Patent Application
20090297479
1. Field of the Invention
The present invention relates to fields of immunology, oncology and medicine. More particularly, the present invention relates to the use of toxin-conjugates that preferentially target activated T-cells. In particular, such conjugates may find use in the treatment of diseases such as T-cell cancers, inflammation and autoimmune diseases.
2. Description of Related Art
T-cell activation is dependent on signals delivered by antigen-presenting cells (APCs) to the antigen (Ag)-specific T-cell receptor (TCR) and accessory receptors on T-cells (Chambers and Allison, 1997). The principal stimulatory accessory signal is transmitted by B7-1 (CD80) or B7-2 (CD86) on APCs to the CD28 receptor on T-cells (Acuto and Michel, 2003). Interestingly, engagement of the same B7-1 or B7-2 ligand to CTLA-4 (CD152) on T-cells markedly attenuates T-cell responses (Walunas et al., 1994; Krummel and Allison, 1995). The importance of CTLA-4 as an inhibitory regulator of T-cell activation is illustrated by death of CTLA-4-deficient mice within 4 weeks of birth because of massive lymphocytic infiltration destroying critical organs (Tivol et al., 1995).
More recently, other inhibitory regulators of T-cell activation were identified, including PD-L1 (B7-H1) and PD-L2 (B7-DC) on APCs and PD-1 on T-cells (Okazaki et al., 2002), BTLA on B cells and T helper 1 (Th1) effector cells and its ligand (herpes virus entry mediator) on T-cells (Sedy et al., 2005; Watanabe et al., 2003), and Tim-3 on APCs and Th1 effector cells and Tim-3 ligand on CD4 T-cells (Monney et al., 2002; Sabatos et al., 2003; Sanchez-Fueyo et al., 2003). The T-cell ligands possess a single immunoglobulin (Ig)-like variable (IgV) domain, and the APC receptors contain both IgV and Ig constant (IgC) domains.12 Interactions between ligand-receptor pairs are mediated predominantly by residues of Ig-like domains (Carreno and Collins, 2002). Because of their structural and functional similarities to B7 molecules, these ligands/receptors are considered members of the B7 receptor superfamily (Carreno and Collins, 2002).
Ligation of PD-1 on T-cells leads to inhibited T-cell responses that can be rescued by exogenous IL-2 or CD28 costimulation (Latchman et al., 2001; Tseng et al., 2001; Freeman et al., 2000), although one report showed that binding of PD-L1 (B7-H1) to PD-1 stimulated T-cell proliferation and IL-10 secretion (Dong et al., 1999; Dong and Chen, 2003; Subudhi et al., 2004). PD-1 deficiency leads to exaggerated autoimmunity since PD-1 knockout mice develop splenomegaly, increased numbers of B and myeloid cells, increased serum IgG and IgA, and a lupus erythematosus-like disease with age (Nishimura et al., 1999; Nishimura et al., 1998). These mice are also markedly susceptible to Ag-induced experimental autoimmune encephalomyelitis (EAE) (Nishimura et al., 1999; Nishimura et al., 1998). BTLA knockout mice do not exhibit developmental Tor B-cell defects, but their lymphocytes have heightened responses to anti-CD3 antibody (Ab) and to anti-IgM Ab (Watanabe et al., 2003) these mice are also prone to developing EAE (Watanabe et al., 2003). In the case of the Tim-3 pathway, its blockade by monoclonal Ab (mAb), Fc-fused soluble receptor, or gene disruption leads to exacerbated Th1-mediated autoimmune diabetes mellitus in nonobese diabetic (NOD) mice (Sabatos et al., 2003; Sanchez-Fueyo et al., 2003).
T-cell expression of PD-1, BTLA or Tim-3 resembles CTLA-4 in that it is not constitutive, but is induced by activation (Liang and Sha, 2002). Moreover, the costimulation delivered by each appears to be mediated through the TCR (Carreno and Collins, 2002). By contrast, expression of PD-1, BTLA, Tim-3, or their ligands differ from CTLA-4 in that it is not restricted to T-cells, but is expressed more widely to include B cells and APCs. Indeed, some of these ligands (PD-L1 and PD-L2) are also expressed in nonlymphoid tissues (Carreno and Collins, 2002). Such broad expression profiles suggest that these molecules can modulate immune responses in secondary lymphoid organs and peripheral tissues (Okazaki et al., 2002), consistent with the observation that IFN-γ can induce expression of PD-1 ligands on non-lymphoid cells (Latchman et al., 2001).
Previously, the inventors identified DC-HIL as a highly glycosylated type I transmembrane protein of 125 and 95 kDa containing an extracellular Ig-like domain (Shikano et al., 2001). They also showed that DC-HIL is expressed constitutively at high levels on the surface of all dendritic cell subsets, including plasmacytoid dendritic cells and Langerhans cells and at lower levels on macrophages (Shikano et al., 2001), and that its expression can be induced in non-lymphoid cells (keratinocytes) following IFN-γ treatment. In human, DC-HIL is expressed constitutively at high levels by CD14+ monocytes and dendritic cells (but not by other leukocytes). They have also shown that DC-HIL is a negative regulator of T-cell activation (Chung et al., 2007a; Chung et al., 2007b) through binding to syndecan-4 on activated T-cells, indicating that interaction of DC-HIL with syndecan-4 attenuates T-cell activation triggered by anti-CD3 Ab or by APCs in a manner resembling the inhibitory function of PD-L1/PD-L2.
Thus, in accordance with the present invention, there is provided a method of reducing T-cell induced inflammation in a subject comprising administering to said subject a conjugate comprising (a) DC-HIL or a syndecan-4-binding fragment thereof; and (b) a toxin. The T-cell induced inflammation may be host-versus-graft disease, psoriasis, atopic dermatitis, contact hypersensitivity, autoimmune disease, or skin graft rejection. The subject may be a human, a mouse, a rat, a dog or a cat. The syndecan-4-binding fragment of DC-HIL comprising the DC-HIL Ig-like domain. The toxin may be saporin, ricin, botulinum toxin or diptheria toxin. Administration may comprise intravenous, intra-arterial, topical, intralesional, subcutaneous, intraperitoneal, intradermal, or intranasal administration. The method may further comprise administering to said subject a second anti-inflammatory treatment, such as an immunosuppressant, a steroid or an NSAID. The method may further comprise a second administration of said conjugate.
In another embodiment, there is provided a method of inhibiting a syndecan-4-positive T-lymphoma or T-leukemia cell in a subject comprising administering to said subject a conjugate comprising (a) DC-HIL or a syndecan-4-binding fragment thereof; and (b) a toxin. Inhibiting comprises reducing the viability or proliferation of said cell. The subject may be a human, a mouse, a rat, a dog or a cat. The syndecan-4-binding fragment of DC-HIL may comprise the DC-HIL Ig-like domain. The toxin may be saporin, ricin, botulinum toxin or diptheria toxin. Administration may comprise intravenous, intra-arterial, intra-lymphatic, intralesional, subcutaneous, intraperitoneal, intradermal or intranasal administration. The method may further comprise administering to said subject a second anti-lymphoma or -leukemia treatment, such as chemotherapy, radiotherapy, IFNα and/or anti-CD20 antibody. The method may further comprise a second administration of said conjugate.
In yet another embodiment, there is provided a conjugate comprising (a) DC-HIL or a syndecan-4-binding fragment thereof; and (b) a toxin. The syndecan-4-binding fragment of DC-HIL may comprise the DC-HIL Ig-like domain. The toxin may be saporin, ricin, or diptheria toxin. The conjugate may be disposed in a pharmaceutically acceptable buffer, carrier or diluent.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIGS. 1A-B—DC-HIL-Fc binds to activated (but not resting) T-cells. (
FIGS. 2A-J—Immobilized DC-HIL-Fc inhibits T-cell activation triggered by anti-CD3 Ab. CD4+ (
FIGS. 3A-B—Cell-cycle analyses of CD4+ T-cells treated with DC-HIL-Fc. (
FIGS. 4A-H—Soluble DC-HIL-Fc enhances responses of CD4+ T-cells by APCs. Effects of soluble DC-HIL-Fc on T-cell activation were examined in MLR (
FIGS. 5A-C—Soluble DC-HIL-Fc enhances elicitation of Ox-induced contact hypersensitivity in mice. Sensitization of BALB/c mice (n=5) with Ox for CH (
FIGS. 6A-D—Ox/DC-HIL-Fc-treated LN cells display hyperactivation phenotypes. In an independent experiment, draining LN (DLN) cells prepared from BALB/c mice treated similarly (as in
FIGS. 7A-D—Mutant analyses of DC-HIL-Fc function. (
FIGS. 8A-F—Among HSPG, SD-4 is likely a ligand of DC-HIL. (
FIGS. 9A-E—DC-HIL binds SD-4 on T-cells. (
FIGS. 10A-E—SD-4 inhibits anti-CD3 responses. Function of SD-4 was analyzed using DO11.10 T-cell lines (
FIGS. 11A-C—T-cells knocked-down for SD-4 respond more strongly to DC. (
FIGS. 12A-H—Infusion of anti-SD-4 or SD4-Fc in mice enhances elicitation of contact hypersensitivity. Sensitization of BALB/c mice (n=5) with oxazolone (Ox) for contact hypersensitivity (CH) (
FIG. 13—Kinetics of SD-4 expression in LN T-cells during CH. LN cells of untreated BALB/c mice (Ct) or mice at different days after challenge with Ox (day 0, just prior to challenge) were doubly-stained with PE-anti-SD-4 Ab (or PE-anti-PD-1 Ab) and FITC-anti-CD4 or CD8, followed by FACS for expression of SD-4 and PD-1 in CD4+ and CD8+ T-cells. Each value is shown as frequency (%) of SD-4+ or PD-1+ cells in the total number of CD4+ or CD8+ T-cells, with standard errors derived from 3 independent experiments.
FIGS. 14A-E—Effects of DC-HIL-SAP on T-cells in vitro. Conjugation with SAP (saporin): DC-HIL-Fc or control Ig was biotynylated (one protein molecule has 1-2 biotin molecules) and then coupled with streptavidin-SAP (Advanced Targeting System) at a molecular ratio of 5:1. (
FIGS. 15A-C—Effects of DC-HIL-SAP on contact hypersensitivity(CH). CH was induced in BALB/c mice (6-10 wks; female): On day 0, mice (n=4) were sensitized by painting 2% oxazolone (Ox) on shaved abdominal skin. On day 6, CH was elicited in sensitized mice by painting 1% Ox or solvent control to right or left ears, respectively (Challenge). Ear thickness was measured daily from day 1 through day 3 or 4 following challenge. (
FIGS. 16A-C—Ox-unresposiveness lasts 3 weeks. CH was induced in BALB/c mice as described previously. (
FIG. 17—Infusion of DC-HIL-SAP shifts from Th1 to Th2-like response. BALB/c mice were sensitized and challenged as described previously. Mice were also given intravenous injection of 40 nM Ig-SAP or DC-HIL-SAP 3 h prior to challenge. One day post-challenge, draining lymph node cells (1×106 cells/well) were cultured with immobilized anti-CD3 Ab (1 μg/ml) for 3 days. Production of IFN-γ (Th1 cytokine) and IL-4 (Th2 cytokine) was measured by ELIZA.
FIGS. 18—Antigen-specific unresponsiveness induced by infusion of DC-SAP. BLAB/c mice were sensitized, challenged with OX, and received intravenous injection of PBS, Ig-SAP or DC-HIL-SAP (40 nM). Mice injected with DC-HIL blocked elicitation of CH response by nearly 90%. One week after the challenge (all mice had no ear swelling), mice were sensitized (day 12 after Ox sensitization) and challenged (day 18) with DNCB contact allergen (1%). Ear thickness was measured daily.
FIGS. 19A-L—Binding of DC-HIL to activated T cells leads to attenuated anti-CD3 response. (
FIGS. 20A-H—SD-4 is the T cell ligand of DC-HIL. (
FIGS. 21A-D—SD-4 acts as a negative regulator of anti-CD3 response. (
FIGS. 22A-F—Expression of DC-HIL by human leukocytes. (
FIGS. 23A-F—DC-HIL expression correlates inversely with allostimulatory capacity of CD14+ monocytes. (
FIG. 24—DC-HIL inhibits cytokine production by activated T cells on a per cell basis. CD4+ or CD8+ T cells (2×105 cells/well, in triplicate) purified from PBMCs were cultured for 3 d in microculture wells precoated with anti-CD3 Ab (0.3 μg/ml) and DC-HIL-Fc or control Ig (10 μg/ml). Cells were harvested from 3 wells, pooled, permeabilized, and then stained with PE-conjugated anti-IL-2, anti-IFN-γ, pooled, permeabilized, and then stained with PE-conjugated anti-IL-2, anti-IFN-γ, and anti-TNF-α. Numbers in quadrants indicate the percentage of cytokine-positive cells. Data shown are representative of 2 independent experiments.
FIGS. 25A-D—Human DC-HIL α and β isoforms possess heparin-binding activity. (
FIGS. 26A-C—Expression of SD-4 by CTCL lines. (
FIGS. 27A-C—CD4+ T cells in PBMCs of CTCL patients express SD-4 at markedly upregulated levels as compared to those of normal donors. PBMCs isolated from CTCL patients with different stages of lympho-malignancy (see Table 2) or normal donors were examined by flowcytometry for surface expression of SD-4. (
FIG. 28A-D—DC-HIL binds to MJ and HUT-78 CTCL through particular types of heparin sulfate saccharide. (
FIGS. 29A-B—SD-4+HUT-78 cells respond to inhibitory function of DC-HIL by suppressing IL-2 secretion. HUT-78 cells (
FIGS. 30A-B—Treatment with DC-HIL has no effect on proliferation of HUT-78 and HH cells triggered by anti-CD3 Ab. (
FIGS. 31A-B—Anti-CTCL activity of saporin-conjugated DC-HIL. HUT-78 (
The inventors have previously demonstrated that human DC-HIL on antigen presenting cells inhibited T-cell functions on activated T-cells, but not resting T-cells, via syndecan-4 (SD-4), a transmembrane (type I) heparan sulfate proteoglycan. Here, they now show that a DC-HIL conjugate of ribosome-inactivating toxin saporin (DC-HIL-SAP) bound to activated SD-4+ effector T-cells, but not to SD-4− regulatory T-cells, nor did it bind to other SD-4+ cell types, including B-cells which express SD-4 constitutively at high level. After binding to SD-4 on activated T-cells, DC-HIL-SAP is internalized and exhibits cytotoxic activity.
In an oxazolone-sensitized mouse model, administration of DC-HIL-SAP suppressed ear-swelling to almost baseline level when compared to SAP alone. The DC-HIL-SAP recipient mice survived without visible adverse effects and displayed suppressed contact hypersensitivity responses to additional oxazolone challenges up to 6 weeks. Twenty percent of CD69+ (a T-cell activation marker) cells from draining lymph nodes (DLN) of oxazolone-challenged mice are also SD-4+. Infusion of DC-HIL-SAP suppressed T-cell activation as the number of CD69+ cells in DLN was reduced by 50%. There was also a 30% drop of SD-4+ cells. No difference in the numbers of CD4+, CD8+ or B220+ cells between DC-HIL-SAP and SAP treated DLN was observed. SD-4+ cells almost disappeared in DC-HIL-SAP-treated mouse skin where oxazolone was applied directly. The inventors further showed that DC-HIL binds to heparan sulfate saccharide on syndecan-4 on activated T-cells but not to syndecan-4 on other cell-types, which is likely to be produced only by activated T-cells.
