Patent application title: REDIRECTED, GENETICALLY-ENGINEERED T REGULATORY CELLS AND THEIR USE IN SUPPRESSION OF AUTOIMMUNE AND INFLAMMATORY DISEASE
Zelig Eshhar (Rehovot, IL)
Eran Elinav (Jerusalem, IL)
YEDA RESEARCH AND DEVELOPMENT CO. LTD.
IPC8 Class: AA61K3512FI
Class name: Whole live micro-organism, cell, or virus containing animal or plant cell leukocyte
Publication date: 2010-06-03
Patent application number: 20100135974
A redirected Treg cell is endowed with specificity toward a selected
target antigen or ligand. The cell contains a chimeric receptor
polypeptide that is expressed in a single, continuous chain, with an
extracellular recognition region displayed on the surface of the cell, a
transmembrane region and an intracellular signaling region. The
extracellular recognition region is specific for the selected target
antigen or ligand. The intracellular signaling region includes a
combination of T-cell signaling polypeptide moieties, which combination,
upon binding of the extracellular recognition region to the selected
target antigen or ligand, triggers activation of the redirected Treg
cells to cause suppression of T-cell mediated immunity. Such redirected
Treg cells may be used to suppress undesired activity of T effector cells
thereby mediating an immune or inflammatory response. They are
particularly useful in treating T effector cell-mediated diseases, such
as inflammatory bowel disease, transplant rejection and GVH disease.
1. A redirected regulatory T lymphocyte (Treg cell) endowed with
specificity toward a selected target antigen or ligand, which cell
comprises a chimeric nucleic acid that encodes a chimeric receptor
polypeptide that comprises, expressed in a single, continuous chain, an
extracellular recognition region, a transmembrane region and an
intracellular signaling region, and is expressed in the redirected Treg
cell so as to display the extracellular region on the cell surface,
wherein(a) said extracellular recognition region of said chimeric
receptor is specific for the selected target antigen or ligand; and(b)
said intracellular signaling region comprises a combination of T-cell
signaling polypeptide moieties, which combination of moieties, upon
binding of the extracellular recognition region to the selected target
antigen or ligand, triggers activation of the redirected Treg cells to
cause suppression of T-cell mediated immunity.
2. A redirected Treg cell in accordance with claim 1, wherein said extracellular recognition region comprises an antibody-derived scFv domain that is specific for a selected antigen.
3. A redirected Treg cell in accordance with claim 1, wherein said extracellular recognition region comprises a member of a ligand-receptor pair, which is specific for the other member of that pair.
4. A redirected Treg cell in accordance with claim 1, wherein said extracellular recognition region does not comprise an MHC protein extracellular domain.
5. A redirected Treg cell in accordance with claim 1, wherein said extracellular recognition region is linked to said transmembrane region through a flexible spacer.
6. A redirected Treg cell in accordance with claim 5, wherein said flexible spacer is a hinge from a molecule of the immunoglobulin superfamily.
7. A redirected Treg cell in accordance with claim 1, wherein said intracellular region comprises a signaling moiety from (i) a polypeptide chain of an antigen-specific receptor of a T-cell and/or (ii) a polypeptide chain of a receptor of a T-cell that has a region comprising an immunoreceptor tyrosine-based activation motif (ITAM).
8. A redirected Treg cell in accordance with claim 7, wherein said intracellular region comprises a signaling moiety from (ii).
9. A redirected Treg cell in accordance with claim 7, wherein said intracellular region comprises a signaling moiety from a polypeptide chain selected from the group consisting of a chain of the TCR/CD3 complex, and the γ chain of an Ig Fc receptor (FcRγ).
10. A redirected Treg cell in accordance with claim 9, wherein said polypeptide chain of an antigen-specific receptor of a T-cell is the CD3-.zeta. chain or the FcRγ chain.
11. A redirected Treg cell in accordance with claim 1, wherein said intracellular region includes a signaling moiety of a T cell costimulatory-receptor protein.
12. A redirected Treg cell in accordance with claim 11, wherein said costimulatory-receptor protein is at least one protein selected from the group consisting of CD28, OX40, CD40L, 4-1BB and PD-1.
13. A redirected Treg cell in accordance with claim 11, wherein said costimulatory-receptor protein is CD28.
14. A redirected Treg cell in accordance with claim 11, wherein said costimulatory-receptor protein is 4-1BB.
15. A redirected Treg cell in accordance with claim 1, wherein said intracellular region comprises at least two different ones of said costimulatory-receptor protein signaling moieties.
16. A redirected Treg cell in accordance with claim 15, wherein said intracellular region comprises an intracellular signaling moiety from CD28 and from 4-1BB.
17. A redirected Treg cell in accordance with claim 1, wherein said intracellular region comprises a signaling moiety from a T cell cytokine receptor.
18. A redirected Treg cell in accordance with claim 17, wherein said cytokine receptor is the IL-2 receptor.
19. A redirected Treg cell in accordance with claim 17, wherein said cytokine receptor is the TGF-.beta. receptor.
20. A redirected Treg cell in accordance with claim 1, wherein said intracellular signaling region includes a signal-transducing enzyme that (a) is an enzyme in the signal transduction pathway of an antigen-specific receptor of a T-cell or (b) is an enzyme with corresponding specificity and activity as the enzyme of (a), derived from a non-T-cell lymphocyte.
21. A redirected Treg cell in accordance with claim 20, wherein said signal-transducing enzyme is a member of the Syk-kinase family.
22. A redirected Treg cell in accordance with claim 1, wherein said chimeric nucleic acid further includes a nucleotide sequence that will cause the redirected Treg to express Foxp3.
23. A redirected Treg cell in accordance with claim 1, wherein said selected target antigen or ligand is one that is present or expressed at a site or target tissue of an autoimmune or inflammatory response mediated by effector T-cells.
24. A redirected Treg cell in accordance with claim 23, wherein said autoimmune or inflammatory response comprises an autoimmune response or disease, an allograft or xenograft rejection, or graft-vs. host (GVH) disease.
25. A redirected Treg cell in accordance with claim 1, wherein said target antigen or ligand is an autoantigen or an antigen that is cross-reactive with an autoantigen.
26. A redirected Treg cell in accordance with claim 25, wherein the autoantigen is a pathogenic antigen in the pathophysiology of said autoimmune disease.
27. The redirected Treg cell of claim 1, wherein said autoimmune or inflammatory response and said target antigen or ligand are selected from the group consisting of:(a) inflammatory bowel disease (IBD), wherein said antigen or ligand is one that is expressed in diseased colon or ileum;(b) rheumatoid arthritis, wherein said antigen or ligand is an epitope of collagen or an antigen present in joints;(c) Type I diabetes mellitus or autoimmune insulitis, wherein said antigen or ligand is a pancreatic β cell antigen;(d) multiple sclerosis, wherein said antigen or ligand is a myelin basic protein antigen, MOG-1, MOG-2 or another neuronal antigen;(e) autoimmune thyroiditis, wherein said antigen or ligand is a thyroid antigen;(f) autoimmune gastritis, wherein said antigen or ligand is a gastric antigen;(g) autoimmune uveitis or uveoretinitis, wherein said antigen or ligand is S-antigen or another uveal or retinal antigen;(h) autoimmune orchitis, wherein said antigen or ligand is a testicular antigen;(i)) autoimmune oophoritis, wherein said antigen or ligand is an ovarian antigen;(j) psoriasis; wherein said antigen or ligand is a keratinocyte antigen or another dermal or epidermal antigen;(k) vitiligo, where said antigen or ligand is a melanocyte antigen;(l) autoimmune prostatitis, wherein said antigen or ligand is a prostate antigen;(m) any undesired immune response, wherein said antigen or ligand is an activation antigen expressed on T effector cells present at the site of the undesired immune response;(n) tissue rejection, wherein said antigen or ligand is the MHC molecule having the haplotype of the transplanted tissue, or a portion of that MHC molecule; and(o) an inflammatory condition, wherein said antigen or ligand is one that is expressed on nonlymphoid cells of the hemopoietic lineage that participate in inflammation.
28. A redirected Treg cell in accordance with claim 27, wherein said autoimmune disease is IBD and said antigen or ligand is carcinoembryonic antigen (CEA), or an antigen of intestinal bacterial flora.
29. A redirected Treg cell in accordance with claim 27, wherein the activation antigen of (m) expressed on T effector cells is CD69 or CD107a.
30. A redirected Treg cell in accordance with claim 27, wherein said target antigen or ligand of (o) is one that is expressed on a dendritic cell, macrophage/monocyte, granulocyte or eosinophil present at said inflammation site.
31. An immunoregulatory pharmaceutical composition for suppressing a T effector cell-mediated immune/inflammatory response or treating a T effector cell-mediated immune/inflammatory disease or condition, comprising:(a) a Treg cell according to claim 1, and(b) a pharmaceutically and immunologically acceptable carrier, excipient or diluent.
32. A method of producing the redirected Treg cell of claim 1, comprising:(a) obtaining from a subject and, optionally enriching or isolating and propagating, a population of lymphocytes or T-cells;(b) inducing a Treg cell phenotype in said cells by suitably stimulating or activating the cells by exposure to TGF-.beta. or another cytokine that induces Foxp3 expression thereby inducing the Treg phenotype;(c) before or after step (b), transducing the cells ex vivo with an expression vector encoding said chimeric receptor to be expressed on said Treg cell; and(d) optionally, growing or expanding the cells obtained as above in vitro.
33. A method of producing the redirected Treg cell of claim 1, comprising:(a) obtaining from a subject and, optionally enriching or isolating and propagating, a population of lymphocytes or T-cells;(b) transducing the cells ex vivo with a vector encoding the chimeric receptor;(c) before, after, or concomitantly with step (b), transducing the cells ex vivo with a recombinant nucleic acid expression construct encoding Foxp3; and(d) optionally, growing or expanding the cells obtained as above in vitro.
34. A method of suppressing undesired activity of T effector cells in mediating an immune or inflammatory response, comprising delivering to a site of T effector cells to be suppressed an amount of Treg cells according to claim 1 effective to suppress said T effector cell activity.
35. The method of claim 34 wherein said Treg cells are administered systemically.
36. The method of claim 34 wherein the redirected Treg cells are administered regionally or locally to a site of inflammation.
37. The method of claim 34 wherein said undesired activity of T effector cells is an autoimmune inflammatory response or disorder, rejection of a transplant or GVH disease.
38. A method for treating or ameliorating symptoms of a disease or condition in a mammalian subject that is mediated by undesired activity of T effector cells, said method comprising administering to said subject in need thereof an effective amount of Treg cells in accordance with claim 1, wherein said target antigen or ligand is one that is present at the site of said undesired T effector cell activity.
39. The method of claim 38 wherein the redirected Treg cells are produced by obtaining a population of T-cells from the mammalian subject to be treated, transfecting said cells with said chimeric nucleic acid and causing the cells to express Foxp3 by(i) stimulating the transfected cells to induce Foxp3 expression, or(ii) introducing an exogenous Foxp3-encoding construct into the transfected cells.
40. The method of claim 38 wherein the redirected Treg cells are produced by obtaining a mixed population of T-cells from the mammalian subject to be treated, enriching or purifying Treg cells from the mixed population of T-cells on the basis of the Treg cells' expression of CD4 and CD25 and/or Foxp3, and transfecting said enriched or purified Treg cells with said chimeric nucleic acid.
41. The method of claim 40 wherein the redirected Treg cells are produced by:(a) obtaining(i) peripheral blood mononuclear cells,(ii) peripheral blood lymphocytes,(iii) T-cells enriched or purified from (i) or (ii), or(iv) a subset of T-cells enriched or purified from (iii);(b) exposing the cells obtained in (a), ex vivo, to an amount of TGF-.beta. or other Treg-inducing cytokine or agent that is effective to induce expression of Foxp3 and convert T-cells to a Treg phenotype,(c) optionally, culturing and expanding said exposed cells of (a); and(d) before after or between said steps (a) and (b), transfecting said cells with said chimeric nucleic acid.
42. The method of claim 38 wherein said disease or condition comprises an autoimmune response or disease, an allograft or xenograft rejection, or graft-vs. host (GVH) disease.
43. The method of claim 38, wherein said disease or condition is IBD and said target antigen or ligand is CEA, or an antigen of intestinal bacterial flora.
44. A method of claim 38, wherein said target antigen or ligand is LPS.
45. A method of claim 44, wherein said extracellular recognition region comprises an LPS-binding domain of MD-2 or an LPS-binding domain of CD-14 or both an LPS-binding domain of MD-2 and an LPS binding domain of CD-14.
46. The method of claim 38, further including the step of, either before, concomitantly with, or after administration of the redirected Treg cells, introducing to a subject to the site or target tissue of said immune or inflammatory response, an exogenous antigen or ligand and said target antigen or ligand is said exogenous antigen or ligand.
47. A chimeric DNA molecule comprising:(a) a first DNA segment comprising a sequence encoding an extracellular recognition region specific for a selected target antigen or ligand, which region does not comprise an MHC protein extracellular domain, said selected target antigen or ligand being one that is present or expressed at a site or tissue of a pathogenic or undesired immune response mediated by effector T-cells;(b) a second DNA segment comprising a sequence encoding a transmembrane region; and(c) a third DNA segment comprising a sequence encoding an intracellular signaling region comprising a combination of T-cell signaling polypeptide moieties, which combination of moieties trigger activation of the Treg cells to cause suppression of T-cell mediated immunity when the chimeric DNA is transfected into a Treg cell and said extracellular recognition region binds to the selected target antigen or ligand,wherein, upon transfection of the chimeric DNA into a Treg cell, the Treg cell expresses said extracellular recognition region on its surface, said transmembrane region and said intracellular signaling region in one single, continuous chain.
48. A chimeric receptor polypeptide encoded by the chimeric DNA molecule of claim 47.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention in the field of immunology and medicine relates to genetic modification of T regulatory cells with chimeric receptors with antibody-type specificity, and the use of such cells to suppress the action of T effector cells and treat any of a number of diseases and conditions in which such suppression is beneficial, primarily autoimmune and inflammatory diseases such as inflammatory bowel diseases (IBD), organ-specific autoimmune diseases, allograft rejection and Graft-vs. Host disease.
2. Description of the Background Art
Regulatory T-Cells (Tregs)
One line of research that led to discovery of Treg cells was the observation that thymectomy of mice of certain susceptible strains on postnatal day 3 results in a spectrum of organ-specific autoimmune effects, which were preventable by "reconstitution" of these animals early in life with normal adult lymphocytes (Asano M et al., J Exp Med 1996; 184:387-96). The effectors and suppressors of autoimmunity in this model of multi-organ autoimmunity were CD4+T-cells. It was subsequently shown that the regulatory CD4+Tregs that prevent disease coexpressed CD25. CD4+CD25+ Tregs represent 5-10% of total peripheral CD4+ cells in mice and 3-6% of total peripheral blood CD4+T-cells in humans (Jonuleit H et al., J Exp Med 2001; 193:1285-94).
Over the past decade, CD4+CD25+ Tregs have been studied for their function in autoimmune disease. CD4+CD25+ Tregs suppressed disease induced by cloned autoantigen-specific T effector cells (Sufi-Payer, E et al., J. Immunol., 1998; 160:1212-18). The CD4+CD25+ Treg cells appeared to be members of a unique lineage of regulatory T-cells. These authors noted that, although the target antigen(s) and mechanism of action of the CD4+CD25+T-cells remained to be determined, they likely played an important role in modulating other autoimmune diseases that are mediated by activation of self-reactive T-cells. Tregs prevent organ-specific autoimmune diseases including autoimmune thyroiditis, autoimmune gastritis, insulitis and arthritis.
More recently, it was discovered that Tregs express a transcription factor known as Foxp3 intracellularly. The absence of the transcription factor called Scurfin (also forkhead box P3) and encoded by the gene Foxp3 was known to cause a rapidly fatal lymphoproliferative disease, similar to that seen in mice lacking cytolytic T lymphocyte-associated antigen 4 (CTLA-4). Khattri R et al. (Nat Immunol. 2003; 4:337-42) showed that Foxp3 was highly expressed by Treg cells and was associated with their activity and phenotype. Foxp3-deficient mice lacked Treg cells whereas mice that overexpressed Foxp3 possess more Treg cells. Tregs constitutively express the transcription factor Foxp3 and the inhibitory costimulatory molecule CTLA-4 (Chen W et al., J Exp Med 1998; 188:1849-57.
Foxp3 is believed to act through negative transcriptional regulation of cytokine genes, including IL2, IL4 and IFN-γ (Kasprowicz D J et al., J Immunol. 2003; 171:1216-23, 2003), though many aspects of Foxp3 activity and regulation of its expression remain obscure.
Loser K et al., Gene Ther 2005; 12:1294-304, generated Tregs in vitro by infecting naive CD4+CD25-T-cells with a retrovirus encoding Foxp3. Foxp3-infected T-cells were similar to naturally occurring Treg cells as evidenced by surface marker expression and function. These authors investigated the effects of Foxp3-infected T-cells on contact hypersensitivity responses mediated by T effector cells by injecting into sensitized mice Foxp3- or control virus-infected T-cells. Only injection of Foxp3-infected T-cells significantly inhibited CHS compared to controls, indicating that Foxp3-infected T-cells are suppressive in vivo. The authors then used Foxp3-infected T-cells to treat autoimmune-prone CD40L transgenic (Tg) mice, which develop a severe systemic autoimmune disease including autoreactive T-cells and autoantibodies. Injection of Foxp3-infected T-cells into these mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8+T-cells. This treatment also reduced serum concentrations of antinuclear antibodies, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function. The authors concluded that newly in vitro-generated regulatory T-cells can be used to treat inflammatory and ongoing autoimmune disorders successfully.
Suri-Payer E and Fritzsching B (Springer Semin Immunopathol. 2006; 28:3-16) recently summarized evidence for a role for Treg in suppression of innate and adaptive immune responses in experimental models of autoimmunity including arthritis, colitis, diabetes, autoimmune encephalomyelitis, lupus, gastritis, oophoritis, prostatitis, and thyroiditis. A common observation from such studies is that Tregs are activated in an antigen-specific manner, but exert their suppressive function in an antigen-independent manner, mainly by producing and secreting suppressive cytokines such as IL-10 and TGF-β. Tregs can suppress "conventional" T-cells in vitro by direct cell contact. It is appreciated, however, that down-regulation of antigen-presenting cell (APC) function, such as that of dendritic cells, and attenuation of secretion of inhibitory cytokines such as IL-10 and TGF-β might be important for Treg function in vivo. The final outcome of autoimmunity vs. tolerance depends on the balance between stimulatory signals to T effector cells and inhibitory signals from Treg. Whereas earlier studies analyzed the capacity of Tregs to prevent onset of autoimmune disease, more recent reports indicate successful treatment of ongoing disease.
In vivo, adoptive transfer of Tregs achieved the following effects:
(1) suppressed development of autoimmunity;
(2) suppressed acute rejection of transplanted solid organs; and
(3) suppressed anti-tumor immunity,
A review of Sakaguchi S et al., Immunol Rev. 2006; 212:8-27, noted that naturally arising CD25+CD4+ Treg cells play key roles in the maintenance of immunologic self-tolerance and negative control of a variety of physiological and pathological immune responses. Most of these cells are produced by the normal thymus as a functionally mature T cell subpopulation. Natural Tregs specifically express Foxp3, a transcription factor that plays a critical role in their development and function. Complete depletion of Foxp3-expressing natural Tregs (whether they are CD25+ or CD25-) activated even weak or rare self-reactive T effector cell clones, inducing severe and widespread autoimmune/inflammatory diseases. Natural Tregs are highly dependent on exogenously provided interleukin (IL)-2 for their survival in the periphery. In addition to Foxp3 and IL-2/IL-2 receptor, a deficiency or functional alteration of other molecules (expressed by T-cells or non-T-cells), may affect the development or function of Tregs, self-reactive T effector cells, or both, and consequently tip the balance between the two populations in the periphery toward autoimmunity. Thus, Tregs suppress the activity of T effector cells that are a major cause of antigen-specific autoimmune inflammatory disorders. Tregs induce anergy and promote suppression by a process that involves both cell-cell contact, and probably more importantly, by their secretion of TGF-β and IL-10.
Sakaguchi et al., supra, stated that elucidation of the molecular and cellular basis of Treg-mediated active maintenance of self-tolerance will facilitate (1) our understanding of the pathogenesis of autoimmune disease and (2) development of novel methods of autoimmune disease prevention and treatment.
The present invention is directed to one such novel approach to prevent or treat autoimmune disease and related immune/inflammatory conditions by imposing Treg-mediated control over T effector cells.
Engineering of Chimeric Receptors with Antibody Specificity into T-Cells
Efforts to confer antibody-like specificity to T lymphocytes arose as a response to certain basic discoveries and the failure to convert them to therapeutic success. Human tumor-associated antigens have been shown to exist as peptides associated with major histocompatibility complex (MHC) proteins. The lack of success in vaccinating tumor patients with a large variety of tumor-associated antigen vaccines were thus frustrating (Rosenberg S A et al., Nat Med 2004; 10:909-15). Passive vaccination with anti-tumor antibodies (Abs), tumor infiltrating lymphocytes (TILs), or lymphokine activated killer (LAK) cells showed very limited effectiveness (primarily for vascular tumors). More recent successes with the adoptive transfer of TILs into stage 1V melanoma patients ((Dudley M E, et al., J Immunother 2001; 24:363-73; Dudley M E, et al., Science 2002; 298:850-4) were still burdened by the fact that the results were limited to a few cancers and to individuals from whom it is possible to derive specific TILs.
To overcome such limitations in adoptive cellular immunotherapy of cancer, and in order to develop an approach that was not restricted to individual patients or limited to a specific form of cancer, one of the present inventors and his colleagues developed the "T-body" approach (Gross G et al., Proc Natl Acad Sci USA, 1989; 86:10024-8; Gross G and Eshhar, Z, FASEB J 1992; 6:3370-8), See also the following patent publications by Eshhar and colleagues, all of which are incorporated by reference in their entirety: US Pat Pub 2002-0137697, U.S. Pat. No. 5,912,172, U.S. Pat. No. 5,906,936, International Patent publications WO00/31239, WO97/15669, WO95/14710 and WO93/19163. In these approaches, a chimeric receptor (CR), was made, for example, by fusing the variable portion of an antibody, such as an anti-tumor monoclonal antibody (mAb), to a lymphocyte intracellular triggering domain, so as to be expressed by the T-cell into which the gene has been transfected as the extracellular domain of that T-cell triggering molecule. Following the expression of such CR genes in immune effector cells (T-cells and natural killer (NK) cells), the resulting engineered cells (nicknamed "T-bodies") recognized their tumor targets and efficiently killed them. Thus, the chimeric immune receptor confers redirected antigenic specificity coupled to direct, MHC-independent triggering of cellular activation in response to binding of pre-defined target antigen.
Originally, the heterodimeric CR configuration comprised the two T cell receptor (TCR) α and β chains in which each pair of TCR variable domains (V.sub.α and V.sub.β) was replaced with a pair of VH and VL domain derived from a selected antibody. These two Ab-derived coding sequences were co-transfected into T cell lines and were found to confer antibody specificity (Gross et al., 1989, supra). Thus, T-cells could be activated to effector function such as cell killing or cytotoxic activity against an immunologically specific target in a manner that was MHC independent (and thereby non-restricted) (Gross et al., 1992, supra).
