims.

Both cynomolgus and rhesus macaque CD4+ effector memory T-cells lack CCR7 expression; however, a population of CD28+CCR7- cells was noted that have previously been described as a transitional central memory subset in rhesus macaques.

“As for human cells, we found that both cynomolgus and rhesus macaque CD4 + effector memory T-cells lack CCR7 expression; however, a population of CD28 + CCR7 -were noted that have previously been described as a transitional central memory subset in rhesus macaques (Picker et al., 2006).”

• cites Picker et al., 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

TGN1412 stimulates cytokine release from CD4+ T-cells, whereas Mabthera and Campath-1H stimulate from CD4- cells.

“In contrast, TGN1412 stimulated a large increase in the frequency of IL-2producing lymphocytes of which the majority were CD4 + (Figure 3A; data from two representative donors) and a small proportion were CD4 -, from the same two representative donors (Figure 3A).”

• Also stated in: § Cytokine release from CD4 + T-cells is characteristic of a TGN1412-type response · plain

  “In contrast, stimulation with Mabthera or Campath-1H stimulated increases in the frequency of CD4 -, but not CD4 + TNF-aproducing lymphocytes (Figure 3B).”

The quote only partly states this claim.

TGN1412 stimulates substantial release of IL-2 and IFN-g, unlike other therapeutic mAbs.

“Substantial quantities of IFN-g were detected in culture supernatants following 24 h in vitro stimulation with immobilized TGN1412 and much more after 48 and 72 h (Figure 2B).”

• Also stated in: § Substantial IL-2 and IFN-g release is characteristic of a TGN1412-type response · plain

  “Substantial concentrations of IL-2 were detected in culture supernatants following 24 h in vitro stimulation with immobilized TGN1412, and these decreased at 48 and 72 h (Figure 2C).”

May combine two claims.

IL-2 expression after in vitro TGN1412 stimulation showed kinetics similar to its appearance in humans, but supernatant concentrations were lower than expected.

“Similarly, the expression of IL-2 following in vitro TGN1412 stimulation showed similar kinetics to its appearance in humans, but the concentrations in cell supernatants were lower than expected.”

May combine two claims.

PBMCs enriched for CD4+ effector memory T-cells and stimulated with TGN1412 produced high levels of TNF-a, IL-2 and IFN-g, with many cells simultaneously producing all three cytokines.

“In marked contrast, PBMCs enriched for CD4 + effector memory T-cells produced high levels of TNF-a, IL-2 and IFN-g, when stimulated with TGN1412, with a large proportion of cells simultaneously producing all three cytokines.”

May combine two claims.

Despite similar in vitro expression kinetics for IFN-g after TGN1412 stimulation, IFN-g concentrations were far lower than TNF-a in vitro and an order of magnitude lower than IFN-g concentrations predicted from in vivo trial responses.

“However, despite similar in vitro expression kinetics for IFN-g following TGN1412 stimulation, the concentrations of IFN-g were far lower than TNF-a responses in vitro, and an order of magnitude lower than concentrations of IFN-g predicted from in vivo responses in trial participants (Suntharalingam et al., 2006).”

• cites Suntharalingam et al., 2006 sourceNot checked — no full text of the source

May combine two claims.

TGN1412-mediated cytokine release predominantly came from CD4+ memory T-cells, which produced high levels of TNF-a, IL-2 and IFN-g.

“TGN1412-mediated cytokine release predominantly originated from CD4 + memory T-cells, which produced high levels of TNF-a, IL-2 and IFN-g.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The cytokine gene expression that showed the greatest increase following TGN1412 stimulation was TNF-a, followed by IL-2 and then IFN-g.

“The cytokine gene expression that showed the greatest increase following TGN1412 stimulation was TNF-a, followed by IL-2 and then IFN-g (data not shown).”

Not yet classified.

Within 90 min, both immobilized TGN1412 and Fc-immobilized TGN1412 stimulated significant increases above control antibody in the frequency of IL-2-producing lymphocytes.

“Within 90 min, both immobilized TGN1412 and Fc-immobilized TGN1412 stimulated significant increases (above those seen with the control antibody) in the frequency of IL-2-producing lymphocytes (both P < 0.0001; Figure 1C).”

Not yet classified.

Human CD4+ effector memory T-cells express high levels of CD28 on their surface and therefore can be stimulated by TGN1412.