The inventors also found that SD-4 is not expressed by regulatory T cells (Treg) that can protect host against graft-versus-host disease caused by bone marrow transplantation. Treatment of Treg in vitro with DC-HIL-SAP has null effect on their function. Injection of DC-HIL-SAP in mice has no deterious effect on Treg in spleen and lymph nodes. These results indicate that injection of DC-HIL-SAP in mice deactivates function of effector T cells while preserving Treg function. Finally, the inventors found that T cells of patients with cutaneous T cell lymphoma (mycosis fungoides and Sezary syndrome) express syndecan-4 on the cell surface and the expression level correlates positively with the stages of Sezary syndrome (the degree of peripheral blood involvement). Further characterization of SD-4 expression by T cells, SD-4 is not expressed by regulatory T cells (Treg) that play a critical role in suppressing immune responses. In consistent with this absence, DC-HIL-SAP does not have deterious effect on Treg in vitro and in mice. SD-4 is expressed at elevated levels by malignant T cells of patients with cutaneous T cell lymphoma (CTCL). In in vitro culture, DC-HIL-SAP kills a cell line HUT-78 derived from Sezary syndrome, an aggressive form of CTCL.
Based on these results, the inventors propose the use of DC-HIL-toxin conjugates for the treatment of various T-cell inflammatory diseases. In addition, examination of clinical pathologic specimen from cutaneous lymphoma patients revealed SD-4+ T-cell in the form that can be bound by DC-HIL, thereby extending the use of these toxin conjugates to the treatment T-cell cancers such as CTCL. No effect of DC-HIL-toxin on Treg indicates that the toxin can be useful to deactivate effector T cell function while sparing immune suppression, thereby extending the use to the suppression of graft-versus-host disease, which hampers the therapeutic success of bone marrow transplantation. These and other aspects of the invention are set forth in detail below.
DC-HIL has a leader sequence (aa 1-19), a long extracellular domain (ECD, aa 20-499), a transmembrane domain (aa 500-523), and a cytoplasmic domain (aa 524-574). The ECD contains 11 potential N-glycosylation sites (NX(S/T)) and several putative O-glycosylation sites based on the stretch of proline-, serine-, and threonine-rich region, and a proline-rich region (aa 320-352) that presumably forms a hinge, as seen in proteins like IgA, which can mediate protein-protein interactions. Other functional motifs are an RGD sequence (aa 64-66), an integrin-binding sequence, and a KRFR (SEQ ID NO: 1) sequence (aa 23-26) that matches a heparin-binding motif composed of a stretch of basic residues (BBXB, where B represents a basic residue). The cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif (ITAM (SEQ ID NO:2), YXXI, aa 529-532, where X represents all other amino acid residues) and two lysosomal targeting di-leucine motifs (LL, aa 548-549 and 566-567).
Human DC-HIL has a leader sequence (aa 1-19), a long extracellular domain (ECD, aa 20-495), a transmembrane domain (aa 496-518), and a cytoplasmic domain (aa 519-572). The ECD contains 11 potential N-glycosylation sites (NX(S/T)) and several putative O-glycosylation sites based on the stretch of proline-, serine-, and threonine-rich region, and a proline-rich region (aa 320-349) that presumably forms a hinge, as seen in proteins like IgA, which can mediate protein-protein interactions. Other functional motifs are an RGD sequence (aa 64-66), an integrin-binding sequence, and a KRFH (SEQ ID NO:3) sequence (aa 23-26) that matches a heparin-binding motif composed of a stretch of basic residues (BBXB, where B represents a basic residue). The cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif (ITAM (SEQ ID NO:2), YXYI, aa 525-528) and two lysosomal targeting di-leucine motifs (LL, aa 516-517 and 562-563).
The present invention contemplates the use of various fragments of DC-HIL, in particular those that retain the ability to bind selectively to the syndecan-4 molecule expressed by activated T-cells, but not the syndecan-4 expressed by other cells, in including B-cell. A particular fragment of DC-HIL would include the Ig-like domain found in the extracellular portion of DC-HIL.
In particular, fragments of DC-HIL would thus include aa 259-319 in the human form:
and aa 256-319 in the mouse form:
Substitutional Variants. It also is contemplated in the present invention that variants or analogs of DC-HIL may also bind preferentially to syndecan-4 on activated T-cells. Sequence variants of DC-HIL, primarily making conservative substitutions, may provide improved compositions. Substitutional variants typically contain the exchange of one amino acid or amino acid analog for another at one or more sites within the molecule, and may be designed to modulate one or more properties of the molecule, in particular the affinity of the molecule for the target, without the loss of other functions or properties.
Altered Amino Acids. As shown above, peptides or proteins may employ modified, non-natural and/or unusual amino acids. A table of exemplary, but not limiting, modified, non-natural and/or unusual amino acids is provided herein below. Chemical synthesis may be employed to incorporated such amino acids into the peptides of interest.
Mimetics. In addition to the variants discussed above, the present inventors also contemplate that structurally similar compounds may be formulated to mimic the key portions of DC-HIL, such as the Ig-like domain. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents.
Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
Other approaches have focused on the use of small, multidisulfide-containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins (Vita et al., 1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and high permissive for mutation. This motif is composed of a β sheet and an α-helix bridged in the interior core by three disulfides.
β-II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids (Weisshoff et al., 1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties.
Methods for generating specific structures have been disclosed in the art. For example, α-helix mimetics are disclosed in U.S. Pat. Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
Methods for generating conformationally restricted beta turns and beta bulges are described, for example, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. β-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures. Other types of mimetic turns include reverse and γ turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.
It may be desirable to purify proteins, peptides and conjugates (below) according to the present invention. Purification techniques are well known to those of skill in the art. These techniques typically involve chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure protein or peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis, isoelectric focusing. A particularly efficient method of purifying peptides or proteins is fast protein liquid chromatography or even HPLC.
Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of a protein. The term “purified protein” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its normally-obtainable state. A purified protein therefore also refers to a protein free from the environment in which it may normally occur.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition by weight.
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of protein or peptide within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the peptide/protein exhibits a detectable activity.
Various techniques suitable for use in peptide/protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified peptide or protein.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
The present invention, one aspect, provides for conjugates between DC-HIL or syndecan-4-binding fragments thereof and a toxin that can kill or impair the function of an activated T-cell that expresses syndecan-4 on its surface. The following discussion of toxins and linking technologies is exemplary and in no way limiting with respect to implementation of this embodiment of the invention.
A. Toxins
A toxin is a poisonous substance produced by living cells or organisms that is active at very low concentrations. Toxins can be small molecules, peptides, or proteins and are capable of causing disease on contact or absorption with body tissues by interacting with biological macromolecules such as enzymes or cellular receptors.
Biotoxins vary greatly in purpose and mechanism, and can be highly complex (the venom of the cone snail contains dozens of small proteins, each targeting a specific nerve channel or receptor), or relatively small protein. Examples include cyantoxins, produced by cyanobacteria, hemotoxins, which target and destroy red blood cells, and are transmitted through the bloodstream, necrotoxins, which cause necrosis in the cells they encounter and destroy all types of tissue, neurotoxins, which primarily affect the nervous systems of animals, and plant toxins.
Ricin. Ricin is a protein toxin that is extracted from the castor bean (Ricinus communis), and is poisonous if inhaled, injected, or ingested, acting as a toxin by the inhibition of protein synthesis. While there is no known antidote, the US military has developed a vaccine. Symptomatic and supportive treatment is available. Long term organ damage is likely in survivors. Ricin causes severe diarrhea and victims can die of shock. Abrin is a similar toxin. Deaths caused by ingestion of castor oil plant seeds is rare. Eight beans are considered toxic for an adult. A solution of saline and glucose has been used to treat ricin overdose.
Ricin consists of two distinct protein chains (almost 30 kDa each) that are linked to each other by a disulfide bond. Ricin A is an N-glycoside hydrolase that targets and depurinates an adenine base in the 28S rRNA molecule of the ribosome, resulting in an inhibition of protein biosynthesis. Ricin B is a lectin that binds galactosyl residues and is important in assisting ricin A's entry into a cell by binding with a cell surface component. Many plants such as barley have the A chain but not the B chain. People do not get sick from eating large amounts of such products, as ricin A alone is of extremely low toxicity.
Diptheria toxin. The diphtheria toxin causes the death eucaryotic cells and tissues by inhibition protein synthesis in the cells. Although the toxin is responsible for the lethal symptoms of the disease, the virulence of C. diphtheriae cannot be attributed to toxigenicity alone, since a distinct invasive phase apparently precedes toxigenesis. However, it has not been ruled out that the diphtheria toxin plays an essential role in the colonization process due to short-range effects at the colonization site.
Botulinum toxin. Botulinum toxin is a neurotoxin protein produced by the bacterium Clostridium botulinum. It is one of the most poisonous naturally occurring substances in the world, and it is the most toxic protein. Though it is highly toxic, it is used in minute doses both to treat painful muscle spasms, and as a cosmetic treatment in some parts of the world. It is sold under the brandnames Myobloc, Botox and Dysport.
Saporin. Saporin is a protein that is useful in biological research applications, especially studies of behavior. Saporin is a so-called ribosome-inactivating protein (RIP), due to its N-glycosidase activity, from the seeds of Saponaria officinalis (common name: soapwort). It was first described by Fiorenzo Stirpe and his colleagues in 1983 in an article that illustrated the unusual stability of the protein.
B. Linkers
Any of a wide variety of linkers may be utilized to effect the joinder of proteins/peptide of DC-HIL to toxins. Certain linkers will generally be preferred over other linkers, based on differing pharmacologic characteristics and capabilities.
Cross-linking reagents are used to form molecular bridges that tie together functional groups of two molecules. Any linking/coupling agents known to those of skill in the art can be used to combine to molecules of the present invention, such as, avidin-biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphoester linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions.
An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhlydryl group) of the other protein (e.g., the selective agent).
It is particular that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is “sterically hindered” by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1986). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
U.S. Pat. No. 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Preferred uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
U.S. Pat. No. 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single-chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Pat. No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
Peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment also are contemplated. Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
Host-versus-graft. Host-versus-graft rejection is defined as the normal lymphocyte mediated reactions of a host against allogeneic or xenogeneic cells acquired as a graft or otherwise, which lead to damage or/and destruction of the grafted cells. The opposite of graft-versus-host reaction. The common basis of graft rejection.
“Acute rejection” is generally acknowledged to be mediated by T-cell responses to proteins from the donor organ which differ from those found in the recipient. Unlike antibody-mediated hyperacute rejection, development of T-cell responses first occurs several days after a transplant if the patient is not taking immunosuppressant drugs. Since the development of powerful immunosuppressive drugs such as cyclosporin, tacrolimus and rapamycin, the incidence of acute rejection has been greatly decreased, however, organ transplant recipients can develop acute rejection episodes months to years after transplantation. Acute rejection episodes can destroy the transplant if it is not recognized and treated appropriately. Episodes occur in around 60-75% of first kidney transplants, and 50-60% of liver transplants. A single episode is not a cause for concern if recognised and treated promptly and rarely leads to organ failure, but recurrent episodes are associated with chronic rejection of grafts. The bulk of the immune system response is to the Major Histocompatibility Complex (MHC) proteins. MHC proteins are involved in the presentation of foreign antigens to T-cells, and receptors on the surface of the T-cell (TCR) are uniquely suited to recognition of proteins of this type. MHC are highly variable between individuals, and therefore the T-cells from the host recognize the foreign MHC with a very high frequency leading to powerful immune responses that cause rejection of transplanted tissue. Identical twins and cloned tissue are MHC matched, and are therefore not subject to T-cell mediated rejection.
The diagnosis of acute rejection relies on the clinical data including patient signs and symptoms, laboratory testing and ultimately a liver biopsy. The biopsy is interpreted by a pathologist who notes changes in the tissue that suggest rejection. Histologically acute rejection is characterized by three main features. First, a predominately T-cell rich lymphocytic infiltrate is often present and may be accompanied by a heterogeneous infiltrate including eosinophils, scattered plasma cells and neutrophils. Of note, an abundance of eosinophils within the mixed infiltrate is a helpful feature of acute rejection. Secondly, evidence of injury to the bile ducts is often seen, manifested by the presence of intrepithelial lymphocytes and loss of epithelial cell polarity. Lastly, injury to the vessels may be seen as endothelialitis. Typically this involves portal vein branches, but may include central veins and sinusoids. For a pathologist who evaluates biopsies for liver disease following transplantation, it is important to be aware of the disorders that commonly occur in this setting and their histologic differences. These include autoimmune hepatitis, which will often have a large number of plasma cells; post-transplant lymphoproliferative disorder with its characteristic monotonous infiltrate and primary biliary cirrhosis which may have focal injury to bile ducts unlike the more monotonous process seen in acute rejection.
Rejection is prevented with a combination of drugs including Calcineurin inhibitors such as Cyclosporin and Tacrolimus; mTOR inhibitors such as Sirolimus and Everolimus; anti-proliferatives such as Azathioprine and Mycophenolic acid; corticosteroids such as Prednisolone and Hydrocortisone; and antibodies such as Basiliximab, Daclizumab, Anti-thymocyte globulin (ATG), and Anti-lymphocyte globulin (ALG)
Generally, a triple therapy regimen of a calcineurin inhibitor, an anti-proliferative, and a corticosteroid is used, although local protocols vary. Antibody inductions can be added to this, especially for high-risk patients and in the U.S. mTOR inhibitors can be used to provide calcineurin-inhibitor or steroid-free regimes in selected patients.
An FDA approved immune function test from Cylex has shown effectiveness in minimizing the risk of infection and rejection in post-transplant patients by enabling doctors to tailor immunosuppressant drug regimens. By keeping a patient's immune function within a certain window, doctors could adjust drug levels to prevent organ rejection while avoiding infection. Such information could help physicians reduce the use of immunosuppressive drugs, lowering drug therapy expenses while reducing the morbidity associated with biopsies, improve the daily life of transplant patients, and could prolong the life of the transplanted organ.
Acute rejection is normally treated initially with a short course of high-dose methylprednisolone, which is usually sufficient to treat successfully. If this is not enough, the course can be repeated or ATG can be given. Acute rejection refractory to these treatments may require plasma exchanges to remove antibodies to the transplant. The monoclonal anti-T-cell antibody OKT3 was formerly used in the prevention of rejection, and is occasionally used in treatment of severe acute rejection, but has fallen out of common use due to the severe cytokine release syndrome and late post-transplant lymphoproliferative disorder, which are both commonly associated with use of OKT3; in the United Kingdom it is available on a named-patient use basis only.
Acute rejection usually begins after the first week of transplantation, and most likely occurs to some degree in all transplants (except between identical twins). It is caused by mismatched HLA antigens that are present on all cells. HLA antigens are polymorphic therefore the chance of a perfect match is extremely rare. The reason that acute rejection occurs a week after transplantation is because the T-cells involved in rejection must differentiate and the antibodies in response to the allograft must be produced before rejection is initiated. These T-cells cause the grafT-cells to lyse or produce cytokines that recruit other inflammatory cells, eventually causing necrosis of allograft tissue. Endothelial cells in vascularized grafts such as kidneys are some of the earliest victims of acute rejection. Damage to the endothelial lining is an early predictor of irreversible acute graft failure. The risk of acute rejection is highest in the first 3 months after transplantation, and is lowered by immunosuppressive agents in maintenance therapy. The onset of acute rejection is combatted by episodic treatment.