In "second generation" CR's, the single chain configuration of the CR was further manipulated to obtain a configuration that would be useful particularly for cancer or antiviral therapy. Here, a single chain Fv (scFv) of an antibody was linked to transmembrane and cytoplasmic domains of lymphocyte triggering moieties such as the TCR/CD3 complex-associated ζ chain, or the Fc receptor γ chain (Eshhar Z et al., Proc Natl Acad Sci USA 1993; 90:720-4). This single chain configuration, which combined antibody recognition and T cell signaling in a single continuous protein, was a modular structure with functional domains that are simple to manipulate, and could be readily expressed in human lymphocytes using retrovirus-based vectors (Eshhar Z et al., J Imm Meth 2001; 248:67-76). Other such receptors designed by some of the present inventors and colleagues, and discussed in more detail below, are referred to as tripartite chimeric receptors (TpCR) that also include a costimulatory domain(s) (e.g. CD28, 4-1BB).
With time, it has emerged that redirection of the specificity of T effector cells using single chain CRs has become a valid therapeutic option for cancer. Many investigators have adapted this "T-body" approach to endow T-cells with various specificities and functions (Gross & Eshhar, supra; Willemsen R A et al., Hum Immunol 2003; 64:56-68; Baxevanis C N et al., Cancer Immunol Immunother 2004; 53:893-903). For a recent review, see: Eshhar, Z. "The T-Body Approach Redirecting T Cells with Antibody Specificity," in Therapeutic Antibodies. Handbook of Experimental Pharmacology 181, Chemajovsky & Nissim (eds.), Springer-Verlag, 2008, pp. 329-342.
In the present invention, the inventors have conceived expanded uses of such CR's for the treatment of undesired immune/inflammatory conditions such as autoimmune diseases (with a particular initial emphasis on inflammatory bowel disease, IBD) and graft rejection.
Chronic Inflammatory Disease and Animal Models
Inflammatory conditions, particularly chronic inflammatory diseases, are of particular importance in clinical medicine. These diseases, caused by actions of the immune system, involve inappropriate or excessive activation of certain T-cells, expression of regulatory cytokines and chemokines, loss of immune tolerance, and the like. Examples of autoimmune and/or chronic inflammatory diseases are multiple sclerosis, inflammatory bowel diseases (IBD), joint diseases such as rheumatoid arthritis, and systemic lupus erythematosus. Some of these diseases are rather organ/tissue-specific as follows: intestine (Crohn's Disease), skin (psoriasis), myelinated nerves (multiple sclerosis or MS), pancreatic islet or β cells (insulin dependent diabetes mellitus (IDDM) or Type I Diabetes), salivary glands (Sjogren's disease), skeletal muscle (myasthenia gravis), the thyroid (Hashimoto's thyroiditis; Graves' Disease), the anterior chamber of the eye (uveitis), joint tissue (rheumatoid arthritis), and various cardiovascular diseases.
Inflammatory bowel disease (IBD) is a collective term used to describe two intestinal disorders whose etiology is not completely understood: Crohn's disease and ulcerative colitis. IBD occurs worldwide and afflicts several million people (0.3% of people in Western countries), and its incidence is on the rise (Tsironi E, et al., Am J Gastroenterol. 2004; 99:1749-55). The course and prognosis of IBD varies widely. Onset of IBD is predominant in young adulthood and presents typically with diarrhea, abdominal pain, and fever; anemia and weight loss are also common signs. Between 10% and 15% of people with IBD require surgery over a ten year period. Patients with IBD are also at increased risk for the development of intestinal cancer. These diseases are accompanied by a high frequency of psychological symptoms, including anxiety and depression.
Although the pathogenesis of this common T cell-mediated disorder remains uncertain, it is believed to result from loss of tolerance in the intestinal immune system due to the presence of constant antigenic stimulation provided by the very large numbers of resident bacteria (Podolsky D K, N Engl J Med 2002; 347:417-29). Unfortunately, new therapies for IBD are few, and both diagnosis and treatment have been hampered by a lack of detailed knowledge of the etiology. A combination of genetic factors, exogenous triggers and endogenous microflora can contribute to the immune-mediated damage of intestinal mucosa. Bacteria have been implicated in initiation and progression of Crohn's Disease since intestinal inflammation frequently responds to antibiotics. Common intestinal colonists and novel pathogens have been implicated, either because of direct detection or disease-associated anti-microbial immune responses. In many genetically susceptible animal models of chronic colitis, luminal microorganisms appear to be a necessary cofactor for disease.
The initiating step in autoimmune disease pathology is often obscure in humans where the diseases are largely sporadic, and symptoms may appear years after the first pathogenic T cell is activated. It has therefore been difficult to design effective therapies to block induction of disease. In contrast, there are common features in many of the later stages of these diseases. Inflammation at the disease site/target organ is typically present, caused by the release of inflammatory, also termed "proinflammatory," cytokines (e.g. TNF-α and interferons) by T-cells and by other cells that contribute to the activation steps and effector pathways of immune/inflammatory processes. These cells include (among others) macrophages, dendritic cells and their precursors, B lymphocytes and plasma cells and NK cells (including NKT-cells). These reactions often involve destruction of "target" cells and tissue damage.
Studies using murine models of experimental chronic inflammation are helping to define nature of the immunological dysregulation that initiates inflammation and leads to destruction of specific end organs as well as for testing therapies. See, for example, Mombaerts et al. Cell, 1993; 75:274-82; Tarrant et al., 2998; J Immunol, 161:122-7; Powrie et al., Immunity, 1994; 1:553-62; Hong et al., J Immunol, 1999; 162:7480-91; Horak, Clin Immunol Immunopathol, 1995, 76(3 Pt 2):5172-173; Ehrhardt et al. J Immunol, 1997; 158:566-73; Davidson et al., J Immunol, 1998; 161:3143-9; Kuhn et al. Cell, 1993. 75:263-74; Neurath et al., J Exp Med, 1995. 182:1281-90. W. Strober, 2002; Annu. Rev. Immunol. 20:495-54 reviews mucosal models of inflammation, and is incorporated by reference in its entirety. One hallmark of the better of these models is that the histopathology and pathophysiology resembles that of the parallel human conditions, further enhancing the models' utility in testing novel treatment strategies. In the case of IBD this development has not been uniform. Most emphasis has been placed on modulation of immune mechanisms (Blumberg R S et al., Curr Opin Immunol. 1999; 11:648-56; Strober et al., supra) and recently of the enteric flora (Sartor R B, Curr Opin Gastroenterol. 2001; 4:324-330).
Bhan A K et al., 1999 Immunol Rev 169:195-207 reviewed studies of colitis in transgenic (Tg) and knockout (KO) animal models for mucosal inflammation in IBD. Genetics and the environment, particularly the normal enteric flora, were factors in the development of mucosal inflammation, as stated above. Normal mucosal homeostasis was disrupted by cytokine imbalance, abrogation of oral tolerance, breach of epithelial barriers, and loss of immunoregulatory cells. Some but not all immunodeficiencies, in the appropriate setting, led to colitis. CD4+T effector cells have been identified as the pathogenic lymphocytes in colitis, and can mediate inflammation by either the Th1 or the Th2 pathway. The Th1 pathway dominates in most colitis models (and human Crohn's Disease). In contrast, in the colitis observed in mice, the T cell receptor (TCR) a chain knockout mice (TCRα KO mice) shared many features of ulcerative colitis including the dominance of Th2 pathway in colon inflammation. Such models are important for the development of therapeutic strategies to treat IBD. In a later review, the same group (Mizoguchi A et al., 2003, Inflamm Bowel Dis. 9:246-259) noted that exaggerated immune responses to normal enteric microflora are involved in the initiation and perpetuation of chronic intestinal inflammation. A major pathway involves development of "acquired" immune responses by the interactions of CD4+TCRαβ+T-cells with antigen-presenting cells (APC), particularly dendritic cells. CD4+CD25+ Treg cells attenuated activated T cell responses.
The progression from the acute to the chronic phase of IBD has not been well characterized in animal models and cannot be easily evaluated in patients. Spencer D M et al. Gastroenterol. 2002; 122:94-105 reported changes in the mucosal immune response over time in experimental colitis. Severity of colitis, body mass, stool consistency and blood content, serum amyloid A, and tissue histology were examined in mice deficient in interleukin-10 (IL-10) over 35 weeks. The corresponding production of IL-12, IL-18, IFNγ, TNFα, IL-4, and IL-13 by lamina propria mononuclear cells in the inflamed intestine was measured. Administration of a neutralizing anti-IL-12 monoclonal antibody (mAb) at distinct times during disease progression permitted evaluation of the therapeutic potential of this agent. Lamina propria mononuclear cells from mice with early disease synthesized progressively more IL-12 and IFNγ, whereas production of both cytokines declined dramatically and returned to pre-disease levels in the late phase. Consistent with this pattern, the neutralizing anti-IL-12 reversed early, but not late, disease. In contrast, IL-4 and IL-13 production increased progressively from pre- to early to late disease. It was concluded that colitis developing in IL-10-deficient mice evolves into two distinct phases. IL-12 plays a pivotal role in early colitis, whereas other immune mechanisms, presumably mediated by IL-4 and IL-13, predominate in late disease to sustain chronic inflammation.
IL-10 and Chronic Inflammatory Disease
It has been known for some years that IL-10 affects the growth and differentiation of many hemopoietic cell types in vitro and is a particularly potent suppressor of macrophage and T cell functions. These observation were based in part from use of IL-10-deficient (knockout, KO) mutant mice by gene targeting (Kuhn R et al., Cell 1993; 75:263-74). In these mice, lymphocyte development and antibody responses are normal, but most animals are growth retarded, anemic and suffer from chronic enterocolitis. Alterations in the intestine include extensive mucosal hyperplasia, inflammation, and aberrant epithelial expression of major histocompatibility complex (MHC) class II molecules. In contrast, IL-10 KO mutants kept under specific pathogen-free conditions, develop only localized inflammation (limited to the proximal colon). It was concluded that (1) bowel inflammation in these mutants originated from uncontrolled immune responses stimulated by enteric antigens and (2) IL-10 is an essential (negative) regulator in the intestinal tract.
In a study validating this IL-10 KO mouse model of colitis, T. Scheinin, T et al. (Clin Exp Immunol 2003; 133:38-43) emphasized that a valuable animal model must respond to existing therapy in a way that resembles the response of human disease. Since refractory Crohn's Disease responded well to anti-TNFα antibody therapy, the investigators examined responses of IL-10 KO mice to anti-TNFα therapy, using a new scoring system similar to the Crohn's Disease "Activity Index" in humans. Stool samples were tested for cytokines and the findings compared with histology. Results showed that anti-TNF antibody therapy starting at 4 weeks markedly ameliorated the disease (as judged by the clinical score or by gut histology). A marked diminution of inflammatory cytokines in stool samples was noted, adding a further accurate measure of clinical improvement. The authors concluded that this model is useful for evaluating other therapeutic modalities of relevance to Crohn's Disease.
Treg Cells and Inflammatory Bowel Disease
One of the commonly used animal models of IBD involves adoptive transfer of CD45RBhiCD4+T-cells into SCID mice, leading to the development of massive colon mononuclear cell infiltrates, epithelial cell hyperplasia and ulceration (Thornton A M, et al., J Immunol 2000; 164(1):183-90). Cotransfer of large numbers of CD4+CD25+ Tregs prevented the development of colitis or cured established colitis, an affect that required signaling through CTLA-4 (Read S, et al., J Exp Med 2000; 192:295-302; 2000; Morrissey P J, et al., J Exp Med 1993; 178:237-44). Even after the development of immune-mediated colitis, adoptive transfer of 106 CD4+CD25+ cells caused significant improvement of intestinal inflammation (Fantini M C et al., Gut 2006; 55:671-80; Mottet C, et al., J Immunol 2003; 170(8):3939-43; Uraushihara K, et al., J Immunol 2003; 171:708-16).
In humans suffering from IBD, peripheral regulatory CD4+CD25+ cells retain suppressive activity. However, in contrast to other intestinal inflammatory disorders, the number of these regulatory cells decreases in peripheral blood during active inflammation and only slightly increases in intestinal lesions (Maul J, et al., Gastroenterology 2005; 128(7):1868-78). This aberration suggests that Treg homing defects, as well as dysregulated in situ activation contribute to the pathogenesis of IBD
There is therefore a recognized need in the art to find modalities to suppress autoimmune/inflammatory reactions and diseases, including but not limited to IBD, as well as to suppress rejection of organ and tissue grafts and prevent Graft vs. Host (GVH) disease. The present invention provides a novel approach, that of redirecting Treg cells, as a means to recruit Tregs to sites of inflammation, and activate them to suppress such immune/inflammatory reactions and protect against, alleviate and even cure such disease as IBD.
SUMMARY OF THE INVENTION
The present invention is based on the inventors' conception that CR-mediated redirection and activation of Treg cells at sites of inflammation results in suppression of inflammatory conditions, commonly part of organ-specific autoimmune disease and exemplified herein as inflammation in the colon in experimental IBD. The inventors have further conceived of using these cells to overcome rejection of mismatched cells and tissues by T effector cells that arise in transplant recipients or to inhibit the pathogenic action of transplanted immunocompetent cells in the case of GVH disease.
The invention relies on the inventors' innovative T-body approach that has thus far proven useful for immunotherapy of cancer (and is currently in phase I/II clinical trials). The invention provides a new approach to the exploitation of Treg cells for amelioration of pathologic and undesired immune responses, particularly immunotherapy of autoimmune and inflammatory conditions, including various organ-predominant autoimmune diseases, and other pathologic or undesirable immune responses such as graft rejection and graft vs. host disease.
According to this invention, Treg cells are endowed with CRs that are specific for a selected target antigen or ligand. Such modification causes activation of redirected Tregs at sites of inflammation to suppress the proinflammatory effector-type immune responses. Based on the present inventors' (and their colleagues') results with redirecting antitumor effector lymphocytes, it is expected that Tregs, endowed with predefined specificity, will migrate/home to and accumulate in, a targeted site, such as the inflamed colon, where they will suppress disease-mediating T effector cells. To avoid the necessity of migration or homing to the targeted site, the Tregs may, where possible, be administered directly at or to such site, where they will become activated and suppress disease-mediating T effector cells.
Such redirected Treg cells, also referred to as "T-bodies," are Treg cells that have been genetically engineered to express a CR, preferably a tripartite chimeric receptor (TpCR) that is made of a single chain extracellular recognition unit, a transmembrane region, and an intracellular signaling region.
The extracellular recognition region is specific for a selected target antigen or ligand and may preferably be a single chain antibody variable (scFv) region or another ligand that is capable of binding to the target antigen or ligand. The extracellular recognition region preferably does not comprise an MHC protein extracellular domain. The redirected Tregs of the present invention are sometimes referred to herein as "T-bodies" despite the fact that the extracellular recognition region is not necessarily an antibody domain. Thus, this term is not intended to be limited to Tregs with antibody-like specificity, but also includes Tregs with ligand-receptor-like specificity or otherwise.
A flexible spacer region may be present between the extracellular recognition region and the transmembrane region. Such flexible spacer is preferably an immunoglobulin (Ig)-like hinge, such as any hinge region derived from the Ig superfamily.
The intracellular region includes a combination of T-cell signaling polypeptide moieties, fused in tandem, which combination of moieties, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the Treg cells to cause suppression of T-cell mediated immunity. The T-cell signaling moieties preferably include one or more cytoplasmic domains of a costimulatory molecule (e.g., CD28) and a cytoplasmic T-cell stimulatory domain, e.g., of FcRγ or a CD3-ζ chain. The redirected Treg cells become specifically activated, upon binding of the extracellular recognition region of the CR to its target antigen or ligand, in a manner that is preferably (1) not restricted by, or dependent upon, the binding of the target antigen or ligand to an MHC, nor is it otherwise dependent in any way on the MHC (HL-A) haplotype of the recipient and (2) independent of engagement of costimulatory ligand(s) on a target cell.
A preferred target disease of this invention is an IBD such as ulcerative colitis, in which the present methods, as used successfully in an animal model, will permit Treg cells to reach bowel lesions in IBD patients and become efficiently activated at the inflammation site. The present invention results in site-specific Treg accumulation, ultimately resulting in CR-mediated, antigen-specific activation that results in the production of suppressive cytokines which in turn suppress effector autoimmune T-cells in an antigen-nonspecific manner, leading to alleviation of symptoms and thereby treating the disease.
The present therapeutic approach has several unique advantages. In contrast to other immunotherapeutic models, it involves T-bodies redirected with a CR, and preferably a TpCR, that combines antibody/antigen or ligand/receptor recognition with stimulatory and costimulatory motifs. Thus, T-bodies can be fully activated in a way that is not restricted by the MHC and is independent of a requirement for costimulation. The second advantage of the present invention stems from the fact that, although Treg activation is antigen-dependent, the suppressive action of these cells is antigen-, TCR-, and MHC-independent. By exploiting this property, one can construct a chimeric receptor that is specific for one or more tissue-associated antigens rather than requiring specificity for an unknown number of yet undefined autoimmune disease-specific antigens. Expression of such chimeric receptors in Tregs redirects these cells and their activation to the appropriate target tissue (in a preferred embodiment, the colon) so that they are activated in an antigen-specific manner, where their potent suppressive effects take place without a need for further recognition of disease-associated-antigens (the "bystander effect"). By using specifically-activated Tregs, many fewer cells are required to treat autoimmune inflammatory conditions; such as IBD, or allograft-associated reactions in patients than would have been possible prior to this invention when much higher numbers of non-specific Tregs would have been needed.
The present inventors have constructed strains of transgenic (Tg) mice whose T-cells and natural killer (NK) cells express an antigen-specific TpCR. For exemplification of the invention, the inventors selected the trinitrophenyl (TNP) hapten as the specific target of the TpCRs. TNP specific Tregs isolated from these Tg mice suppressed TNP-specific effector T-cells in vitro and in vivo and were able to suppress trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. In this embodiment, the target antigen for Treg and the pathogenic antigen--the hapten TNP--are the same. In another example, TNP-specific Tregs suppressed oxazolone-induced colitis in mice in which a low dose of TNP was introduced into the colon together with the oxazolone challenge. However, the TNP-specific Tregs had no effect on the oxazolone-induced colitis in the absence of TNP introduction. This establishes the "bystander" effect of the present invention, i.e., that the target antigen need not be the pathogenic antigen, as long as the redirected Tregs are activated in the vicinity of the pathogenic or undesired immune response.
One distinct advantage of the present invention is that it provides cells and methods that permit antigen-specific activation and antigen-nonspecific action of Treg cells used to suppress effector T cell responses (and treat consequent pathologies) in a way that does not require identity between the ligand (e.g., the antigen) recognized by the TpCR (e.g., by its target recognition portion) and the ligand/antigen that plays a pathogenic role in the disease process. Thus, the antigen that is pathogenic does not have to be recognized by the T effector cells being suppressed, and, indeed, may be unrelated to the disease or condition being treated. Thus, the invention exploits the "bystander" effect. As long as the Treg is in the correct vicinity where T effector cells are located and mediating their undesired effects, the redirected Tregs of the present invention can be triggered or activated at that location to release of suppressive cytokines (e.g., IL-10 and TGF-β), that will result in suppression of any "bystander" effector T-cells, and by this mechanism, quell an ongoing inflammatory/autoimmune response.
The unique characteristics of the Tg system used in the present examples enables evaluation of the suppressive effect of antigen-specific Tregs in IBD both in vitro and in vivo. Different means are used to induce Tregs, allowing those of skill in the art to select the optimal method for generating efficient numbers of antigen-specific redirected Tregs for a desired antigen or disease/condition. According to this invention, redirected human Tregs constitute an effective cell-based therapeutic modality for IBD or ulcerative colitis and, more broadly, for any T effector cell-mediated disease or condition.
To overcome the scarcity of antigen-specific Tregs, the present invention includes methods to induce these cells using cytokines (e.g. TGF-β) or by expression of transgenes (e.g. encoding the Foxp3 transcription factor) that will, together with the TpCR, allow antigen-specific Treg expansion.
According to the present invention, human Treg cells, derived from either the subject with the autoimmune/inflammatory disease or condition to be treated, or from an HLA-matched healthy donor (or a universal cell that is not recognized by the recipient's immune system), are endowed with antigen/ligand-specificity, by transduction with the antigen/ligand-specific TpCR as disclosed herein. Alternatively, the cells being endowed with antigen-specificity are the entire T-cell population and the nucleic acid construct including the sequence encoding the TpCR further includes a Foxp3 transgene that is present as an independently transcribed cistron. In another such alternative, a Foxp3 transgene is separately transfected into the T-cell population, to turn the T-cells into Treg cells. Examples provided below include studies using murine colonoscopy, in vivo imaging and immunofluorescence, and provide the basis for a novel cell-based therapeutic modality for IBD, and, by extension, for other pathologic and undesired immune responses mediated by antigen specific T effector cells.
Various embodiments of the invention are described more specifically below. The present invention is directed to a redirected regulatory T lymphocyte (Treg cell) endowed with specificity toward a selected target antigen or ligand, which cell comprises a chimeric nucleic acid that encodes a chimeric receptor (CR) polypeptide that comprises, expressed in a single, continuous chain, an extracellular recognition region, a transmembrane region and an intracellular signaling region, and is expressed in the Treg cell so as to display the extracellular region on the cell surface, wherein (a) the extracellular recognition region of the chimeric receptor is specific for the selected target antigen or ligand, and does not comprise an MHC protein extracellular domain; and (b) the intracellular region comprises a combination of T-cell signaling polypeptide moieties which combination of moieties, upon binding of the extracellular recognition region to the selected target antigen or ligand, triggers activation of the Treg cells to cause suppression of T-cell mediated immunity.
In preferred embodiments of the present invention, the extracellular recognition region is an antibody-derived scFv domain that is specific for a selected antigen. In another preferred embodiment, the extracellular recognition region is a member of a ligand-receptor pair, which is specific for the other member of that pair.
Preferably, the extracellular recognition region is linked to the transmembrane region through a flexible spacer, which, more preferably, is a hinge from a molecule of the immunoglobulin family.
The intracellular signaling region preferably includes a signaling moiety from a chain of an antigen-specific T-cell receptor, which more preferably is one having a polypeptide region comprising an immunoreceptor tyrosine-based activation motif (ITAM). Non-limiting examples of antigen-specific T-cell receptors are chains of the TCR/CD3 complex, a TCR α, β, γ or δ chain, and the γ chain of an Ig Fc receptor (FcRγ). The chain of an antigen-specific T-cell receptor is preferably the CD3/ζ chain or an FcRγ subunit.
The intracellular signaling region further preferably includes a signaling moiety of a costimulatory-receptor protein of a T-cell. The costimulatory-receptor protein is preferably selected from CD28, OX40, CD40L (gp39), 4-1BB and PD-1 (or preferably the human form or homolog of these costimulatory molecules). Most preferred among these is CD28 or 4-1BB. In another preferred embodiment, the intracellular signaling region includes more than one of the costimulatory-receptor protein signaling moieties. For example, the combination of T-cell signaling polypeptide moieties in the intracellular signaling region may include both CD28 and 4-1BB. In a particularly preferred embodiment, the extracellular hinge and transmembrane regions of CD28 are used as the extracellular hinge and transmembrane regions of the chimeric receptor.
The intracellular signaling region may also include a signaling moiety from a cytokine receptor of a T-cell, such as the IL-2 receptor or the TGF-β receptor. The latter may help to induce the T-cell containing the chimeric receptor to adopt the characteristics of a Treg cell.
The intracellular region may also include a signal-transducing enzyme that (a) is an enzyme in the signal transduction pathway of an antigen-specific T-cell receptor or (b) is an enzyme with corresponding specificity and activity as the enzyme of (a), derived from a non-T-cell lymphocyte. Such enzyme is preferably a kinase, such as the Syk kinase.
The chimeric nucleic acid encoding the CR may also include a nucleotide sequence that encodes Foxp3 arranged such that Foxp3 is expressed by the Treg cell independently of the chimeric receptor. In other words, the chimeric nucleic acid may be bicistronic such that the Foxp3 transgene is present as an independently transcribed cistron.
