A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8(+)CD28(-) Tregs.

A group of 15-aa-long Trichosanthin-derived peptides was synthesized and screened based on their differential abilities to induce low-responsiveness in mouse strains with high and low susceptibility. One of them was conjugated to form a homo-tetramer Tk-tPN. At concentrations of 0.1-50 µg/ml, Tk-tPN activated CD8(+)CD28(-) Tregs in vitro to induce immune suppression as effectively as the native Trichosanthin but did not exhibit cytotoxicity. In EAE mice which were pre-treated with Tk-tPN or Tk-tPN-activated CD8(+) T cells, a marked attenuation of clinical scores was recorded together with an expansion of the CD8(+)CD28(-) Treg from 2.2% to 36.1% in vivo. A pull-down assay and signal transduction analyses indicated that the ability of Tk-tPN to convert the CD8(+)CD28(-) Treg-related cytokine secretion pattern from type 1 to type 2 depends on the TLR2-initiated signaling in macrophages. The high production of IL-4/IL-10 by the Tk-tPN-activated CD8(+)CD28(-) Treg suggests the value of using Tk-tPN as a therapeutic reagent for Th1-dominant immunological diseases.

Trichosanthin-stimulated dendritic cells induce a type 2 helper T lymphocyte response through the OX40 ligand.

BACKGROUND: Trichosanthin (TCS) induces a type 2 helper T lymphocyte (T(H)2) immune response that leads to the production of TCS-specific immunoglobulin (Ig) E and a subsequent allergic reaction in vivo. However, events immediately following treatment with TCS are poorly understood. OBJECTIVE: We aimed to investigate whether dendritic cells (DCs) are the initial mediators of T(H)2 cell polarization induced by TCS and to investigate potential causative mechanisms METHODS: DCs were cultured from purified human peripheral monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL) 4. Flow cytometry was used to analyze cell surface antigen, intracellular cytokines, DC endocytic capacity, and apoptosis. The transcriptional profile for 120 genes in DCs was detected using oligonucleotide microarray analysis. RESULTS: TCS exerted a cytotoxic effect on DCs in a concentration-dependent manner. Although TCS alone did not induce full maturation of DC, it did so in the presence of tumor necrosis factor alpha and IL-1beta in vitro. TCS-stimulated DCs induced a decreased ratio of T(H)1/T(H)2 cells derived from naïve T cells and showed selective expression of OX40 ligand (OX40L) at both mRNA and protein levels. This induction was partially blocked by anti-OX40L antibody. CONCLUSION: Our results imply that TCS-stimulated DCs polarize the development of T(H)2 cells partially by means of the OX40L signal. Modulation of these DC will favor the alleviation of TCS-induced allergy.

Trichosanthin enhances anti-tumor immune response in a murine Lewis lung cancer model by boosting the interaction between TSLC1 and CRTAM.

Trichosanthin (TCS), extracted from the Chinese medicinal herb Trichosanthes kirilowi, has shown promise for the inhibition of tumor growth. However, its immunomodulatory effect on tumor-host interaction remains unknown. In this study, we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved. In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor, TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice. This reflected the fact that the host immune system was involved in tumor eradication. Using FACS analysis, we found that TCS increased the percentage of effector T cells, particularly Interferon-gamma (IFN-γ) producing CD4(+) and CD8(+) T cells from tumor-bearing mice. TCS also promoted the vigorous proliferation of antigen-specific effector T cells, markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice, consequently enhancing the anti-tumor response and inducing immune protection. Furthermore, we found that TCS upregulated the expression of tumor suppressor in lung cancer 1 (TSLC1) in 3LL tumor cells and the expression of its ligand, class I-restricted T cell-associated molecule (CRTAM), in effector T cells. Blocking TSLC1 expression with small interfering RNA (siRNA) significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells, suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM. Collectively, our data demonstrate that TCS not only affects tumor cells directly, but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM. These findings may lead to the development of a novel approach for tumor regression.

Effect of site-directed PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with multiple biological and pharmacological activities. It has been approved effective in the clinical treatment of AIDS and tumor, but its strong immunogenicity and short plasma half-life have limited the clinical administration. To reduce the immunogenicity and prolong the plasma half-life of this compound, three TCS muteins (M(1), M(2) and M(3)) and two PEGylated TCS muteins (PM(1) and PM(2)) were constructed by site-directed mutagenesis and PEGylation, respectively. Compared with the unmodified TCS, both PEGylated TCS showed a 3- to 4-fold decrease in immunogenicity, a 0.5- to 0.8-fold decrease in non-specific toxicity, and a 4.5- to 6-fold increase in plasma half-life. But there is a problem of activity reduction. The increased circulating half-life in vivo may compensate for the reduced activity. Together with the other benefits of PEGylation such as reduced immunogenicity and toxicity, it is worthwhile to further explore the potential application of the PEGylated TCS as a better therapeutic agent for AIDS and tumor.