Contact hypersensitivity/dermatitis. Contact dermatitis (contact hypersensitivity) is a term for a skin reaction resulting from exposure to allergens (allergic contact dermatitis) or irritants (irritant contact dermatitis). Phototoxic dermatitis occurs when the allergen or irritant is activated by sunlight. Contact dermatitis is a localized rash or irritation of the skin caused by contact with a foreign substance. Only the superficial regions of the skin are affected in contact dermatitis. Inflammation of the affected tissue is present in the epidermis (the outermost layer of skin) and the outer dermis (the layer beneath the epidermis). Unlike contact urticaria, in which a rash appears within minutes of exposure and fades away within minutes to hours, contact dermatitis takes days to fade away. Even then, contact dermatitis fades only if the skin no longer comes in contact with the allergen or irritant. Contact dermatitis results in large, burning, and itchy rashes, and these can take anywhere from several days to weeks to heal. Chronic contact dermatitis can develop when the removal of the offending agent no longer provides expected relief. In North/South America, the most common causes of allergic contact dermatitis are plants of the Toxicodendron genus: poison ivy, poison oak, and poison sumac. Common causes of irritant contact dermatitis are harsh (highly alkaline) soaps, nickel, detergents, and cleaning products and rubbers.
There are three types of contact dermatitis: irritant contact, allergic contact, and photocontact dermatitis. Photocontact dermatitis is divided into two categories: phototoxic and photoallergic. Chemical irritant contact dermatitis is either acute or chronic, which is usually associated with strong and weak irritants respectively (HSE MS24).
Common chemical irritants implicated include solvents (alcohol, xylene, turpentine, esters, acetone, ketones, and others); metalworking fluids (neat oils, water-based metalworking fluids with surfactants); latex; kerosene; ethylene oxide; surfactants in topical medications and cosmetics (sodium lauryl sulfate); alkalies (drain cleaners, strong soap with lye residues).
Physical irritant contact dermatitis is a less researched form of ICD (Maurice-Jones et al) due to its various mechanisms of action and a lack of a test for its diagnosis. A complete patient history combined with negative allergic patch testing is usually necessary to reach a correct diagnosis. The simplest form of PICD results from prolonged rubbing, although the diversity of implicated irritants is far wider. Examples include paper friction, fiberglass, and scratchy clothing.
Many plants cause ICD by directly irritating the skin. Some plants act through their spines or irritant hairs. Some plant such as the buttercup, spurge, and daisy act by chemical means. The sap of these plants contains a number of alkaloids, glycosides, saponins, anthraquinones, and (in the case of plant bulbs) irritant calcium oxalate crystals—all of which can cause CICD (Mantle et al., 2001).
Allergic Contact Dermatitis (ACD) is a condition that is the manifestation of an allergic response caused by contact with a substance. Although less common than ICD, ACD is accepted to be the most prevalent form of immunotoxicity found in humans. By its allergic nature, this form of contact dermatitis is a hypersensitive reaction that is atypical within the population. The mechanisms by which these reactions occur are complex, with many levels of fine control. Their immunology centers around the interaction of immunoregulatory cytokines and discrete subpopulations of T lymphocytes.
ACD arises as a result of two essential stages: an induction phase, which primes and sensitizes the immune system for an allergic response, and an elicitation phase, in which this response is triggered. As such, ACD is termed a Type IV delayed hypersensitivity reaction involving a cell-mediated allergic response. Contact allergens are essentially soluble haptens (low in molecular weight) and, as such, have the physico-chemical properties that allow them to cross the stratum corneum of the skin. They can only cause their response as part of a complete antigen, involving their association with epidermal proteins forming hapten-protein conjugates. This, in turn, requires them to be protein-reactive.
Sometimes termed “photoaggravated,” and divided into two categories, phototoxic and photoallergic, PCD is the eczematous condition which is triggered by an interaction between an otherwise unharmful or less harmful substance on the skin and ultraviolet light (320-400 nm UVA), therefore manifesting itself only in regions where the sufferer has been exposed to such rays. Without the presence of these rays, the photosensitiser is not harmful. For this reason, this form of contact dermatitis is usually associated only with areas of skin which are left uncovered by clothing. The mechanism of action varies from toxin to toxin, but is usually due to the production of a photoproduct. Toxins which are associated with PCD include the psoralens. Psoralens are in fact used therapeutically for the treatment of psoriasis, eczema and vitiligo. Photocontact dermatitis is another condition where the distinction between forms of contact dermatitis is not clear cut. Immunological mechanisms can also play a part, causing a response similar to ACD.
Allergic dermatitis is usually confined to the area where the trigger actually touched the skin, whereas irritant dermatitis may be more widespread on the skin. Symptoms of both forms include the following:
Immediately after exposure to a known allergen or irritant, wash with soap and cool water to remove or inactivate most of the offending substance. Weak acid solutions, such as lemon juice, vinegar, can be used to counteract the effects of dermatitis contracted by exposure to basic irritants. If blistering develops, cold moist compresses applied for 30 minutes 3 times a day can offer relief. Calamine lotion and cool colloidal oatmeal baths may relieve itching. Oral antihistamines such as diphenhydramine (Benadryl, Ben-Allergin) can also relieve itching. For mild cases that cover a relatively small area, hydrocortisone cream in nonprescription strength may be sufficient. Avoid scratching, as this can cause secondary infections.
If the rash does not improve or continues to spread after 2-3 of days of self-care, or if the itching and/or pain is severe, the patient should contact a dermatologist or other physician. Medical treatment usually consists of lotions, creams, or oral medications. A corticosteroid medication similar to hydrocortisone may be prescribed to combat inflammation in a localized area. This medication may be applied to your skin as a cream or ointment. If the reaction covers a relatively large portion of the skin or is severe, a corticosteroid in pill or injection form may be prescribed. Prescription antihistamines may be given if nonprescription strengths are inadequate.
Since contact dermatitis relies on an irritant or an allergen to initiate the reaction, it is important for the patient to identify the responsible agent and avoid it. This can be accomplished by having patch tests, a method commonly known as allergy testing. The patient must know where the irritant or allergen is found to be able to avoid it. It is important to also note that chemicals sometimes have several different names.[14]
Atopic dermatitis. Atopic dermatitis, also known as atopic eczema, is an atopic, hereditary, and non-contagious skin disease characterized by chronic inflammation of the skin. The skin of a patient with atopic dermatitis reacts abnormally and easily to irritants, food, and environmental allergens and becomes red, flaky and very itchy. It also becomes vulnerable to surface infections caused by bacteria. The skin on the flexural surfaces of the joints (for example inner sides of elbows and knees) are most commonly affected regions in people.
Since the twentieth century, many mucosal inflammatory disorders have become dramatically more common; atopic eczema (AE) is a classic example of such a disease. It now affects 10-20% of children and 1-3% of adults in industrialized countries, and its prevalence there has more than doubled in the past thirty years.
Although it is an inherited disease, eczema is primarily aggravated by contact with or intake of allergens. It can also be influenced by other “hidden” factors such as stress or fatigue. Atopic eczema consists of chronic inflammation; it often occurs in people with a history of allergy disorders such as asthma or hay fever.
Atopic dermatitis often occurs together with other atopic diseases like hay fever, asthma and conjunctivitis. It is a familial and chronic disease and its symptoms can increase or disappear over time. Atopic dermatitis in older children and adults is often confused with psoriasis. Atopic dermatitis afflicts humans, particularly young children; it is also a well-characterized disease in domestic dogs. Although there is no cure for atopic eczema, and its causes not well understood, it can be treated very effectively in the short term through a combination of prevention (learning what triggers the allergic reactions) and drug therapy.
The primary treatment involves prevention, which includes avoiding or minimizing contact with (or intake of) known allergens. Once that has been established, topical treatments can be used. Topical treatments focus on reducing both the dryness and inflammation of the skin.
To combat the severe dryness associated with eczema, a high-quality, dermatologist approved moisturizer should be used daily. Moisturizers should not have any ingredients that may further aggravate the condition. Moisturizers are especially effective if applied within 5-10 minutes after bathing.
Most commercial soaps wash away the oils produced by the skin that normally serve to prevent drying. Using a soap substitute such as aqueous cream helps keep the skin moisturized. A non-soap cleanser can be purchased usually at a local drug store. Showers should be kept short and at a lukewarm/moderate temperature.
If moisturizers on their own don't help and the eczema is severe, a doctor may prescribe topical steroid ointments or creams. Steroid creams have traditionally been considered the most effective method of treating severe eczema. Disadvantages of using steroid creams include stretch marks and thinning of the skin. Higher-potency steroid creams must not be used on the face or other areas where the skin is naturally thin; usually a lower-potency steroid is prescribed for sensitive areas. Along with creams, antibiotics are often prescribed if an infection is suspected. If the eczema is especially severe, a doctor may prescribe prednisone or administer a shot of cortisone. If the eczema is mild, over-the-counter hydrocortisone can be purchased at the local drugstore.
The immunosuppressant Tacrolimus or pimecrolimus can be used as a topical preparation in the treatment of severe atopic dermatitis instead of traditional steroid creams. However, there can be unpleasant side effects in some patients such as intense stinging or burning. Some alternative medicines may (illegally) contain very strong steroids. Others are completely harmless, such as Oolong tea.
A more novel form of treatment involves exposure to broad or narrow-band ultraviolet light. UV radiation exposure has been found to have a localized immunomodulatory effect on affected tissues, and may be used to decrease the severity and frequency of flares. In particular, some researchers have suggested that the usage of UVA1 is more effective in treating acute flares, whereas narrow-band UVB is more effective in long-term management scenarios. However, UV radiation has also been implicated in various types of skin cancer, and thus UV treatment is not without risk.
If ultraviolet light therapy is employed, initial exposure should be no longer than 5-10 minutes, depending on skin type. UV therapy should only be moderate, and special care should be taken to avoid sunburn (sunburn will only aggravate the eczema). It does not necessarily have to be administered in a hospital, it can be done at a tanning salon or in natural sunlight, so as long as it is performed under the direction and supervision of a dermatologist.
Many of the same types of treatment are used in domestic dogs with atopic dermatitis. In addition, domestic dogs may be successfully managed with allergen-specific immunotherapy; many are treated with low-dose cyclosporine lipid emulsion.
Psoriasis. Psoriasis is a disease which affects the skin and joints. It commonly causes red scaly patches to appear on the skin. The scaly patches caused by psoriasis, called psoriatic plaques, are areas of inflammation and excessive skin production. Skin rapidly accumulates at these sites and takes a silvery-white appearance. Plaques frequently occur on the skin of the elbows and knees, but can affect any area including the scalp and genitals. Psoriasis is hypothesized to be immune-mediated and is not contagious.
The disorder is a chronic recurring condition which varies in severity from minor localised patches to complete body coverage. Fingernails and toenails are frequently affected (psoriatic nail dystrophy)—and can be seen as an isolated finding. Psoriasis can also cause inflammation of the joints, which is known as psoriatic arthritis. Ten to fifteen percent of people with psoriasis have psoriatic arthritis. The symptoms of psoriasis can manifest in a variety of forms. Variants include plaque, pustular, guttate and flexural psoriasis.
The cause of psoriasis is not known, but it is believed to have a genetic component. Several factors are thought to aggravate psoriasis. These include stress, excessive alcohol consumption, and smoking. Individuals with psoriasis may suffer from depression and loss of self-esteem. As such, quality of life is an important factor in evaluating the severity of the disease. There are many treatments available but because of its chronic recurrent nature psoriasis is a challenge to treat.
There are two main hypotheses about the process that occurs in the development of the disease. The first considers psoriasis as primarily a disorder of excessive growth and reproduction of skin cells. The problem is simply seen as a fault of the epidermis and its keratinocytes. The second hypothesis sees the disease as being an immune-mediated disorder in which the excessive reproduction of skin cells is secondary to factors produced by the immune system. T-cells (which normally help protect the body against infection) become active, migrate to the dermis and trigger the release of cytokines (tumor necrosis factor-alpha (TNFα), in particular) which cause inflammation and the rapid production of skin cells. It is not known what initiates the activation of the T-cells.
Psoriasis is a fairly idiosyncratic disease. The majority of people's experience of psoriasis is one in which it may worsen or improve for no apparent reason. Studies of the factors associated with psoriasis tend to be based on small (usually hospital based) samples of individuals. These studies tend to suffer from representative issues, and an inability to tease out causal associations in the face of other (possibly unknown) intervening factors. Conflicting findings are often reported. Nevertheless, the first outbreak is sometimes reported following stress (physical and mental), skin injury, and streptococcal infection. Conditions that have been reported as accompanying a worsening of the disease include infections, stress, and changes in season and climate. Certain medicines, including lithium salt and beta blockers, have been reported to trigger or aggravate the disease. Excessive alcohol consumption, smoking and obesity may exacerbate psoriasis or make the management of the condition difficult.
There can be substantial variation between individuals in the effectiveness of specific psoriasis treatments. Because of this, dermatologists often use a trial-and-error approach to finding the most appropriate treatment for their patient. The decision to employ a particular treatment is based on the type of psoriasis, its location, extent and severity. The patient's age, gender, quality of life, comorbidities, and attitude toward risks associated with the treatment are also taken into consideration.
Medications with the least potential for adverse reactions are preferentially employed. If the treatment goal is not achieved then therapies with greater potential toxicity may be used. Medications with significant toxicity are reserved for severe unresponsive psoriasis. This is called the psoriasis treatment ladder. As a first step, medicated ointments or creams, called topical treatments, are applied to the skin. If topical treatment fails to achieve the desired goal then the next step would be to expose the skin to ultraviolet (UV) radiation. This type of treatment is called phototherapy. The third step involves the use of medications which are taken internally by pill or injection. This approach is called systemic treatment.
Over time, psoriasis can become resistant to a specific therapy. Treatments may be periodically changed to prevent resistance developing (tachyphylaxis) and to reduce the chance of adverse reactions occurring. This is called treatment rotation.
Bath solutions and moisturizers help soothe affected skin and reduce the dryness which accompanies the build-up of skin on psoriatic plaques. Medicated creams and ointments applied directly to psoriatic plaques can help reduce inflammation, remove built-up scale, reduce skin turn over, and clear affected skin of plaques. Ointment and creams containing coal tar, dithranol (anthralin), corticosteroids like Topicort Desoximetasone), vitamin D3 analogues (for example, calcipotriol), and retinoids are routinely used. Argan oil has also been used with some promising results. The mechanism of action of each is probably different but they all help to normalise skin cell production and reduce inflammation. Activated vitamin D and its analogues are highly effective inhibitors of skin cell proliferation.
The disadvantages of topical agents are variably that they can often irritate normal skin, can be time consuming and awkward to apply, cannot be used for long periods, can stain clothing or have a strong odour. As a result, it is sometimes difficult for people to maintain the regular application of these medications. Abrupt withdrawal of some topical agents, particularly corticosteroids, can cause an aggressive recurrence of the condition. This is known as a rebound of the condition.
Some topical agents are used in conjunction with other therapies, especially phototherapy. It has long been recognized that daily, short, non-burning exposure to sunlight helped to clear or improve psoriasis. Niels Finsen was the first physician to investigate the therapeutic effects of sunlight scientifically and to use sunlight in clinical practice. This became known as phototherapy.