The target antigen or ligand is preferably one that is present or expressed at a site or target tissue of an immune or inflammatory response mediated by effector T-cells. The autoimmune or inflammatory response may comprise an autoimmune response or disease, an allograft or xenograft response or rejection, or graft vs. host (GVH) disease. In an alternative preferred embodiment, the target antigen or ligand may be an autoantigen or an antigen that is cross-reactive with an autoantigen, i.e., is also bound by an antibody that is specific to the autoantigen. The autoantigen may be a pathogenic antigen in the pathophysiology of the autoimmune disease.
The antigen is not necessarily an autoantigen, but can be, for example, an antigen that is part of the bacterial flora, such as LPS derived from the bacteria native to the colon.
The autoimmune disease or graft response and the antigen/ligand or antigens/ligands against which the Treg is specific is preferably selected from the following group: (a) inflammatory bowel disease (IBD), wherein the antigen or ligand is one that is expressed in diseased colon or ileum; (b) rheumatoid arthritis, wherein the antigen or ligand is an epitope of collagen or an antigen present in joints; (c) Type I diabetes mellitus or autoimmune insulitis, wherein the antigen or ligand is a pancreatic β cell antigen; (d) multiple sclerosis, wherein the antigen or ligand is, for example, a myelin basic protein (MBP) antigen or MOG-1 or MOG2-2, or a neuronal antigen. (e) autoimmune thyroiditis, wherein the antigen or ligand is a thyroid antigen; (f) autoimmune gastritis, wherein the antigen or ligand is a gastric antigen; (g) autoimmune uveitis or uveoretinitis, wherein the antigen or ligand is S-antigen or another uveal or retinal antigen (h) autoimmune orchitis, wherein the antigen or ligand is a testicular antigen; (i)) autoimmune oophoritis, wherein the antigen or ligand is an ovarian antigen; (j) psoriasis, wherein the antigen or ligand is a keratinocyte antigen or another antigen present in dermis or epidermis; (k) vitiligo, where the antigen or ligand is a melanocyte antigen such as melanin or tyrosinase; (l) autoimmune prostatitis, wherein the antigen or ligand is a prostate antigen; (m) any undesired immune response, wherein the antigen or ligand is an activation antigen or other antigen expressed on T effector cells present at the site of the undesired response; (n) tissue rejection, wherein the antigen or ligand is the MHC specific to the transplanted tissue; and (o) an inflammatory condition, wherein the antigen or ligand is one that is expressed on nonlymphoid cells of the hemopoietic lineage that participate in inflammation.
Most preferred is a Treg cell that is able to act and suppress IBD or ulcerative colitis, and may be specific for an antigen associated with IBD such as carcinoembryonic antigen (CEA) or an antigen of intestinal bacterial flora such as bacterial lipopolysaccharide (LPS) or a component thereof, preferably a Lipid A component.
The Treg may be specific for an activation antigen expressed on T effector cells such as CD69 or CD107a. The Treg may be specific for an antigen expressed on a dendritic cell, macrophage/monocyte, granulocyte or eosinophil present at the inflammation site.
In a preferred embodiment, the above Treg cell is specific for an antigen that is introduced exogenously to a subject to the site or target tissue of the immune or inflammatory response, either before, concomitantly with, or after administration of the Treg cell.
The above Treg cell preferably is one that expresses CD4 or CD8, along with CD25 on its surface and expresses the Foxp3 transcription factor intracellularly. The Foxp3 transcription factor may be expressed in the cell endogenously (i.e., from the cells' own Foxp3 gene); this expression is enhanced by exposure of cells to TGF-β or another cytokine that induces Foxp3 expression and induces a Treg phenotype in T-cells. In another embodiment of the above Treg cell, Foxp3 is expressed from a nucleic acid that has been introduced into the cell exogenously (i.e., transduced) as a recombinant nucleic acid expression construct encoding Foxp3 and regulating its expression. The above Treg may be obtained from a mammalian subject prior to introduction of the chimeric nucleic acid and prior to stimulation that induces Foxp3 expression or prior to transducing the exogenous Foxp3-encoding construct. The chimeric nucleic acid encoding the chimeric receptor and the nucleic acid construct encoding Foxp3 may be co-transduced into the cell. In one embodiment, co-transduction is achieved using a bicistronic vector that includes, in a single vector, a sequence of (i) the chimeric nucleic acid encoding the chimeric receptor and (ii) the nucleic acid construct encoding Foxp3, under the control of a common (or separate) promoter and regulatory sequences.
The above Treg cells may be enriched or purified from a mixed population of lymphocytes or T-cells on the basis of the Treg cells' expression of CD4 (or CD8) and CD25 and/or Foxp3. The cell may be subjected to the following treatment: (a) exposure ex vivo of: (i) peripheral blood mononuclear cells, (ii) peripheral blood lymphocytes, (iii) T-cells enriched or purified from (i) or (ii), or (iv) a subset of T-cells enriched or purified from (iii); to an amount of TGF-β or other Treg-inducing cytokine or agent that is effective to convert T-cells to a Treg phenotype and to induce expression of Foxp3; and (b) optionally, culturing and expanding the exposed cells of step (a).Preferred Treg cells comprise the above cell that has been transduced with an expression vector encoding Foxp3.
Also provided herein is an immunoregulatory pharmaceutical composition for suppressing a T effector cell-mediated immune/inflammatory response or treating a T effector cell-mediated immune/inflammatory disease or condition, comprising a redirected Treg as described above and a pharmaceutically and immunologically acceptable carrier, excipient or diluent.
This invention is also directed to a method for producing the above redirected Treg that expresses a chimeric receptor as described. This method preferably comprises: (a) obtaining from a subject and, optionally, enriching or isolating and propagating, a population of lymphocytes or T-cells; (b) inducing the Treg phenotype in these lymphocytes by suitably stimulating or activating the cells by exposure to TGF-β or another cytokine or agent that induces Foxp3 expression and a Treg phenotype; (c) before or after step (b), transducing the cells ex vivo with an expression vector encoding the chimeric receptor to be expressed in the Treg; and (d) optionally, growing or expanding in vitro the cells obtained as above.
In another embodiment, this method comprises: (a) obtaining from a subject and, optionally, enriching or isolating and propagating, a population of lymphocytes or T-cells; (b) transducing the cells ex vivo with a vector encoding the chimeric receptor; (c) before, after, or concomitantly with step (b), transducing the cells ex vivo with a recombinant nucleic acid expression construct encoding Foxp3; and (d) optionally, growing or expanding in vitro the cells obtained as above.
This invention further is directed to a method of suppressing undesired activity of T effector cells in mediating an immune or inflammatory response, comprising delivering to a population of T effector cells to be suppressed (or to a site where such T effector cells are present) an amount/number of redirected Tregs as above, effective to suppress activity of the T effector cells.
Also intended is a method of suppressing undesired activity of T effector cells as indicated above, which method comprises delivering to a population of T effector cells to be suppressed (or to a site where such T effector cells are present) an amount/number of redirected Tregs produced according to the above methods that are effective for suppressing the T effector cell activity.
This delivering is preferably in vivo. The redirected Treg cells are delivered by injection or infusion to a subject in whom the T effector cell activity is to be suppressed, preferably by a route selected from intravenous, intramuscular, subcutaneous, intraperitoneal, intra-articular, intrathecal, intraluminal, intracerebroventricularly, rectal, and topical. In one embodiment, the Treg cells are delivered regionally or locally to a site of inflammation.
The above method is intended for use in situations wherein the T effector cells mediate an autoimmune inflammatory response or disorder, rejection of a transplant or GVH disease.
In one embodiment, the method for treating or ameliorating symptoms of a disease or condition in a subject that is mediated by undesired activity of T effector cells comprises administering to the subject in need thereof an effective amount/number of Treg cells as described above, or a pharmaceutical composition described above, wherein the target recognition domain of the redirected Treg cells is specific for an antigen/ligand present in the subject in the vicinity of the T effector cells so that, upon recognizing and binding the antigen, these redirected Treg cells are activated to secrete suppressive cytokines that suppress the T effector cells in an antigen-nonspecific manner. As noted above, the Treg cell activation occurs in a manner that is not restricted by the MHC and does not require costimulation by a ligand for the costimulatory signaling protein.
Also included is a method for treating or ameliorating symptoms of a disease or condition in a subject that is mediated by undesired activity of T effector cells, the method comprising: (a) producing redirected Treg cells using the production methods described above; (b) administering to the subject in need thereof an effective amount/number of these Treg cells, thereby treating or ameliorating symptoms of the disease or condition.
Stated more generally, the invention is directed to a method for suppressing a T effector cell-mediated immune/inflammatory process in a subject in need thereof, comprising administering to the subject an effective amount/number of redirected Treg cells that express on their surface an antigen-specific chimeric receptor that includes portions that activate Treg cells upon contact with the antigen for which the receptor is specific, the antigen being one that is present in the vicinity of the immune/inflammatory activity. The disease or condition to be treated or ameliorated is preferably: (a) IBD; (b) rheumatoid arthritis; (c) Type I diabetes mellitus or autoimmune insulitis; (d) multiple sclerosis; (e) thyroiditis; (f) gastritis; (g) uveitis or uveoretinitis; (h) orchitis; (i)) oophoritis; (j) psoriasis; (k) prostatitis; (l) encephalomyelitis; (m) vitiligo; (n) rejection of a mismatched cell, tissue or organ graft; or (o) GVH disease.
The present method is used to inhibit the rejection of transplanted cells, tissue, or an organ (alto- or xeno-) that is, for example, mismatched for a major and/or one or more minor histocompatibility antigens. In the case of GVH disease, the recipient generally has received a transplant of allogeneic, semi-allogeneic or non-MHC-mismatched bone marrow cells or enriched or isolated hematopoietic stem cells that are responsible for mediating pathogenic effects.
The present invention is further directed to the novel chimeric DNA that can be used to produce the redirected T-cells described above, as well as to the chimeric receptor protein encoded thereby. Such chimeric DNA comprises:
a first DNA segment encoding an extracellular recognition region specific for a selected target antigen or ligand, which does not comprise an MHC protein extracellular domain, the selected target antigen or ligand being one that is present or expressed at a site or target tissue of a pathogenic or undesired immune response mediated by effector T-cells;
a second DNA segment encoding a transmembrane region; and
a third DNA segment encoding an intracellular signaling region comprising a combination of T-cell signaling polypeptide moieties, which combination of moieties, upon transfection of the chimeric DNA into a regulatory T lymphocyte (Treg cell) and binding of the extracellular recognition region to the selected target antigen or ligand thereof, triggers activation of the Treg cells to cause suppression of T-cell mediated immunity, which chimeric DNA, upon transfection into a Treg cell, expresses the extracellular recognition region, the transmembrane region and the intracellular signaling region in one single, continuous chain on the surface of the transfected cell such that the transfected Treg is triggered to activate and cause suppression of T-cell mediated immunity when the expressed extracellular recognition region binds to its selected target antigen or ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Schematic diagram of TNP-specific TPCR structure. (A) Schematic presentation of the TNP-specific chimeric receptors. The TNP-specific CR encompasses a scFv derived from the anti-TNP mAb, Sp6. In the tripartite configuration, the scFv is joined in tandem to a short portion of CD28 (lacking the ligand-binding site) of the extracellular and including the transmembrane, and cytoplasmic domains fused to the FcRγ ITAM domain. (B) Chimeric receptor transgene constructs. Constructs used to generate the transgenic mice were placed under the control of the human CD2 promoter/enhancer that directs expression only in T and NK cells. CYT indicates cytoplasmic domain; H, hinge domain; L, immunoglobulin leader; LCR, locus control region; P, promoter; pL, plasmid sequence; TM, transmembrane domain; VH and VL, immunoglobulin heavy and light-chain variable domains, respectively; ACD28, truncated CD28 containing part of the extracellular and the transmembrane domain, and lacking the cytoplasmic signaling moiety.
FIG. 2: Flow cytometry results of Foxp3 staining of TNP-specific Tregs. Splenocytes isolated from WT and TNP-Tg mice were stained for intracellular Foxp3 and for TNP-specific chimeric receptor using antiidiotypic antibody to the Sp6 scFv. Representative flow cytometry analyses are shown for an individual mouse out of five tested mice. Percentages indicate double-stained cells.
FIG. 3: Graph showing the ratio of CD4+CD25+ cells to CD4+ cells in splenocytes of wildtype and Tg mice. The groups are: wildtype mice, mice Tg for a chimeric receptor specific for an "irrelevant" antigen, ErbB2 (also referred to as ErbB2-Tg mice), TNP-Tg mice that have been transfected with a vector lacking the transgenic costimulatory CD28 domain (also referred to as TNPΔCD28-Tg mice), and TNP-Tg. TNP-Tg Tregs fully express the TNP-specific TpCR
FIG. 4: Graph (left) showing Foxp3+/CD4 cell ratio in wildtype, ErbB2-Tg, TNPΔCD28-Tg, and TNP-Tg mice. Flow cytograms (right) showing splenic Foxp3 expression. Results compare wildtype, ErbB2-Tg, TNP-Tg and TNPΔCD28-Tg mice.
FIG. 5: Flow cytograms showing Foxp3 staining in sorted wildtype, ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg CD4+CD25+ effector T-cells.
FIG. 6: Graph (left) showing ratio of Foxp3+ to CD25+/Foxp3+ cells. Flow cytogram (right) showing co-staining of Foxp3 and CD25. Results compare wildtype, ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg splenocytes.
FIG. 7: Graph showing percentage of Foxp3+ splenocytes in the total CD3+T-cell population following induction of TNBS colitis. Splenic lymphocytes were isolated from WT and TNP-Tg mice prior to or 48 hours following induction of TNP colitis, and double-stained with anti-Foxp3 and anti-CD3 antibodies.
FIG. 8: Graph showing percentage of Foxp3+ lymphocytes extracted from colonic lamina propria following induction of TNBS colitis. Lymphocytes were isolated from WT and TNP-Tg mice prior to and 48 hours following induction of TNP colitis, and double-stained as in FIG. 7. The percentage of Foxp3+ lymphocytes in the CD3+ population is presented as the average Foxp3/CD3 ratio ±s.d. of each five-mouse group. Data shown are averages of two independent experiments performed. Differences in ratios between naive and colitis-induced TNP-Tg mice were significant (P<0.05).
FIG. 9: Series of 6 graphs showing stimulation of proliferation of redirected Tregs (left) and T effector cells (right) by an antigen-nonspecific stimulus (anti CD3 and anti CD28 mAbs) and antigen-specific (TNP) stimulus.
FIG. 10: Graph showing polyclonal activation with Concanavalin A (Con A) of co-cultures of Tregs and T effectors cells (and control cultures of individual cell populations)
FIG. 11: Graphs showing Specific activation of TNP-Tg Tregs and their suppression of effector T-cells requires TNP and CD28-co-stimulation. In the left panel, specific (TNP) activation of Tregs is shown. WT or TNP-Tg Tregs (5×104) were cocultured with WT or TNP-Tg Teff (5×104) in the presence of irradiated, T-cell depleted, TNPylated splenic APC (1.5×105). Teff proliferation was measured after 48 hours by 3H-Thymidine incorporation. Right panel: TNP-loaded APCs as stimulus.
FIG. 12: Graph showing dose-response of TNP-specific stimulation of Treg cell+T effector cell cocultures. APCs were TNP-modified stimulator P815 mastocytoma cells, which do not express B7 (P815-TNP) or TNP-modified P815 cells into which the B7 gene was stably transfected (B7-TNP).
FIG. 13: Graph showing Specific activation of TNP-Tg Tregs and their suppression of effector T-cells requires TNP and CD28-co-stimulation. To establish whether costimulatory signaling is required for TNP-Tg Treg activation, coculture experiments were repeated using as APC irradiated P815 mastocytoma cells (1.5×105) that were either stably transfected (or not) with B7 cDNA. Teff cell proliferation was measured after 48 hours by 3H-Thymidine incorporation. Each group was cultured in triplicate and the experiment was repeated three times. The data shown represent mean (+s.d.) of triplicate cultures of a representative experiment. Differences in stimulation index between Teff+WT Tregs and Teff+TNP-Tg Tregs were significant (P<0.01).
FIG. 14: Photograph of colon of wildtype mice (left) and TNP-Tg mice (right) four days after induction of high-dose TNBS colitis by intrarectal instillation of TNBS at day 0.
FIG. 15: Mortality curve of wild-type (WT), TNP-Tg, ErbB2-Tg and TNP-ΔCD28-Tg mice following induction of TNBS colitis by intrarectal instillation of TNBS at day 0.
FIG. 16: Photomicrograph of stained tissue (H&E, 40×) of the colons from FIG. 14.
FIG. 17: Mortality curve of WT, TNP-Tg, ErbB2-Tg and TNP-ΔCD28-Tg mice following induction of colitis with oxazolone (OXA; a hapten/antigen that is distinct from TNP). These results serve as a control for Treg specificity in the experiment the results of which are shown in FIGS. 14-16. Colitis was induced using the unrelated hapten, oxazolone, which was intrarectally instilled in similar strains of mice (n=10).
FIG. 18: Flow cytograms of Foxp3 staining of wildtype, ErbB2-Tg, TNP-ΔCD28-Tg and TNP-Tg T effector cells after a week of culture in the presence of the following "stimuli" (across top): anti-CD3, TGF-β, anti-CD3+TGF-β, TNP, or TNP+TGF-β.
FIG. 19: Graphs showing mortality or survival rate of wildtype mice subjected to induction of moderate (left panel) and severe (right panel) TNBS colitis following adoptive transfer of the following Treg populations: WT, ErbB2-Tg, TNP-Tg and TNP-ΔCD28-Tg.
FIG. 20: Graph showing Wallach Colitis Severity Score of wildtype mice subjected to induction of TNBS colitis after adoptive transfer of cells from WT, ErbB2-Tg or TNP-Tg donors. TNBS colitis was induced in WT mice (n=8) on day 0. After 16 hours, Tregs (1×105) from TNP-Tg, ErbB2-Tg or WT mice were adoptively transferred to the recipient mice. Each experiment was repeated three times. The data shown represent the average of a representative experiment.
FIG. 21: Photograph of excised colons of wildtype mice in which TNBS colitis was induced, following adoptive transfer of wildtype, ErbB2-Tg and TNP-Tg mice in the experiment described in FIG. 20.
FIG. 22: Photomicrograph of stained colon tissue sections (H&E, 40×) from wildtype mice with TNBS colitis after adoptive transfer of Tregs from the following donors: (A) wildtype (B) ErbB2-Tg and (C) TNP-Tg. Panel D shows normal control colon.
FIG. 23: Localization of Tregs in the colon. Flow cytograms of fluorescent staining Tregs labeled with the intracellular dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the colonic lamina propria of naive mice or mice with TNBS-colitis. Labeled Tregs were intraperitoneally injected 24 hours following induction of TNBS colitis. Lymphocytes from lamina propria were obtained 16 hrs after adoptive transfer of 106 wildtype or TNP-Tg Tregs to indicated recipients. CFSE-labeled Tregs were 9-fold more abundant in diseased colons. Data shown represent the percentages of CFSE-positive cells in the corresponding gates of one representative mouse of each four-mouse group. Each experiment was repeated twice.
FIG. 24: Localization of Tregs in the colon. In-vivo imaging of WT mice receiving DiR-labeled wildtype and TNP-Tg Tregs (1×106) 16 hours following induction of TNP colitis (n=3). Mice were subjected to a whole body imaging (IVIS® 100 Series Imaging System) at 12 hour intervals. A single representative mouse out of three in each group is shown at all time points. Two independent experiments were performed, with similar results. (
FIG. 25: Localization of Tregs in the colon. In-situ fluorescent microendoscopic (Cell Vizio) evaluation of CFSE-labeled Tregs accumulating at the colonic pre-luminal mucosal layer. The experimental design is identical to the one described in FIG. 23. The figure shows representative frames taken 48 hours following adoptive transfer. Each group consisted of four mice, and each experiment was repeated twice.
FIG. 26: Intrarectal administration of TNBS results in TNP-Tg Treg-mediated protective effect from oxazolone colitis. (a) Mortality rates of wildtype and TNP-Tg mice administered oxazolone±low doses of TNBS, 1 week following presensitization with oxazolone only. (b) Murine colonoscopy images of representative WT and TNP-Tg mice. (c) Macroscopic appearance of representative colons from various mouse groups. (d) Microscopic appearance of colons shown in c. (e) Adoptive transfer of Tregs (Tr) to oxazolone (O) pre-sensitized mice induced one week later with oxazolone (O) colitis in the presence of low dose of TNBS (T). WT or TNP-Tg Tregs were administered to mice (n=8) 16 hours after the induction of colitis.
FIG. 27 shows 8 schematic drawings of T cells which are transduced with a retroviral vector that carried one of 8 CR constructs that include the detectable fluorescent protein, eGFP. Those depicted in the lower half of the Figure are bicistronic constructs that encode a fusion of GFP and the transcription factor Foxp3. Light and fluorescence microscopy were used to follow expression of the GFP in the cytoplasm of nucleus of the transduced cells. The constructs are labeled as follows (where "TPCR" refers to "tripartite chimeric receptor" even though, some of these CR's were "more" than tripartite). a. TNP-TPCR: extracellular recognition region comprised an scFv of a TNP-specific mAb. b. MD2-TPCR: extracellular recognition region comprised an LPS binding fragment or motif of the human MD2 protein, an LPS co-receptor (that interacts with TLR4 receptors). The fragment of MD2 corresponds to residues 120-132 of SEQ ID NO:5. The sequences of such chimeric nucleic acids used here are SEQ ID NO:8 and 9. c. CD14-TPCR: extracellular recognition region comprised an LPS binding fragment of the human CD14 protein, a known LPS receptor. The fragment of CD14 corresponds to residues 100-119 of SEQ ID NO:4. The sequences of such chimeric nucleic acids used here are SEQ ID NO:6 and 7. d. MD2-CD14-TPCR: the extracellular recognition region comprised both the MD2 fragment and the CD14 fragments described above. The sequences of such chimeric nucleic acids used here are SEQ ID NO:10 and 11.Each of the constructs encoded as stimulatory and costimulatory moieties, tandemly linked sequences encoding CD28 and FcRγ. Results are shown as side-by-side light and fluorescence micrographs.
FIG. 28. An annotated nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of a TNP-specific tripartite CR as used herein. The annotations include the origin of the regions (scFv, here the "Sp6" mAb), the "CD28" region, and the FcRγ regions (indicated as "GAMMA"), as well as restriction sites, leader sequence, etc. The mature protein begins at amino acid residue 23.
FIG. 29. An annotated nucleotide sequence (SEQ ID NO:3) of a pBullet plasmid that includes a CR-encoding construct that comprises a nucleotide sequence encoding the scFv of mAb HB 9081 (i.e., produced by a hybridoma given ATCC Accession No. HB9081) fused to C28/FcRγ. This mAb and, hence, the scFv, is specific for LPS. Annotations show various restriction enzyme recognition sites, the leader sequence, and plasmid sequences.
FIG. 30A-30B. FIG. 30A is an annotated amino acids sequence of Human CD14 (SEQ ID NO:4). See GenBank Accession No. P08571. A signal sequence and an LPS-binding motif (residues 100-119) are noted. This protein serves as an LPS receptor on cells. FIG. 30B is an annotated amino acids sequence of Human MD-2 protein (SEQ ID NO:5). See GenBank Accession No. NP--056179. A signal sequence and an LPS-binding motif (residues 120-132) are noted. This LPS-binding protein interacts with TLR-4 as a co-receptor.