Novel IgE peptide-based vaccine prevents the increase of IgE and down-regulates elevated IgE in rodents.

BACKGROUND: Immunotherapy with anti-IgE antibodies for treatment of allergy is promising but a short half-life and extremely high cost limit its application. OBJECTIVE: We sought to develop IgE vaccines that induce longer-lasting auto-antibodies to neutralize self-IgE as an alternative therapy. METHODS: The vaccine was made by conjugating three synthetic peptides corresponding to human IgE receptor-binding sites to a carrier, hepatitis B surface antigen. To test the immunogenicity of the vaccine, rats were immunized with the vaccine or hepatitis B surface antigen as control. Serum IgG titres to human IgE and the IgE of other species were measured. The inhibition by rat antisera of the binding of human IgE to its receptor was assessed by ELISA, flow cytometry analysis, and passive cutaneous anaphylaxis (PCA), and its ability to recognize receptor-bound IgE was examined. The in vivo effect of the vaccine was evaluated in trichosanthin-sensitized mice and rats. In the preventative study, vaccination started before sensitization commenced, while in the treatment study, vaccination started after sensitization. Sensitized mice and rats receiving injections of the carrier served as controls. Trichosanthin-specific IgE was measured using PCA. RESULTS: Sera from vaccine-immunized rats contained high titre antibodies that reacted with soluble and plate-bound but not with receptor-bound human IgE; they also reacted with mouse, rat, and dog IgE. Furthermore, the sera inhibited the binding of human IgE to its receptor in a dose-dependent manner. In preventative and treatment studies, serum trichosanthin-specific IgE levels were significantly reduced in vaccinated groups compared with controls. CONCLUSION: Antibodies against self-IgE can be induced by IgE peptide-based vaccines, which are effective in preventing the increase of IgE and in down-regulating IgE in sensitized animals.

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81-83 and KR173-174) were identified by computer modeling, and then three TCS mutants namely TCS(YFF81-83ACS), TCS(KR173-174CG), and TCS(YFF-KR) were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD(50) of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.

Y55 and D78 are crucial amino acid residues of a new IgE epitope on trichosanthin.

Trichosanthin (TCS) possesses many biological and pharmaceutical activities, but its strong immunogenicity limits its clinical application. To reduce the immunogenicity of TCS, we modified the reported method for the prediction of antigenic site and identified two crucial amino acid residues (Y55 and D78) for a new epitope. We mutated these two residues into glycine and serine, respectively, and obtained three mutants, Y55G, D78S, and Y55G/D78S. These mutants induced less amount of Ig and IgG antibodies in C57BL/6J mice than wild-type TCS (wTCS) (p<0.01) and almost lost the ability to induce IgE antibody production. The mutants stimulated fewer TCS-specific B cells in C57BL/6J mice than wTCS (p<0.01). Compared with wTCS, Y55G, D78S, and Y55G/D78S lost 26.9%, 17.9%, and 98.7% specific binding ability to anti-TCS monoclonal antibody TCS4E9, respectively. These mutants still retained RNA N-glycosidase activity. In conclusion, Y55 and D78 are two crucial amino acid residues of a new IgE epitope on TCS, and their mutation reduces the immunogenicity of TCS, but still retained the enzymatic activity.

Inhibition of Th1- and enhancement of Th2-initiating cytokines and chemokines in trichosanthin- treated macrophages.

Trichosanthin (TCS), the major effective component from Chinese herb Trichosanthes Kirilowii Maxim, is also a potent allergen. Our previous work has shown that TCS can upregulate interleukin-4 (IL-4) and interleukin-13 (IL-13) while inhibit interferon-gamma (IFN-gamma) in mesenteric lymph node cells after TCS immunization. Thus, TCS can arouse a T helper 2 (Th2) response in the draining lymph node. However, little is known about the early effects of TCS on antigen-presenting cells, the initiator of T cell response. In the current study, the effects of TCS on macrophage cytokines and chemokine expression were investigated. Peritoneal macrophages were treated with or without TCS in the presence of lipopolysaccharide (LPS). We found that TCS increased macrophage interleukin-10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) expression, whereas it decreased interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) expression. Our study clearly demonstrated that TCS, as an allergen, has differential effects on macrophage Th1/Th2 initiative factors, effects that are likely to facilitate its inducing of Th2 and immunoglobulin E (IgE) response.

Identification of epitopes of trichosanthin by phage peptide library.