Sunlight contains many different wavelengths of light. It was during the early part of the 20th century that it was recognised that for psoriasis the therapeutic property of sunlight was due to the wavelengths classified as ultraviolet (UV) light. Ultraviolet wavelengths are subdivided into UVA (380-315 nm) UVB (315-280 nm), and UVC (<280 nm). Ultraviolet B (UVB) (315-280 nm) is absorbed by the epidermis and has a beneficial effect on psoriasis. Narrowband UVB (311 to 312 nm), is that part of the UVB spectrum that is most helpful for psoriasis. Exposure to UVB several times per week, over several weeks can help people attain a remission from psoriasis.
Ultraviolet light treatment is frequently combined with topical (coal tar, calcipotriol) or systemic treatment (retinoids) as there is a synergy in their combination. The Ingram regime, involves UVB and the application of anthralin paste. The Goeckerman regime combines coal tar ointment with UVB.
Psoralen and ultraviolet A phototherapy (PUVA) combines the oral or topical administration of psoralen with exposure to ultraviolet A (UVA) light. Precisely how PUVA works is not known. The mechanism of action probably involves activation of psoralen by UVA light which inhibits the abnormally rapid production of the cells in psoriatic skin. There are multiple mechanisms of action associated with PUVA, including effects on the skin immune system. PUVA is associated with nausea, headache, fatigue, burning, and itching. Long-term treatment is associated with squamous-cell and melanoma skin cancers.
Psoriasis which is resistant to topical treatment and phototherapy is treated by medications that are taken internally by pill or injection. This is called systemic treatment. Patients undergoing systemic treatment are required to have regular blood and liver function tests because of the toxicity of the medication. Pregnancy must be avoided for the majority of these treatments. Most people experience a recurrence of psoriasis after systemic treatment is discontinued.
The three main traditional systemic treatments are methotrexate, cyclosporine and retinoids. Methotrexate and cyclosporine are immunosupressant drugs; retinoids are synthetic forms of vitamin A. Other additional drugs, not specifically licensed for psoriasis, have been found to be effective. These include the antimetabolite tioguanine, the cytotoxic agent hydroxyurea, sulfasalazine, the immunosupressants mycophenolate mofetil, azathioprine and oral tacrolimus. These have all been used effectively to treat psoriasis when other treatments have failed. Although not licensed in many other countries fumaric acid esters have also been used to treat severe psoriasis in Germany for over 20 years.
Biologics are manufactured proteins that interrupt the immune process involved in psoriasis. Unlike generalised immunosuppressant therapies such as methotrexate, biologics focus on specific aspects of the immune function leading to psoriasis. These drugs (interleukin antagonists) are relatively new, and their long-term impact on immune function is unknown. They are very expensive and only suitable for very few patients with psoriasis. Ustekinumab (IL-12 and IL-23 blocker) shows hopeful results for psoriasis therapy.
A new natural systemic option, XP-828L, for mild to moderate psoriasis relief has been developed by a Canadian life science and technology company. This oral product with clinically proven efficacy and safety is extracted through a patented process from whey and has immuno-modulatory effects. Antibiotics are not indicated in routine treatment of psoriasis. However, antibiotics may be employed when an infection, such as that caused by the bacteria Streptococcus, triggers an outbreak of psoriasis, as in certain cases of guttate psoriasis.
A variety of autoimmune disorders may be treated in accordance with the present invention, including ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory disease, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile onset diabetes mellitus (also known as Type I diabetes mellitus), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, Graves Disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, Alzheimer's disease, demyelating diseases, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangtasia), adult onset diabetes mellitus (also known as Type II diabetes mellitus), mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, anti-phospholipidsyndrome, erythema multiforme, Cushing's syndrome, autoimmune chronic active hepatitis, allergic disease, allergic encephalomyelitis, transfusion reaction, leprosy, malaria, leshmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, shistosomiasis, gianT-cell arteritis, eczema, lymphomatoid granulomatosis, Kawasaki's disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, Epstein-Barr virus infection, mumps, echovirus infection, cardiomyopathy, parvovirus infection, rubella virus infection, anthrax infection, small pox infection, hepatitic C viral infection, tularemia, sepsis, periodic fever syndromes, pyogenic arthritis, Familial Mediterrenan Fever, TNF-receptor associated periodic syndrome (TRAPS), Muckle-Wells syndrome, hyper-IgD syndrome, familial cold urticaria, Hodgkin's and Non-Hodgkin's lymphoma, renal cell carcinoma, or multiple myeloma.
There has also been no known cause for autoimmune diseases such as systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disease characterized by deposition in tissues of autoantibodies and immune complexes leading to tissue injury (Kotzin, 1996). In contrast to autoimmune diseases such as MS and type 1 diabetes mellitus, SLE potentially involves multiple organ systems directly, and its clinical manifestations are diverse and variable (reviewed by Kotzin & O'Dell, 1995). For example, some patients may demonstrate primarily skin rash and joint pain, show spontaneous remissions, and require little medication. At the other end of the spectrum are patients who demonstrate severe and progressive kidney involvement that requires therapy with high doses of steroids and cytotoxic drugs such as cyclophosphamide (Kotzin, 1996).
The serological hallmark of SLE, and the primary diagnostic test available, is elevated serum levels of IgG antibodies to constituents of the cell nucleus, such as double-stranded DNA (dsDNA), single-stranded DNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNA antibodies play a major role in the development of lupus glomerulonephritis (GN) (Hahn & Tsao, 1993; Ohnishi et al., 1994). Glomerulonephritis is a serious condition in which the capillary walls of the kidney's blood purifying glomeruli become thickened by accretions on the epithelial side of glomerular basement membranes. The disease is often chronic and progressive and may lead to eventual renal failure.
The mechanisms by which autoantibodies are induced in these autoimmune diseases remains unclear. As there has been no known cause of SLE, to which diagnosis and/or treatment could be directed, treatment has been directed to suppressing immune responses, for example with macrolide antibiotics, rather than to an underlying cause. (e.g., U.S. Pat. No. 4,843,092).
In order to increase the effectiveness of the DC-HIL-toxin therapy, it may be desirable to combine these compositions with another agent effective in the treatment of T-cell inflammatory disorders. The terms “contacted” and “exposed,” when applied to a cell, tissue or organism, are used herein to describe the process by which a therapy and/or other agent are delivered to a target T-cell, tissue or organism or are placed in direct juxtaposition with the target T-cell, tissue or organism. Appropriate “combination” therapies for the various disease states are discussed above, and relevant timing of administration issues are the same as discussed below for combination treatments for T-cell malignancies.
In one aspect of the present invention, one may utilize toxin conjugates in the treatment of cancer. Any of a variety of T-cell related cancers are contemplated as suitable for treatment with the present invention. The cancer may also be primary, metastatic, multi-drug resistant or recurrent. In one embodiment, the subject will be administered toxin conjugates through a variety of routes including, but not limited to, intravenous, intra-arterial, intra-lymphatic, intralesional, subcutaneous, intraperitoneal, intradermal or intranasal routes. Particular routes include intravenous injection and intralymphatic injection. Repeated or continuous therapy over a period of time (weeks to months) also is contemplated.
A. T-cell Lymphoma
The following is a list of various T-cell lymphomas that may be treated according to the present invention:
B. T-cell Leukemias
The following is a list of various T-cell leukemias that may be treated according to the present invention:
C. Combined Therapy
In order to increase the effectiveness of the DC-HIL-toxin therapy, it may be desirable to combine these compositions with another agent effective in the treatment of T-cell-related cancers. The terms “contacted” and “exposed,” when applied to a cell, tissue or organism, are used herein to describe the process by which a therapy and/or other agent are delivered to a target T-cell, tissue or organism or are placed in direct juxtaposition with the target T-cell, tissue or organism. Other anti-cancer agents include, but are not limited to, chemotherapeutics, radiotherapeutics or biologicals (anti-CD20 Abs, INFα).
The toxin conjugate treatment may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the toxin conjugate treatment and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the toxin conjugate and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the toxin conjugate. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 4 weeks, about 5 weeks, about 6 weeks, about 7 week or about 8 weeks or more, and any range derivable therein, prior to and/or after administering toxin conjugate.
Various combination regimens of the toxin conjugate treatment and one or more other anti-cancer agents may be employed. Non-limiting examples of such combinations are shown below, wherein a toxin conjugate is “A” and the other anti-cancer agent is “B”:
Other combinations are contemplated. Again, to achieve cancer cell inhibition, both agents are delivered to a cell in a combined amount effective to achieve the desired inhibition, which may include cell stasis or cell death.
Administration of the toxin conjugate to a cell, tissue or organism may follow general protocols for the administration of pharmaceuticals. It is expected that the treatment cycles would be repeated as necessary. In particular embodiments, it is contemplated that various additional agents may be applied in any combination with the present invention.
Radiation agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as “chemotherapeutic agents,” function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., doxorubicin, verapamil, podophyllotoxin, adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. Biologicals include cytokines, interferons, and antibodies.
Pharmaceutical formulations of the present invention comprise an effective amount of a toxin conjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refer to compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of such pharmaceutical compositions are known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The pharmaceuticals of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art.
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
The pharmaceuticals may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
In certain embodiments, the compositions are prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Mice and cell culture. Female BALB/c and C57BL/6 (5-8 weeks old) mice were obtained from the Animal Breeding Center at The University of Texas Southwestern Medical Center (Dallas, Tex.). BALB/cTac-TgN(DO11.10)-Rag2tm1 mice (Hsieh et al., 1993) were purchased from Taconic (Hudson, N.Y.). Following National Institutes of Health guidelines, these animals were housed and cared for in the pathogen-free facility, and all animal studies were approved by the Institutional Animal Care Use Center of the same institution.
Production of DC-HIL-Fc protein. The Fc-fusion proteins (DC-HIL-Fc, its mutants, and Fc alone) were produced in COS-1 cells and purified as described previously (Shikano et al., 2001). Isolation of T-cells and binding of DC-HIL. Following manufacturer's recommendations, CD3+, CD4+, and CD8+ T-cells were purified from spleen cells of BALB/c mice using pan-T-cell, CD4+, and CD8+ T-cell isolation kits (Miltenyi Biotec, Auburn, Calif.), respectively.
Splenic CD3+ T-cells (1×106) were activated by immobilized anti-CD3 Ab (1 or 3 μg/mL) or concanavalin A (10 μg/mL; Sigma, St Louis, Mo.) for 3 days. Freshly isolated and activated CD3+ T-cells were then treated with 5 μg/mL Fc blocker (BD Pharmingen, San Diego, Calif.) on ice for 30 minutes to block Fc-binding activity of Fc receptors on T-cells and incubated with 5 μg/mL PE-anti-CD3 Ab (BD Pharmingen) and 10 μg/mL DC-HIL-Fc or control human IgG (hIgG) plus 5 μg/mL FITC-anti-human IgG (both from Jackson ImmunoResearch, West Grove, Pa.). In some experiments, activated and Fc-blocked CD3+ T-cells were doubly-stained with 5 μg/mL FITC-labeled anti-CD4 or anti-CD8 Ab (BD Pharmingen) and Fc proteins/PE-anti-human IgG (BD Pharmingen). The treated cells were also stained with anti-CD69 Ab or isotypic control IgG to evaluate activation levels. After staining, binding of Fc proteins to T-cells and expression of marker molecules were analyzed by fluorescence-activated cell sorting (FACS).
T-cell proliferation and IL-2 assay. Purified CD4+ or CD8+ T-cells (2×105/well) were cultured for 2 days in enzyme-linked immunosorbent assay (ELISA) wells (in triplicate) precoated with indicated doses of anti-CD3 Ab and Fc proteins or anti-CD28 mAb (BD Pharmingen). After pulsing with 3H-thymidine (1 μCi/well [0.037 MBq/well]) for 20 to 22 hours, cells were collected and evaluated for 3H radioactivity. Culture supernatant was used to measure IL-2 production using the mouse IL-2 ELISA kit (BD Pharmingen). To examine the effects of DC-HIL on reactivation of previously activated T-cells, spleen cells (1×106/mL) isolated from Tac-TgN(DO11.10)-Rag2tm1 mice were cultured for 3 days in the presence of the ovalbumin OVA323-339 peptide (1 μg/mL).24 After purifying CD4+ T-cells from the culture, cells (1×106/mL) were cultured for another day without stimuli and then subjected to T-cell proliferation and IL-2 assays.
Cell-cycle analyses. Cell cycles of CD4+ T-cells treated with anti-CD3 Ab and Fc protein were examined using carboxy-fluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Oreg.). Purified CD4+ T-cells (1×106) were labeled by 1 μM CFSE/DPBS for 15 minutes at 37° C. After another 30 minutes of incubation in culture medium, labeled T-cells were cultured in ELISA wells coated with anti-CD3 Ab (0.3 μg/mL) and control IgG or DC-HIL-Fc (5 μg/mL). At different time points, cells were harvested to examine asynchronous cell division by FACS. Cell cycles of treated T-cells were also analyzed using FITC-BrdU flow kit (BD Pharmingen), following the manufacturer's recommendations. BrdU incorporation and DNA content on a per-cell basis were analyzed by FACS and presented as dot plots.
MLR. BALB/c T-cells and C57BL/6 spleen cells served as responders and stimulators, respectively. C57BL/6 spleen cells (5×104) were y-irradiated (2000 Gy) and mixed with CD4+ T-cells (2×105) purified from BALB/c spleen in 96-microwell plates. Fc fusion protein or control hIgG was added to the mixed leukocyte reaction (MLR) culture and incubated for varying periods. After 3H-thymidine pulsing for 20 hours, cells were harvested and the cell-incorporated 3H radioactivity was measured. T-cell proliferation was expressed as radioactivity left after subtracting background counts per minute (cpm; 3H cpm of control culture in which y-irradiated responders and stimulators were mixed) from experimental cpm.
In vitro antigen presentation by DCs. BM cells were prepared from the femurs of BALB/c mice and cultured with 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, N.J.).25 After culturing for 6 days, DCs were harvested, seeded on 96-well plates at a density of 5×105 cells/well, and cultured with OVA323-339 peptide (2 μg/mL)24 synthesized by the Protein Chemistry Technology Center, UT Southwestern. After 6 hours of antigen pulsing, DC cultures (5×104 cells) were added with CD4+ T-cells (1×105/well) purified from the spleens of BALB/cTac-TgN(DO11.10)-Rag2tm1 mice (Hsieh et al., 1993). After coculture (2 days), cells and supernatant were harvested: cells were stained with FITC-anti-CD4 and PE-anti-CD69 Ab and determined by FACS for frequency of CD69+ cells in CD4+ T-cells. The supernatant was assayed by ELISA for IL-2 production.