FIG. 31A-31B. FIG. 31A is an annotated nucleotide sequence (SEQ ID NO:6) showing the nucleotide sequence of a Chimeric Receptor comprising CD14 motif-CD28-FcRγ. FIG. 31B is an annotated nucleotide sequence (SEQ ID NO:7) showing the nucleotide sequence of a chimeric, bicistronic receptor: CD14 motif-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the amino acid sequence (single letter code) of the CD14 motif (residues 110-119 of SEQ ID NO:4). Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
FIG. 32A-32B. FIG. 32A is an annotated nucleotide sequence (SEQ ID NO:8) showing the nucleotide sequence of a Chimeric Receptor comprising MD2 motif-CD28-FcRγ. FIG. 32B is an annotated nucleotide sequence (SEQ ID NO:9) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2 motif-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the amino acid sequence of the MD2 motif (residues 120-132 of SEQ ID NO:4. Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
FIGS. 33A and 33B. FIG. 33A is an annotated nucleotide sequence (SEQ ID NO:10) showing the nucleotide sequence of a Chimeric Receptor comprising MD2 motif-CD14 motif-CD28-FcRγ. FIG. 33B is an annotated nucleotide sequence (SEQ ID NO:11) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2 motif-CD14 motif-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the amino acid sequence of the MD2 motif (residues 120-132 of SEQ ID NO:4) and the amino acid sequence of the CD14 motif (residues 100-119 of SEQ ID NO:3). Nucleotides 106-148 of SEQ ID NO:11 (double underlined) encode a flexible linker (14 amino acids, SEQ ID NO:12, also double underlined). Annotations show various restriction sites, beginnings and ends of protein regions, IRES region, etc.
FIG. 34 is an annotated nucleotide sequence (SEQ ID NO:13) showing the nucleotide sequence of a Chimeric Receptor comprising MD2-CD28-FcRγ (SEQ ID NO:13). Also shown is the amino acid sequence of the full length MD2 protein (SEQ ID NO:4). The LPS-binding region of this amino acid sequence is underscored. Annotations show various restriction sites, beginnings and ends of protein regions, etc.
FIG. 35 is an annotated nucleotide sequence (SEQ ID NO:14) showing the nucleotide sequence of a chimeric, bicistronic receptor: MD2-CD28-FcRγ-IRES-GFP-Foxp3. Also shown is the amino acid sequence of the full length MD2 protein (SEQ ID NO:4). The LPS-binding region of this amino acid sequence is underscored. Annotations show various restriction sites, beginnings and ends of protein coding regions, IRES, vector sequence, etc.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
"Regulatory T lymphocyte" or "Treg cell" or "Treg," as used in the present specification and claims are synonymous and are intended to have its standard definition as used in the art. Treg cells are a specialized subpopulation of T cells that act in a "regulatory" way to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have sometimes been referred to suppressor T-cells. Treg cells are characterized by expression of the forkhead family transcription factor Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They usually also express CD25. As used in the present specification and claims, and unless otherwise specified, Tregs include natural Tregs and induced or adaptive Tregs and Tregs that have been created using recombinant DNA technology. Naturally-occurring Treg cells (CD4+CD25+Foxp3+) arise like all other T cells in the thymus. In contrast, adaptive Treg cells (also known as Trl cells or Th3 cells) may originate during a normal immune response. Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of the activated effector cells, and this subgroup can develop a regulatory (Treg) phenotype. One way to induce Tregs is by prolonged exposure of T effector cells to TGF-β. T-cells may also be converted to Treg cells by transfection or transduction of the Foxp3 gene into a mixed population of T-cells. A T-cell that is caused to express Foxp3 adopts the Treg phenotype and such recombinant Tregs are also defined herein as "Tregs".
"Redirected Treg" is intended to be a comprehensive term for Tregs carrying a chimeric receptor (CR) as described and claimed herein which confers on the cells the ability to bind to and be activated by a target antigen or ligand that is different from that to which a Treg population may have been previously specific (as controlled by its endogenous antigen-specific TCR). Redirected Tregs are "MHC-independent" and "non-MHC restricted" in the process of their activation and in their actions as they do not require association of a peptide derived from their target antigen or ligand with MHC in order to recognize it. However, for special purposes, it may be possible to design a redirected Treg that recognizes a specific epitope of an MHC molecule per se, e.g., functioning as a transplantation antigen. In such a case these redirected Tregs are still non-MHC restricted.
The term "selected target antigen or ligand" means a molecule to which the extracellular recognition region of the redirected Treg is intended to bind so as to activate that Treg. If the selected target is an antigen, then an antibody can be raised against it and the binding regions of such an antibody used to construct the extracellular recognition region of the redirected Treg. If the target molecule is a member of a receptor/ligand pair (defined below), then the other member of that pair can be used as part of the extracellular recognition region of the redirected Treg. Generally, in designing a redirected Treg for use according to this invention, the intended target tissue or site where the Treg is to be employed is first identified, and then, an antigen or ligand that is present on or near this intended target tissue or site is identified. An antibody or ligand/receptor that binds thereto is then identified, or, if necessary, created or constructed for use on the redirected Treg.
The term "ligand" as used herein, and particularly as part of the term "target antigen or ligand" or the term "receptor/ligand pair" refers to a molecule that is able to bind to and form a complex with another biomolecule to serve a biological purpose. Often the binding partner of a ligand is called a receptor so that the two binding partners are termed a "receptor/ligand pair." For the purpose of the present specification and claims, the term "receptor," when used in the sense of a "receptor/ligand pair," has a broader meaning than, for example, a typical definition of a "receptor" as a protein in or on a cell that binds to a specific ligand. It is rather intended to mean any binding partner for a ligand. Either member of a binding pair can be considered the "ligand" while the other member is considered the "receptor." Thus, a classical receptor may qualify as a "ligand" when used herein in the term "target antigen or ligand" as it is one member of a receptor/ligand binding pair. For example, IL-2 can be a ligand because it binds to and forms a complex with another biomolecule, i.e., an IL-2 receptor (IL-2R), to serve a biological purpose. However, IL-2R is also a "ligand" because it is a molecule that binds to and forms a complex with another biomolecule, i.e., IL-2, to serve a biological purpose. Thus, under the present usage, if IL-2R is considered a ligand in the IL2R/IL-2 binding pair, IL-2 may be considered the receptor, and vice versa.
A "chimeric receptor," as used in the present specification and claims, is a recombinant polypeptide that includes an extracellular recognition region that is derived from one molecule and at least one intracellular signaling moiety that is derived from a different molecule. In that sense it is chimeric.
A "chimeric nucleic acid" is a recombinant polynucleotide that includes a sequence that encodes a chimeric receptor.
The terms "recombinant" or "recombinantly" when applied to a polynucleotide, polypeptide or cell means that the molecule or cell is made using genetic engineering techniques and would not exist but for the hand of man.
The term "T-cell signaling polypeptide moiety" means that portion of a molecule endogenous to a T-cell that mediates signaling. It may be a portion of a T-cell receptor molecule that mediates signaling, or a downstream signal-transducing enzyme or a portion thereof that mediates signaling, i.e., that has enzymatic activity.
The term "antibody-derived scFv domain" means a single-chain antibody in which the VL of a specific antibody is linked to the VH thereof by a flexible linker or spacer.
The term "an MHC protein extracellular domain" refers to the disclosure of Meal D J et al. (Proc Nall Acad Sci USA 2005; 102:11817-22), discussed below, and Jodi M D et al., Nat. Biotechnol., 2002, 20:1215-1220. These publications describe Treg cells redirected against T-cells in a murine system. They used the class II MHC Is α and Is β chains as extracellular regions of two separate chimeric receptors for use in a redirected Treg. The term MHC protein extracellular domain is defined so as to encompass what was used in the Meal et al. and Jodi et al. publications and any analogs or fractions thereof that would have been obvious to a person of ordinary skill in the art to substitute for such domains for the purpose disclosed by Meal et al. and by Jodi et al., i.e., to cause binding of the redirected Treg to a T lymphocyte specifically directed to a particular autoantigen.
The term "flexible spacer" means any flexible peptide moiety that will facilitate the functionality of the extracellular recognition region. When this region is not rigidly attached to the transmembrane region, but is allowed some degree of flexibility with respect to the cell membrane, the ability of the recognition region to recognize and bind to its target antigen or ligand is facilitated. Small neutral amino acids, such as glycine and serine, confer such flexibility. Examples are Gly4Ser and Gly4Ser3.
The "immunoglobulin superfamily" (Igs) means the large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Molecules are categorized as members of this superfamily based on shared structural features with immunoglobulins (also known as antibodies); they all possess a domain known as an immunoglobulin domain or fold. Members of the Igs include various cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules and certain cytokine receptors. They are commonly, though not exclusively, associated with roles in the immune system.
The term "hinge" when referring to a region of a molecule of the Igs means the region between the CH1 and CH2 domains consisting of a small number of amino acids. The hinge is flexible and allows the binding region to move freely relative to the rest of the molecule. At the hinge region are the disulfide bridges which link the two dimers, creating the tetramer structural unit. Examples of such immunoglobulin hinge sequences may be found in U.S. Pat. No. 6,165,476, which is incorporated herein by reference.
The term "antigen-specific receptor of a T-cell" refers to a receptor that is found on a T-cell that is antigen-specific, i.e., naturally has an extracellular region that binds specifically to a particular antigen in preference to another. Examples of such antigen-specific receptors of a T-cell are the TCR α, β, γ or δ chains, the TCRαβ dimer and TCR dimer.
The term "TCR/CD3 complex" is sometimes called the "TCR complex." CD3 is a protein complex composed of four chains in mammals (CD3γ, CD3δ and two CD38 chains), that associate with molecules known as the T cell receptor (TCR; see above) and with the ζ-chain and η-chain (as homo- or heterodimers) to generate an activation signal in T lymphocytes. The intracellular tails of these CD3 molecules contain a single conserved motif known as an "immunoreceptor tyrosine-based activation motif" or ITAM for short, which is essential for the signaling capacity of the TCR. The CD3-γ, -δ, and -ε chains and the ζ- and η-chains, also known as CD3-ζ and CD3-η chains, together with the TCR, form what is known as the T cell receptor complex.
The term "T-cell costimulatory-receptor protein" means a receptor of the T-cell that provides a costimulatory signal. During the activation of T cells, costimulation is often crucial to the generation of an effective immune response. T cells require two signals to become fully activated. A first, antigen-specific, signal is provided through the T cell receptor/CD3 complex. A second signal, the costimulatory signal, is antigen-nonspecific and is provided by costimulatory molecules expressed on the T cell membrane. Examples of T-cell costimulatory-receptor proteins are CD28, OX40, CD40L, 4-1BB and PD-1.
The present invention is based on the conception that regulatory T-cells (Treg) that have been modified to possess antibody-type antigen specificity, can be harnessed to suppress T effector cells function in vivo. The action of these Treg cells is mediated in an antigen-nonspecific manner, primarily by release of suppressive cytokines in the vicinity where the Tregs are activated or stimulated by an antigen recognized by their TpCR. Once activated, Tregs can suppress bystander T cell responses. Thus, transfer of these cells that have been engineered to express the CRs (as described herein) to a subject in whom it is desired to suppress a T effector cell response and its attendant or consequent inflammation, and their delivery to, and activation at, the site of such inflammatory activity, results in therapeutic effects.
Thus, preferred target diseases or conditions for this invention are autoimmune diseases, more preferably, organ-specific, T cell-mediated autoimmune diseases. Other examples of undesired immune responsiveness to be targeted herein are graft rejection of solid tissue and organ grafts as well as grafts of suspended cells (e.g. bone marrow (BM) transplants or hemopoietic stem cell (HSV) transplant). Another target disease is graft-vs-host disease (GVH) that is a common consequence of a mismatched BM or HSV transplant. An additional condition targeted by this invention is transplant rejection (e.g., of a mismatched kidney) where the recipient's immune effector cells reject the graft.
Because the Foxp3 transcription factor (a member of the forkhead family) appears to be essential for Treg development and function, and is a distinctive marker for these cells (along with CD4 and CD25), the present invention provides methods for producing Tregs, as well as providing the Tregs produced by those methods, that are based on induction of Foxp3 in T-cells in a process of driving cells along the pathway to Treg status.
In another embodiment, DNA encoding Foxp3 is transduced or transfected into T-cells using any suitable expression vector as a delivery vehicle in a process to drive these cells to become Tregs. Further support for this conception is found in reports that prevention of Foxp3 expression in vivo results in animals with a propensity for development of autoimmune and lymphoproliferative disorders (Sakaguchi S, et al., J Immunol 1995; 155:1151-64; Hori S et al., Science 2003; 299:1057-61; Khattri R, et al., supra; Fontenot J D et al., Nat Immunol. 2003; 4:330-6.).
The starting population can be total PBL, T-cells that have been enriched or isolated from PBL, or CD4+T-cells that have been enriched or isolated from such T-cells (either expressing CD25 or not). These cells can be redirected by transducing the TpCR prior to, concomitantly with, or after transducing DNA encoding Foxp3 DNA, preferably in the form of a Foxp3 expression vector. Walker M R. et al., 2005, Proc Natl Acad Sci USA. 102:4103-8, have shown that antigen-specific human CD4+CD25+ Treg cells can be generated de novo from CD4+CD25- cells. The advantage of the present invention over the approach described by Walker et al. is that the antigen-specificity and Treg activation requirements are independent of the MHC. This important improvement makes isolation and activation of antigen-specific Tregs simpler and allows for therapeutic methods (described below) in which the antigen can be conveniently administered together with the transferred Treg cells to a desired site, such as an inflammatory site, exemplified by the colon in IBD.
Naturally-Occurring vs. Inducible Tregs
"Classical" naturally-occurring Tregs are thymus-derived, express high levels of Foxp3 and suppress activation of effector lymphocytes. Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of the activated effector cells, which subgroup can develop a regulatory (Treg) phenotype. These induced regulatory T-cells can suppress (independently of cell contact) freshly isolated effector cells (Walker M R., et al., 2005, supra; Walker M R., et al., 2003, J Clin Invest. 112:1437-43). In mice, both in vitro and in vivo induction of Tregs is achieved by prolonged exposure of effector cells to TGF-β (Wan Y R, et al., 2005, Proc Natl Acad Sci USA. 102:5126-31; Mantini M C, et al., 2004, J Immunol. 72:5149-53; Mantini et al., 2006, supra). This small, peripherally generated population of inducible Tregs are believed to play a central role in regulating and containing ongoing immune responses just as the lack of Treg induction is associated with a propensity for autoimmunity.
Genetic Manipulation of CD4+CD25+T Regulatory Cells
According to the present invention, approaches that specifically redirect regulatory T-cells to suppress the activity of pathological T-cells are beneficial in inflammatory conditions by facilitating localization of Tregs to inflammatory sites and their specific activation by inflammation-associated antigens. When specifically activated in inflammatory lesions, such Tregs are expected to attenuate inflammatory disease by suppressing pathogenic effector T lymphocytes in an antigen-nonspecific, MHC-unrestricted, manner.
The MHC-independent activation and action of Treg cells according to the present invention is an important advantage. Such action is contrasted with the report of Meal D J et al. (Proc Natl Acad Sci USA 2005; 102:11817-22) of a study of experimental allergic encephalomyelitis (EAE) which described CD4+25+ Treg cells redirected against myelin basic protein (MBP) epitope 89-101-reactive T cells by a CR that included the MBP epitope linked to MHC class II protein. By enforcing the interaction between a Treg cell and the autoreactive T cells directed against MBP epitope 89-101, the Treg activity is antigen-specifically focused against the autoreactive T-cells. Such a model requires some degree of MHC-dependency as a single CR can only have domains of a single MHC and thus can only be used for patients with that HLA characteristic.
In contrast, the Treg cells of the present invention act to suppress pathogenic T effector cells in an MHC-independent manner, making them more advantageous for treating autoimmune/inflammatory conditions because they can target common target antigens shared among many individuals. In the model of Meal et al., supra, Tregs acted by MHC- and antigen-restricted engagement. As such these Tregs, which express the ligand that is recognized by the TCR of the autoreactive T-cells, are stimulated by such interaction and suppress the effector cells. However, as a clinical approach this suffers from the disadvantage that it would require full donor-recipient MHC compatibility in the human population in which MHC (HLA) diversity is substantial. Moreover, such an approach would be limited to suppression of clones that are autoreactive against a single, recognizable peptide epitope in the context of defined MHC-II (HLA-DR). An additional significant advantage of the present invention is that it overcomes the requirement that the pathogenic antigen be known. Indeed, disease-associated antigens in a large number of human autoimmune disorders, including human IBD, are not yet known and may be multiple in number.
The "T-Body" Approach of the Present Invention
The "T-body" approach was designed by one of the present inventors and his colleagues as a novel modality for specific redirection and activation of effector T lymphocytes towards pre-defined targets, mostly those associated with neoplastic processes (e.g., Pin thus JHU et al., J Clin Invest 2004; 114:1774-81) and infectious diseases (Bitton N, et al., Curr Top Microbiol Immunol 2001; 260:271-300). The T-body approach was intended to overcome the relative inaccessibility of antibodies to certain sites (such as solid tumors) and the general ineffectiveness of tumor-infiltrating lymphocytes to combat solid tumors by combining into one effector cell population the properties of the humoral and cellular arms of the immune system (Gross G et al., 1989; supra Eshhar Z, et al., Br J Cancer Suppl, 1990; 10:27-9).
The preferred T-body chimeric receptors comprise a ligand binding portion, preferably (1) a single chain antibody variable region (scFv) directed against a disease-associated antigen, linked to (2) an optional extracellular spacer and a transmembrane region and (3) one or more intracytoplasmic moieties of T cell costimulatory and stimulator/signaling molecules. Such CRs as initially developed enable non-MHC restricted, specific antibody-type recognition, homing and penetration of neoplastic tissues. Within the target tissues, antigen-specific activation of chimeric-receptor bearing T effector cells enabled T cell-mediated destruction of tumor cells either by direct cytotoxicity or by induction of a local inflammatory response.
While the scFv domain is the preferred recognition unit of the present invention, in other embodiments, it may be substituted by another structure that serves as a targeting ligand (or ligand binding partner) that will facilitate bringing the Treg cells expressing the CR to a selected site or a selected antigen. Capon and colleagues have disclosed a number of CRs of this sort, such as one where a ligand binding partner polypeptide is fused at its C-terminus to the N-terminus of an immunoglobulin constant region. See, for example, Roberts M R. et al., Blood 1994; 84:2878-89; Ashkenazi A et al., Int Rev Immunol. 1993; 10:219-27; Chamow S M et al., Int J Cancer Suppl. 1992; 7:69-72. See also U.S. Pat. Nos. 6,710,169; 6,407,221; 6,406,697; 6,319,494; 6,117,655; 6,103,521; 6,077,947; 5,741,899; 5,714,147; 5,514,582; 5,455,165; 5,428,130; 5,359,046; 5,336,603; 5,225,538; and 5,116,964. All of these documents are incorporated by reference in their entirety.
The CR polypeptide of the present invention is characterized broadly as comprising (1) an extracellular portion or domain capable of binding to a ligand (such as a target antigen) in a non-MHC restricted manner, (2) an optional extracellular spacer and a transmembrane domain and (3) a cytoplasmic region (one or more domains) capable of activating an intracellular signaling pathway.
Examples of preferred T cell CRs comprise a first binding domain, a preferred example of which is an extracellular scFv fragment derived from a monoclonal antibody (mAb) specific for a selected antigen. The foregoing domain is fused to a spacer domain (preferably a hinge domain of the Ig family that provides spacing and flexibility), a transmembrane domain, a costimulatory region, for example parts of a CD28 molecule, and a further intracellular signaling moiety for T-cells. Examples of the latter include a TCR/CD3 complex-associated ζ chain or η chain, or an ITAM-containing cytoplasmic region such as the γ chain of an Ig Fc receptor (FcRγ). An ITAM is an "immunoreceptor tyrosine-based activation motif; for reviews, see Humphrey M B et al., Immunol Rev. 2005 December; 208:50-65; Pitcher L A et al., Trends Immunol. 2003; 24:554-60; Isakov N, Receptors Channels. 1998; 5:243-53; Daeron M, Annu Rev Immunol. 1997; 15:203-34; Isakov N, J Leukoc Biol. 1997, 61:6-16; Cambier J C, J Immunol. 1995; 155:3281-5; Flaswinkel H et al., Semin Immunol. 1995; 7:21-7, all of which are incorporated by reference in their entirety. It is also possible to use the intracellular portions of TCR α, β, γ or δ receptor molecules in the CR for this purpose. The signaling moiety of a cytokine receptor may also be present in the chimeric receptor chain for use in the present invention. For example, adding the signaling portion of the IL-2 receptor will cause the Treg cell to further act as if it had been subjected to external IL-2 upon binding of the extracellular targeting domain to the selected target antigen or ligand. Furthermore, adding the signaling moiety of the TGFβ receptor will induce a T effector cell to become a Treg cell and thus this may also be a useful addition to the chimeric receptor chain of the present invention. Such CRs expressed on T-cells are known to be functional and, upon exposure to antigen, promote cytokine production (and, when expressed on appropriate effector cell type in the prior art, promoted lysis of antigen-bearing target cells (Stancovski I, et al., J Immunol 1993; 151:6577-82)).
An early configuration of a scFv-based CR comprised an extra-cellular recognition domain and an intracellular signaling moiety. Full activation of such T-bodies through the CR required either pre-stimulation of the T-body or activation of a costimulatory pathway by exposure to CD08/CD86 (B7)-bearing antigen presenting cells (APCs).
The creation of a tripartite chimeric receptor (TpCR) by one of the present inventors and by others (Pule M A, et al., Mol. Ther. 2005; 12:933-41)), in which the signaling domain of the costimulatory CD28 molecule was added to the cytoplasmic domain of the CR, enabled antigen-mediated activation of both the stimulatory and costimulatory signaling pathways independent of B7-CD28 interactions (Eshhar et al., 2001, supra). This approach facilitates full activation of scFv-expressing lymphocytes, resulting in improved effects (in the case of T effector cells, improved anti-tumor; Pin thus JHU et al., supra).
Another useful intracellular signaling domain for the present invention is all or part of the cytoplasmic domain of a phosphotyrosine kinase (e.g., a molecule of the Syk family) which is fused to the CR. See, for example, Eshhar Z & Fitzer-Attas C J, Adv Drug Deliv Rev. 1998; 31:171-82; Fitzer-Attas C J et al., J Immunol. 1998; 160:145-54.; and Eshhar Z et al., Springer Semin Immunopathol. 1996; 18(2):199-209. Use of such a signaling moiety bypasses membrane-proximal signaling events that are often defective in T-cells of subjects with acute or chronic inflammation or cancer.
Costimulatory Domains/Regions and Signals in the Tripartite Chimeric Receptor
Retroviral-mediated expression of CRs in T-cells in general requires T cell activation which activation is commonly achieved by combined use of anti-CD3 and anti-CD28 antibodies. Such pre-activation was sufficient to prime the T-cells to respond to a signal mediated through the CR upon interaction with the antigen for which the CR is specific--both in vitro and in vivo (e.g., Schwartz R H; Annu Rev Immunol 2003; 21:305-34). A costimulatory signal is advantageous for optimal and sustained T cell function and antigen-driven re-activation, even by targets that often lack ligands for costimulatory molecules.
Antigen stimulation alone of CRs that lack a structure or mechanism for costimulatory signaling is generally inadequate to activate resting or naive lymphocytes (Brocker T et al., J Exp Med 1995; 181:1653-9). Thus, in the absence of costimulatory signaling by CD28, resting T lymphocytes typically undergo anergy or apoptosis (Boussiotis V A et al., Immunol Rev 1996; 153:5-26). For further discussion of CD28 and its interactions with B7, see also, L. Chen (ed.) The B7-CD28 Family Molecules, Landes Bioscience, 2003, which is incorporated by reference in its entirety.
