An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions

Ch, Nam; Mn, Lobato; Appert, Alexandre; Lf, Drynan; Tanaka, Tomoyuki; Rabbitts, Terence H.

doi:10.1038/onc.2008.130

Cited by 38 publications

(46 citation statements)

References 25 publications

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“…S3 and Fig. 4B) (11). VH#576 binding to LMO2 was abolished with LIM finger 2, 3, or 4 mutations, and VL#551 does not bind to LIM finger 3 or 4 mutants.…”

Section: Iac 3 -Selected Single Domains Function In Mammalian Cells-tmentioning

confidence: 86%

“…S3) is described elsewhere (11). pBD-LMO2 was constructed by subcloning the EcoRI and PstI fragment from pBTM116-LMO2 into EcoRI/PstI sites of pBD-Gal4-Cam (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…Antibody fragments cloned into the SfiI and NotI sites of the pEFVP16 mammalian prey vector (12) were anti-LMO2 VH#576 and VL#551 (isolated from yeast IAC 3 single domain library screening), anti-LMO2 scFv ALR3 (11), anti-ATF2 VH#27 (4), or anti-RAS VH#6 (13). Bait clones expressing Gal4-DBD-antigen fusions were cloned in pM (16), and the various forms of LMO2 bait and Gal4-DBD-RAS have been described previously (4,11). For transient transfection, pM bait, pEFVP16-prey, pG5-Fluc, and pRL-CML plasmids were co-transfected into CHO cells using Lipofectamine 2000 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…VH#576 Hinders LMO2-dependent T-cell Neoplasia-LMO2 was originally discovered because it is activated in T-cell acute leukemia by chromosomal translocations (28,29) and, using macrodrugs such as scFv and peptide aptamers, we have shown that it is required for tumor growth in transgenic mouse models of Lmo2-dependent neoplasia (11,30). We sought to further validate the anti-LMO2 VH#576 utilizing FIGURE 3.…”

Section: Iac 3 -Selected Single Domains Function In Mammalian Cells-tmentioning

confidence: 99%

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Single Domain Intracellular Antibodies from Diverse Libraries

Tanaka

Sewell

Waters

et al. 2011

Journal of Biological Chemistry

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Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC 3 ) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Cocrystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC 3 offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.Antibodies and derivative fragments are molecules that can capture a huge variety of antigens with high specificity and affinity and are indispensable tools and reagents in bioscience and in medicine (1). The utilities of intracellular application of antibody fragments is that, unlike gene knock-out and interfering RNAs or antisense, these molecules interfere with cellular functions directly by binding to target proteins and can therefore target specific properties of proteins such as particular signal transduction via blockading protein-protein interaction (2, 3). This not only provides valuable and robust tools in biological analysis such as functional genomics and proteomics but also serve as drug surrogates to establish the target validity for drug development by ascertaining the biological effects from targeting the protein. To make these options possible on a broad basis, robust screening methods with high quality libraries are needed to allow selection of target-specific intracellular antibodies.Antibody binding sites comprise heavy chain variable domain (VH) 4 and light chain variable domains (VL), each with three complementarity determining regions (CDRs), directly contacting antigen and concerned in binding affinity and specificity. Single domain antibody fragments (DAbs), comprising either VH or VL, encompass a minimal antigen recognition can also be used for intracellular applications, so-called intracellular domain antibodies (iDAbs) (4). Structural analysis of V regions based on a consensus framework sequence have shown that essentially no diff...

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“…S3 and Fig. 4B) (11). VH#576 binding to LMO2 was abolished with LIM finger 2, 3, or 4 mutations, and VL#551 does not bind to LIM finger 3 or 4 mutants.…”

Section: Iac 3 -Selected Single Domains Function In Mammalian Cells-tmentioning

confidence: 86%

“…S3) is described elsewhere (11). pBD-LMO2 was constructed by subcloning the EcoRI and PstI fragment from pBTM116-LMO2 into EcoRI/PstI sites of pBD-Gal4-Cam (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Iac 3 -Selected Single Domains Function In Mammalian Cells-tmentioning

confidence: 99%

See 2 more Smart Citations

Single Domain Intracellular Antibodies from Diverse Libraries

Tanaka

Sewell

Waters

et al. 2011

Journal of Biological Chemistry

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“…8 Inhibition of Lmo2, using a specific intracellular antibody or peptide targeted toward the protein, blocks both Lmo2-induced erythropoiesis and tumor formation in a mouse explant model of leukemia. 9,10 Lmo2 is a 158-residue protein that contains two closely spaced LIM domains and very little other sequence. LIM domains (named for the first three genes in which the motif was identified (Lin-11, Isl-1, and Mec-3) are zinc fingers that coordinate two zinc ions, and that function as protein-protein interaction motifs (reviewed in Ref.…”

mentioning

confidence: 99%

Solution structure of a tethered Lmo2_LIM2/Ldb1_LID complex

Dastmalchi¹,

Wilkinson-White²,

Kwan³

et al. 2012

Protein Science

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LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein-protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (Ldb1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of Ldb1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to Ldb1 LID . The ordered regions of Ldb1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2 LIM2 -Ldb1 LID structure with previously determined structures of the Lmo2/Ldb1 LID complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in Ldb1 is accompanied by several disorder-to-order transitions and/ or conformational changes in both proteins.

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Nuclear JAK2: Form and Function in Cancer

Qian

Yao

2011

The Anatomical Record

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The conventional view of Janus kinase 2 (JAK2) is a nonreceptor tyrosine kinase which transmits information to the nucleus via the signal transducer and activator of transcriptions (STATs) without leaving the cytoplasm. However, accumulating data suggest that JAK2 may signal by exporting from cytoplasm to nucleus, where it guides the transcriptional machinery independent of STATs protein. Recent studies demonstrated that JAK2 is a crucial component of signaling pathways operating in the nucleus. Especially the latest landmark discovery confirmed that JAK2 goes into the nucleus and directly interacts with nucleoproteins, such as histone H3 at tyrosine 41 (H3Y41), nuclear factor 1-C2 (NF1-C2) and SWI/SNF-related helicases/ATPases (RUSH)-1a, indicating that JAK2 has a fresh nuclear function. Nuclear JAK2 is linked to a variety of cellular functions, such as cell cycle progression, apoptosis and genetic instability. The balance between these functions is an essential factor in determining whether a cell remains benign or becomes malignant. The aim of this review is intended to summarize the state of our knowledge on nuclear localization of JAK2 and nuclear JAK2 pathways, and to highlight the emerging roles for nuclear JAK2 in carcinogenesis.

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An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions

Cited by 38 publications

References 25 publications

Single Domain Intracellular Antibodies from Diverse Libraries

Single Domain Intracellular Antibodies from Diverse Libraries

Solution structure of a tethered Lmo2_LIM2/Ldb1_LID complex

Nuclear JAK2: Form and Function in Cancer

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An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions

Cited by 38 publications

References 25 publications

Single Domain Intracellular Antibodies from Diverse Libraries

Single Domain Intracellular Antibodies from Diverse Libraries

Solution structure of a tethered Lmo2LIM2/Ldb1LID complex

Nuclear JAK2: Form and Function in Cancer

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Product

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About

Solution structure of a tethered Lmo2_LIM2/Ldb1_LID complex