The phage displayed random peptide library has recently emerged as a powerful technique for analyzing Ab-Ag interactions. In this study, the method was employed to identify epitopes of trichosanthin. Two monoclonal Abs (4B5, 2E9) which recognized different epitopes of trichosanthin (TCS) were selected and a phage-peptide library with nine amino acids (9 aa) was used to screen the positive phage clones that have high affinity to the mAbs. Two groups of phage clones that carried peptide-specific binding to mAbs were identified by the screen. The identified phage clones carried peptide-specific binding to 4B5 and 2E9 mAbs were immunized in mice. To evaluate mimotope of selected phages, the specific binding activity to TCS was measured in the serum from phage-immunized mice. They all showed positive results. The conserved interaction motifs were deduced from the peptide sequences of each group of selected phage clones. When compared the motif sequence with the sequence of TCS, it was predicted that 4B5-corresponding epitope was located at 27-37 aa of TCS protein and 2E9-corresponding epitope was located at 41-48 aa of TCS. The predicted sequence of 4B5-corresponding epitope was further confirmed by site-directed mutation of TCS protein. The data showed that the expressed TCS protein mutated in 4B5-corresponding epitope was unable to bind 4B5 mAb. The results suggested that the phage display peptide library is useful to identify Ag epitopes and to raise Ab in disease diagnosis and treatment.

Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C-terminal amino acid residues.

Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity.

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.

Lowering of trichosanthin immunogenicity by site-specific coupling to dextran.

Trichosanthin is a type I ribosome-inactivating protein possessing a broad spectrum of biological and pharmacological activities. Therapeutic use of this compound is hampered by its immunogenicity. It was shown earlier that coupling of dextran to trichosanthin can increase plasma half-life and reduce antigenicity. However, the site where dextran attaches to trichosanthin cannot be controlled; ideally, it should be at or near the antigenic determinant. The present study attempted to couple dextran to trichosanthin at a potential antigenic site. By site-directed mutagenesis, two sites, R29 and K173, were replaced by cysteine, and dextran was coupled to the newly created cysteine residues. The dextran-trichosanthin complex retained 50% of abortifacient activity and had a mean residence time in rats 27-fold longer than natural trichosanthin. Acute hypersensitivity reaction in guinea pigs was reduced greatly after coupling of K173C (a trichosanthin mutant with lysine-173 replaced by cysteine) to dextran. Compared with natural trichosanthin, dextran-K173C had a decrease in IgG and IgE response, whereas the coupling of R29C (a trichosanthin mutant with arginine-29 replaced by cysteine) to dextran did not show significant reduction of immunogenicity. This suggests that K173 but not R29 is located at or near an antigenic determinant. This study has demonstrated an alternative approach for mapping of antigenic determinants. The information obtained is also useful in producing an improved trichosanthin derivative for therapeutic use.

Effect of donor antigen-trichosnthin conjugate in induction of mouse to rat cardiac xenografting tolerance.

OBJECTIVE: To investigate the special effect of donor antigen-trichosnthin (TCS) conjugate on depleting the immune reactive cells that respond to xenograft, and thus inducing specific xenograft tolerance. METHODS: Rat anti-mouse antibodies coupled sepharose-4B beads were used to purify mouse H-2 antigens by affinity chromatography. Those purified H-2 antigens were conjugated with TCS by heterobifunctional reagents SPDP [3-(2-pyridyldithio) propionic acid-hydroxysuccinimide ester] and 2-IT (2-iminothiolane). The specific inhibition function of Ag-TCS to recipient's immune cells was measured in vitro. Ag-TCS conjugate (200 micrograms/kg, i.v.) was administered to the recipients (rats) 4 days prior to cardiac xenotransplantation of mouse to rat. RESULTS: In vitro, the proliferation of immune reactive cells in the recipients pretreated with Ag-TCS was inhibited. The Ag-TCS conjugate significantly prolonged the cardiac survival time (6.88 +/- 1.36 days) compared with cyclosporine A (CsA) group (2.83 +/- 0.75 days) (P < 0.01). CONCLUSION: Donor Ag-TCS conjugate can induce specific tolerance to xenograft.

The antigenic sites of trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties.

Trichosanthin (TCS) fragments were produced by Tn1000 deletion mutagenesis and by cyanogen bromide (CNBr) cleavage and their immunoreactivity was examined by incubating with various antibodies. Twelve C-terminally truncated TCS variants were successfully synthesized under the control of a T7 RNA driven promoter. The smallest antigenic fragment mapped corresponded to the N-terminal 20 amino acids (aa). Six CNBr fragments of TCS were created and identified by electrospray mass spectrometry and N-terminal sequencing. Three antigenic fragments corresponding to aa 1-72, 101-152 and 153-246, respectively were mapped. Fragments corresponding to aa 1-72 and 153-246 were immunoreactive to the same monoclonal antibody showing they are components of a discontinuous epitope. On the other hand, the fragment containing aa 73-100 was not detected by any of the antibodies used.