To overcome these limitations in the CR's used in the present invention, the first (recognition) domain, preferably an scFv domain, is linked through an Ig hinge spacer and transmembrane segments to the intracellular segment of a costimulatory signaling molecule, preferably CD28, and then to an intracellular activation region, such as from the CD3ζ chain or the FcRγ chain. Co-expression of two CRs, each with the same scFv, the first linked to CD3ζ and the second to CD28, was found to provide the requisite stimulatory and costimulatory signals for T cell activation (Beecham E J et al., J Immunother 2000; 23:631-42).
Thus, in one preferred embodiment herein, an extracellular recognition site, preferably an antibody-based recognition site such as an scFv, is linked to a CD28 intracellular domain "in series" and further linked to the intracellular signaling region of the TCR complex ζ chain. Such a construct was 20-fold more potent in stimulating IL-2 production upon exposure to solid phase antigen (compared with transfectants expressing CR's lacking the CD28 domain (Finney H M et al., J Immunol 1998; 161:2791-7)). Intracellularly, this domain in the CR binds the p85 subunit of phosphatidylinositol 3'-kinase.
One of the present inventors designed a novel tripartite CR composed of a scFv recognition moiety fused to the non-ligand binding part of the extracellular domain (ECD) of CD28, the entire transmembrane and intracellular domains of CD28, and the intracellular stimulatory domain of FcRγ ("scFv-CD28-γ") (Eshhar et al., 2001, supra). Human PBL transduced with a nucleic acid construct encoding this CR were specifically stimulated to produce IL-2. Activation was dependent on CD28 costimulatory activity.
The present inventors' laboratory has generated several lines of Tg mice expressing CRs under control of T cell-specific regulatory sequences. T lymphocytes from unprimed, naive mice that are Tg for the scFv-CD28-γ TpCR manifested potent responses (proliferation, IL-2 secretion, and rescue from apoptosis) upon stimulation solely by the cognate antigen in immobilized form (Friedmann-Morvinski D et al., supra).
According to the present invention, molecules other than, or in addition to, CD28 are exploited to provide costimulatory signals when included in the present CR configuration. Preferred examples of these are the members of the "inducible co-stimulator" (ICOS) family, including OX40 (CD134), CD40 ligand (CD40L, CD154), PD-1 ("programmed death receptor-1), and 4-1BB (CD137). Each of these ligand/receptor pairs possess distinct functions that differ according to the nature of the stimulus and the "antigenic history" of the T-cells on which they are expressed. For example, CD28 signaling is accompanied by induction of ICOS, which, in turn, co-stimulates CD4+T cell activation. The engagement of OX40 (studied in the context of tumor-specific adoptive immunotherapy) improved survival and anti-metastatic actions of T effector cells by CD4+T helper cell-dependent mechanism (Weinberg A D, Trends Immunol 2002; 23:102-9). Activation of OX40 promotes expression of anti-apoptotic proteins Bcl-XL and Bcl-2 and, accordingly, enhances the survival and hence the number of antigen-specific CD4+ T-cells, resulting in strong antigen-specific CD4+T cell memory. Engagement of 4-1BB (CD137) costimulatory receptor with its ligand, 4-1BBL, increased TCR-induced proliferation, survival, and cytokine production in both CD4+ and CD8+T-cells (Cheuk A T et al., Cancer Gene Ther 2004; 11:215-26). Cell survival was associated with increased expression of the anti-apoptotic genes bcl-XL and bfl-1. In general, the interacting ligand/receptor pair 4-1BB/4-1BBL acts to amplify existing costimulatory signals, particularly those emanating from CD28 (Guinn B A et al., J Immunol 1999; 162:5003-10). Human CD4+T-cells express PD-1 and its ligands, PD-L1 and PD-L2, upon activation. Antibodies to the receptor can be agonists or antagonists of the apoptotic pathway. PD-1 engagement can promote ICOS- or CD28-mediated costimulation. (e.g., Bennett F et al., J Immunol. 2003; 170:711-8.
The activity of costimulatory domains of CD28, ICOS, OX40 (CD134), and 4-1BB (CD137) in CRs is also known in human CD4+ and CD8+T-cells (Finney H M et al., J Immunol 2004; 172:104-13). In that study, the tripartite genes were electroporated into cells to avoid pre-activation of the cells. When CR-bearing T-cells were stimulated by their specific antigen (CD33), cytokine release and cytotoxic activity were dramatically enhanced compared to cells in which the CRs lacked costimulatory signaling structures. Inclusion of the 4-1BB signaling domain as the costimulatory moiety in a TpCR on human T-cells with specificity against the CD19 antigen (anti-CD19-1BB-ζ) led to potent cytotoxicity against CD19-bearing acute lymphoblastic leukemia target cells in vitro (Imai C, et al., 2004; 18:676-84).
While the present invention includes the use of an intracellular domain or part of any of these costimulatory sequences in the CR, it is not certain that signaling evoked by these molecules has practical advantages over use of the CD28 costimulatory sequences alone. So, even though the performance of CD28 appears thus far to be quite satisfactory both in vitro and in vivo, the present invention includes within its scope the use of additional or alternative costimulatory systems to CD28 for generating Treg cells that perform optimally in suppressing T effector cells and treating autoimmune/inflammatory and other conditions as described herein. 4-1BB has been used successfully as an alternative to CD28 in T-bodies. See Zhang et al., J. Immunol., 2007; 179:4910-4918.
Transfer of Redirected Tregs to Recipient Subjects
Use of transferred T-cells in vivo in adoptive therapy requires that transferred cells survive, overcome the host's homeostatic control mechanisms that may serve to hinder the acceptance of these cells, and migrate to (home to, or traffic to) and accumulate or localize at, the desired target site(s).
The immune system utilizes internal stimuli to regulate the total size of lymphocyte pools. The total number of peripheral T lymphocytes remains fairly constant, despite production of new cells, turnover of existing cells, and clonal expansion of antigen-specific cells during an immune response (Jameson S C. Nat Rev Immunol 2002; 2:547-56.). These "internal stimuli," include cytokines and self-peptide-MHC ligands for the TCR. At least two general mechanisms are believed to be responsible for homeostatic effects of bystander T-cells in limiting proliferation: (1) inhibition by physical T cell-T cell interactions; and/or (2) competition for limited "resources" (e.g., IL-7 and access to APCs with suitable self-MHC ligands). The most prominent cytokines in this process are those that signal through receptors containing a common γ chain, termed collectively "γC cytokines." These include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Homeostatic control of naive T cell expansion (examined in vitro) is supported by IL-4, IL-7, IL15 and IL21 through the CD28 transmembrane region, whereas only IL-7 appears to be required in vivo (Jameson, supra).
Lymphodepletion or "lymphoablation" is preferably performed to condition a recipient of the transduced Tregs of the present invention. Any method known in the art may be used, for example, irradiation, treatment with certain antimetabolites such as fludarabine, etc. Such treatments have been used in conjunction with adoptive T cell therapy in other contexts. Lymphodepletion in vivo performed as a precursor to adoptive cell transfer is known to boost antitumor immunotherapeutic activity in mice and in humans (as studied particularly with autologous, tumor-reactive T effector cells; Klebanoff C A et al., Trends Immunol 2005; 26:111-7). In clinical trials, objective response rates of 50% were seen in patients with solid metastatic tumors who had first been subjected to lymphodepletion. The mechanisms that are believed to underlie such effects include: the elimination of cellular cytokine `sinks` for homeostatic γC-cytokines (such as IL-7, IL-15 and possibly IL-21 (which serve to activate and expand effector T-cells)), induction of tumor cell apoptosis and necrosis in conjunction with APC activation, and, most important for the present invention, the impairment of CD4+CD25+ Treg cells that suppress T effector cells.
As noted, treatment with homeostatic cytokines may be used to maintain the Treg populations in the recipient.
The present inventors' group found that activation of T-cells in general and T-bodies in particular (such as that required during the ex vivo manipulations to express the CR with certain vectors) down regulated expression of the chemokine receptor CXCR4, thereby impairing T cell homing in response to the chemokine SDF-1, for example. SDF-1 is a chemoattractant for T-cells that express the CXCR4 (Bleul C C et al., J Exp Med 1996; 184:1101-9; Beider K et al., Blood 2003; 102:1951-8). Using ErbB2-specific human T-bodies; these investigators showed that this homing is an essential step for the T effector cells to act in vivo, measured as inhibition of advanced prostate cancer progression and even cure (Pin thus et al., supra). Based on the foregoing knowledge, according to the present invention, redirected Treg cells must either home/migrate to the desired target sites or be administered to such sites.
Persistence of Responses of TpCR-Bearing T-Cells
A key factor for success of adoptively transferred T cell therapy (which thus far has been examined most thoroughly with T effector cells in cancer) is maintenance of the transduced T-cells' (effector) function. In one embodiment of the present invention, it is desired to maintain the function of Tregs that have been administered to perform a suppressive function. In another embodiment, it may be preferred that the Tregs act in shorter "bursts" to curtail a more acute (rather than a chronic) T effector response.
Because lymphocytes found in tumor patients include CD4+CD25+ Treg cells that suppress T effector cells (Wang H Y et al., Immunity 2004; 20:107-18; Curiel T J, et al., Nat Med 2004; 10:942-9), such "endogenous" suppressive activity must be overcome to optimize the action of redirected T effector cells. In the present invention, the objective is the converse: redirected Treg cells are administered to a subject in need thereof to quell or otherwise inhibit immune/inflammatory responses that characterize autoimmune conditions, transplant rejection, etc.
Examples of Clinical Trials Using Redirected T-Cells
While clinical trials using Tregs in accordance with the present invention have not yet been carried out, a number trials using redirected, CR-bearing T effector cells are described below. Advantage may be taken of various lessons learned in those trials in practicing the present invention.
In a Phase I trial in HIV infected subjects, autologous lymphocytes bearing a CD4-ζ CR were administered (Mitsuyasu R T, et al., 2000, Blood 96:785-93). Out of 24 patients, 11 also received concurrent IL-2 infusions for 5 days. The treatment was well tolerated. In some patients, a transient decrease of the viral load was observed in plasma and rectal mucosa (the tissue reservoir for HIV). All subjects tested negative for replication-competent retrovirus (the delivery vector) for up to 1 year after infusion.
Cell Genesys, Inc. conducted phase I clinical trials in colorectal cancer patients using an anti-TAG72-ζ CR made from the humanized CC49 mAb (Warren R et al., In: 7th International Conference on Gene Therapy of Cancer; 1998).
The group of Junghans tested 24 doses of CR-bearing lymphocytes the antigen-specificity of which was directed to CEA in colorectal patients. Up to 1011 cells/patient were given. The treatment was adequately tolerated (Junghans R et al., Proc Am Assoc Can Res, 2000, 41:543).
Hwu and co-workers at the National Cancer Institute conducted a phase I clinical trial in ovarian cancer patients using T-bodies expressing a CR directed against the MoV18-murine anti-folate-binding protein. Large doses of the modified cells were infused into patients together with controlled administration of IL-2. No adverse side effects were reported. Neutralizing antibodies specific to murine MoV18 mAb determinants were found in the sera of several patients (Kershaw M H et al., Clin Canc Res, 2006; 12:6106-15.
A Phase I clinical trial in renal cell cancer (RCC) employed autologous G250-specific genetically modified T lymphocytes (Lamers C H J et al., Daniel den Hoed Cancer News, 2004, 2:8-10). Infusions of these cells were clinically well-tolerated. After 4-5 infusions, patients began to develop liver enzyme abnormalities, a finding explained by the reactivity of the infused T-cells with G250L expressed on bile duct epithelium, albeit at low levels. Treatment was thus limited to only low doses of CR-expressing T-bodies. The results showed in any case that the redirected T-cells did exert CR-dictated functions in vivo.
Two other Phase I clinical trials have been initiated though their results have not yet been reported to the best of the inventors' knowledge. One Phase I trial treated neuroblastoma patients with PBLs and Epstein Barr virus-specific CTLs, both expressing GD-2 specific chimeric T cell receptors (Brenner M K. World wide web URL clinicaltrials.gov/ct/show/NCT00085930, 2005). The other trial employs genetically modified CD20-specific CD8+CTLs for relapsed follicular lymphoma (Wang J, et al., Mol Ther 2004; 9:577-86
The present inventors recognize that certain events may interfere with the efficacy of the therapy using Treg cells expressing CR's in vivo in humans, for example: (1) formation of neutralizing anti-idiotypic antibodies directed to an idiotope of the scFv part of the CR that could reduced the life-span or effectiveness of the Tregs; (2) the low proportion of engineered cells that eventually reached the targeted sites and; (3) the potential damage to healthy tissue that expresses the targeted antigen.
The use of Tregs according to the present invention has a much lower risk of (3). As described herein, direct administration of Tregs to sites of inflammation should overcome the limitation of (1)-(3). Adjustment of dose regimens (number of cells, frequency of administration) using routine clinical considerations are expected to limit the impact of the above limiting factors.
According to the present invention, an effective amount of redirected Treg cells are administered to a subject. Preferred carriers for the Treg cells are phosphate buffer, preferably 0.01-0.1M, more preferably 0.05M, or 0.8% saline. Acceptable diluents or carriers for various routes of administration are well-known.
While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Determination of the effective amounts can readily be made empirically by those of ordinary skill in the art without undue experimentation.
Typical dosages are between about 106 and about 1011 Treg cells per injection or infusion, more preferably, about 107 to about 1010 cells. If an antigen is to be administered with the cells (or separately, but to a site where it is intended to activate the cells), a dose of about 0.01 to 100 mg/kg/body preferably, 0.1 to 50 mg/kg/body wt is preferred.
An effective amount of Treg cells is that needed to induce a measurable change, generally a decrease, in the severity of any measurable symptom of the disease, preferably more than one symptom, and most preferably, would result in cessation of symptoms and cure of the disease or condition. For example, without limiting the invention, the above decrease may be at least about 10%, more preferably at least about 20%, more preferably at least about 30%, even more preferably, at least about 40%, and more preferably, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%. It is within the skill of the clinical arts to determine when such therapeutic goals have been achieved, and to adjust the dose or frequency of administration accordingly, or to cease further treatment.
The Treg cells of the invention may be given once, or on multiple occasions, via a single or multiple routes. The cells may be administered daily, or preferably on alternate days, preferably weekly or biweekly. Administration can range over an interval of several days to weeks, or even months or years. The frequency and duration of administration can be determined empirically, or based on the clinical history and experience of the subject.
The cellular compositions of the present invention can be administered by any of a number of means and routes known in the art. Administration is preferably parenteral. Preferred routes include, intravenous, intramuscular, subcutaneous, intraperitoneal, intra-articular, intracerebroventricular, intraluminal (preferably into the lumen of the ileum or colon), rectal or the topical route. Also included is the "intrathecal" route, which is intended to encompass injection, infusion or instillation directly into a cavity or space (thecum) surrounding an organ or body region in which an undesired immune/inflammatory response is occurring. Such spaces include the pleural space, peritoneum, subarachnoid space or dural space, or pericardial space. The generic term for administration into a sheath encasing an organ is termed "intrathecal (see, for example, definition in Dorland's Medical Dictionary 29th Edition, WB Saunders (2000) and Stedman's Medical Dictionary, 27th Edition, Lippincott, Williams & Wilkins (2000)) as meaning "within a sheath." As used herein, this term is intended to be broader than a more commonly used definition which is limited to intracranial spaces.
The compositions, methods, and products of this invention are applicable to human and veterinary uses. The preferred subject is a human.
Transgenic Mice Expressing TNP-Specific Chimeric Receptors
Several Tg strains of mice that express the TNP-specific TpCR, that were recently produced by the present inventors and their colleagues (Friedmann-Morvinski D, 2005) are described herein. These mice are the source of TNP-specific T effector and T regulatory cells and are used as experimental animals in which the induction of colitis is evaluated using the `classical` reactive hapten, TNBS. As a control for these CR-bearing cells, cells from erbB-2-specific TpCR Tg mice that were produced in the present inventors' laboratory are used as they express a CR specific for an irrelevant antigen.
All mature T-cells and NK cells in these Tg mice express the scFv-CD28-FcRγ construct. Naive Tg T-cells can be fully activated by plastic-immobilized TNP without the need for pre-sensitization. (Friedmann-Morvinski D, et al., supra). Results in the Examples herein show that splenic CD4+CD25+ Tregs isolated from such mice specifically suppress proliferation and cytokine secretion by TNP-specific effector T-cells. Moreover, these Tregs are responsible for the delayed development and attenuation of TNBS-induced colitis in these animals. Of importance is the fact that the level of Tregs in the periphery of the TNP-specific TpCR-expressing strains is higher than in wild-type (WT) mice and that the Tregs do not require pre-activation to exhibit their suppressive activity in vivo. This is believed to result from the cross-reactivity of the SP6 mAb, from which the scFv of the TpCR was derived.
Delivery of DNA Encoding the CR into T-Cells
Genetic modification of human peripheral T-cells is achieved in one embodiment using retroviral vectors (Eshhar Z, et al., 2001, supra). As a non-limiting example, the pBullet vector is used, into which the CR-encoding cDNA (Weijtens M E, et al., 1998) is introduced. A bicistronic expression construct is used in which the TpCR and eGFP cDNA are expressed under control of the LTR. This serves to generate a packaging cell based on PG13 that is being used to pseudotype the retroviral vector with the gibbon ape leukemia virus (GALV). Flow cytometric sorting is done on the basis of eGFP expression, and packaging cells producing high-titer virions are selected to achieve high transduction efficacy. To transduce human lymphocytes from healthy donors, lymphocytes are activated in culture with plate-bound anti-CD3 and anti-CD28 mAbs (or using commercial microbeads coated with these antibodies; e.g. from Invitrogen, Miltenyi Biotec, Inc.,) and are transferred to plates coated with Retronectin® (fibronectin fragment CH-296) plates together with fresh supernatants taken from the packaging cells. At the end of the process that takes 5-8 days, cells are propagated in the presence of IL-2 and then harvested and used. Following this ex vivo procedure, 45-70% of the cells are positive for CR (and GFP) expression.
Useful additional reagents are anti-idiotypic antibodies against idiotopes of the scFv of the TpCR. These enable direct labeling and visualization of TpCR on cell membranes. Such antibodies against the SP6 scFv (exemplified below) have been made and used by the present inventors.
An annotated nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of the TNP-specific TpCR used herein is shown in FIG. 27. The mature protein begins at amino acid residue 23 of SEQ ID NO:2.
A preferred sequence that excludes the scFv above, and that can be linked to any other appropriate ligand binding region, preferably a different scFv specific for another antigen, is that defined by the above sequences beginning at the CD28 region. Thus, a preferred nucleotide coding sequence is nucleotides 2203-2523 of SEQ ID NO:1 and amino acids 260-367 of SEQ ID NO:2. Additional nucleotides comprising a 5' restriction site, and amino acids "inadvertently" encoded thereby, may also be included in a preferred sequence. Additional coding sequence added at the 3' end of 2203-2523 of SEQ ID NO:1, or additional amino acids encoded thereby and added to at the C-terminus of 260-367 of SEQ ID NO:2, may be present, provided that they permit the encoded sequence, as expressed on the redirected Tregs, to function as a TpCR in ways described herein. Those skilled in the art of cloning and recombinant DNA technology will understand how to modify theses sequences to achieve the desired objective without undue experimentation.
Expression vectors comprising the foregoing sequences are also used in the present invention, in the production of redirected, TpCR-expressing Tregs.
Generation and Expression of TpCR and Foxp3-GFP Fusion Gene and its Expression
Redirected T-cells are "converted" to Tregs by causing them to express both Foxp3 transcription factor and the antigen-specific TpCR. Such manipulation permits production of large numbers of Tregs for evaluation and therapeutic use. Successful co-transduction or co-expression is tested by including a Foxp3-GFP fusion gene in the same construct as a TpCR to express both in the same cells. This approach is particularly useful when the starting cell populations are human PBL in which Tregs constitute only about 3-5% of CD4+T-cells. This avoids the complications of another approach, also within the scope of the invention, in which large scale Treg propagation is required for effective transduction with retroviral vectors. Moreover it will simplify the isolation of the Tregs and assessment of their fate in vivo.
In one non-limiting example, messenger RNA (mRNA) for Foxp3 is cloned from purified Tregs using PCR. Foxp3 cDNA is cloned into an eGFP Clontech plasmid to create a Foxp3-GFP fusion protein. The fusion protein is cloned into the pBullet vector containing TpCR inserted after an IRES to create a bicistronic expression vector. Both a Foxp3-GFP single gene retroviral vector and a bicistronic TpCR--IRES-Foxp3-GFP double gene retroviral vector are transduced into isolated CD4+CD25- human peripheral blood T-cells following their activation with anti-CD3 and anti-CD28 antibodies. The resulting cells are tested for expression of the three genes by FACS using (1) antiidiotypic antibodies specific for the scFv idiotype, or anti-hinge region antibodies and (2) intracellular GFP and Foxp3 by staining fixed cells with primary antibodies specific for Foxp3 (Alexis Biochemicals, Lausanne, Switzerland).
In another embodiment, sequential expression protocols are used (first TpCR and then Foxp3-GFP genes) or co-expression protocols. Once the genes are expressed, relatively large number of Tregs can be obtained and separated using cell sorter (FACSaria fluorescence-activated cell sorting (Becton Dickinson, Mountain View, Calif.), sorting for GFP and TpCR co-expressing cells.
The Foxp3 construct may be in the form of a bicistronic vector that includes DNA encoding a reporter molecule such as a fluorescent protein. Suitable reporter molecules are well-known in the art and include fluorescent, chemiluminescent or chromogenic proteins, for example Green fluorescent protein (GFP) or enhanced yellow fluorescent protein (EYFP) or a fluorescent homologue thereof, firefly luciferase protein (encoded by the Luc gene) the enzymes chloramphenicol acetyl-transferase (CAT), or bacterial LacZ, (β-galactosidase) or the thymidine kinase gene (encoded by the HSV1 TK gene. GFP and EYFP are detected by fluorimetry or fluorescence histochemistry; enzymes are detected by use of a chromogenic substrate that is converted into a colored product which can be used in histochemical colorimetric detection of enzymatic activity. Luciferase is measured by activation of luciferin which emits light at a known wavelength. Reporter molecules may be detected in vivo by non-invasive detection techniques such as fluorescence optical imaging (FOI), bioluminescence optical imaging (BOI), cooled charged coupled device (CCD) camera optical imaging (CCOI) and positron emission tomography (PET).
Infection of human CD4+CD25- T lymphocytes with retroviral vectors carrying the Foxp3 gene was shown to convert these cells into ones with a Treg phenotype (Walker et al., 2005, supra; Wan et al., 2005, supra).
Any method for introducing DNA into a cell and expressing it may be used in the present invention, including, but not limited to vectors such as retroviral or lentiviral vectors, electroporation, lipofection, and the like.
The functionality of redirected Tregs can be determined using co-culture tests as described in the Examples. If APCs are to be used in such tests, a preferred source is irradiated monocytes. The antigen is loaded into irradiated human APCs which will present it to T effectors and Tregs. In the case of antigens such as CEA, human colon carcinoma cells stably transfected with the CEA epitope may be used. In such coculture tests, one may detect specific activation of TpCR-bearing Tregs through the TpCR. Treg activation is assessed by examining these cells' action on T effector cell (1) proliferation and (2) cytokine secretion profile, focusing on IL2, IL4, IL10, IFN-γ and TGF-β (using commercial ELISA kits, e.g., Ready-Set Go ELISA kit, Ebioscience CA). It is preferred to assay TGF-β and/or IL-10 as an indication of the cells' Treg phenotype.
In a preferred embodiment, the present invention redirects Tregs to sites of colonic inflammation, by introducing into such cells CRs with antibody-type specificity. In sites of inflammation the redirected Tregs are activated to suppress IBD-associated immune response. Tregs endowed with predefined specificity migrate and home to inflamed sites in the colon where they undergo activation and, as a result, suppress T effector cells that mediate the disease processes.