Knockdown of DC-HIL expression. A total of 2 different DC-HIL-targeted siRNAs (21 nucleotides long) were synthesized and purified by Qiagen (Valencia, Calif.): DC-HIL siRNA no. 3, sense 5′-r(AACUUGUCUGAUGAGAUCU)dTdT-3′ (SEQ ID NO:6) and antisense 5′-r(AGAUCUCAUCAGACAAGUU)dTdT-3′ (SEQ ID NO:7); DC-HIL siRNA no. 10, sense 5′-r(GCGUACAAGCCAAUAGGAA)dTdT-3′ (SEQ ID NO:8) and antisense, 5′-r(UUCCUAUUGGCUUGUACGC)dTdT-3′ (SEQ ID NO:9). Shuffled sequences of these 2 siRNAs were used as controls. A mixture of siRNA no. 3 and no. 10 (each 1.5 μg) or of the shuffled siRNAs was treated with 5 μL of Gene Silencer (Genlantis, San Diego, Calif.) in the serum-free RPMI for 20 min at room temperature and then added to BM-DCs (2×106 cells). After culturing for 4 hours at 37° C., 20% FCS-RPMI was added to the culture and allowed to incubate for another 24 hours. These transfected DCs were harvested and examined by Western blotting for protein expression of DC-HIL or mixed with OVA-specific T-cells as described in “In vitro antigen presentation by HSCs.”
CH assays. BALB/c mice (n=5) were sensitized for contact hypersensitivity (CH) on day 0 by painting 2% oxazolone (Ox; Sigma) in acetone-olive oil (4:1 in volume) on shaved abdominal skin (sensitization) (Cruz et al., 1988). Mice were challenged on day 6 by painting 1% Ox and solvent control onto right and left ears, respectively (elicitation). Thereafter, CH was assessed daily through day 12 by measuring ear thickness and calculating changes in ear swelling (thickness of right ear minus thickness of control left ear) (Cruz and Bergstresser, 1988). Different panels of mice were injected intraperitoneally with DC-HIL-Fc or the control hIgG (10 mg/kg each) or DPBS on days −1, 1, and 3 (before and after sensitization) or on days 5, 7, and 9 (before and after elicitation). The Student t test was used to determine statistically significant differences in ear-swelling responses.
Histologic examination of skin and phenotyping of LN cells. After painting Ox on ears of Ox-sensitized mice treated with Fc protein (2 days), ear skin and draining lymph nodes (LNs) were procured. Ear skin was embedded in paraffin, thin-sectioned, and stained with hematoxylineosin (Sigma). Histologic examination was carried out under light microscopy using an Olympus BH2 microscope (Olympus, Center Valley, Pa.) at a magnification of ×10. In independent experiments, unsensitized mice were treated similarly and their draining LNs excised 2 days after ear challenge. LN cells were counted and examined for spontaneous proliferation and frequency of CD69+ cells.
For proliferation, LN cells (4×105/well) from untreated or treated mice were cultured without stimulation for 3 days and pulsed with 3H-thymidine (1 μCi/well [0.037 MBq/well]) for 20 hours. For CD69 expression, LN cells (5×105) were stained with FITC-anti-CD69 mAb (eBioscience, San Diego, Calif.) or FITC-isotypic control hamster IgG (BD Pharmingen) (2.5 μg/mL each) in the presence or absence of Ab directed at T-cell or B-cell surface markers (CD4, CD8, and B220; 2.5 μg/mL each) and examined by FACS for surface expression of CD69 in each leukocyte subpopulation.
Generation of mutants DC-HIL-Fc carrying the RAA mutant (replacement of RGD sequence with RAA) was generated as before (Shikano et al., 2001). PRR and PKD mutants (lacking a region between amino acids 301 to 334 and 230 to 355, respectively) were produced by polymerase chain reaction (PCR)-based mutagenesis. Resulting nucleotides coding extracellular domains of DC-HIL mutants were inserted in-frame to the coding sequence of the human IgG1 Fc in pSecTagA plasmid (Invitrogen, Carlsbad, Calif.) using 3 restriction enzyme sites (from the 5′ end, HindIII, EcoRI, and XbaI). The mutant DC-HIL-Fc proteins were produced as described previously (Shikano et al., 2001). The yield for each mutant was very similar to the wild-type and all preparations showed a single band reactive to anti-human IgG Ab.
Activated T-cells express ligands of DC-HIL. To study the function of DC-HIL, the inventors created soluble DC-HIL receptors (DC-HIL-Fc) consisting of the extracellular domain fused with the Fc portion of human IgG1 (hIgG) ((Shikano et al., 2001), and used FACS analysis to examine binding to T-cells. DC-HIL-Fc did not bind to T-cells freshly isolated from spleen of naive mice, but did so after the T-cells were activated by concanavalin A (
Immobilized DC-HIL inhibits T-cell activation triggered via the TCR. The inventors next used immobilized anti-CD3 Ab as a surrogate stimulator of TCR-dependent T-cell activation. CD4+ T-cells were cultured in microculture wells precoated with increasing concentrations of anti-CD3 Ab and a constant concentration of DC-HIL-Fc or control hIgG. T-cell activation was assessed by proliferative capacity measured by 3H-thymidine incorporation. Treatment with immobilized anti-CD3 Ab led to activation of CD4+ T-cells in a dose-dependent manner (
By contrast, coimmobilization of control hIgG with anti-CD3 Ab had almost no effect on CD4+ T-cell activation. An irrelevant Fc fusion protein dectin-2-Fc (Sato et al., 2006), a C-type lectin receptor, had little to no effect on T-cell activation. The ability of DC-HIL to inhibit T-cell activation was also documented by little to no IL-2 secreted by T-cells treated with DC-HIL-Fc (
Because ligation of CD28 on T-cells amplifies TCR-mediated T-cell activation (Linsley and Ledbetter, 1993), the inventors questioned whether CD28 co-stimulation can rescue DC-HIL-induced inhibition (
The inventors also examined whether DC-HIL exerts inhibitory effects on previously activated T-cells (
Binding of DC-HIL to T-cells induces cell-cycle arrest. Since PD-1-mediated signals prevented anti-CD3 Ab-treated T-cells from entering the cell cycle (Latchman et al., 2001; Freeman et al., 2000), the inventors questioned whether inhibition of T-cell activation by DC-HIL-Fc is similarly achieved. CD4+ T-cells were cultured in wells coated with anti-CD3 Ab plus DC-HIL-Fc or control hIgG; at different time points thereafter, the T-cells were assayed for asynchronous cell division using CFSE labeling and FACS analysis (
Soluble DC-HIL enhances T-cell activation triggered by APCs. The inventors next examined the effect of soluble DC-HIL-Fc on the MLR, in which alloreactive T-cells are activated by APCs. In this MLR, spleen cells from C57BL/6 mice served as stimulators, CD4+ T-cells from BALB/c mice served as responders, and the proliferative capacity of responder cells was measured by 3H-thymidine incorporation (
Because the enhancing effect of soluble DC-HIL on the MLR contrasted with the inhibitory effect of immobilized DC-HIL-Fc on anti-CD3 Ab-induced T-cell proliferation (
To more rigorously examine the effects of soluble DC-HIL-Fc on T-cell activation, the inventors used an in vitro antigen presentation assay in which BM-DCs were pulsed with OVA peptide (OVA323-339) (Demotz et al., 1993) and allowed to activate CD4+ T-cells prepared from unprimed BALB/cTac-TgN(DO11.10)-Rag2tm1 mice. Different doses of DCHIL-Fc or control hIgG were added to the assay, and T-cell activation was measured by IL-2 production and frequency of CD69+/CD4+ cells (
DCs with knockdown DC-HIL display enhanced immunostimulatory capacity. To evaluate the function of DC-associated DC-HIL, the inventors prepared DCs with genetically modified (knockdown) expression of DC-HIL and examined immunostimulatory capacity. Transfection of DC-HIL siRNA inhibited most of the DC-HIL protein expression in DCs when compared with DCs with control siRNA (
In vivo injection of soluble DC-HIL augments CH responses. To ascertain the biological significance of the DC-HIL/DC-HIL-L pathway, the inventors examined the effects of soluble DC-HIL on CH, an experimental model of delayed-type, T-cell-mediated skin inflammation. BALB/c mice were sensitized by topical application of the hapten Ox at a dose of 2% on abdominal skin (day 0) and then challenged/elicited by painting ears with 1% Ox (day 6) (
By contrast, mice injected with DC-HIL-Fc just before and after hapten elicitation displayed significantly greater ear swelling that persisted longer (up to 6 days after elicitation), compared to those of control mice (
To determine the activation status of T-cells in Ox-sensitized mice injected with DC-HIL-Fc, the inventors compared draining LNs and LN cells of DC-HIL-Fc-treated versus control mice 2 days after Ox challenge, which was the time of greatest ear swelling. LNs of DC-HIL-treated mice were 3 times larger than hIgG-treated mice, contained 3 times the number of cells (
PKD domain is required for the inhibitory function of DC-HIL on T-cell activation. The inventors reported previously that the extracellular domain of DC-HIL contains an RGD motif required for integrin-mediated cell adhesion; an Ig-like polycystic kidney disease (PKD) domain (Bycroft et al., 1999), and a proline-rich region (PRR) involved in protein-protein interactions (Kay et al., 2000). To determine whether some or all are required for the inhibitory function of DC-HIL on T-cell activation, the inventors created extracellular domains of mutant DC-HIL lacking PKD or PRR, or containing RAA (instead of RGD), and fused these mutants to the IgG-Fc (
RAA and PRR mutants bound to T-cells as efficiently as the wild-type, whereas the PKD mutant failed to bind to T-cells (correlating with results of inhibitory function). These findings indicate that the Ig-like PKD domain is required for binding of DC-HIL to T-cells and for its inhibitory function. By contrast, neither proline-mediated interaction nor RGD-dependenT-cell adhesion appears necessary for DC-HIL's inhibitory function.
Subtractive cDNA cloning of mouse XS52 DCs minus J774 macrophages (Shikano et al., 2001) led to the inventors' discovery of DC-HIL, also known as human nmb glycoprotein or gpnmb (Anderson et al., 2002; Weterman et al., 1995) and rat osteoactivin (Safadi et al., 2001). The extracellular domain of DC-HIL contains a putative heparin-binding site (Cardin and Weintraub, 1989), many N-glycosylation sites, an RGD cell-adhesion motif (Ruoslahti, 1996), a PRR (involved in O-glycosylation (Wilson et al., 1991) and/or protein-protein interactions (Kay et al., 2000)), and an Ig-like PKD domain conserved among 14 repeats in the extracellular region of the PKD-susceptible gene product, product, polycystin-1 (Bycroft et al., 1999; Ponting et al., 1999). Moreover, the inventors showed that DC-HIL is highly N-glycosylated, that it recognizes heparan sulfate (especially on small-vessel endothelial cells [SVECs]) (Shikano et al., 2001) and that its RGD motif is responsible for integrin-mediated cell adhesion (Shikano et al., 2001). The have also uncovered a new function for DC-HIL, as a potent inhibitor of TCR-induced T-cell activation for both primary and secondary responses.
Compared with known pairs of inhibitory regulators of T-cell activation, DC-HIL and its putative ligand (DC-HIL-L) best resemble of PD-L1/PDL2 and its ligand, PD-1. For example, unlike BTLA8 or Tim-3 (Sabatos et al., 2003; Sanchez-Fueyo et al., 2003) whose expressions are restricted strictly to leukocytes, DC-HIL or PD-L1/PD-L2 expression can be induced in non-leukocytes by proinflammatory stimuli like IFN-γ (Freeman et al., 2000). Moreover, in contrast to B7-1/B7-2, whose T-cell ligand CD28 is present on resting T-cells, the T-cell ligand of DC-HIL or of PD-L1/PD-L2 is not expressed constitutively, requiring activation of the T-cells for expression. Engagement of DC-HIL or of PD-L1/PD-L2 with their respective T-cell ligand attenuates T-cell activation, suppresses IL-2 secretion, and arrests T-cell proliferation, all of which can be rescued by costimulation of CD28. By contrast, interference with binding of DC-HIL to DC-HIL-L or of PD-L1/PD-L2 to PD-139 leads to enhanced T-cell responses in MLR and Ag-specific reactions in vitro and in CH in vivo. This antagonism by soluble DC-HIL is not unusual, since other Fc-tagged recombinant proteins have been used to block the endogenous functions of CD200, TREM-1, PD-1, DIgR2, and other receptors, respectively (Brown et al., 2003; Gorczynski et al., 2004; Gibot et al., 2004; Shi et al., 2006).
As cited previously, all known pairs of T-cell inhibitory regulators on APCs and their ligands on T-cells (including CD80 and CD86, which bind to CTLA-4 (Krummel and Allison, 1995; Tsushima et al., 2003); and PD-L1/PD-L2, which bind to PD-1 (Latchman et al., 2001; Dong et al., 1999); possess Ig domains that allow their categorization as members of the B7 receptor superfamily. Indeed, the Ig domains are responsible for ligation of each pair. DC-HIL differs from these inhibitors in not possessing an Ig domain typical of the B7 receptor family, instead containing a PKD domain that can fold into an Ig-like tertiary structure critical to its binding and inhibitory functions.
An important task ahead is the identification of the DC-HIL-L on activated T-cells. Given the previous finding that DC-HIL recognizes heparin sulfate on endothelial cells (Shikano et al., 2001), the inventors speculate that heparan sulfate is involved in the binding of DC-HIL to DC-HIL-L. However, heparan sulfate alone is not likely to be the complete ligand since the PKD-deficient DC-HIL mutant the inventors tested bound heparan sulfate but not activated T-cells. Rather, the inventors hypothesize the putative ligand to bear heparan sulfate plus a peptide with affinity for the PKD domain.
Mice. Female BALB/c and C57BL/6 (5-8 wk old) mice were purchased from HarLan (Indianapolis, Ind.) and BALB/cTac-TgN(DO11.10)-Rag2tm1 (or DO11.10) mice from Taconic (Hudson, N.Y.). Following National Institutes of Health guidelines, mice were housed and cared for in the pathogen-free facility, and subjected to experimental procedures approved by Institutional Animal Care Use Center at The University of Texas Southwestern Medical Center, Dallas, Tex.
Construction of plasmid vectors. The previously constructed pSTB-DC-HIL-Fc, which encodes the extracellular domain of DC-HIL fused to the Fc portion of human IgG1. A plasmid pSTB-SD4-Fc was constructed by replacing the extracellular domain with that of SD-4 obtained by RT-PCR. The V5 epitope sequence was inserted just after the leader sequence of the SD-4 encoding sequence (V5-SD4) and introduced into a lentiviral vector plasmid, pHR-SIN-CSGW (GFP)-Ub-Em (gift from Y. Ikeda, Mayo Clinic, Rochester, Minn.). This recombinant lentivirus co-expresses Emerald GFP and V5-SD4. Infectious particles were prepared and their titration performed according to established protocols.
Generation of DO11.10 expressing V5-SD4. AT-cell hybridoma DO11.10 line (provided by J. Kappler and P. Marrack, National Jewish Medical and Research Center, Denver, Colo.) was infected with V5-SD4 lentiviruses at a multiplication of infection (MOI) of 10. Two days after infection, GFP-positive cells were enriched 3 times by flow cytometric sorting.
T-cell proliferation assays. To assay effects of heparin on DC-HIL-mediated inhibition, DC-HIL-Fc or control Ig (5 μg/ml) was incubated with heparin at increasing concentrations at room temperature for 30 min, followed by coating ELISA wells (in triplicate) that were precoated with anti-CD3 Ab (0.01-0.3 μg/ml). Purified CD4+ T-cells (2×105/well) were added to the coated wells and cultured for 2 d. After pulsing with 3H-thymidine (1 μCi/well) for the last 20-22 h of the culture period, cells were collected and evaluated for 3H-radioactivity. Culture supernatant was stored at −85° C. until needed for IL-2 assay using mouse IL-2 ELISA kit (BD Pharmingen).