“In contrast, the CD4 + effector memory T-cells of humans express high levels of CD28 on their surface (Sallusto et al., 1999;2004), so therefore can be stimulated by TGN1412.”

• Also stated in: § Figure 6

  “Human, cynomolgus and rhesus macaque naive CD4 + T-cells (CCR7 + CD28 + ) and central memory T-cells (CCR7 + CD28 + ), macaque transitional memory T-cells (CCR7 -CD28 + ) and human effector memory CD4 + T-cells (CCR7 -CD28 + ) all express CD28.”

• cites Sallusto et al., 1999 sourceNot checked — no full text of the source
• cites [2004]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Increased levels of TNF-a gene expression were detected 30 min after stimulation with TGN1412, and increased IL-2 and IFN-g gene expression at 60 min.

“Increased levels of TNF-a gene expression were detected 30 min after stimulation with TGN1412, and increased IL-2 and IFN-g gene expression at 60 min.”

May combine two claims.

Human CD4+ T-cells constitutively express CD28, so IFN-g staining after PMA/ionomycin stimulation stems only from the CD28+ population.

“Human CD4 + T-cells constitutively express CD28, so IFN-g staining stems only from the CD28 + population.”

May combine two claims.

Macaque T-cells are not less responsive to in vitro TGN1412 stimulation but are non-responsive.

“However, it should be noted that macaque T-cells are not 'less responsive' to in vitro stimulation with TGN1412, but 'non-responsive'.”

May combine two claims.

Macaque CD4+ T-cells can be subdivided into CD28+ (naive, central and transitional memory) and CD28- (effector memory) populations, both of which produce IFN-g after PMA/ionomycin stimulation.

“Macaque CD4 + T-cells can be subdivided into CD28 + (naive, central and transitional memory T-cell) and CD28 -(effector memory T-cell) populations, both of which produce IFN-g following PMA and ionomycin stimulation for 4 h.”

May combine two claims.

ANC28.1 is a superagonist in vitro in both aqueous phase and when immobilized, whereas TGN1412 was found to be a superagonist only when immobilized.

“However, ANC28.1 is a superagonist in vitro in both aqueous phase and when immobilized, whereas we found that TGN1412 was only a superagonist when immobilized.”

May combine two claims.

In 2006, a life-threatening cytokine storm occurred in all six healthy volunteers during the phase I trial of TGN1412, beginning about 1 h after infusion and making all critically ill within 12-16 h.

“In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412.”

• Also stated in: § Introduction · plain

  “A near-fatal systemic inflammatory immune response, termed a 'cytokine storm', began 1 h after infusion of TGN1412 in all six healthy recipients, and all became critically ill within 12-16 h (Suntharalingam et al., 2006).”

• cites Suntharalingam et al., 2006 sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

TGN1412 is a superagonist CD28-specific monoclonal antibody developed as an immunotherapeutic for balancing the immune system.

“TGN1412 is a superagonist CD28-specific monoclonal antibody (mAb) developed as an immunotherapeutic for the purpose of balancing the immune system (TeGenero, 2005).”

• cites TeGenero, 2005Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

A species difference in CD28 expression on the CD4+ effector memory T-cell subset is almost certainly responsible for the failure of pre-clinical safety testing of TGN1412 in cynomolgus macaques and for the disparate immunopharmacology.

“Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology.”

• Also stated in: § Introduction · hedged

  “Third, we identify a species difference in CD28 expression on the CD4 + effector memory T-cell subset as being most likely responsible for the failure of pre-clinical safety testing of TGN1412 in cynomolgus macaques.”

• AssumptionRests on an assumption: No unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established.

May combine two claims.

Macaque CD4+ transitional memory T-cells are functionally different from macaque CD4+ effector memory T-cells; IL-2 preferentially induces proliferation of transitional memory T-cells, whereas effector memory T-cells are poorly responsive, and the reverse is true for IL-15.

“However, macaque CD4 + transitional memory T-cells are functionally different to macaque CD4 + effector memory T-cells; IL-2 preferentially induces macaque transitional memory T-cells to proliferate, whereas macaque effector memory T-cells are poorly responsive and vice versa for IL-15 (Picker et al., 2006).”

• cites Picker et al., 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Immobilizing mAb to a plastic surface is a highly artificial stimulation method unlikely to simulate what happens inside the body.