The present redirected Tregs represent a novel form of the `T-bodies` discussed above and are employed as a novel therapeutic modality in IBD. These T-bodies are T-cells that have been genetically engineered to express TpCR in which an antibody variable region is the recognition unit linked to T-cell costimulatory and stimulatory domains that enable specific activation of these T-cells but in a manner that is MHC independent and not MHC-restricted. Based on previous studies using tumor models described above, these redirected Tregs are tested in murine models of IBD models.
An important aspect of this invention is the inventors' conception that, in the context of treating IBD, the colon-associated antigen(s) to which the T-bodies are redirected and targeted are not necessarily the pathogenic autoantigens recognized by the autoaggressive T effector cells. Thus, this invention can exploit the phenomenon of "bystander" reactivity--where the presence of the relevant antigens at the sites of the inflammatory reactions serve to attract and "hold" or localize the redirected Tregs, permitting them to be activated and to exert their suppressive effects in a paracrine manner--acting on target effector cells in the vicinity irrespective of differences in the T effector cells' and Treg cells' antigen specificity.
CEA and LPS-Colonic Antigens as Targets for Redirected Human Tregs in IBD
Advantage was taken of a hapten-specific IBD model that is based on specificity to the hapten TNP to study the suppressive effects of Tregs. In human disease, other antigens that are expressed in intestinal or colonic tissue either normally or in the relevant disease state are preferred targets. The include carcinoembryonic antigen, CEA, and bacterial floral antigens such as lipopolysaccharide, LPS.
Human IBD is idiopathic to the extent that pathogenic antigen(s) remain unknown. Lack of knowledge of the antigen would appear to be an obstacle to implementing the T-bodies clinically. Nevertheless, according to the present invention, there is no requirement that a pathogenic antigen must also be the target antigen for Treg redirection and activation. Treg activation is indeed antigen-specific and thus depends on TCRs, or in the present Tregs, on antibody-based specificity, associated with costimulation together with the activation/mediated by the intracellular signaling moieties of the present constructs. However, once the Tregs are activated, their suppressive action is antigen-independent, and is carried out by secretion of suppressive cytokines (e.g., TGF-β and IL-10) even after the activating antigen has been eliminated. Thus, inducing colonic Treg activation by any local colon-associated antigen will promote potent Treg activation and proliferation, while the action of these cells in inhibiting local inflammatory processes proceeds independently of antigen. CEA is significantly over-expressed in diseased colon tissue in patients with active ulcerative colitis compared to normal individuals and to patients with quiescent IBD (Smithson J E et al., J Pathol. 1996; 180:146-51; Pavelic Z P et al., Anticancer Res. 1991; 11:1671-5). This enhanced tissue expression of CEA was independent of dysplastic changes and is a result of the mucosal reaction to the inflammatory process itself. Thus, CEA is a preferred candidate for Treg TpCR targeting in active ulcerative colitis.
A second candidate antigen (or "non-antigen" ligand) to which Tregs may be redirected is endotoxin or LPS, derived from the outer membrane of Gram-negative bacteria resident in the colon. In one embodiment, the antibody-like part (scFv) the CR's extracellular recognition region may be derived from an anti-LPS antibody, such as the mAb produced by the hybridoma with ATCC Accession No. HB9081. The nucleotide sequence of an scFv made from this mAb is shown as an annotation in FIG. 29 as part of the full sequence of a plasmid (pBullet) comprising this scFv--SEQ ID NO:3. Thus, a Treg expressing a TpCR that displays this scFv extracellularly will, at a site where LPS is present such as inflamed colon tissue (whether the gut lumen, the lamina propria or even regional lymph nodes and other gut-associate lymphatic tissue) bind the LPS and be activated to cause suppression of any T effectors cells in the vicinity in an antigen-nonspecific and MHC-independent manner.
Several types of non-antibody LPS receptors are known in the art. CD14 (SEQ ID NO:4) is a class of LPS receptor that is a GPI-anchored 356 aa glycoprotein. It contains a 19aa signal peptide, an extracellular domain which contain 11 leucine-rich repeat (LRR) domains, 4 N-glycosylation sites and an unknown number of O-glycosylation sites. At least 2 soluble forms of CD14 have been described, one retains GPI and is released from the cell surface which results in an approximately 48 kDa molecule and the other is released prior to the addition of the GPI anchor resulting in a higher molecular weight (>48 kDa).
While LPS interacts with CD14, CD14 is not capable of initiating a transmembrane activation signal because it is a glycosylphosphatidylinositol (GPI)-anchored protein. Thus, LPS must interact with a transmembrane receptor(s) that is responsible for signal transduction. LPS is recognized by the toll-like receptor TLR4 and MD-2 (SEQ ID NO:5; human), a molecule associated with the extracellular domain of TLR4. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, apparently by LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. (Akashi S, et al., J. Exp. Med. 198:1035-42 (2003)).
Interaction of LPS with MD-2 in a TLR4-MD-2 complex triggers an intracellular signal transduction cascade that leads to the production and release of proinflammatory cytokines, particularly TNF-α (Dauphinee S M et al., 2006, Lab. Invest. 86, 9-22). Patients with IBD show increased colon and serum levels of endotoxin, LBP, CD14, and MD-2 (Pastor Rojo O, et al., 2006, Inflamm Bowel Dis., December 19 (epub); Amati L et al., Curr Pharm Des. 2003; 9:1937-45; Cario E et al., J Immunol. 2006; 176:4258-66). This change correlates with disease activity, and proinflammatory cytokine levels return to normal after treatment.
A motif of human MD-2, for example, from amino acids 119-132 (14 residues) of SEQ ID NO: ______ can substitute for MD-2 in MD-2-TLR4 complex binding to the lipid A moiety of LPS, which (Mancek M et al., Biochem Biophys Res Comm 2002; 292: 880-5; Kobayashi M et al., J Immunol. 2006; 176:6211-8).
Thus, in one preferred TpCR of the present invention, the extracellular recognition region comprises, in place of an antibody-like structure (e.g., an scFv), a receptor that binds to a ligand that is not acting as an "antigen." A preferred ligand in the present invention is LPS. Thus, the extracellular recognition region may comprise any of the following receptor structures: (a) CD14 (SEQ ID NO:4), (b) an LPS-binding motif of CD14, such as residues 100-119 of SEQ ID NO:4, (c) full length MD-2 (SEQ ID NO:5), (d) an LPS-binding motif of MD-2 (residues 120-132 of SEQ ID NO:5), (e) a combination of a CD14 and MD-2 or (f) a combination of a CD14-motif and an MD-2 motif (as is encoded by the relevant segment of the chimeric nucleic acid of SEQ ID NO:10.
Any of these constructs, when displayed on a Treg surface, will allow the redirected Treg to bind to, and be activated by LPS molecules, for example, at colon inflammatory sites, and thereby exert their suppressive activities in that vicinity. Again, this is an example of receptor-ligand binding/recognition that is not "antibody-like" but nevertheless permits the TpCR to act in accordance with this invention and activate Tregs in an antigen-nonspecific (and MHC-independent) manner.
The present invention includes an embodiment in which redirected Tregs bearing a TpCR are designed to be specific for an antigen, referred to herein as "AgX," that may have no inherent relationship with the tissue being targeted or the disease being treated. In this embodiment, the Tregs specific for AgX are activated specifically in a selected site by administering them together with AgX to that site. The site is one where T effector cells are situated and active, where the ongoing inflammation is to be suppressed. The AgX-specific antibody-like receptor of the Tregs will recognize AgX without a need for antigen presentation, MHC, etc., and the linked signaling moieties on the TpCR will serve to activate the Tregs to release inhibitory cytokines at that site. This process will lead to nonspecific suppression of the ongoing T effector cell and inflammatory activity.
The methods and compositions described herein are useful for any of a number of autoimmune diseases which involve undesired effector T-cells activity as an underlying cause or as a consequence of the pathophysiology. Such diseases include, but are not limited to, IBD, rheumatoid arthritis, Type I diabetes, multiple sclerosis, autoimmune thyroiditis, autoimmune uveoretinitis, autoimmune orchitis, autoimmune insulitis, autoimmune oophoritis, psoriasis, autoimmune polymyositis and the like. See, for example, Theofilopoulos, A., In: Stites, D P et al., eds., Basic and Clinical Immunology, Lange Medical Publications, Los Altos, Calif., 1988)).
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
Materials and Methods
The following materials and methods are used in various of the Examples that follow, as well as in carrying out certain embodiments of the invention.
Cell Fractionation and Isolation
CD4+CD25+ Tregs were purified from splenic lymphocytes or peripheral blood mononuclear cell populations using several methods. One method utilized magnetic bead separation (MACS). Spleens are mashed gently into HBSS/5% FCS to prepare single cell suspensions.
CD4+T-cells were purified by negative selection by incubation with biotin-conjugated CD4 MACS beads (Miltenyi Biotec, Inc., Auburn, Calif.). Further purification of CD4+CD25+ cells was conducted by incubation with phycoerythrin (PE)-conjugated anti-CD25 antibodies or anti-CD45RBhigh, followed by incubation with anti-PE microbeads (Miltenyi Biotec, Inc., Auburn, Calif.). Magnetic separation was conducted using magnetic columns according to manufacturer's instructions. For highly-purified (>99%) Treg and effector T lymphocyte subpopulation, high-speed cell sorting is be applied, using BD FACSaria (®) cell-sorting system (BD Bioscience)
Lamina propria lymphocytes from colon were isolated as previously described (Han X et al., Gastroenterology. 2005; 129:185-203). Briefly, colonic mucosa was dissected, followed by incubation with Ca2+--Mg2+-free Hanks' balanced salt (HBSS) solution containing 1 mM dithiothreitol (Sigma-Aldrich, St. Louis, Mo.) for 30 min to remove mucus, and then serially incubated twice times in medium containing 0.75 mM EDTA (Sigma-Aldrich) for 60 min at each incubation. The supernatants from these incubations containing epithelium and intraepithelial lymphocyte population are discarded, and the residual fragments pooled and treated with 2 mg/mL collagenase A (Worthington Biomedical, Freehold, N.J.) and 0.01% DNase (Worthington) in humidified air at 37° C. for 2 hours. The cells are then be pelleted twice through a 40% isotonic Percoll solution, after which they are purified further by Ficoll-Hypaque density gradient centrifugation (40%/75%).
In Vitro Induction of Tregs
Naturally-occurring Tregs are thymus derived, express high levels of Foxp3 forkhead transcription factor and suppress activation of effector lymphocytes. It has been discovered that antigen-specific activation of human effector T-cells may induce expression of Foxp3 in a subgroup of the activated effector cells, which in turn develop a regulatory phenotype. These induced regulatory T-cells were shown to be capable of cell-contact-dependent suppression of freshly isolated effector cells (Walker et al., 2003, supra). In mice, prolonged exposure of effector cells to TGF-β induces Tregs both in vitro and in vivo (Fantini et al., J Immunol. 2004 and 2006, supra). This small, peripherally generated population of inducible Tregs may be central in regulation and containment of ongoing immune response, while the inability to induce such Tregs may be responsible for a propensity to develop autoimmunity.
To test whether such induction occurred after stimulation of T effector cells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and TNP-CD28A-Tg T effector cells were isolated by FACS sorting and cultured for 7 days in the presence of either (1) anti CD3 Ab, (2) murine TGF-β, (3) mAb to TNP, (4) anti CD3 Ab+TGF-β, or (5) anti TNP Ab+TGF-β. Induction of Foxp3 in cells "developing" from these effector T-cells was assessed after seven days of culture using intracellular Foxp3 staining
Antigen-specific activation of human effector T-cells leads to inducible expression of Foxp3 in a subgroup of activated effector cells, which in turn develop regulatory phenotype. These induced regulatory T-cells are capable of cell-contact-dependent suppression of freshly isolated effector cells. In mice, both in vitro and in vivo induction of Tregs can be achieved with prolonged exposure of effector cells to TGF-β (Fantini et al., 2004, 2006, supra). The present inventors adopted this technology to induce murine redirected Tregs from redirected effector T-cells (see FIG. 3).
Several mouse strains were used in the studies described below and are used in various other embodiments of the invention. These include transgenic mouse lines that specifically expresses anti-TNP or anti-Erb B2 TpCRs (bearing CD28-FcR signaling chains) under the control of a CD2 promoter, as well as a transgenic mouse line expressing human CEA (Saha A et al., Immunology 2006, 118:483-496)
All transgenic mice were back-crossed to Balb/c. Balb/c wild-type mice serve routinely as controls and recipients of adoptively transferred cells.
One cell-transfer colitis model is used in immune deficient Rag.sup.-/- and SCID mice.
All invasive procedures were and are conducted under Ketamine and Xylazine general aesthesia (127.5 and 4.5 mg/kg, respectively). Subcutaneous (S.C.) injections are conducted under local anesthesia with 10% Xylocalne spray.
Colitis Induction and Assessment:
To induce TNP hapten-mediated colitis mice were sensitized with 15 μl of the haptenating agent 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a concentration of 2.5% v/v in 50% ethanol by skin painting on day 1. On day 8, 150 μl of 1% TNBS in 50% ethanol was administered intrarectally via a 3.5 F catheter under general anesthesia. OXA-induced colitis was induced by sensitizing mice with oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich) at a concentration of 3% v/v in 100% ethanol by skin painting on day 1, followed by intrarectal administration of 150 μl at a concentration of 1% v/v in 50% ethanol on day 8.
In one preferred cell transfer colitis models, CD45RBhigh (naive) T-cells are transferred to immune deficient mice from syngeneic background (Powrie F et al., J Exp Med. 1994; 179:589-600. This model of mucosal inflammation allows separating T effector and Treg cell function within an inflammatory site.
In all models, colitis is assessed following induction using the following parameters: degree of colon ulcerations, intestinal and peritoneal adhesions, wall thickness, and degree of mucosal edema. Each parameter is graded on a scale from 0 (completely normal) to 4 (most severe) by two experienced, blinded observers. For histological evaluation of inflammation, distal colon tissue (last 10 cm) is removed and fixed in 10% formaldehyde. Five paraffin sections from each mouse are stained with hematoxylin-eosin using standard techniques. The degree of inflammation is graded semiquantitatively on microscopic cross sections of the colon from 0 to 4 as follows: Grade 0: Normal with no signs of inflammation; Grade 1: very low level of leukocyte infiltration; Grade 2: Low level of leukocyte infiltration; and Grade 3: High level of infiltration with high vascular density, and bowel wall thickening; Grade 4: Transmural infiltrates with loss of goblet cells, high vascular density, wall thickening, and disruption of normal bowel architecture.
For continuous monitoring of colitis pathology, a newly-developed, high resolution mouse video endoscopic system has been used Becker C et al., Gut. 2005; 54:950-4. The experimental endoscopy system (from Karl Storz, Tuttlingen, Germany) consists of a miniature endoscope (1.9 mm outer diameter), a xenon light source, a triple chip camera, and an air pump. Parameters for grading of colitis include bowel wall thickening, granularity, fecal consistency, fibrin deposition and vascular pattern. Whole colon methylene blue chromoendoscopy staining is used, when appropriate, to visualize crypt pattern. A 3fr. Flexible biopsy forceps is used for biopsy-taking. Biopsies are either placed in formalin for paraffin embedding, sectioning and subsequent immunohistochemistry, frozen in liquid nitrogen for cryosections, or obtained and used for RNA isolation. A typical yield of a biopsy specimen is approximately 2 μg RNA
In Vivo Imaging:
To follow migration (also referred to as homing or trafficking) of redirected Tregs in mice, a whole body CCD camera (IVIS® 100 Series Imaging System, Xenogen, Alameda Calif.). was used. Redirected Tregs were labeled with the near-infrared (NIR) lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindotricarbocyanine iodide (DiR, Invitrogen, USA). This dye has absorption and fluorescence maxima at 750 and 782 nm, respectively, enables the safe direct labeling of membranes of human lymphoid cells with very low light absorption and autofluorescence levels in living tissues (Miller M J et al., Proc Natl Acad Sci USA, 2003; 100:2604-9; Kalchenko V et al., submitted for publication, 2007). Additional in vivo visualization of Tregs labeled with carboxy fluorescein diacetate succinimide ester (CFSE) at colonic mucosa was performed by intrarectal insertion of a 300 and 650 μm diameter confocal microendoscope (Cell Vizio, MKT, Paris, France). This unique modality, previously untested in colitis models, allows repeated in vivo assessment of homing of CFSE-labeled redirected Tregs to the most inner layers of colon tissue following induction of inflammation.
Determination of Colon Cytokine Levels
Colon mRNA expression of selected cytokines is determined to allow assessment of redirected Treg effects on local intestinal immune response. in particular, levels of pro-inflammatory (TNFα and IFNγ) and anti-inflammatory cytokines (TGFβ and IL10), as well as levels of the TH1 transcription factor Tbet and the TH2 transcription factor GATA-3. Colon cytokine levels are assessed by measuring mRNA expression and protein levels.
Samples for mRNA isolation are removed from colons of mice using in vivo colonoscopy or during sacrifice. Total RNA is isolated and processed and cDNA produced by RT-PCR. In all experiments, mice are divided into the following groups: naive mice, colitis-induced mice, and colitis-induced mice adoptively transferred with Tregs (naturally occurring, induced, or redirected, see detailed adoptive transfer experiments herein). The following sets of oligonucleotides and amplification conditions are used:
TABLE-US-00001 SEQ ID Amplification SEQUENCE NO: conditions TNF-α sense 5'-AGTCCGGGCAGGTCTACTTT-3' 15 60°/30 cycles antisense 5'-GAGGCAACCTGACCACTCTC-3' 16 IFN-γ sense 5'-TCTGGAGGAACTGGCAAAA-3' 17 63°/35 cycles antisense 5'-TGAGCTCATTGAATGCTTGG-3' 18 TGF-β sense 5'-TACAGGGCTTTCGATTCAGC-3' 19 63°/35 cycles antisense 5'-CGCACACAGCAGTTCTTCTC-3' 20 IL-10 sense 5'-TCCTTGGGAAGCAATTGAAG-3' 21 63°/35 cycles antisense 5'-AACTGGCCACAGTTTTCAGG-3' 22 T-bet sense 5'-CTAAGCAAGGACGGCGAATGT-3' 23 60°/35 cycles antisense 5'-GGCTGGGAACAGGATACTGG-3' 24 GATA-3' sense 5'-GCCTGCGGACTCTACCATAA-3' 25 54.8°/30 cycles antisense 5'-CAGGGATGACATGTGTCTGG-3' 26 GAPDH sense 5'-GTGTTCCTACCCCCAATGTG-3' 27 60°/25 cycles Antisense 5'-CTTGCTCAGTGTCCTTGCTG-3' 28
The relative mRNA expression compared to the housekeeping GAPDH is assessed using NIH image software and averaged from mice in each group.
IL-10 and IFN-γ protein expression levels in colon tissue are quantified by a cytofluorimetry-based ELISA system. In brief, whole proteins are isolated from colon specimens in the absence of detergent. Proteins (100 μg) are immediately used for cytokine determination according to manufacturer's instructions.
Foxp3 Immunohistochemistry of Colon Samples:
Foxp3 immunofluorescence is performed to estimate in situ the targeting of Treg to diseased colon, using TSA Cy3 and a fluorescence microscope (Olympus). In brief, cryosections are fixed in cold acetone for 10 minutes, followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories, CA), and protein blocking reagent to eliminate nonspecific background staining. Slides are then incubated overnight with primary antibodies specific for Foxp3 (e.g., from Alexis Biochemicals, Lausanne, Switzerland). Subsequently, slides are incubated for 30 minutes at room temperature with biotinylated secondary antibodies, and treated with streptavidin-horseradish peroxidase and stained with Tyramide (Cy3 or FITC). Before examination, nuclei are counterstained with Hoechst 3342 (Molecular Probes, Ohio).
Phenotypic Characterization of TNP-Specific Tregs
The inventors have produced transgenic (Tg) mice expressing a TNP-specific tripartite chimeric receptor (TpCR) that serve as a source of redirected Treg cells specific for the trinitrophenyl (TNP) hapten. This hapten has served as a "classical" antigen for years in studying both antibodies and T cell-mediated immunity. A chemically reactive form of this hapten, TNBS, is a contact sensitizing agent that induces and evokes delayed-type hypersensitivity (DTH) responses as well as inducing colitis in animals, as described herein.
Generation of TNP-specific Tregs was achieved by the creation of Tg mice that express TNP-specific TpCR that comprises an scFv from the TNP-specific mAb Sp6 mAb linked to a truncated CD28 molecule which was inserted between the scFv and the cytoplasmic part of the FcRγ chain (abbreviated as y herein (see FIG. 1). This construct includes the hinge region, transmembrane region, and cytoplasmic region of CD28 but lacks the B7 (ligand) binding site.
For the truncated form of CD28 (TpCR/CD28, FIG. 1) that does not include the CD28 intracellular signaling domain, the inventors cloned the vector at the same site. As a control, a Tg mouse expressing TpCR specific for another, irrelevant antigen (Erb-B2) was used.
For expression of TpCR in T-cells of Tg mice, a construct comprising an anti-TNP (Sp6-derived scFv-CD28-γ was cloned into a human CD2 promoter/enhancer minigene-based vector. Tg mice were generated at the Weizmann Institute's Department for Veterinary Resources by pronuclear microinjection of (BALB/c×C57BL/6)F1 fertilized eggs derived from hyperovulated donor females. Founder mice were screened by PCR of DNA from tail samples. Several founder strains were obtained that express high level of the TpCR on their cell surfaces. These were backcrossed for more than nine generations to either BALB/c or C57BL/6 mice to obtain MHC-homogeneous mice.
The studies below describe the characterization of various Treg subpopulations in the different strains of TNP-CR transgenic mice, and the expression of TpCR on these Tregs.
Isolation of Trees in which TNP-Specific TpCR are Highly Expressed
Tregs were isolated using double magnetic bead separation (Miltenyi Biotech) or by fluorescent cell sorting in which fluorescently labeled CD4+CD25+ cells were sorted using the FACSARIA cell sorting system.
Treg expression of TNP-specific TpCR was assessed by containing cells for Foxp3 (considered the "gold standard" marker of Tregs) and PE-labeled mAb specific for TNP antibody (generated in the inventors' laboratory). Controls included groups stained with the appropriate isotype controls. As is shown in FIG. 2, Tregs from TNP-Tg mice, but not from wild-type mice, expressed high levels of TNP-specific TpCR.
TNP-Tg Mice Posses Increased Numbers of Foxp3+Tree Population
Peripheral lymphocytes from the spleen as well as gut-associated lymphocytes from the lamina propria of the colon were stained. As shown in FIG. 3, a CD4+CD25+ cell population (represented as the ratio of CD4+CD25+ cells among CD4+T-cells) was elevated modestly in TNP-Tg mice in comparison to control mice (wildtype, ErbB2-Tg and TNP-CD28 null-Tg mice). In contrast, higher numbers of Foxp3+ cells were observed in TNP-Tg animals compared to the control animals in comparison to all other mouse types (FIG. 4).
To resolve what may have appeared to be an inconsistency between the highly elevated Foxp3+Treg population in TNP-Tg mice and the modestly elevated CD4+CD25+ Treg population in these mice, effector CD4+CD25- cells were isolated by cell sorting to a level of 99% purity. Isolated cells were stained for Foxp3 (FIG. 5). As expected, no positive Foxp3 staining was noted in T effector cells from wildtype, ErbB2-Tg and TNP-CD28null-Tg mice. In contrast, TNP-Tg T effector cells featured a significant population of Foxp3+ cells. This observation was further validated in whole spleen cell populations that were co-staining for Foxp3 and CD25 (FIG. 6). The presence of a significantly greater Foxp3+CD25- Treg population in TNP-Tg mice is supported by other recent results by the inventors' laboratory showing that the Sp6 mAb from which the scFv of the TNP-specific TpCR was derived recognizes cross-reactive endogenous thymic antigens. This results in either deletion or early release from the thymus to the periphery before several other immature T cell subsets, including immature CD25- Tregs.