Effects of anti-SD-4 Ab on T-cell activation were examined as follows: CD4+ T-cells (2×105/well) were treated with biotinylated anti-CD3 Ab at varying concentrations plus biotinylated anti-SD-4 Ab or biotinylated control IgG (10 μg/ml) on ice for 30 min. After adding anti-biotin microbeads (1 μl, Miltenyi Biotec), treated T-cells were incubated in the 96-well-plate and examined for proliferation as described above. For V5-SD4-DO11.10 T-cells, cells (3×104/well) were cultured in 96-wells coated with DC-HIL-Fc (or anti-SD-4 Ab) and anti-CD3 Ab, followed by IL-2 assay.
Expression of SD. Expression of SD was determined by RT-PCR (for mRNA) and flow cytometry (for surface expression). Total RNA was extracted from freshly isolated (or resting) CD4+ or CD8+ T-cells or from T-cells activated 3 d after treatment with immobilized anti-CD3 Ab, and then subjected to RT-PCR analysis using primers for SDs and β-actin as described previously. Primers for SD-1: 5′-CCCTCCCGCAAATTGTGGCTGTAA-3′ (5′-primer) (SEQ ID NO:10) and 5′-CCCCGTGCGGATGAGATGTGAC-3′ (3′-primer) (SEQ ID NO:11); primers for SD-2: 5′-CCGGGGCGCAGGGAGAA-3′ (SEQ ID NO: 12) and 5′-TTTGGGGGAAGCAGCACTA-3′ (SEQ ID NO:13); primers for SD-3: 5′-CTTGGACACAGAGGCCCCGACACC-3′ (SEQ ID NO:14) and 5′-CGCCCACCACCCCACCCACGAT-3′ (SEQ ID NO:15); and primers for SD-4: 5′-CCTCCCCGACGACGAAGATGC-3′ (SEQ ID NO:16) and 5′-AACGCCCGCCACCCACAAC-3′ (SEQ ID NO:17). The inventors also examined mRNA expression of all members of the glypican family using primers reported previously.
At different time points after activation of CD4+ or CD8+ T-cells with immobilized anti-CD3 Ab, T-cells were pretreated with Fc blocker and stained with biotinylated anti-SD-4 Ab or biotinylated isotypic control IgG (5 μg/ml) plus PE-streptavidin. Cells were also stained with Ab raised against SD-1 (eBioscience), SD-2, and SD-3 (Santa Cruz Biotechnology, Santa Cruz, Calif.). Finally, PE-labeling intensity was measured by FACS.
Immunoblotting and immunoprecipitation Whole cell extracts were prepared from resting or activated T-cells and subjected to SDS-PAGE/Western blotting using anti-SD-1 or anti-SD-4 Ab (each 5 μg/ml) as described previously. For immunoprecipitation, the protein extracts were incubated with DC-HIL-Fc or control Ig (5 μg/ml) for 3 h at 4EC. Resulting immunocomplexes were precipitated with Protein A-agarose (50 μl of 50% slurry) overnight at 4° C., and washed extensively with PBS. The complexes were dissociated by boiling and then analyzed for SD-4 expression by Western blotting using rat anti-SD-4 Ab (5 μg/ml) and HRP-anti-rat IgG (1:10,000 dilution).
Heparin antagonizes DC-HIL function. Having shown that DC-HIL binds to heparin/heparan sulfated polysaccharides, the inventors considered a role for these polysaccharides in the interaction between DC-HIL and activated T-cells. Pretreatment of DC-HIL-Fc with heparin blocked binding of this soluble receptor to activated T-cells in a dose-dependent fashion (
Syndecan-4 on activated T-cells is a ligand of DC-HIL. Because the syndecan (SD) and glypican families of transmembrane heparin/heparan sulfate proteoglycans (HSPG) are major sources of cell surface-heparan sulfate, the inventors questioned whether these molecules are involved in binding of DC-HIL to T-cells. The inventors first examined expression by resting versus activated CD4+ or CD8+ T-cells of all known SDs and glypicans. At the mRNA level, all 4 known SDs were expressed by resting and activated T-cells at differing levels, but only SD-4 expression was upregulated by T-cell activation (
To determine whether DC-HIL binds directly to SD-4 on activated T-cells, the inventors extracted proteins from activated CD4+ T-cells, immunoprecipitated these with DC-HIL-Fc or control Ig, and analyzed precipitants by Western blotting using anti-SD-4 Ab (
Engagement of SD-4 leads to inhibition of T-cell activation. The inventors examined the function of SD-4 on T-cells again using transfected DO11.10 T-cells. V5-SD4-expressing DO11.10 T-cells (V5-SD4-DO) or those expressing GFP (GFP-DO) were treated with immobilized anti-CD3 Ab in the presence/absence of DC-HIL-Fc, followed by measurement of IL-2 production (
The inventors also examined the inhibitory function of SD-4 on CD4+ T-cells (
Soluble SD-4 enhances T-cell activation. The inventors next compared effects of SD4-Fc and DC-HIL-Fc on activation of alloreactive T-cells (mixed lymphocyte reaction or MLR), in which added Fc-fusion proteins were used to block endogenous binding of DC-HIL on APC with SD-4 on T-cells (
T-cells with knocked-down SD-4 display enhanced responses to DC. To better study the role of SD-4 in regulating T-cell activation, the inventors knocked-down SD-4 expression on splenic CD4+ T-cells and examined their response to antigen presentation by DC. The inventors first determined the efficacy of siRNA's ability to block SD-4 expression in COS-1 cells co-transfected with SD-4 gene and SD-4-targeting SC-siRNA or shuffled control siRNA (Sf-siRNA), using Western blotting for protein expression of SD-4 transgene or endogenous β-actin (as control) (
Blockade of endogenous SD-4 augments contact hypersensitivity (CH). To evaluate SD-4 function in vivo, the inventors employed CH, an experimental model of delayed-type, T-cell-mediated, skin inflammation. Mice were sensitized by topical application of oxazolone (Ox) on abdominal skin and challenged by painting ear skin with Ox (
Histological examination confirmed enhanced ear swelling and larger numbers of infiltrating leukocytes in mice treated with Ox and anti-SD-4 Ab (
Expression of SD-4 on LN T-cells. The time-dependent effect of anti-SD-4 Ab in mice (
These results demonstrate that SD-4 is the ligand through which DC-HIL inhibits T-cell activation. Among known co-inhibitory receptors, SD-4 resembles PD-1 in that expression requires TCR activation and appears during a later phase of T-cell activation (e.g., elicitation of CH). Unlike all known co-inhibitory receptors including PD-1 that bind their counter-receptors via protein-protein interaction, binding of SD-4 to DC-HIL appears to require involvement of heparin/heparan sulfate residues.
Because binding of DC-HIL to activated T-cells involves its Ig-like PKD domain and because DC-HIL does not bind to CD44, another HSPG expressed on resting and activated T-cells (data not shown), the inventors hypothesize that DC-HIL/SD-4 binding requires simultaneous recognition of heparin/heparan sulfate and of a peptide epitope of SD-4. This circumstance may resemble selectins, which bind the carbohydrate moiety, sialyl-Lewis×(SLex), and a peptide sequence on the backbone of its ligands.
Because endothelial cells and B cells consitutively express SD-4, it is possible such cells are also stimulated by DC-HIL. The inventors have shown DC-HIL to bind endothelial cells in an heparin-dependent fashion. By contrast, B cells do not bind DC-HIL (data not shown); reflecting a discordance between expression and binding activity that may be due to diverse heparan sulfate structures expressed by disparate cells, in turn corresponding to different binding activities. DC-HIL may recognize a unique heparan sulfate structure on SD-4 synthesized by activated T-cells, similar to T-cell-specific glycosylation again exhibited by the selectin ligands.
The enhancing effect of anti-SD-4 Ab or soluble recombinant SD4-Fc on CH indicates interference with endogenous binding of DC-HIL to SD-4. A good question is whether such enhancement is due entirely to blockade of DC-HIL/SD-4 binding since SD-4 participates in leukocyte rolling and migration. If SD-4 antagonists block the latter processes, these experiments should have produced down-regulated CH instead. That these outcomes were the reverse indicate that the primary target of the inventors' interventions is endogenous binding of DC-HIL to SD-4, and thus modulators of DC-HIL and/or SD-4 may be used to treat T-cell-mediated diseases.
BTLA, CTLA-4, and PD-1 possess a typical ITIM, an ITIM-like motif, and an immunoreceptor tyrosine-based switch motif (ITSM), respectively, that can activate the tyrosine phosphatases, SHP-1 and SHP-2, responsible for mediating negative T-cell effector function. By contrast, SD-4 does not contain any of these inhibitory motifs. Rather, ligated SD-4 is known to induce serine and tyrosine autophosphorylation, which may regulate intracellular interactions of SD-4 with other cell surface receptors. Although the inventors have no direct evidence connecting these events to the SHP-1/SHP-2 pathway in T-cells, activated SD-4 has been shown to complex with other intracellular proteins like syntenin that can bind directly to the intracellular domain of CD148, a membrane protein tyrosine phosphatase (PTPη) known to inhibit CD3-mediated T-cell activation. Moreover, SD-4 can regulate activation of PKCα, that in turn can modulate phosphorylation of CD148. Finally, the inventors speculate that ligated SD-4 on T-cells partners with CD148 to activate tyrosine phosphatases, which can lead to neutralization of TCR-induced activation signals.
Plasmids and preparation of DC-HIL-Fc. A plasmid vector encoding SD-1 or SD-4 was constructed by inserting the full-length cDNA into pcDNA3.1 vector (Invitrogen, Carlsbad, Calif.) using HindIII and XbaI restriction enzyme sites. The inventors also constructed pcDNA-SD-4-V5 by attaching the V5 epitope sequence (Sato et al., 2006) to the 3′-end of the SD-4 cDNA insert. A plasmid vector encoding the extracellular domain of human DC-HIL fused to the Fc portion of mouse IgG2a (DC-HIL-Fc) was constructed by replacing the extracellular domain of mouse homolog in pSTB-DC-HIL-Fc (Chung et al., 2007a) with the corresponding of human homolog. Fc proteins were produced in COS-1 cells and purified using protein-A-agarose (Shikano et al., 2001).
Ab and immunofluorescence labeling. Mouse anti-human DC-HIL mAb was generated by immunizing BALB/c mice with human DC-HIL-Fc at 2 week-intervals. A week after the last immunization, spleen cells from mice with highest Ab titer were fused with the F/0 myeloma cell line. One 3D5 IgG1 clone was purified from mouse ascites using protein-A affinity chromatography.
mAb against CD1a (HI149), CD3 (UCHT1), CD14 (61D3), CD28 (CD28.2), CD69 (FN50), CD80 (2D10.4), CD86 (FUN-1), HLA-DR (LN3), PD-1 (MIH4) and SD-1 (DL-101) were purchased from eBiosciences (San Diego, Calif.); Ab against SD-2 (M-140), SD-3 (M-300), SD-4 (H-140 and 5G9) and p-SD-4 (Ser 179) from Santa Cruz Biotechnology (Santa Cruz, Calif.); anti-V5 Ab from SeroTec (Raleigh, N.C.); and secondary Ab from Jackson ImmunoResearch (West Grove, Pa.). For flow cytometric analysis, cells (5−10×105) were incubated with 5-10 μg/ml primary Ab for 30 min on ice, followed by addition of secondary Ab (2.5 μg/ml). After washing, cell-bound fluorescence was analyzed by FACSCalibur (BD Biosciences, San Jose, Calif.).
Binding of DC-HIL to T cells. After providing informed consent, blood was collected from healthy donors and PBMC isolated by Ficoll-Hypaque gradient centrifugation. Following manufacture's recommendations, CD4+ or CD8+ T cells (1×106) were isolated from PBMC using respective isolation kits (Myltenyi Biotec, Auburn, Calif.) and cultured with concanavalin A (Con A, 2 μg/ml), phytohemagglutinin (PHA, 5 μg/ml), phorbol 12-myristate 13-acetate (PMA, 5 ng/ml) plus ionomycin (250 ng/ml) (all from Sigma, St. Louis, Mo.), or anti-CD3 Ab (2 μg/ml) plus anti-CD28 Ab (0.5 μg/ml). At indicated time points after culturing, activated T cells (1×106) were pretreated with 5 μg/ml human IgG on ice for 30 min to block Fc-binding activity on T cells prior to incubation with 10 μg/ml DC-HIL-Fc or control Ig plus 2.5 μg/ml PE-anti-mouse IgG F(ab′)2.
T cell activation. Freshly-isolated CD4+ or CD8+ T cells (2×105/well) were cultured for 2 d in ELISA wells (in triplicate) precoated with indicated doses of anti-CD3 Ab and together with Fc proteins or Ab. CD4+ T cells were also incubated in ELISA wells precoated with DC-HIL-Fc (10 μg/ml)/anti-CD3 Ab (0.3 μg/ml) and increasing doses of anti-CD28 Ab. T cell activation was measured by 3H-thymidine incorporation (pulsing with 1 μCi/well in the last 20 h of the culture period) or by IL-2, TNF-α, and IFN-γ production using ELISA kit (eBioscience).
For the mixed lymphocyte reaction (MLR), PBMC were γ-irradiated (2,000 Gy) and mixed with CD4+ T cells (2×105/well) in 96-microwell-plate (in triplicate) at indicated ratios (PBMC vs. T cells) in the absence/presence of DC-HIL-Fc or control Ig (5 μg/ml) for 4 d (Chung et al., 2007a).
For allostimulatory assays, CD14+ monocytes were isolated from PBMC of a healthy donor using anti-CD14 Ab magnetic beads (Miltenyi Biotec) and cultured with/without TGF-β (10 ng/ml) for 2 d. Cells were then harvested and cocultured in 96-well plates with CD4+ T cells (from a different donor) at the indicated cell ratio for 6 d [35]. In some experiments, the cell mixture was cultured in the presence of DC-HIL-Fc or control Ig (20 μg/ml).
Cell cycle analysis. Cell cycles of CD4+ T cells treated with anti-CD3 Ab (0.3 μg/ml) and Fc protein (5 μg/ml) were analyzed using FITC-BrdU flow kit (BD Pharmingen) (Chung et al., 2007a).
RT-PCR. Total RNA was extracted from leukocytes, reverse-transcribed to cDNA, and PCR-amplified using primers for DC-HIL (5′-primer, 5′-GTGGAGCTTCGGGGATAATACT-3′ (SEQ ID NO:18); and 3′-primer, 5′-CTACTCAGCTCCAGGGGGTTGT-3′ (SEQ ID NO:19)), SD members, and GAPDH (Wegrowski et al., 2006).