Construction and diversity analysis of a murine IgE phage surface display library.

To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses, a murine IgE phage surface display library was constructed (3.0 x 10(5) independent clones). We first constructed the V epsilon cDNA library (4.6 x 10(5) independent clones) and V kappa cDNA library (3.0 x 10(5) independent clones). Then, the V epsilon and V kappa gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs was thus constructed. The diversity of V epsilon from this library was analyzed and proved. Fab clones with high specificity to TCS have been screened out.

Structural analysis and molecular modeling of two anti-trichosanthin IgE clones from phage antibody library.

Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS). In this work, the V epsilon and V kappa genes of the two clones were sequenced and their putative germline gene usages were studied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carried out to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments were also analyzed respectively.

Molecular characterization of the V regions of four IgE antibodies specific for trichosanthin.

We report here the cDNA cloning and variable region nucleic acid sequences of the heavy and the light chains of four monoclonal IgE and one monoclonal IgG1 antibodies against trichosanthin (TCS), all of which recognized a similar or related antigenic determinant. Sequence analysis revealed that the VH regions of the four IgE antibodies use different V gene segments from the 7183, 36-60 and J558 families. The DH genes varied extensively in nucleotide sequence and length, with D segments DSP2, DFL16 and DQ52 being used with equal frequency. All JH gene segments except JH1 were present in the epsilon transcripts. In contrast, there appeared to be restricted L-chain utilization. All four L-chain V genes cloned belonged to the V kappa 21 family and a bias in the use of J kappa 1 gene was also observed. The VL sequences of the anti-TCS monoclonal antibodies showed much less diversity than the VH sequences. We propose that the L-chain rather than H-chain may play a more significant role in specific binding with allergenic determinant on TCS.

Importance of lysine and arginine residues to the biological activity of trichosanthin, a ribosome-inactivating protein from Trichosanthes kirilowii tubers.

Lysine and arginine residues in trichosanthin were modified with ethyl acetimidate and phenylglyoxal, respectively. The effects of these chemical modifications on the cell-free protein-synthesis-inhibitory activity and the antigen-antibody (Ag-Ab) interaction between trichosanthin and its antibody were examined. Ethyl acetimidate modification abolished the protein-synthesis-inhibitory activity of trichosanthin. The inactivation process followed simple first-order kinetics with an inactivation half-life of about 18.5 min. Modification of one lysine residue seemed to be responsible for such inactivation. Arginine modification also inactivated trichosanthin. The inactivation process, however, was biphasic with inactivation half-lives of approximately 15 and 540 min. Arginine modification of trichosanthin had little or no effect on Ag-Ab interaction, whereas lysine modification slightly but significantly weakened Ag-Ab binding.

Immunological relatedness of ribosome-inactivating proteins from the Cucurbitaceae family.

The immunological relatedness of various ribosome-inactivating proteins (RIPs) from the Cucurbitaceae family was examined using enzyme-linked immunosorbent assay (ELISA). The proteins studied included trichosanthin (TCS), beta-trichosanthin (beta-TCS), alpha-momorcharin (alpha-MMC), beta-momorcharin (beta-MMC), momorcochin (MCC), luffaculin (LFC) and Mirabilis antiviral protein (MAP). In general, an antigen exhibited the highest immunoreactivity in an assay using it own antibodies when compared with others RIPs, and MAP demonstrated the lowest immunoreactivity in most ELISAs employing antibodies raised against the other RIPs. LFC also exhibited very low immunoreactivity in most assays, TCS and beta-TCS are immunologically disparate in a number of ELISAs. alpha-MMC and beta-MMC are immunologically related but not identical. MCC manifested immunological relatedness to the momorcharins to a certain extent.

Modulation of trichosanthin antigenicity by coupling to dextran.

Trichosanthin (TCS) is a plant-derived type I ribosome-inactivating protein with a wide spectrum of biological and pharmacological activities. Recently, it was covalently coupled to dextran in order to prolong its half-life in plasma. The major biological activities were generally retained but at lower potency. The immunogenicity of the dextran-trichosanthin (DX-TCS) was compared to that of TCS itself in this study. The results showed that mice immunized with TCS produced 8 times as much TCS-reactive IgE than those immunized with DX-TCS. However, both TCS and DX-TCS immunization produced similar titers of TCS-reactive IgG. A trace of dextran-reactive IgG was detected in mice immunized with DX-TCS. Thus, coupling of TCS to dextran reduced its antigenicity but slightly enhanced that of dextran, and the conjugate elicited less IgE than TCS.