Induction of TNBS Colitis in TNP-Tg Mice Significant Elevated the Numbers of Foxp3+ Expressing Cells in Peripheral and Colon-Derived Lymphocyte Populations
Induction of TNBS colitis results in further elevation in splenic (FIG. 7) and colon (FIG. 8) Foxp3+Tregs in TNP-Tg (FIGS. 7 & 8, respectively). These results demonstrated that TNP-specific Treg expansion occurred following induction of colitis in Tg mice, reflecting Treg proliferation following antigen-specific activation by TNP.
In Vitro Functional Characterization of Redirected Tregs
A key prerequisite for the utility of Tregs expressing TNP-specific TpCR in the treatment of autoimmunity is verification of their regulatory activity, namely an ability to suppress T effector cell proliferation in a dose-dependent manner. Also examined was whether such Treg activation occurs as a result of TpCR signaling, and whether it was indeed independent of CD28-B7 interaction. A series of coculture experiments examined Tregs from the different Tg strains, as is outlined below.
Tregs Bearing the TNP-Specific Chimeric Receptor Specifically Suppressed the Activity of T Effector Cells
To characterize whether TNP-Tg Tregs retained their anergic properties, CD4+CD25+ Treg cells and CD4+CD25- T effector cells from different Tg mouse founders (anti-TNP, anti-Erb-b2 control and wildtype (WT) mice) were purified from bulk splenocytes. 105 cells were incubated in vitro for 24 h, 48 h or 72 hrs (FIG. 9) and activated non-specifically with anti CD3 and anti-CD28 Abs, or specifically with Fowl gamma globulin-modified TNP (FyG-TNP). T cell proliferation was measured using either the uptake of a dye (tetrazolium salt XTT) or radiolabeled Thymidine. IL2 secretion was measured using XTT staining of the IL-2-dependent CTLL-2 cell line.
All effector cell populations showed significantly increased proliferation and IL2 secretion following non-specific stimulation with anti-CD3+ anti-CD28 Abs. Specific stimulation by FyG-TNP resulted in proliferation and IL2 secretion by T effector cells bearing TNP-chimeric receptor, but not by such T-cells from WT or anti-Erb-b2 Tg mice. In contrast, Tregs from wildtype mice, TNP-chimeric receptor Tg mice and Erb-b2 Tg mice retained their anergic properties: they did not undergo measurable proliferation or IL2 secretion when subjected to the non-specific stimulus or specific Ag.
To characterize whether polyclonal activation could trigger the suppressive action of TNP-Tg Tregs, these Tregs were cocultured in 96-well microplates (0.2 ml) with irradiated antigen presenting cells (APCs) and T effector cells (CD4+CD25-) at 1:1 ratios. Cells in these culture were activated either by (1) immobilized antigen "mimic" (anti-CD3+ anti-CD28) or (2) soluble Concanavalin A (ConA). T cell proliferation was measured as Thymidine uptake and IL2 secretion was measured as growth of cells of the IL-2-dependent CTLL-2 cell line (XTT staining).
FIG. 10 shows a ConA experiment. Non-specific (polyclonal) stimulation of Tregs induced these cells to exhibit potent inhibition of T effector cell proliferation and IL2 secretion, irrespective of the origin of the Tregs or the presence of the chimeric receptor. Thus, genetic manipulation of Tregs of the type described here preserves their suppressive properties.
Antigen-Specific Stimulation of Redirected Tregs Cells with TNP Results in Suppression of T Effector Cell Proliferation
To study the antigen-specific Treg stimulation through the TpCR, coculture experiments were done in which TNP-loaded APCs provided the Ag presentation (FIG. 11). Comparisons of TNP-specific Treg stimulation was performed, comparing wildtype vs. TNP-Tg Tregs (FIG. 11, left panel) or ErbB2-Tg and TNP-Tg Tregs (FIG. 11, right panel). In the absence of TNP stimulation, T effector cell proliferation did not occur (left-most bars in both graphs). In contrast, incubation with TNP-modified APC's resulted in:
(1) marked proliferation of TNP-Tg but not of wildtype or ErbB2-Tg effector T-cells in the absence of Tregs; and(2) activation of TNP-Tg, but not of WT or Erb-b2-Tg Tregs, manifest as suppression of effector cell proliferation by TNP-specific Tregs only.
These results proved the antigen-specific manner of activation and function of TNP specific TpCR Tregs cells in response to the antigen, TNP.
Co-culture of varying ratios of TNP-specific Tregs and TNP-specific effector T-cells (FIG. 12) demonstrated successful antigen-specific inhibition by Tregs at a ratio of 1 Treg to 8 T effector cells.
Studies supporting the existence of the bystander effects were carried out. Colitis was induced in mice as above using OXA as described in Example I. Adoptive transfer of TNP-specific Tregs alone did not protect these animals from colitis. However, in the presence of trace amounts of TNP applied to the colon, animals were protected from this OXA-induced colitis.
Suppressive Activity of TpCR-Redirected Tregs is Independent of Costimulatory Receptors
To assess the role of costimulatory signaling in the above TpCR-Tg model, coculture experiments as above were performed using as APC's (a) TNP-modified P815 cells, a cell line that does not express B7, or (b) TNP-loaded genetically modified P815 cells stably expressing the B7 gene (FIG. 13). Stimulation of TNP-Tg effector T-cells with TNP-P815 cells induced proliferation, which was markedly suppressed by TNP-Tg Tregs. Expression of B7 on these APC's did not promote any further Treg-mediated suppression. It was concluded that maximal Treg suppression occurred independently of B7. Some suppression was also noted with wildtype Tregs. This was explained by the pre-activation of these cells prior to their harvesting. Based on these results, it could be concluded that inclusion of the intra-cytoplasmic signaling domain of CD28 in the TpCR of redirected Treg cells results in full activation of their suppressive activity when stimulated by Ag irrespective of the presence of B7-CD28 costimulation.
Functional Characterization of TNP-Specific Treg Activity In Vivo in Murine Colitis
TNBS is a potent inducer of T-cell responses such as DTH/contact sensitization. This reactive hapten also induces autoimmune colitis when applied to the colon of pre-sensitized mice. To determine whether TpCR-bearing Tregs could suppress autoimmunity, the acute TNBS-mediated colitis model was employed. Intra-rectal administration of TNBS leads to its binding to colon proteins, rendering these modified proteins immunogenic so that they elicited a T cell mediated immune response. The suppressive effect of endogenous or exogenously transferred Tregs on autoimmune inflammatory disease was tested in this model. A different hapten, oxazolone (OXA) with similar sensitizing properties and which induces experimental colitis was used as a specificity control in vivo.
Transgenic Mice Whose Entire Treg Population Expresses the Chimeric Anti-TNP Receptor are Resistant to TNBS-Induced Colitis
TNP hapten-mediated colitis was induced in Tg and WT mice by first sensitizing the animals with 150 μl of the 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich) at a concentration of 2.5% in 50% ethanol painted on the skin on day 1. On day 8, the antigen was administered rectally (150 μl of 1% TNBS in 50% ethanol; high dose colitis). WT mice developed severe colitis within 2-5 days of rectal TNBS administration (FIG. 14, left panel). In contrast, 90% of the TNP-Tg mice had normal looking colons (FIG. 14, right). Colitis severity scores were as follows:
TABLE-US-00002 Colitis score Animals (Arbitrary Units) Mortality Wildtype 12 ± 3.1 90 ± 20% TNP-ΔCD28-Tg 11.1 ± 4 ErbB2-Tg 12.7 ± 3.2 TNP-Tg 2 ± 2 (p < 0.05) 20 ± 20% (p < 0.01)
To produce mortality curves the above experiments were repeated with lower doses of TNBS (75 μl of 1% TNBS in 50% ethanol). Similar differences in colitis severity and in mortality were noted (FIG. 15). Microscopically, colons of wildtype, TNP-ΔCD28-Tg and ErbB2-Tg mice showed severe inflammation, necrosis, hemorrhage and in some cases perforation, while those of TNP-Tg mice appeared normal or near normal (FIG. 16).
Evidence of antigen specificity of the protection from hapten-mediated colitis came from studies of OXA-induced colitis. As shown in FIG. 17, no differences in mortality were noted between wildtype, TNP-Tg, TNP-ΔCD28-Tg and ErbB2-Tg mice. The same was true for macroscopic and microscopic colitis scores. From these in vivo experiments, it was concluded that the presence of a Treg cell population that uniformly expresses the anti-TNP chimeric receptor results in high grade protection against TNBS-induced inflammation, manifest as reduced colon inflammation and significantly improved survival. It is noteworthy that inclusion of CD28 costimulatory signaling in the CR significantly enhances TNP-Tg Treg suppressive function.
Prolonged Stimulation with TNP Combined with TGF-β Promotes Conversion of Leads to TNP-Specific Effector T-Cells to TNP-Specific Trees
Naturally-occurring Tregs are thymus derived, express high levels of Foxp3 and suppress activation of effector lymphocytes. Antigen-specific activation of human effector T-cells may induce expression of Foxp3 in a subgroup of the activated effector cells, which in turn develop a regulatory phenotype. These induced regulatory T-cells were shown to be capable of cell-contact-dependent suppression of freshly isolated effector cells (Walker et al., 2003, supra). In mice, it has been demonstrated that prolonged exposure of effector cells to TGF-β induces Tregs both in vitro and in vivo (Fantini et al., 2004, 2006, supra). This small, peripherally generated population of inducible Tregs may be central in regulation and containment of ongoing immune response, while the inability to induce such Tregs may be responsible for a propensity to develop autoimmunity.
To test whether such induction occurred after stimulation of T effector cells through the TpCR, wildtype, TNP-Tg, Erbb2-Tg and TNP-CD28null-Tg T effector cells were isolated by FACS sorting and cultured for 7 days in the presence of either (1) anti CD3 Ab, (2) murine TGFβ, (3_mAb to TNP, (4) anti-CD3 Ab+TGF-β, or (5) anti-TNP Ab+TGF-β. Induction of Foxp3 in cells "developing" from these effector T-cells was assessed after seven days of culture using intracellular Foxp3 staining (FIG. 18).
At time 0 to the time of T effector cell sorting, no Foxp3 staining was noted. A week of stimulation with anti-CD3+TGF-β, but not with TNP+TGF-β, resulted in a 2-fold increase in Foxp3+ cells in wildtype, ErbB2-tg and TNP-CD28null-Tg T effector cells. In contrast, a dramatic 30-fold increase in Foxp3+ cells was observed in TNP-Tg effector cells following exposure to TNP+TGF-β. Interestingly, no Foxp3 induction was noted in TNP-Tg T effector cells following incubation with anti CD3 Ab or with TGF-β, probably due to significant attenuation of CD3 expression in TNP-Tg T-cells (Morvinsky-Friedman et al, in press).
The foregoing results demonstrated that antigen-specific stimulation through the TpCR in the presence of TGF-β, led to induction of Ag-specific Tregs from T effector cells, further contributing to Treg expansion. This change was dependent both on the antigen-specificity of the Ab recognition unit of the TpCR and the intra-cytoplasmic CD28 signaling moiety.
According to the present invention, induction of Tregs in this manner permits the generation of large populations of TpCR-bearing Tregs that can be used in cell-based therapy of autoimmunity.
Adoptive Transfer of TNP-Tg Tregs to WT Mice with TNBS Colitis Ameliorates Symptoms and Improved Survival
Studies were conducted to establish that TNP-Tg Tregs are responsible for the resistance of TNP-Tg mice to TNBS colitis and to evaluate their therapeutic capacity in autoimmunity. Wildtype, TNP-Tg and Erb-b2-Tg Tregs were isolated and administered in varying numbers to wildtype mice a day after induction of TNBS colitis. As was previously described, adoptive transfer of large numbers of Tregs of any origin (>2×105) caused nonspecific attenuation of TNBS colitis. This is believed to result from the presence of a sufficiently large population of pre-activated Tregs that can exert their suppressive activity in the absence of antigen stimulation or specificity. In contrast, adoptive transfer of smaller numbers (5×104) of TNP-Tg Tregs but not of wildtype or Erbb2-Tg Tregs, prolonged survival (FIG. 19), improved Wallach colitis severity scores (FIG. 20), and significantly improved macroscopic (FIG. 21) and microscopic (FIG. 22) appearance of colon tissue. FIG. 21 shows marked bowel shortening, a manifestation of colon inflammation (in WT and ErbB2-Tg, but not in TNP-Tg mice). FIG. 22 shows the severe transmural inflammation, necrosis, mucosal bleeding and loss of normal architecture in colons of WT mice with TNBS colitis that had received control (WT and ErbB2-Tg), but not in TNP-Tg colons.
Migration/Trafficking of Redirected `Tregs to Sites of Inflammation: Adoptively-Transferred TNP-TG Tregs Localize to Colons in TNBS Induced Colitis
Studies were done to garner additional support for the role of TNP-Tg Tregs in attenuating TNBS colitis by showing that these cells indeed localize to inflamed colon tissue. WT and TNP-Tg Tregs were isolated and stained with the fluorescent intracellular dye, carboxyfluorescein diacetate succinimidyl ester (CFSE). Following staining, 106 Tregs were administered intraperitoneally (ip) to control WT mice or to WT mice in which TNBS colitis had been induced 12 hours earlier. Sixteen hours after this treatment, mice were sacrificed and lamina propria lymphocytes isolated from their colons. The protocol used was described above to isolate lamina propria lymphocytes. Thereafter, the cells were examined for the presence of CFSE-positive cells by FACS analysis. As shown in FIG. 23, very small numbers of adoptively-transferred WT or TNP-Tg Tregs reached the colons of normal mice. Induction of colitis led to a small increase (0.5% to 0.8%) in the number of WT CFSE-stained Tregs in colon tissue. In contrast, adoptive transfer of CFSE-labeled TNP-Tg Tregs to mice with colitis led to a significant increase in colon Treg population, ranging from 0.4% to 3.6%. This demonstrates that TNP-Tg Tregs localize in a TNBS-exposed target organ, where they exert their suppressive function.
Accumulation of Adoptively Transferred TNP-Tg Tregs within the Mucosal Layer of Colons of Mice with TNBS Colitis
An important aspect of understanding the role of Tregs for adoptive therapy of autoimmune inflammation of the Treg in diseased organs, where they are expected to exert their suppressive effects. To demonstrate TNP-Tg-Treg localization, WT and TNP-Tg Tregs were labeled with CFSE and transferred to WT mice 24 hours following induction of TNBS colitis. While very small numbers of CFSE-labeled WT Tregs were observed in cell extracted from colonic lamina propria of naive or TNBS colitis-induced mice, a nine-fold increase in TNP-Tg Tregs was noted (FIG. 23).
To study the kinetics of TNP-Tg Treg localization of in the living animal, the IVIS® 100 Series Imaging System was employed (Xenogen, Alameda Calif.). Wildtype and TNP-Tg Tregs, 1.5×106, labeled with the near-infrared lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR, Invitrogen USA), were administered ip to WT mice with or without TNBS colitis, who were monitored daily with the IVIS whole body CCCD camera (FIG. 24). A strong anterior-abdominal fluorescent signal, reflecting the bulk of injected Tregs, was noted in naive mice 24-48 hours following ip injection of Tregs and disappeared thereafter due to Treg redistribution. In mice with TNBS colitis, a faint abdominal signal was noted for 72 hours, probably reflecting inflammation-related auto-fluorescence. In mice with TNBS colitis that received labeled WT Tregs, a week-moderate abdominal fluorescent signal could be recognized up to 96 hours following Treg transfer. In contrast, TNP-Tg Treg-administered to WT mice with TNBS colitis featured a distinct abdominal fluorescent signal for up to a week following cell transfer, substantially stronger than that of wildtype Tregs at all time points. These results reflect persistent TNP-Tg Treg localization within colons during the colitis.
To determine whether TNP-Tg Tregs reach the inner colonic mucosal layer, the location where most of TNBS-induced mucosal damage takes place, the Cell Vizio confocal microendo-scopy system was employed (Cell Vizio, MKT, Paris, France). An intrarectally-inserted 650 μm diameter confocal microendoscope enabled visualization of CFSE-labeled cells in up to a 150 μm bowel wall thickness (FIG. 25). Numerous adoptively transferred CFSE-labeled TNP-Tg, but no WT Tregs could be visualized in the inner mucosal layer of WT mice with TNBS colitis as early as 12 hours following systemic Treg transfer. This result indicates that TNP-Tg Tregs localize in response to colonic TNBS, within hours of their administration, and that they reach the deepest colonic mucosal layers, where they exert their suppressive functions.
Administration of TNP-Tg Tregs Specific to a Bystander Antigen (TNBS) Cures Colitis Mediated by a Pathogenic (Oxazolone) Antigen
In contrast to hapten-mediated colitis, in which the eliciting antigen is predefined, the disease-causing antigen in inflammatory bowel disease (IBD) is unknown. To enable application of the `T-body` approach in IBD, naive TPCR-bearing Tregs were tested to determine whether they can be activated by a predetermined `bystander` colon- or colitis-associated antigen, to perform their antigen-nonspecific suppressive action. To this end, WT and TNP-Tg mice were pre-sensitized to oxazolone only. A mixture of oxazolone and low doses of TNBS were introduced intrarectally. As is shown in FIG. 26a, concomitant challenge of WT mice with TNBS and oxazolone, was associated with a 100% one-week mortality rate, as compared to only a 15% one-week mortality of TNP-Tg mice (P<0.01). Similarly, significant mucosal inflammation was evident in both WT and TNP-Tg mice with oxazolone colitis (not shown), and was most severe in wild-type mice given TNBS+oxazolone (FIG. 26b, box I) resulting in severe bleeding, fibrin deposition and sloughing off of colonic mucosa.
In utter contrast, TNP-Tg mice administered TNBS+oxazolone featured normal-appearing colonic mucosa with scattered areas of mild colitis (FIG. 26b, box II). Macroscopically and microscopically, colons of concomitantly TNBS-treated and oxazolone-treated WT mice featured severe colitis, as opposed to the near-normal colons in TNP-Tg mice (FIGS. 26c and 26d, respectively.)
Notably, this "bystander" protective effect also occurred when TNP-Tg Tregs were adoptively transferred to oxazolone-presensitized wild-type mice which were intrarectally boosted with a mixture of oxazolone and low doses of TNBS (FIG. 26e, P<0.01)). In contrast, adoptively transferred WT Tregs did not have this curative effect, and the very low TNBS doses in the absence of pre-sensitization were insufficient by themselves to induce TNBS colitis. These results demonstrate that Treg activation by a bystander antigen (TNBS) cause an improvement in colitis that has been induced by a different non-cross-reactive antigen (oxazolone).
Delivery of Foxp3 to Cell Nucleus by Vectors Comprising Chimeric Receptors
An experiment was conducted to verify that the Foxp3 can be expressed following transduction of A273 cells with retroviral vector constructs designed to transduce Treg cells. A fused gene was generated that included eGFP sequences encoding green fluorescent protein (referred to as eGFP or GFP). This was engineered as a bicistronic construct with the GFP sequence alone or linked with a Foxp3-encoding sequence (after an IRES) into vectors that comprised a chimeric receptor construct with the following extracellular recognition regions: See description of FIG. 27 for discussion of the chimeric receptor constructs used. 273 cells. transduced with vectors comprising the same chimeric receptors but with a bicistronic eGFP gene only (without Foxp3) served as controls for Foxp2 expression.
The results are shown in FIG. 27. The upper half of the Figure shows the GFP-only controls, whereas the lower half of the Figure shows GFP-Foxp3 constructs. The two-paneled rectangles in the Figure show light microscopic (left half) and fluorescence microscopic (right half) images of the same material (to visualize and localize the GFP).
All the control group expressed the eGFP in their cytoplasm only. In contrast, in cells that were transduced with the eGfP-Foxp3-fusion constructs, the nuclei were fluorescent (appearing as bright nuclear images) due to the transport to and expression of the Foxp3 transcription factor in the nuclei.
In another experiment not shown here, expression of chimeric receptor made of the full length MD2 protein (SEQ ID NO:5) or the CD14 protein (SEQ ID NO:4) was confirmed by the ability of transduced cells, which expressed the extracellular region of the CR on their surface, to bind the ligand of MD2 and CD14, bacterial LPS, which was provided in biotinylated form and revealed by secondary binding of fluorescent avidin.
Vectors Comprising Chimeric Receptors with LPS-Binding Extracellular Regions
Nucleic acid constructs and vectors that encode extracellular regions that comprise an anti-LPS antibody domain (e.g., SEQ ID NO:3 or the scFv-coding portion thereof) have been made and others can be made. Such vectors express extracellular polypeptide domains that are shown to bind LPS, for example in an assay using biotinylated LPS and detectably labeled (e.g., fluorescently labeled) avidin. See also Example XVI above.
Nucleic acid constructs and vectors that encode extracellular regions that comprise a LPS-binding nonantibody polypeptide have been made (e.g., SEQ ID NO:6-11, 13 and 14). Such constructs include bicistronic ones that also comprise Foxp3. Other such constructs can be made using the method described above along with methods well-known in the art. Such constructs (such as SEQ ID NO: 13 and 14) include those encoding full length CD14 (SEQ ID NO:4) or MD2 (SEQ ID NO:5) protein, and constructs encoding LPS-binding motifs therefrom (such as SEQ ID NO:6-9) and combinations (such as SEQ ID NO:10 and 11). The constructs that are made include those with CD28-FcRγ intracellular stimulatory/costimulatory regions and those that utilize others of the type disclosed herein.
Treg cells are redirected as described herein using the above constructs, including those that have been made and tested and those that can be made.
Such Treg cells are administered into subjects suffering from IBD, such as ulcerative colitis. Treg cells are administered in numbers in accordance with the above examples, or in numbers that are readily determined to be effective by those skilled in the art using only routine experimentation, and via routes of administration as exemplified above and disclosed throughout this document. These redirected Treg cells that express an LPS binding antibody region or another LPS-binding moiety on their surface (CD14, MD2, fragments thereof, or combinations of these) as part of their CR's are able to reduce the symptoms, intensity, severity and duration of the IBD in the subject to a significant degree compared to untreated control subjects or control subjects administered with control Tregs. (Such control Treg cells are ones not transduced to express the present CR's, or those redirected to be specific to antigens or ligands not present at the site of the IBD.) Introduction of LPS-related molecules or epitopes that bind to these same extracellular receptors on the redirected Treg cells to the sites of administration (and/or expected action) of the redirected Treg cells further facilitates their therapeutic activity.