Stable transfectants. Jurkat T cell E6-1 line (1×106 cells) was transfected with an empty vector, pcDNA-SD-1 or pcDNA-SD-4 (2 μg), using the Amaxa Nucleofector System (Gaithersburg, Md.). 2 d post-transfection, cells were allowed to grow in the selection media containing 600 μg/ml G418 (Invitrogen) for 2 weeks. Jurkat cells expressing SD-1 or SD-4 were then enriched by FACS-sorting until more than 90% of cells are positive for surface expression. Control Jurkat cells (transfected with vector alone) were established by culturing with G418 more than 3 weeks. For binding of DC-HIL, Jurkat cells were activated with Con A (2 μg/ml) for 2 d prior to the binding assay. For experiments examining involvement of heparin, binding of DC-HIL-Fc (10 μg/ml) to Con A-activated Jurkat cells was performed in the presence of heparin, or Jurkat cells (5×105) were treated with heparinase I (0.1 U/ml) and III (0.2 U/ml) (Sigma) at 37° C. for 2 h before binding assay. To examine specificity of SD-4 binding to DC-HIL, activated Jurkat cells (5×105) were pretreated with anti-SD-4 or control IgG at indicated concentrations for 30 min at room temperature before binding. For activation assay, Jurkat T cells (3×104/well) were cultured for 2 d in ELISA wells (in triplicate) precoated with indicated doses of anti-CD3 Ab and DC-HIL-Fc or control Ig (each 10 μg/ml).
Immunoprecipitation and tyrosine phosphorylation assay. Whole cell extracts (1×107 cells/ml) were prepared from activated CD4+ T cells (Sato et al., 2006) and incubated with DC-HIL-Fc or control Ig (5 μg) for 3 h at 4° C. Resulting immunocomplexes were precipitated with protein A-agarose (50 μl of 50% slurry) overnight at 4° C., and washed extensively with PBS. To assay phosphorylation of SD-4, Jurkat cells were transfected with pcDNA-SD4-V5 and immediately cultured with Con A (2 μg/ml) for 2 d. After culturing for another 1 d without Con A, cells (2×106) were cultured in a petri dish precoated with DC-HIL-Fc (20 μg/ml) at 37° C. for different time period. Whole cell extracts were prepared, incubated with anti-V5 Ab (2 μg/ml) at 4° C. for 3 h, and then precipitated with 50 μl of 50% slurry protein G-agarose (Pierce, Rockford, Ill.) by overnight incubation. After washing, agarose-beads were then left untreated (to detect phosphoserine) or treated (to detect phosphotyrosine) with a mixture of heparinase I (0.1 U/ml) and III (0.2 U/ml), and chondoroitinase ABC (Sigma) (Charnaux et al., 2005) prior to immunoblotting using anti-phosphorylated Ser-179 of SD-4 or biotinylated anti-phospho-tyrosine (0.5 μg/ml) (4G10, Upstate, Lake Placid, N.Y.) and HRP-streptavidin (1:10,000). Blotted membranes were also stripped and re-probed with mouse anti-SD-4 Ab (1 μg/ml) and HRP-anti-mouse IgG (1:10,000).
Culture of monocytes and DC. PBMC were cultured in 24-well plates (1×106 cells/well in triplicate) for 2 d with 10% FCS-RPMI supplemented with IL-2 (100 U/ml), IL-4, IL-6, IL-10, IL-11 (each at 10 ng/ml), IL-13 (100 U/ml), IFN-γ (200 U/ml), TNF-α, TGF-β (each at 10 ng/ml) (all from PeproTech Inc, Rocky Hill, N.J.), or LPS (1 μg/ml) (Sigma). For generation of monocyte-derived immature DC [37], PBMC were seeded onto tissue culture flasks. After culturing for 1 h, non-adherent cells were washed off and remaining adherent cells cultured in DC culture media (10% FCS-RPMI supplemented with 800 U/ml GM-CSF and 250 U/ml IL-4) for 6 d. Resulting non-adherent cells were used as immature DC. For induction of maturation, DC harvested from the day 4 culture of adherent PBMC were cultured in 24-well plates (1×106/well) for another 2 d with DC culture media added with IL-1β (10 ng/ml), TNF-α (10 ng/ml), and prostaglandin E2 (PGE2, 1 μg/ml). For LC, epidermal cells were prepared from foreskin and LC identified as CD 1a+ epidermal cells (Takao et al., 2002).
Knock-down of DC-HIL expression. DC-HIL-targeted siRNA (Cat#sc-60721) and control siRNA (Cat#sc-37007, Santa Cruz Biotechnology) (each 2 μg) was treated with 15 μl of Metafetene™Pro (Biotex, Martinsried, Germany) in the serum-free RPMI for 30 min and then added to 1×106 CD 14+ cells. After culture for 4 h at 37° C., cells were washed and cultured in 10% FCS-RPMI for another 2 d before experiments.
Statistical analysis. Results are presented as means±s.d. of n independent experiments. Significance was assessed using the Student's t test at p<0.05.
Binding of DC-HIL to human T cells attenuates responses to anti-CD3 Ab. To examine the function of human DC-HIL, the inventors created a soluble DC-HIL receptor (DC-HIL-Fc) consisting of the extracellular domain fused to the Fc portion of mouse IgG, and examined binding to T cells (
The inventors next examined effects of DC-HIL on cytokine production. Co-treatment of T cells with DC-HIL markedly inhibited production of IL-2 and TNF-α induced by anti-CD3 Ab (
SD-4 is the T cell ligand of DC-HIL Having identified SD-4 as the ligand of DC-HIL in mice (Chung et al., 2007a), the inventors wished to ascertain a similar circumstance in humans. The inventors first examined mRNA and protein expression of the 4 known syndecans (SDs) by RT-PCR and flow cytometry, respectively. By RT-PCR analysis (
The inventors next examined binding of DC-HIL to SD-4 and SD-1. Protein extracts from activated T cells were treated with DC-HIL-Fc or control Ig, and immunoprecipitates blotted with anti-SD-1, anti-SD-4 Ab, or control IgG (
Taking advantage of the Jurkat T cell line naturally devoid of endogenous SD-1 and SD-4, the inventors transfected the SD-1 or SD-4 gene into these cells, and confirmed high cell surface expression of either gene in the respectively engineered cells (
Binding of DC-HIL to SD-4 induces serine and tyrosine autophosphorylation. Because SD-4 autophosphorylates its intracellular tyrosine and serine residues following binding to ligands (Horowitz and Simons, 1998), the inventors ascertained a similar scenario for binding to DC-HIL. Jurkat cells were transfected transiently with a gene for V5-tagged SD-4 (SD4-V5) and stimulated with Con A before incubation with immobilized DC-HIL-Fc. At different time points after incubation, Jurkat cells were assayed for phosphorylation using immunoprecipitation of SD4-V5 with anti-V5 Ab, followed by immunoblotting with Ab to phosphorylated serine of SD-4 (
Engagement of SD-4 attenuates anti-CD3 response of T cells. To study the function of SD-4 on T cells, Jurkat transfectants were incubated with co-immobilized anti-CD3 Ab (different doses) and DC-HIL-Fc or control Ig (a constant dose). Activation was measured by IL-2 production (
To evaluate the effect of anti-SD-4 Ab on the anti-CD3 response of CD4+ T cells, cells were cultured in microculture wells precoated with anti-SD-4, anti-SD-1, DC-HIL-Fc, or control Ig (
Among blood leukocytes, CD14+ monocytes display highest DC-HIL expression. Although a previous study showed high mRNA expression of DC-HIL by the human histiocytic lymphoma line U937 (Weterman et al., 1995), DC-HIL expression by normal leukocytes has not been examined. Human PBMC were sorted into CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes. Total RNA from these cells was examined by RT-PCR for DC-HIL or GAPDH mRNA expression (
By flow cytometry, 9% of PBMC stained with 3D5 anti-DC-HIL mAb, and 70% of these DC-HIL+ cells co-expressed the CD 14 marker (
DC-HIL is expressed preferentially by immature DC. To study correlation of DC-HIL expression levels with maturation status of APC, the inventors assayed the expression on immature vs. mature DC. Epidermal LC are known to represent the immature form of DC, and these cells were distinguished from other epidermal cells by expression of CD1a. In fact, Ficoll-enriched epidermal cell suspension contained 10% of CD1a+ LC, most of which expressed DC-HIL at very high levels (mean fluorescent intensity, MFI, of 524) (
DC-HIL expression correlates inversely with allostimulatory capacity of CD14+ monocytes. The inventors next examined a role for DC-HIL in the mixed lymphocyte reaction (MLR), in which y-irradiated PBMC from one donor are mixed with purified CD4+ T cells from a different donor; T cell activation was assayed by 3H-thymidine incorporation. DC-HIL-Fc added to the MLR blocked the endogenous function of DC-HIL and augmented T cell proliferation 2-fold (
Because CD14+ monocytes possess APC capacity (Bhardwaj and Colston, 1988) and because TGF-β treatment amplified DC-HIL expression (
These findings document the T cell inhibitory function of the DC-HIL/SD-4 pathway in humans. Human DC-HIL binds to SD-4 on activated (but not resting) T cells and this binding strongly blocks anti-CD3 responses of CD4+ and CD8+ T cells, including production of cytokines (IL-2, TNF-α, and IFN-γ) and entry into the S-phase. The inventors also showed that binding of DC-HIL to T cells transduces SD-4-dependent signaling. DC-HIL is expressed constitutively at high levels by CD14+ monocytes and immature DC (particularly epidermal LC) and this expression is regulated by TGF-β. The inventors also found DC-HIL to bind heparinase-sensitive structures on SD-4 (but not SD-1). Finally, SD-4 is a more potent inhibitor of the anti-CD3 Ab response than CTLA-4 and PD-1.
Whereas DC-HIL in mice is expressed highest by DC and macrophages and this expression is unaffected by treatment with cytokines or LPS (unpublished data), DC-HIL in human blood leukocytes is expressed highest by CD 14+ moncoytes and this expression is upregulated by various cytokines especially TGF-β. LPS, which is an activator of CD14+ cells, had almost no effect on DC-HIL expression, which is a feature in stark contrast with PD-L1, whose expression is increased markedly by the stimulus [22]. Note that TGF-β is the critical cytokine responsible for differentiation of CD14+ monocytes into epidermal LC (Geissmann et al., 1998), and the inventors have shown human LC to constitutively express DC-HIL at very high levels. Moreover, TGF-β can convert DC from immunostimulatory to tolerogenic phenotype, and it has been implicated as key to the ability of CD8+ regulatory T cells to suppress experimental autoimmune encephalomyelitis in mice (Rutella et al., 2006). Indeed, regulatory T cells produce TGF-β, which inhibits APC capacity of monocytes (Ersquerre et al., 2008), and malignant cells can secrete large amounts of TGF-β touted to play a role in suppressing anti-tumor immune responses (Teicher, 2007). Thus, the upregulated expression of DC-HIL on APC and the inhibitory effects of TGF-β may be interrelated.
Very recently, human CD4+ T cells were shown to express SD-2 and SD-4 upon activation, with both SD molecules capable of negatively regulating T cell activation induced by anti-CD3 Ab (Teixe et al., 2008). The inventors showed SD-4 expressed by activated T cells and anti-SD-4 Ab to inhibit the anti-CD3 Ab response of T cells. They were also able to show SD-1 on T cells, but not SD-2. These inconsistencies may be due to differences in PCR (real time vs. conventional PCR) and staining methods (intracellular vs. surface staining).
Unlike other inhibitory molecules (PD-1/PD-L1 and BTLA/HVEM) that bind their ligands via protein-protein interaction, DC-HIL appears to recognize SD-4 through non-peptide structures (heparin/heparan sulfate (HS) side chains), consistent with abrogation of its binding by heparinase treatment. Although SD-4 also bears chondroitin sulfate chains, these are not likely ligands for DC-HIL since chondroitin sulfate failed to block binding of DC-HIL to T cells (unpublished data). On the other hand, HS may not be the sole moiety responsible for DC-HIL/SD-4 binding since DC-HIL does not bind other HS-bearing proteins like SD-1, CD44, and glypicans. The inventors postulate DC-HIL to simultaneously recognize HS and a peptide epitope of SD-4. However, this possibility is confused by the observation that DC-HIL does not bind SD-4 engineered on Jurkat cells until after these cells are activated by Con A. Because different cells are known to express diverse HS structures corresponding to disparate binding activities (Esko and Selleck, 2002), the results suggest that DC-HIL recognizes a unique HS structure of SD-4 present on activated T cells, akin to interaction of selectins with ligands (PSGL-1) bearing T cell-specific glycosylation (Vestweber and Blanks, 1999). Regardless, there remains the question of why DC-HIL binds to SD-4 but not SD-1 on activated T cells especially since the inventors have no evidence of differences in HS chains on these molecules.
Autophosphorylation of serine and tyrosine residues on SD-4 following ligation by DC-HIL provides circumstantial evidence that intracellular signaling is responsible for DC-HIL/SD-4 inhibition of TCR-driven T cell activation. Because TCR signaling can be deleted by dephosphorylating molecules, and because CTLA-4, PD-1, and BTLA associate with protein tyrosine phosphatase (PTP) to mediate inhibitory function (Watanabe et al., 2003; Shlapatska et al., 2001; Chemnitz et al., 2004), the inventors postulate that SD-4 signaling involves some linkage with a PTP. However, unlike the other inhibitory molecules, SD-4 lacks an ITIM or other signaling motifs known to recruit the PTP-like SHP-1 and SHP-2 (Plas and Thomas, 1998). Absent direct linkage with PTP, our current investigation has focused on coupling of SD-4 function with a membrane-type PTP known to attenuate TCR signaling (Tangye et al., 1998).
Documenting the DC-HIL/SD-4 pathway to inhibit T cell activation in humans lays the foundation for future immunopharmacologic manipulation that may benefit patients with T cell-driven diseases like T cell lymphomas/leukemias, psoriasis, rheumatoid arthritis and inflammatory bowel disease.
Toxin-conjugated DC-HIL selectively kills SD-4+ T-cells in vitro. The inventors chose saporin (SAP), an extensively-used and potent type I ribosome-inactivating protein as the toxin conjugated to DC-HIL. The inventors next verified that this immunotoxin retains the ability of DC-HIL to bind selectively to activated (but not resting) T-cells (
Infusion of DC-HIL-SAP blocks elicitation of CH. The inventors examined the effects of DC-HIL-SAP on CH to Ox (
Antigen-specific unresponseviness induced by DC-HIL immunotoxin lasts until 3 weeks. To determine the duration of unresponsiveness to oxazolone in mice treated with DC-HIL-immunotoxin, BALB/c mice were sensitized and given intravenous injection of 20 or 40 mM of DC-HIL-SAP or Ig-SAP 3 h prior to challenge on day 6. Mice were kept for 1 week to completely recover to the baseline (no ear swelling) and then rechallenged with the same contact allergen oxazolone (the second challenge,
The inventors also examined whether this unresponse is achieved in an antigen-specific manner (
Shift from Th1 to Th2 response by DC-HIL-SAP. Acute T-cell-mediated inflammatory is caused by hyper Th2 response. In fact, mice immunized with oxazolone induce Th1-response that is denoted by high levels of IFNα production. To gain more insights into the mechanisms underlying blocking of CH elicitation by DC-HIL-SAP, the inventors analyzed balance of Th1 vs. Th2 response (indicated by IL-4 production) in lymph nodes of mice treated with DC-HIL-SAP. Lymph node cells were isolated from mice treated with DC-HIL-SAP or Ig-SAP (or untreated mice) and stimulated by anti-CD3 Ab. Production of IFN-g and IL-4 was measured in the culture supernatant (FIG. 17). DC-HIL-treated lymph node cells produced reduced levels of IFN-g (about 50% reduction), compared to Ig-SAP-treated mice. By contrast, IL-4 level was increased by mice with DC-HIL. This result shows that DC-HIL-SAP converted the Th1 type response to Th2-type, probably transiently. This fits the recent idea that Th2 response counteracts Th1 response.
