Use of Anti-CD3 × Anti-HER2/neu Bispecific Antibody for Redirecting Cytotoxicity of Activated T Cells Toward HER2/neu + Tumors
Relapse after adjuvant chemotherapy or high-dose chemotherapy with stem cell transplant for high-risk breast cancer remains high and new strategies that provide additional antitumor effects are needed. This report describes methods to generate highly effective HER2/neu-specific cytotoxic T cells by arming activated T cells with anti-CD3 x anti-HER2/neu bispecific antibody (BsAb). OKT3 and 9184 (anti-HER2) monoclonal antibodies (mAb) were conjugated and used to arm T cells that were subsequently tested in binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregated and specifically killed HER2/neu(+) breast cancer cells. Cytotoxicity emerged after 6 days of culture, was higher in armed T cells than unarmed T cells at all effector to target ratios (E/T) tested, and increased as the arming dose was increased. At an E/T of 20:1, the mean cytotoxicity of armed activated T cells (ATC) from 10 normal subjects increased by 59 +/- 11% (+/-SD) over that seen in unarmed ATC (p < 0.001) and the mean cytotoxicity of armed ATC from 6 cancer patients increased by 32 +/- 9% above that seen for unarmed ATC (p < 0.0004). After arming, the BsAb persisted on ATC up to 72 h and armed ATC continued to be cytotoxic up to 54 h. The amount of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted was 1699, 922, and 3092 pg/ml/10(6) cells per 24 h, respectively, when armed T cells were exposed to a HER2/neu(+) breast carcinoma cell line. These studies show the feasibility and clinical adaptability of this approach for generating large numbers of anti-HER2-specific, cytotoxic T cells for clinical trials.
Available from: Hiroshi Yano
- "Bispecific antibodies (BiAbs) were produced by chemical heteroconjugation of OKT3 and Rituxan (a humanized anti-CD20 IgG1, Genentech Inc., South San Francisco, CA) or Erbitux (a humanized anti-epidermal growth factor receptor (EGFR) IgG1, ImClone LLC., Branchburg, NJ) as described
[15,16]. ATC were armed with anti-OKT3 x anti-CD20 BiAb (CD20Bi) or anti-OKT3 x anti-EGFR BiAb (EGFRBi) using a previously optimized concentration (50 ng/106 ATC)
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Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC).
PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt’s lymphoma cell line (Daudi).
In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p = 0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p < 0.0004) and 27.5% (p < 0.0004), respectively, and increased the secretion of chemokines (CCL2, CCL3, CCL4,CCL5, CXCL9, and granulocyte-macrophage colony stimulating factor(GM-CSF)) and cytokines (IFN-γ, IL-2R, IL-12, and IL-13), while reducing IL-10 secretion.
Expansion of ATC in the presence of ipilimumab significantly improves not only the T cell proliferation but it also enhances cytokine secretion and the specific cytotoxicity of T cells armed with bispecific antibodies.
Journal of Translational Medicine 07/2014; 12(1):191. DOI:10.1186/1479-5876-12-191 · 3.93 Impact Factor
Available from: Thomas Kean
- "This bispecific antibody approach has been used to target HSCs to damaged vasculature in the heart, using either anti-VCAM-1 conjugated to anti-c-kit  or anti-CD45-anti-myosin light chain kinase . This method has also been used in the field of cancer therapy to direct activated T-cells to human epidermal growth factor receptor-2+ tumor cells in vitro . This cancer treatment was tested in vivo, yielding reduced tumor size and a significantly increased rate of survival . "
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ABSTRACT: Mesenchymal stem cells (MSCs) are currently being widely investigated both in the lab and in clinical trials for multiple disease states. The differentiation, trophic, and immunomodulatory characteristics of MSCs contribute to their therapeutic effects. Another often overlooked factor related to efficacy is the degree of engraftment. When reported, engraftment is generally low and transient in nature. MSC delivery methods should be tailored to the lesion being treated, which may be local or systemic, and customized to the mechanism of action of the MSCs, which can also be local or systemic. Engraftment efficiency is enhanced by using intra-arterial delivery instead of intravenous delivery, thus avoiding the "first-pass" accumulation of MSCs in the lung. Several methodologies to target MSCs to specific organs are being developed. These cell targeting methodologies focus on the modification of cell surface molecules through chemical, genetic, and coating techniques to promote selective adherence to particular organs or tissues. Future improvements in targeting and delivery methodologies to improve engraftment are expected to improve therapeutic results, extend the duration of efficacy, and reduce the effective (MSC) therapeutic dose.
Stem cell International 08/2013; 2013(5):732742. DOI:10.1155/2013/732742 · 2.81 Impact Factor
Available from: Ian Zitron
- "Anti-CD3-activated ATC were expanded in culture from human peripheral blood mononuclear cells (PBMC) . Briefly, ATC were produced by activating PBMC with 20 ng/ml of soluble anti-CD3 (OKT3, Ortho Pharmaceutical, Raritan, NJ) and expanded in IL-2 (aldesleukin, Prometheus Laboratories Inc., San Diego, CA) (100 IU/ml) in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 1% penicillin-streptomycin for 14 days. "
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Since most glioblastomas express both wild-type EGFR and EGFRvIII as well as HER2/neu, they are excellent targets for activated T cells (ATC) armed with bispecific antibodies (BiAbs) that target EGFR and HER2.
ATC were generated from PBMC activated for 14 days with anti-CD3 monoclonal antibody in the presence of interleukin-2 and armed with chemically heteroconjugated anti-CD3×anti-HER2/neu (HER2Bi) and/or anti-CD3×anti-EGFR (EGFRBi). HER2Bi- and/or EGFRBi-armed ATC were examined for in vitro cytotoxicity using MTT and 51Cr-release assays against malignant glioma lines (U87MG, U118MG, and U251MG) and primary glioblastoma lines.
EGFRBi-armed ATC killed up to 85% of U87, U118, and U251 targets at effector:target ratios (E:T) ranging from 1:1 to 25:1. Engagement of tumor by EGFRBi-armed ATC induced Th1 and Th2 cytokine secretion by armed ATC. HER2Bi-armed ATC exhibited comparable cytotoxicity against U118 and U251, but did not kill HER2-negative U87 cells. HER2Bi- or EGFRBi-armed ATC exhibited 50—80% cytotoxicity against four primary glioblastoma lines as well as a temozolomide (TMZ)-resistant variant of U251. Both CD133– and CD133+ subpopulations were killed by armed ATC. Targeting both HER2Bi and EGFRBi simultaneously showed enhanced efficacy than arming with a single BiAb. Armed ATC maintained effectiveness after irradiation and in the presence of TMZ at a therapeutic concentration and were capable of killing multiple targets.
High-grade gliomas are suitable for specific targeting by armed ATC. These data, together with additional animal studies, may provide the preclinical support for the use of armed ATC as a valuable addition to current treatment regimens.
BMC Cancer 02/2013; 13(1):83. DOI:10.1186/1471-2407-13-83 · 3.36 Impact Factor
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