Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412

Findlay, Lucy; Eastwood, David; Stebbings, Richard; Sharp, Giles; Mistry, Yogesh; Ball, Christine; Hood, John; Thorpe, Robin; Poole, Stephen

doi:10.1016/j.jim.2009.10.013

Cited by 70 publications

(65 citation statements)

References 8 publications

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“…Immobilized TGN1412 stimulated substantial release of TNF-a, IL-2 and IFNg, and massive PBMC proliferation, as observed previously (11,28), with peak responses obtained using 0.25 mg mAb/well (Fig. 4A-D).…”

Section: In Vitro Functional Activity Of Tgn1412 and Fragmentssupporting

confidence: 83%

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Ball

Fox

Hufton

et al. 2012

The Journal of Immunology

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The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab′)2 and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the “wild-type” IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm–exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab′)2 fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

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Section: In Vitro Functional Activity Of Tgn1412 and Fragmentssupporting

confidence: 83%

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Ball

Fox

Hufton

et al. 2012

The Journal of Immunology

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“…Fc-cross-linking or immobilization of TGN1412, respectively, was shown to be a prerequisite to mediate T-cell proliferation and cytokine release in vitro. [10][11][12][13] We used primary HUVECs to study their ability to induce activation of TGN1412-treated T cells. Using an endothelial-like cell line, Stebbings et al showed that endothelial cells indeed can confer TGN1412-mediated T-cell activation.…”

Section: Discussionmentioning

confidence: 99%

“…Immobilized or IgG crosslinked TGN1412 indeed was able to induce cell activation indicating contribution of the antibody's Fc-part to its function. [10][11][12][13] So far, it is not clear which cell and which Fc receptor (FcR) mediates TGN1412 cross-linking, and to which extent FcR expressing cells contribute to cytokine release or T-cell stimulation.Interestingly, Fc ligation by Fc␥Rs was reported to contribute to OKT3-mediated CRS. Here, the interaction of T-cell bound antibody with FcRs on monocytes and other cells critically contributed to antibody-mediated CRS.…”

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confidence: 99%

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation

Weißmüller

Semmler

Kalinke

et al. 2012

Blood

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TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-crosslinking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, FcFc␥R interaction was dispensable for endothelial cell-mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. IntroductionMonoclonal antibodies (mAbs) are widely used for therapeutic applications. Although, therapeutic mAbs are potent and effective agents, they can induce severe adverse events including cytokine release syndrome (CRS), a cascade of systemic cytokine release.The first mAb approved in 1986 as a drug for humans, muromonab (orthoclone OKT3), is a murine anti-human CD3 mAb, indicated for the treatment of acute renal, steroid-resistant cardiac, or steroid-resistant hepatic allograft rejection. In particular during the first infusion, OKT3-mediated T-cell activation can induce massive cytokine release, possibly culminating in a CRS. 1,2 Currently, approximately 29 mAbs are available on the market in the European Union, including those directly influencing T-cell function.For proper T-cell activation, at least 2 signals are required: the first signal is provided by the T-cell receptor (TCR) upon recognition of antigen:MHC (major histocompatibility complex) complexes on the surface of antigen presenting cells (APCs). However, antigen alone is not sufficient to drive activation of naive T cells. To fully activate resting naive T lymphocytes, a second signal, which emerges from triggering of so called costimulatory molecules, must be provided (reviewed by Sharpe 3 ). The transmembrane CD28 homodimer is the most prominent costimulatory molecule. Its ligands B7-1 (CD80) and B7-2 (CD86) are up-regulated on APCs upon triggering of cells with danger signals derived from pathogens, such as bacteria or viruses, or upon cytokine stimulation. 4 As for signal one, triggering of CD28 alone is not capable of inducing T-cell activation, whereas simultaneous engagement of the TCR and CD28 leads to activation of resting T lymphocytes.Another important costimulatory molecule is ICOS (inducible costimulator; CD278) binding to its ligand LICOS (CD275), expressed on APCs, B cells, and endothelial cells. 5,6 H...

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“…assays (28,29). The reports that followed highlighted the need to advance with caution when translating immunostimulatory antibodies to the clinic and to better assess cytotoxicity of such high-risk agents as a means not only to predict mechanism of action but also to better predict cytotoxicity before use in clinical studies.…”

Section: Il-12mentioning

confidence: 99%

Ex Vivo Assays of Dendritic Cell Activation and Cytokine Profiles as Predictors of In Vivo Effects in an Anti-Human CD40 Monoclonal Antibody ChiLob 7/4 Phase I Trial

Chowdhury

Johnson

Glennie

et al. 2014

Cancer Immunology Research

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Immunostimulatory antibodies entering the clinic create challenge in terms of not only pharmacodynamics for monitoring anticipated mechanisms but also predetermining cytotoxicity. We show the use of ex vivo wholeblood samples to predict the activation requirements, cytokine signature, and adverse events of an anti-human-CD40 chimeric IgG1 antibody, ChiLob 7/4. Assessments were initially undertaken on human myeloid (mDC1) and plasmacytoid (pDC) dendritic cells, in which an absolute need for cross-linking was shown through the upregulation of activation markers CD83 and CCR7. Subsequent cytokine secretion evaluations of ex vivo whole blood showed the cross-linked antibody-induced increases in MIP1b, interleukin (IL)-8, IL-12, TNFa, and IL-6. This cytokine signature compared favorably with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS), in which levels of TNFa and IL-6 were significantly higher, suggesting a less intense proinflammatory response and possible modified cytokine release syndrome when used in human trials. Following first-in-human use of this agent within a dose escalation study, in vivo evaluations of dendritic cell activation and secreted cytokines closely matched the predetermined immunomonitoring endpoints. Patients showed a comparable pattern of MIP1b, IL-8, and IL-12 secretion, but no TNFa and IL-6 were identified. Mild symptoms relating to a cytokine release syndrome were seen at an equivalent dosage to that observed for dendritic cell activation and cytokine release. In summary, ChiLob 7/4 induces a distinctive pattern of dendritic cell activation and cytokine secretion in ex vivo assays that can be predictive of in vivo responses. Such preclinical approaches to monoclonal antibody evaluation may inform both the starting dosages and the anticipated cytokine release events that could occur, providing a valuable adjunct for future first-in-human assessments of immunostimulatory antibodies. Cancer Immunol Res; 2(3); 229-40. Ó2013 AACR.

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Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412

Cited by 70 publications

References 8 publications

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

Antibody C Region Influences TGN1412-like Functional Activity In Vitro

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation

Ex Vivo Assays of Dendritic Cell Activation and Cytokine Profiles as Predictors of In Vivo Effects in an Anti-Human CD40 Monoclonal Antibody ChiLob 7/4 Phase I Trial

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