“However, it should be noted that immobilizing mAb to a plastic surface is a highly artificial means of stimulation that is unlikely to simulate what is happening inside the body.”

May combine two claims.

In humans, the TGN1412 cytokine storm involved substantial TNF-a release within the first hour, followed by large increases in IFN-g, IL-2, IL-6, IL-8 and IL-10 plasma concentrations within 4 h.

“One of the most striking features of the TGN1412-mediated 'cytokine storm' observed in humans was substantial TNF-a release within the first hour of infusion followed by dramatic increases in IFN-g, IL-2, IL-6, IL-8 and IL-10 plasma concentrations within 4 h (Suntharalingam et al., 2006).”

• cites Suntharalingam et al., 2006 sourceNot checked — no full text of the source

May combine two claims.

If the explanation were simply differences in cell numbers, all cytokine responses would have been equally lower, but they were not.

“If the explanation were as simple as differences in cell numbers, then all cytokine responses would have been equally lower, but they were not.”

Classified as probably not a substantial claim.

TGN1412 bound to human and macaque CD28 with equal affinity.

“We also did not understand why TGN1412 bound to human and macaque CD28 with equal affinity (TeGenero, 2005), yet caused a 'cytokine storm' only in humans.”

• cites TeGenero, 2005Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Multiple cell types and subsets in blood can release different cytokines with overlapping repertoires that ELISA of mixed cell populations cannot discriminate, whereas flow cytometric phenotypic analysis allowed dissection of different effector mechanisms.

“Within blood, multiple cell types and their subsets can release different cytokines with overlapping repertoires which are not discriminated by ELISA assessment of culture supernatants from mixed cell populations stimulated with mAbs, whereas phenotypic analysis of cytokine-producing cells using flow cytometry, as is shown here, has allowed the dissection of different effector mechanisms.”

May combine two claims.

Macaque CD4+ transitional memory T-cells lack CCR7 expression and express CD28, similar to human CD4+ effector memory T-cells.

“Similar to human CD4 + effector memory T-cells, macaque CD4 + transitional memory T-cells lack CCR7 expression and express CD28 (Picker et al., 2006).”

• cites Picker et al., 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Not yet classified.

Lymph nodes and tonsils are enriched in central memory CD4+ T-cells, whereas lungs and gastrointestinal mucosa contain mainly effector memory CD4+ T-cells.

“The lymph nodes and tonsils are enriched in central memory CD4 + T-cells, whereas the lungs and gastrointestinal mucosa contain mainly effector memory CD4 + T-cells (Campbell et al., 2001;Picker, 2006).”

• cites Campbell et al., 2001 sourceNot checked — no full text of the source
• cites Picker, 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Retention of CD28 expression on human CD4+ effector T-cells would allow required co-stimulatory signals to reduce activation thresholds.

“The evolutionary reason for this subtle comparative immunological difference in receptor expression is unclear, but retention of CD28 expression on human CD4+ effector T-cells would allow the required co-stimulatory signals to reduce activation thresholds (Fraser et al., 1991).”

• cites Fraser et al., 1991 sourceNot checked — no full text of the source

May combine two claims.

The difference in CD28 expression questions the suitability and validity of existing pre-clinical non-human primate models for the safety testing of therapeutic mAbs and other immunomodulatory biological agents.

“This difference not only questioned the suitability and validity of existing pre-clinical non-human primate models for the safety testing of therapeutic mAbs, but also for other immunomodulatory biological agents.”

May combine two claims.

ANC28.1 likely binds to a different region of CD28 from TGN1412, which may explain why lower responses were observed.

“Therefore, it is likely that ANC28.1 binds to a different region of CD28 from that binding TGN1412, which may explain why lower responses were observed.”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

Preclinical safety testing in non-human primate models will still be useful but needs to be interpreted together with subtleties in antigen expression on target cells in preclinical models.

“While much of the immunopharmacology of cytokine release syndrome in humans remains largely unknown, pre-clinical safety testing in non-human primate models will still be useful, but needs to be interpreted in conjunction with subtleties in antigen expression on target cells in pre-clinical models.”

May combine two claims.

ANC28.1 stimulates release of the same pattern of cytokines as TGN1412 and is likely to activate the same pro-inflammatory pathways.

“Nevertheless, ANC28.1 does stimulate release of the same pattern of cytokines as TGN1412 so is likely to activate the same pro-inflammatory pathways.”