Dong-Ki Choi

Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris

The neonatal Fc receptor (FcRn) is a non-covalently associated heterodimeric protein composed of a transmembrane anchored heavy chain (alphaFcRn) and a soluble light chain beta2-microglobulin (beta2m). In addition to its role in the transfer of maternal immunoglobulin Gs (IgGs) to the fetus, FcRn plays a key role in prolonging the serum half-life of IgGs in vivo. Herein, we report a strategy for functional expression of soluble human FcRn (shFcRn) in Pichia pastoris using a two-promoter vector system, where alphaFcRn and beta2m are co-expressed under their respective promoters in a single vector. The purified shFcRn from the culture supernatants correctly assembled to form the heterodimer with the typical secondary structures. At acidic pHs between 5.0 and 6.4, shFcRn exhibited substantial binding to the four subclasses of human IgGs at acidic pHs between 5.0 and 6.4, but at pHs between 6.8 and 8.0, its binding was negligible binding. No cross-reactivity with mouse IgG was exhibited even at acidic pH. This was consistent with the pH-dependent binding profiles of the shFcRn prepared from the mammalian cell expression. Furthermore, the shFcRn exhibited about 10-fold higher binding affinity with the tumor necrosis factor-alpha antagonists of monoclonal antibodies Infliximab and Adalimumab than that of Etanercept, providing a clue to their different serum half-lives in vivo. Our results suggest that the functionally expressed shFcRn from Pichia can be used for the biochemical and biological studies and as a screening probe for Fc engineering of human IgGs.

Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening

Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80–90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

Proinsulin Shares a Motif with Interleukin-1α (IL-1α) and Induces Inflammatory Cytokine via Interleukin-1 Receptor 1

Although it has been established that diabetes increases susceptibility to infections, the role of insulin (INS) in the immune response is unknown. Here, we investigated the immunological function of INS. Proinsulin dimer (pINSd) was a potent immune stimulus that induced inflammatory cytokines, but mature INS was unable to induce an immune response. An affinity-purified rabbit polyclonal antibody raised against mature IL-1α recognized IL-1α and pINS but failed to detect mature INS and IL-1β. Analysis of the pINS sequence revealed the existence of an INS/IL-1α motif in the C-peptide of pINS. Surprisingly, the INS/IL-1α motif was recognized by monoclonal antibody raised against IL-1α. Deleting the INS/IL-1α motif in pINSd and IL-1α changed their activities. To investigate the pINSd receptor, the reconstitution of IL-1 receptor 1 (IL-1R1) in Wish cells restored pINSd activity that was reversed by an IL-1R antagonist. These data suggested that pINSd needs IL-1R1 for inflammatory cytokine induction. Mouse embryo fibroblast cells of IL-1R1-deficient mice further confirmed that pINSd promotes immune responses through IL-1R1.

IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells

The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Interfering transbody-mediated Her2 gene silencing induces apoptosis by G 0 /G 1 cell cycle arrest in Her2-overexpressing SK-BR-3 breast cancer cells

We previously isolated an interfering transbody, 4MH2, which penetrated the cytosol of living cells and preferentially hydrolyzed the target Her2 (ErbB2) mRNA, resulting in Her2 gene silencing followed by apoptotic cell death in Her2-overexpressing breast cancer cells. Here, we report the apoptotic cell death mechanism mediated by 4MH2-induced Her2 gene silencing in Her2-overexpressing SK-BR-3 breast cancer cells, in comparison with a small interfering RNA (siRNA) targeting Her2 mRNA (Her218-siRNA). 4MH2 induced G0/G1 cell cycle arrest to cause apoptotic cell death in SK-BR-3 cells by triggering specific signaling pathways associated with Her2 knockdown, including upregulation of G0/G1 cell cycle arrest-associated p21Cip1 and p27Kip1, downregulation of cyclin D1, inhibition of Akt phosphorylation, and downregulation of antiapoptotic Bcl-xL, which are comparable to those mediated by Her218-siRNA. Our results suggest that 4MH2-mediated Her2 gene silencing can trigger the downstream signaling pathways caused by Her2 downregulation, comparable to those mediated by the corresponding siRNA.

Engineering of a tumor cell–specific, cytosol-penetrating antibody with high endosomal escape efficacy

The main obstacles for practical uses of cytosol-penetrating peptides and proteins include their lack of cell- or tissue-specific targeting and limited cytosolic access owing to the poor endosomal escape ability. We have previously reported a cytosol-penetrating, human IgG1 antibody TMab4-WYW, generally referred to as a cytotransmab (CT), which reaches the cytosol of living cells but nonspecifically because it is endocytosed via a ubiquitously expressed receptor called heparan sulfate proteoglycan (HSPG). Here, our aim was to construct a next-generation CT with tumor cell specificity and improved endosomal escape efficiency. We first substantially reduced the HSPG-binding activity of TMab4-WYW and then fused a cyclic peptide specifically recognizing tumor-associated epithelial cell adhesion molecule (EpCAM) to the N terminus of the light chain for EpCAM-mediated endocytosis, while maintaining the endosomal escape ability in the light chain variable domain (VL), thus generating epCT05. Then, we separately engineered another CT, dubbed epCT65-AAA, with an endosomal escape ability only in the heavy chain variable domain (VH) but not in VL, by functional grafting of the endosomal escape motif of epCT05 VL to the VH. We finally combined the heavy chain of epCT65-AAA and the light chain of epCT05 to create epCT65 with endosomal escape capacity in both the VH and VL. epCT65 effectively localized to the cytosol of only EpCAM-expressing tumor cells and showed approximately twofold improved endosomal escape efficiency, as compared with CTs with endosomal escape motifs in either VH or VL. The full-IgG format CT, epCT65, with a tumor cell-specific cytosol-penetrating activity, has a great potential for practical medical applications, e.g., as a carrier for cytosolic delivery of payloads.