Cell isolation and culture. The human CTCL cell lines MJ (G11), Hut-78, and HH, obtained from American Type Culture Collection (ATCC, Rockville, Md.), were derived from peripheral blood of patients with MF, SS, and non-MF/SS aggressive CTCL, respectively (Gootenberg et al., 1981; Starkebaum et al., 1991; Popovic et al., 1983). Other T cell leukemia lines (Jurkat and Molt-4) were also obtained from ATCC. Samples of peripheral blood were obtained from 3 healthy donors, 6 SS patients, and MF with different malignancy stage (see Table 2). Blood samples were taken at two different time points from one SS patient. Samples were obtained during routine diagnostic assessments. The institutional review board of MD Anderson Cancer Center approved this study and the participants gave written informed consent. This study was conducted according to the Declaration of Helsinki Principles. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors using CD4 T cell isolation kit (Myltenii, Biotec Auburn) according to the manufacturer's instructions. These normal T cells and the T cell lines were maintained in RPMI 1640 medium (Sigma Chemical Co., St Louis, Mo.) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, Utah).
#Indicates positive cells among total PBMCs.
RT-PCR Analysis. Total RNA (1 μg) isolated from cell lines or normal CD4+ T cells was converted to the cDNA by reverse transcriptase (Life Technologies, Inc., Rockville, Md.) (Ariizumi et al., 1995). An aliquot (typically 5%) was used for PCR amplification (Ariizumi et al., 1995) using the primers: for CD160, 5′-GTTCACCATAAGCCAAGTCACACC-3′ (SEQ ID NO:20) and 5′-TTGCCCCAGCTTATATTTCCACAG-3′ (SEQ ID NO:21); for CTLA-4, 5′-GACCTGGCCCTGCACTCTCCT-3′ (SEQ ID NO:22) and 5′-AAAAACAACCCCGAACTAACTGCT-3′ (SEQ ID NO:23); for BTLA, 5′-ATGCCCTGTGAAATACTGTGCTAA-3′ (SEQ ID NO:24) and 5′-TGCCTGGTGCTTGCTTCTGT-3′ (SEQ ID NO:25); for PD-1, 5′-GGGCCCGGCGCAATGACA-3′ (SEQ ID NO:26) and 5′-GCGGGCGGGGGATGAGGT-3′ (SEQ ID NO:27); or for CD148, 5′-CTCCGCTCCAGCACCTTCTACAAC-3′ (SEQ ID NO:28) and 5′-GCACCGTCAGGGCTCTTCCAGTC-3′ (SEQ ID NO:29). Primers for SD-4 and GAPDH were the same as before (Chung et al., 2007a). Following 30-cycles of amplification, PCR products were separated electrophoretically on 1% agarose gel.
Western blot analysis. Whole cell extracts were prepared from T cell lines by lysis with 0.3% Triton X-100/DPBS for 15 min, followed by centrifugation for 20 min at 10,000×g (Sato et al., 2006b). An aliquot (40 μg) of extract was pretreated with heparinase I (0.1 U/ml) and III (0.2 U/ml) (Sigma) at 37° C. for 2 h before applying to SDS/4-20% gradient PAGE, followed by immunoblotting using anti-SD-4 Ab (μg/ml, eBiosciences). Color was developed by HRP-secondary Ab (1:10,000 dilution, Jackson ImmunoResearch, West Grove, Pa.) for 1 h and ECL plus system (Amersham Pharmacia Biotech, Piscataway, N.Y.).
Ab, Flow cytometry and confocal analysis. Abs used for fluorescent staining of cells include anti-SD-4 (5G9, H-140), anti-CD148 (143-41, H-300, CM, G15), anti-CD4 (RPA-T4), anti-CD3 (UGHT1), and anti-CD69 (FN50); all purchased from eBioscience (San Diego, Calif.). Three independent mAb directed against heparan sulfate (F58-10E4, HepSS-1, and F69-3G10) were obtained from Seikagaku Corporation (Japan). For analysis of surface expression, T cells or PBMCs (1×105) were incubated with primary Ab (1-5 μg/ml) (or control IgG) and labeled fluorescently with 5 μg/ml PE- or FITC-conjugated secondary Ab, Jackson ImmunoResearch Laboratories Inc., West grove, PA). After extensively washing, cells were Fluorescence intensity of stained cells was analyzed by FACS Calibur (BD biosciences). For confocal analysis of CTCL lines, CTCL cells were stained with anti-SD-4 mAb (5 μg/ml) and FITC-anti-mouse IgG (5 μg/ml). After extensive washing, an aliquot was spotted on a slide glass, and fixed with 4% PFA at room temperature for 20 min. Fluorescently stained cells and skin tissues were examined using a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, N.Y.).
DC-HIL binding. CTCL lines or normal CD4+ T cells (day 3 after activation with 1 μg/ml immobilized anti-CD3 Ab) (1×105 cells) were incubated with 10 μg/ml DC-HIL-Fc (fused with the Fc portion of mouse IgG) produced in transfected COS-1 cells as described previously (Chung et al., 2007). For experiments examining involvement of heparin, T cell lines (5×105 cells) were pretreated with heparinase I (0.1 U/ml) and III (0.2 U/ml) (Sigma) at 37° C. for 2 h before binding assay. DC-HIL-Fc-treated cells were then fluorescently stained with PE-anti-mouse IgG (Fab) (5 μg/ml) followed by flowcytometry.
Cell cycle analysis. HH or HUT-78 cells were seeded onto a Petri dish (5×105 cells/dish) and starved for 24 h in serum-free media to synchronize. After harvesting, cells were seed on microculture well (1×105 cells/well) precoated with anti-CD3 Ab (increasing doses) plus DC-HIL-Fc or control Ig (each 10 μg/ml). After culturing for 18 h in the presence of 10% FCS-media, cells were subjected to cell cycle analysis using BrdU labeling and the BrdU flow kit (BD Biosciences), following the manufacturer's recommendations. BrdU incorporation (labeled with FITC) and DNA content (stained by 7-AAD) on a per-cell basis were analyzed by flow-cytometry.
T cell activation assay. HH or HUT-78 cells (without starvation) were cultured in microculture wells (1×105 cells/well) precoated with anti-CD3 Ab (increasing doses) plus DC-HIL-Fc or control Ig (each 10 μg/ml) for 2 d. For HH line, cells were stained with anti-CD69 mAb and assayed by flow cytometry for frequency (%) of CD69+ cells in the culture. For HUT-78 line, culture supernatant was recovered and examined by ELIZA for production of IL-2 using IL-2 ELIZA kit (eBiosciences). To measure proliferation, cells were pulsed with 3H-thymidine (1 μCi/well) for the last 20 h of the culture period.
Anti-CTCL activity. DC-HIL-Fc or mouse IgG (as control) was biotinylated using EZ-link™ NHS-BIOTIN (Pierce, Rockford, Ill.) following manufacturer's recommendations. Normally, one Fc-protein molecule has 1-2 biotin molecules. Biotinilated protein was then conjugated with streptavidin-saporin (Advanced Targeting System, San Diego, Calif.) by mixing together at a molecular ratio (protein:saporin) of 1:1. Growing HH or HUT-78 cells were harvested, washed, seeded to microculture wells (5×104 cells/well), and cultured with 10% FCS-media containing **100 U/ml of IL-2 (eBiosciences) and indicated concentration of saporin conjugates. Cells were pulsed with 3H-thymdine (1 μCi/well) for the last 20 h of the culture period (2 d).
Statistical analysis. Comparison of healthy donors and CTCL patients at frequency of SD-4 expression was made using Student's t test. The degree of linear relationship between two variables was evaluated using the Pearson correlation.
SD-4 is a co-inhibitory receptor expressed constitutively at high levels by CTCL. The inventors found previously that SD-4 is expressed primarily by effector/memory T cells in immunized mice. Because cutaneous T cell lymphoma (CTCL) cells also display effector/memory T cell phenotype, the inventors posited these cells to also express SD-4. The inventors examined mRNA expression of SD-4 in total RNA isolated from T cell lines including three CTCL lines (HH, MJ, and HUT-78) and two of acute T cell lymphoma (ATCL) lines (as control) (
Peripheral blood CD4+ T cells of MF and SS patients express SD-4. The inventors next examined whether expression of SD-4 by CTCL lines is also true for CD4+ T cells from MF and SS patients. PBMCs isolated from 10 patients and 3 healthy donors (Table 2) were assayed by flowcytometry for frequency of CD4+ SD-4+ or of CD4+26− cells (a marker for malignant T cells (Bemengo et al., 2001) (
DC-HIL binds certain types of heparan sulfate expressed by CTCL cells. Since SD-4 is a receptor that binds to DC-HIL, the inventors examined binding of DC-HIL to the surface of CTCL cell lines and other T cells (
The inventors previous studies documented that DC-HIL binds to heparan sulfate-like molecules on SD-4. Thus, they addressed whether DC-HIL binding to CTCL cells is achieved in a similar manner. DC-HIL was pretreated with heparin or HUT-78 cells were pretreated with heparinase (a enzyme that remove heparan sulfate from the cell) prior to binding assay. Addition of heparin (data not shown) or heparinase treatment abrogated binding of DC-HIL to HUT-78 eclls (
TCL-associated SD-4 responds to DC-HIL's inhibitory function by blocking secretion of IL-2 without blocking the entry to cell cycle. The inventors next examined whether SD-4 on CTCL cells is sensitive to DC-HIL's inhibitory function (
Since ligation of DC-HIL to SD-4 also inhibits proliferation of normal CD4+ T cells by blocking the entry to the cell cycle, the inventors assessed effect of DC-HIL binding on proliferation of HUT-78 or HH cells (
Toxin-bearing DC-HIL exhibits anti-CTCL activity. Having shown that DC-HIL binds to CTCL cells, the inventors posited that toxin-bearing DC-HIL kills CTCL cells. DC-HIL-Fc recombinant protein (or control Ig) was conjugated to the toxin saporin, an extensively-used and potent type I ribosome-inactivating protein (Flavell, 1998) and evaluated for efficacy to kill HUT-78 cells in vitro using 3H-thymidine incorporation assay (
CTCL-specific surface phenotypes can be identified, thus providing the ability to detect and monitor CTCL cells among circulating CD4+ T-lymphocytes (Nikolova et al., 2002). Such phenotypes include co-expression of the NK receptor (NKR) p140/KIR3DL2, SC5 inhibitory receptor, or CD26− by CD4+ T-lymphocytes, with the latter widely accepted to distinguish malignant from normal T cells. The inventors documented SD-4 to be expressed by some cells from MF and SS patients (but not from non-CTCL patients) at a level comparable to that found on normal CD4+ T cells activated by anti-CD3 Ab in vitro. Note little to no expression of SD-4 by normal resting CD4+ T cells. CD4+ PBMCs freshly isolated from 11 patients with MF or SS express it at markedly increased levels compared to those from normal donors. Importantly, such expression correlates positively and significantly with the degree of peripheral blood malignancy in MF and SS, as assessed by percentage of CD4+ CD26− PBMCs. Thus, SD-4 expression by CTCL cells should augment precision in identifying susceptible CTCL cells and may contribute to new development of diagnosis for the peripheral blood malignancy in future.
There are a variety of therapeutic modalities available for CTCL patients, one of which is a newly developed Denileukin diftitox (or Ontak), a IL-2 recombinant protein fused with diphtheria toxin. This binds to the high affinity IL-2 receptor (or high CD25) and delivers the toxin into target cells, thus killing them. Recent studies have documented that high CD25 expression is associated with advanced CTCL and that in a phase III study, overall, 30% of the 71 patients with CTCL treated with denileukin diftitox had a response (20% partial responses rate; 10% complete response rate) (Zhang et al., 2008; Horwitz, 2008). Response rate and progression-free survival were superior for patients treated with denileukin diftotox compared with patients receiving placebo. However, there were frequent adverse events including constitutional and gastrointestinal symptoms. A conceptual disadvantage of denileukin diftotox is depletion of all activated T cells including those in the memory pool that are important for protective immunity. Like IL-2 protein, DC-HIL binds to activated effector/memory T cells in PBMCs of normal donors through the ligand, particular types of heparan sulfates on SD-4 expressed by T cells but not B cells nor by other leukocytes (Chung et al., 2007). Unlike IL-2 receptor, SD-4 is expressed by only 25% of activated CD25+ T cells. The inventors' finding that toxin-conjugated DC-HIL exhibits efficient anti-CTCL activity provides the rationale for its therapeutic use in CTCL.
SD-4 belongs to the syndecan family of transmembrane receptors heavily laden with heparin/heparan sulfate (HS) chains consisting of alternating disaccharide residues (glucuronic acid or iduronic acid with glucosamine) (Baciu et al., 2000; Charnaux et al., et al., 2005; Ishiguro et al., 2003). The inventors showed that DC-HIL ligands are likely particular types of HS detected by two different mAb clones (F58-10E4 and Hepss-1) that block DC-HIL binding. Limited information for structures of these mAb epitopes does not allow us to identify the exact structures. During studies about HS, the inventors found a distinct difference in the HS expression between normal and CTCL cells. CTCL cells express SD-4 as high as activated CD4+ T cells of normal donors, whereas the former express these two epitopes at high levels but at no to very low levels by normal cells, consistent with the extent of DC-HIL binding. This aberrant (or abnormally increased) glycosylation is a likely carbohydrate marker to discriminate CTCL cells from normal cells (Ohyama, 2008; Ono and Hakomori, 2004).
In normal T cells, ligation of DC-HIL to SD-4 inhibits production of IL-2 triggered by anti-CD3 Ab, accompanied with blocking of proliferation and the entry of T cells to the cell cycle. In CTCL cells, SD-4 is capable of inhibiting the IL-2 production but incapable of blocking proliferation of HUT-78 cells and the entry to the cell cycle. However, failure of blocking proliferation may be due to very low responsiveness to anti-CD3 Ab stimulation. Since CTCL cells have variability in phenotype and function, other CTCL lines may respond to DC-HIL's inhibitory signal to proliferation. Although the inventors showed that CTCL cells express SD-4 but not other known co-inhibitory receptors (PD-1, BTLA, CD160, and CTLA-4), it does not mean that it is only one inhibitory mechanism for TCR-driven activation. A previous report shows that a newly identified SC5 receptor also is expressed by CTCL cells and that it inhibits anti-CD3 Ab response of a CTCL line (inhibition of IL-2 production and proliferation) (Nikolova et al., 2001; Nikolova et al., 2002). It may be likely that CTCL cells retain several inhibitory mechanisms that normally limit activation of normal T cells. Then, there is a critical question of whether retaining of such feedback mechanisms contributes to uncontrolled growth of CTCL cells. Since SD-4 has functions other than adhesion and T cell inhibition (Couchman and Woods, 1999), the inventors speculate that another function of SD-4 may promote oncogenesis of CTCL cells. Thus, SD-4 may be a new surface marker to distinguish some CTCL lymphocytes. Moreover, as an inhibitor of the anti-CD3 Ab response it is a potentially useful target for treatment of CTCL.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims benefit of priority to U.S. Provisional Application Ser. No. 61/040,524, file Mar. 28, 2008, the entire contents of which are hereby incorporated by reference.
The government owns rights in the present invention pursuant to grant number RO1-A164927-01 from the National Institutes of Health.
| Number | Date | Country | |
|---|---|---|---|
| 61040524 | Mar 2008 | US |