The references cited above are all incorporated by reference herein, whether specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
3311176DNAArtificial Sequencesynthetic 1tctagactgc c atg gat ttt cag gtg cag att ttc agc ttc ctg cta atc 50 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile 1 5 10agt gcc tca gtc ata atg tct aga gga gat att gtg atg acc cag tct 98Ser Ala Ser Val Ile Met Ser Arg Gly Asp Ile Val Met Thr Gln Ser 15 20 25cca aaa ttc atg tcc aca tca gta gga ggc agg gtc agc atc acc tgc 146Pro Lys Phe Met Ser Thr Ser Val Gly Gly Arg Val Ser Ile Thr Cys30 35 40 45aag gcc agt cag aat gtg ggt act gct gta gcc tgg tat caa cag aaa 194Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys 50 55 60cca gga caa tct cct aaa cta ctg att tac tcg gca tcc aat cgg tac 242Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr 65 70 75act gga gtc cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc 290Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe 80 85 90act ctc acc atc agc aat atg cag tct gaa gac ctg gca gat tat ttc 338Thr Leu Thr Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Tyr Phe 95 100 105tgc cag caa tat agc agc tat cct ctc acg ttc ggt gct ggc acc aag 386Cys Gln Gln Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys110 115 120 125ctg gaa atw aaa ggg tcg act tcc ggt agc ggc aaa tcc tct gaa ggc 434Leu Glu Xaa Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly 130 135 140aaa ggt cag gtc cag ctg cag cag tct gga cct gag ctg gtg aag cct 482Lys Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 145 150 155ggg gct tca gtg agg ata tcc tgc aag gct tct ggc tac acc ttc aca 530Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 160 165 170agc tac tat ata cac tgg gtg aag cag agg cct gga cag gga ctt gag 578Ser Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu 175 180 185tgg att gga tgg att tat cct gga aat gtt aat act aag tac aat gag 626Trp Ile Gly Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu190 195 200 205aag ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agc aca 674Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr 210 215 220gcc tac atg cag ctc agc agc ctg acc tct gag gac tct gcg gtc tat 722Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 225 230 235ttc tgt gca aga aac tac ggt agt agc tac ggg ctt gct tac tgg ggc 770Phe Cys Ala Arg Asn Tyr Gly Ser Ser Tyr Gly Leu Ala Tyr Trp Gly 240 245 250caa gga act acg gtc acc gtg aaa ggg aaa cac ctt tgt cca agt ccc 818Gln Gly Thr Thr Val Thr Val Lys Gly Lys His Leu Cys Pro Ser Pro 255 260 265cta ttt ccc gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt ggt 866Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly270 275 280 285gga gtc ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt att att 914Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 290 295 300ttc tgg gtg agg agt aag agg agc agg ctc ctg cac agt gac tac atg 962Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 305 310 315aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc 1010Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 320 325 330tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg cga aag 1058Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys 335 340 345gca gct ata acc agc tat gag aaa tca gat ggt gtt tac acg ggc ctg 1106Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu350 355 360 365agc acc agg aac cag gag act tac gag act ctg aag cat gag aaa cca 1154Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro 370 375 380cca cag tag ctttagactc gag 1176Pro Gln2383PRTArtificial SequenceSynthetic Construct 2Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gly Asp Ile Val Met Thr Gln Ser Pro Lys Phe 20 25 30Met Ser Thr Ser Val Gly Gly Arg Val Ser Ile Thr Cys Lys Ala Ser 35 40 45Gln Asn Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val65 70 75 80Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 100 105 110Tyr Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 115 120 125Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly Gln 130 135 140Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser145 150 155 160Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr 165 170 175Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys 195 200 205Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 210 215 220Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala225 230 235 240Arg Asn Tyr Gly Ser Ser Tyr Gly Leu Ala Tyr Trp Gly Gln Gly Thr 245 250 255Thr Val Thr Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro 260 265 270Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 275 280 285Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 290 295 300Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr305 310 315 320Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 325 330 335Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys Ala Ala Ile 340 345 350Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg 355 360 365Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln 370 375 38037881DNAArtificial Sequencesynthetic 3gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 60cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 120gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 180caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 240atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 300acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 360gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 420ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 480acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 540tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 600tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 660gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 720taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 780taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 840cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 900ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 960cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1020ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1080aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1140acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1200acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1260ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1320cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgaa 1380ggctgccgac cccgggggtg gaccatcctc tagactgcc atg gat ttt cag gtg 1434 Met Asp Phe Gln Val 1 5cag att ttc agc ttc ctg cta atc agt gcc tca gtc ata atg tct aga 1482Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser Val Ile Met Ser Arg 10 15 20gga gat att gtg ctc aca cag tct cca tcc tcc ctg gct gtg tca gca 1530Gly Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala 25 30 35gga gag aag gtc act atg agc tgc aaa tcc agt cag agt ctg ctc aac 1578Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn 40 45 50agt aga acc cga aag aac tac ttg gct tgg tac cag cag aaa cca ggg 1626Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 55 60 65cag tct cct aaa ctg ctg atc tac tgg gca tcc act agg gaa tct ggg 1674Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly70 75 80 85gtc cct gat cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc 1722Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 90 95 100acc atc agc agt gtg cag gct gaa gac ctg gca gtt tat tac tgc aag 1770Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys 105 110 115caa tct tat aat ctg tac acg ttc gga ggg ggg acc aag ctg gaa ata 1818Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 120 125 130aaa ggg tcg act tcc ggt agc ggc aaa tcc tct gaa ggc aaa ggt gag 1866Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly Glu 135 140 145gtc cag ctg cag cag tct gga ggt ggc ctg gtg cag cct gga gga tcc 1914Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser150 155 160 165ctg aaa ctc tcc tgt gca gcc tca gga ttc gat ttt agt aga tac tgg 1962Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr Trp 170 175 180atg agt tgg gtc cgg cag gct cca ggg aaa ggg cta gaa tgg att gga 2010Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 185 190 195gaa att aat cca gat agc agt acg ata aac tat acg cca tct cta aag 2058Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys 200 205 210gat aaa ttc atc atc tcc aga gac aac gcc aaa aat acg ctg tac ctg 2106Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 215 220 225caa atg agc aaa gtg aga tct gag gac aca gcc ctt tat tac tgt gca 2154Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala230 235 240 245aga cgt tat ggt aac tac tgg tac ttc gat gtc tgg ggc gca ggg acc 2202Arg Arg Tyr Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 250 255 260acg gtc acc gtg aaa ggg aaa cac ctt tgt cca agt ccc cta ttt ccc 2250Thr Val Thr Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro 265 270 275gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt ggt gga gtc ctg 2298Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu 280 285 290gct tgc tat agc ttg cta gta aca gtg gcc ttt att att ttc tgg gtg 2346Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 295 300 305agg agt aag agg agc agg ctc ctg cac agt gac tac atg aac atg act 2394Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr310 315 320 325ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc tat gcc cca 2442Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 330 335 340cca cgc gac ttc gca gcc tat aga tct caa gtg cga aag gca gct ata 2490Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys Ala Ala Ile 345 350 355acc agc tat gag aaa tca gat ggt gtt tac acg ggc ctg agc acc agg 2538Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg 360 365 370aac cag gag act tac gag act ctg aag cat gag aaa cca cca cag tag 2586Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln 375 380 385ctttagactc gagcgggatc aattccgccc cccccctaac gttactggcc gaagccgctt 2646ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg 2706caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc 2766ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga 2826agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc 2886tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc 2946acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc 3006aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga 3066tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg 3126ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatagc atgctgagca 3186agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 3246acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 3306ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 3366ccttcgccta cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact 3426tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 3486acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 3546tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 3606acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 3666tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 3726agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 3786cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 3846tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga gtgtaaactc 3906gaggatccgc gccgctcgcg actcgagaga tccggattag tccaatttgt taaagacagg 3966atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa gcctatagag 4026tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa 4086gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcag gcatggaaaa 4146atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagctgaat 4206atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag 4266atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct 4326cagggccaag aacagatggt ccccagatgc ggtccagccc tcagcagttt ctagagaacc 4386atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa 4446ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga 4506gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg 4566tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag 4626ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca gcatgtatca 4686aaattaattt ggtttttttt cttaagtatt tacattaaat ggccatagtc tgctcgatcg 4746aggagctttt tgcaaaagcc taggcctcca aaaaagcctc ttcactactt ctggaatagc 4806tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatgcatg 4866gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg 4926tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt 4986ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat 5046tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc 5106aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg 5166gactctagcc catcgatggg aattccggtc tccctatagt gagtcgtatt aatttcgata 5226agccagacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt 5286cctaatgcag gagtcgcata agggagagcg tcgaatggtg cactctcagt acaatctgct 5346ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 5406gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 5466tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 5526gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 5586ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 5646atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 5706tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5766tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 5826gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5886aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5946gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 6006ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6066gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
6126gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 6186atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 6246ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 6306cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 6366cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 6426gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 6486cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 6546cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 6606taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 6666ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgga gaaaagatca 6726aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 6786caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 6846taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 6906gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 6966cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 7026taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 7086agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 7146ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 7206gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 7266acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 7326acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 7386tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 7446ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 7506agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcaatt 7566agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 7626ccgcccattc tccgccccat gcatggtgat gcggttttgg cagtacatca atgggcgtgg 7686atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 7746gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 7806gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 7866ccgcgccagt cctcc 78814375PRTHomo sapiensCD14 protein 4Met Glu Arg Ala Ser Cys Leu Leu Leu Leu Leu Leu Pro Leu Val His1 5 10 15Val Ser Ala Thr Thr Pro Glu Pro Cys Glu Leu Asp Asp Glu Asp Phe 20 25 30Arg Cys Val Cys Asn Phe Ser Glu Pro Gln Pro Asp Trp Ser Glu Ala 35 40 45Phe Gln Cys Val Ser Ala Val Glu Val Glu Ile His Ala Gly Gly Leu 50 55 60Asn Leu Glu Pro Phe Leu Lys Arg Val Asp Ala Asp Ala Asp Pro Arg65 70 75 80Gln Tyr Ala Asp Thr Val Lys Ala Leu Arg Val Arg Arg Leu Thr Val 85 90 95Gly Ala Ala Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val 100 105 110Leu Ala Tyr Ser Arg Leu Lys Glu Leu Thr Leu Glu Asp Leu Lys Ile 115 120 125Thr Gly Thr Met Pro Pro Leu Pro Leu Glu Ala Thr Gly Leu Ala Leu 130 135 140Ser Ser Leu Arg Leu Arg Asn Val Ser Trp Ala Thr Gly Arg Ser Trp145 150 155 160Leu Ala Glu Leu Gln Gln Trp Leu Lys Pro Gly Leu Lys Val Leu Ser 165 170 175Ile Ala Gln Ala His Ser Pro Ala Phe Ser Cys Glu Gln Val Arg Ala 180 185 190Phe Pro Ala Leu Thr Ser Leu Asp Leu Ser Asp Asn Pro Gly Leu Gly 195 200 205Glu Arg Gly Leu Met Ala Ala Leu Cys Pro His Lys Phe Pro Ala Ile 210 215 220Gln Asn Leu Ala Leu Arg Asn Thr Gly Met Glu Thr Pro Thr Gly Val225 230 235 240Cys Ala Ala Leu Ala Ala Ala Gly Val Gln Pro His Ser Leu Asp Leu 245 250 255Ser His Asn Ser Leu Arg Ala Thr Val Asn Pro Ser Ala Pro Arg Cys 260 265 270Met Trp Ser Ser Ala Leu Asn Ser Leu Asn Leu Ser Phe Ala Gly Leu 275 280 285Glu Gln Val Pro Lys Gly Leu Pro Ala Lys Leu Arg Val Leu Asp Leu 290 295 300Ser Cys Asn Arg Leu Asn Arg Ala Pro Gln Pro Asp Glu Leu Pro Glu305 310 315 320Val Asp Asn Leu Thr Leu Asp Gly Asn Pro Phe Leu Val Pro Gly Thr 325 330 335Ala Leu Pro His Glu Gly Ser Met Asn Ser Gly Val Val Pro Ala Cys 340 345 350Ala Arg Ser Thr Leu Ser Val Gly Val Ser Gly Thr Leu Val Leu Leu 355 360 365Gln Gly Ala Arg Gly Phe Ala 370 3755159PRTHomo sapiensMD-2 protein 5Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe Ser Ser Ile Phe Thr1 5 10 15Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser Ser Asp Ala Ser Ile 20 25 30Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro Ile Ser Ile Asn Val 35 40 45Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly Leu Leu His Ile Phe 50 55 60Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr Phe Asn Leu Tyr Ile65 70 75 80Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys Glu Val Ile Cys Arg 85 90 95Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu Thr 100 105 110Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys 115 120 125Gly Lys Tyr Lys Val Val Glu Ala Ile Ser Gly Ser Pro Glu Glu Met 130 135 140Leu Phe Cys Leu Glu Phe Val Ile Leu His Gln Pro Asn Ser Asn145 150 1556504DNAArtificial Sequencesynthetic 6atg gat ttt cag gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga cag gtt cct gct cag cta ctg gta ggc gcc ctg 96Val Ile Met Ser Arg Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu 20 25 30cgt gtg cta gcg tac tcc cgc ctc aag gtc acc gtg aaa ggg aaa cac 144Arg Val Leu Ala Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His 35 40 45ctt tgt cca agt ccc cta ttt ccc gga cct tct aag ccc ttt tgg gtg 192Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val 50 55 60ctg gtg gtg gtt ggt gga gtc ctg gct tgc tat agc ttg cta gta aca 240Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr65 70 75 80gtg gcc ttt att att ttc tgg gtg agg agt aag agg agc agg ctc ctg 288Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu 85 90 95cac agt gac tac atg aac atg act ccc cgc cgc ccc ggg ccc acc cgc 336His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 100 105 110aag cat tac cag ccc tat gcc cca cca cgc gac ttc gca gcc tat aga 384Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 115 120 125tct caa gtg cga aag gca gct ata acc agc tat gag aaa tca gat ggt 432Ser Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly 130 135 140gtt tac acg ggc ctg agc acc agg aac cag gag act tac gag act ctg 480Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu145 150 155 160aag cat gag aaa cca cca cag tag 504Lys His Glu Lys Pro Pro Gln 16573984DNAArtificial Sequencesynthetic 7atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agacaggttc ctgctcagct actggtaggc gccctgcgtg tgctagcgta ctcccgcctc 120aaggtcaccg tgaaagggaa acacctttgt ccaagtcccc tatttcccgg accttctaag 180cccttttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 240gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 300atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 360ccacgcgact tcgcagccta tagatctcaa gtgcgaaagg cagctataac cagctatgag 420aaatcagatg gtgtttacac gggcctgagc accaggaacc aggagactta cgagactctg 480aagcatgaga aaccaccaca gtagctttag actcgagcgg gatcaattcc gccccccccc 540taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt 600ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt 660gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 720cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct 780ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 840ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 900ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa 960ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 1020gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa 1080aacacgataa tagcatgcag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct 1140gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 1200cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 1260ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 1320cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 1380catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 1440gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 1500catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 1560ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 1620ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 1680catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 1740gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 1800cgggatcact ctcggcatgg acgagctgta caagtccggc cggactcaga tctcgagctc 1860aagcttcgaa ttcatgccca accccaggcc tggcaagccc tcggcccctt ccttggccct 1920tggcccatcc ccaggagcct cgcccagctg gagggctgca cccaaagcct cagacctgct 1980gggggcccgg ggcccagggg gaaccttcca gggccgagat cttcgaggcg gggcccatgc 2040ctcctcttct tccttgaacc ccatgccacc atcgcagctg cagctgccca cactgcccct 2100agtcatggtg gcaccctccg gggcacggct gggccccttg ccccacttac aggcactcct 2160ccaggacagg ccacatttca tgcaccagct ctcaacggtg gatgcccacg cccggacccc 2220tgtgctgcag gtgcaccccc tggagagccc agccatgatc agcctcacac cacccaccac 2280cgccactggg gtcttctccc tcaaggcccg gcctggcctc ccacctggga tcaacgtggc 2340cagcctggaa tgggtgtcca gggagccggc actgctctgc accttcccaa atcccagtgc 2400acccaggaag gacagcaccc tttcggctgt gccccagagc tcctacccac tgctggcaaa 2460tggtgtctgc aagtggcccg gatgtgagaa ggtcttcgaa gagccagagg acttcctcaa 2520gcactgccag gcggaccatc ttctggatga gaagggcagg gcacaatgtc tcctccagag 2580agagatggta cagtctctgg agcagcagct ggtgctggag aaggagaagc tgagtgccat 2640gcaggcccac ctggctggga aaatggcact gaccaaggct tcatctgtgg catcatccga 2700caagggctcc tgctgcatcg tagctgctgg cagccaaggc cctgtcgtcc cagcctggtc 2760tggcccccgg gaggcccctg acagcctgtt tgctgtccgg aggcacctgt ggggtagcca 2820tggaaacagc acattcccag agttcctcca caacatggac tacttcaagt tccacaacat 2880gcgaccccct ttcacctacg ccacgctcat ccgctgggcc atcctggagg ctccagagaa 2940gcagcggaca ctcaatgaga tctaccactg gttcacacgc atgtttgcct tcttcagaaa 3000ccatcctgcc acctggaaga acgccatccg ccacaacctg agtctgcaca agtgctttgt 3060gcgggtggag agcgagaagg gggctgtgtg gaccgtggat gagctggagt tccgcaagaa 3120acggagccag aggcccagca ggtgttccaa ccctacacct ggcccctgag gatccgcgcc 3180gctcgcgctc gagagatccg gattagtcca atttgttaaa gacaggatat cagtggtcca 3240ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg agccatagat 3300aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag 3360gtttggcaag ctagcttaag taacgccatt ttgcaggcat ggaaaaatac ataactgaga 3420atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg 3480atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa 3540tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca 3600gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca 3660gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct 3720tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc 3780actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac 3840cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt 3900gattgactac ccgtcagcgg gggtctttca catgcagcat gtatcaaaat taatttggtt 3960ttttttctta agtatttaca ttaa 39848489DNAArtificial Sequencesynthetic 8atg gat ttt cag gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga ttc tcc ttc aag gga ata aaa ttt tct aag gga 96Val Ile Met Ser Arg Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30aaa tac aaa ggt cac ctc gtg aaa ggg aaa cac ctt tgt cca agt ccc 144Lys Tyr Lys Gly His Leu Val Lys Gly Lys His Leu Cys Pro Ser Pro 35 40 45cta ttt ccc gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt ggt 192Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 50 55 60gga gtc ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt att att 240Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile65 70 75 80ttc tgg gtg agg agt aag agg agc agg ctc ctg cac agt gac tac atg 288Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 85 90 95aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag ccc 336Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 100 105 110tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg cga aag 384Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys 115 120 125gca gct ata acc agc tat gag aaa tca gat ggt gtt tac acg ggc ctg 432Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu 130 135 140agc acc agg aac cag gag act tac gag act ctg aag cat gag aaa cca 480Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro145 150 155 160cca cag tag 489Pro Gln93160DNAArtificial Sequencesynthetic 9atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agattctcct tcaagggaat aaaattttct aagggaaaat acaaaggtca cctcgtgaaa 120gggaaacacc tttgtccaag tcccctattt cccggacctt ctaagccctt ttgggtgctg 180gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 240ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 300cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 360gcctatagat ctcaagtgcg aaaggcagct ataaccagct atgagaaatc agatggtgtt 420tacacgggcc tgagcaccag gaaccaggag acttacgaga ctctgaagca tgagaaacca 480ccacagtagc tttagactcg agcgggatca attccgcccc ccccctaacg ttactggccg 540aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc 600gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag 660gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt 720tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa 780ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc 840aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg 900gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat 960gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa 1020acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gataatagca 1080tgcagcgcta ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt 1140gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga 1200gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa 1260gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag 1320ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta 1380cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt 1440gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga 1500ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat 1560catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga 1620ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc 1680cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa 1740cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg 1800catggacgag ctgtacaagt ccggccggac tcagatctcg agctcaagct tcgaattcat 1860gcccaacccc aggcctggca agccctcggc cccttccttg gcccttggcc catccccagg 1920agcctcgccc agctggaggg ctgcacccaa agcctcagac ctgctggggg cccggggccc 1980agggggaacc ttccagggcc gagatcttcg aggcggggcc catgcctcct cttcttcctt 2040gaaccccatg ccaccatcgc agctgcagct gcccacactg cccctagtca tggtggcacc 2100ctccggggca cggctgggcc ccttgcccca cttacaggca ctcctccagg acaggccaca 2160tttcatgcac cagctctcaa cggtggatgc ccacgcccgg acccctgtgc tgcaggtgca 2220ccccctggag agcccagcca tgatcagcct cacaccaccc accaccgcca ctggggtctt 2280ctccctcaag gcccggcctg gcctcccacc tgggatcaac gtggccagcc tggaatgggt 2340gtccagggag ccggcactgc tctgcacctt cccaaatccc agtgcaccca ggaaggacag 2400caccctttcg gctgtgcccc agagctccta cccactgctg gcaaatggtg tctgcaagtg 2460gcccggatgt gagaaggtct tcgaagagcc agaggacttc ctcaagcact
gccaggcgga 2520ccatcttctg gatgagaagg gcagggcaca atgtctcctc cagagagaga tggtacagtc 2580tctggagcag cagctggtgc tggagaagga gaagctgagt gccatgcagg cccacctggc 2640tgggaaaatg gcactgacca aggcttcatc tgtggcatca tccgacaagg gctcctgctg 2700catcgtagct gctggcagcc aaggccctgt cgtcccagcc tggtctggcc cccgggaggc 2760ccctgacagc ctgtttgctg tccggaggca cctgtggggt agccatggaa acagcacatt 2820cccagagttc ctccacaaca tggactactt caagttccac aacatgcgac cccctttcac 2880ctacgccacg ctcatccgct gggccatcct ggaggctcca gagaagcagc ggacactcaa 2940tgagatctac cactggttca cacgcatgtt tgccttcttc agaaaccatc ctgccacctg 3000gaagaacgcc atccgccaca acctgagtct gcacaagtgc tttgtgcggg tggagagcga 3060gaagggggct gtgtggaccg tggatgagct ggagttccgc aagaaacgga gccagaggcc 3120cagcaggtgt tccaacccta cacctggccc ctgaggatcc 316010588DNAArtificial Sequencesynthetic 10atg gat ttt cag gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga ttc tcc ttc aag gga ata aaa ttt tct aag gga 96Val Ile Met Ser Arg Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30aaa tac aaa ggg tcg act tcc ggt agc ggc aaa tcc tct gaa ggc aaa 144Lys Tyr Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys 35 40 45ggt cag gtt cct gct cag cta ctg gta ggc gcc ctg cgt gtg cta gcg 192Gly Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val Leu Ala 50 55 60tac tcc cgc ctc aag gtc acc gtg aaa ggg aaa cac ctt tgt cca agt 240Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His Leu Cys Pro Ser65 70 75 80ccc cta ttt ccc gga cct tct aag ccc ttt tgg gtg ctg gtg gtg gtt 288Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val 85 90 95ggt gga gtc ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt att 336Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile 100 105 110att ttc tgg gtg agg agt aag agg agc agg ctc ctg cac agt gac tac 384Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 115 120 125atg aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac cag 432Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 130 135 140ccc tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg cga 480Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg145 150 155 160aag gca gct ata acc agc tat gag aaa tca gat ggt gtt tac acg ggc 528Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly 165 170 175ctg agc acc agg aac cag gag act tac gag act ctg aag cat gag aaa 576Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys 180 185 190cca cca cag tag 588Pro Pro Gln 195113259DNAArtificial Sequencesynthetic 11atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agattctcct tcaagggaat aaaattttct aagggaaaat acaaagggtc gacttccggt 120agcggcaaat cctctgaagg caaaggtcag gttcctgctc agctactggt aggcgccctg 180cgtgtgctag cgtactcccg cctcaaggtc accgtgaaag ggaaacacct ttgtccaagt 240cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg tggagtcctg 300gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 360agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 420aagcattacc agccctatgc cccaccacgc gacttcgcag cctatagatc tcaagtgcga 480aaggcagcta taaccagcta tgagaaatca gatggtgttt acacgggcct gagcaccagg 540aaccaggaga cttacgagac tctgaagcat gagaaaccac cacagtagct ttagactcga 600gcgggatcaa ttccgccccc cccctaacgt tactggccga agccgcttgg aataaggccg 660gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc 720ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa 780aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag 840acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg 900cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg 960ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa 1020caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg 1080gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca 1140cggggacgtg gttttccttt gaaaaacacg ataatagcat gcagcgctac cggtcgccac 1200catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 1260cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 1320cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac 1380cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa 1440gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 1500cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 1560ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 1620caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa 1680cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc 1740cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 1800ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt 1860cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc 1920cggccggact cagatctcga gctcaagctt cgaattcatg cccaacccca ggcctggcaa 1980gccctcggcc ccttccttgg cccttggccc atccccagga gcctcgccca gctggagggc 2040tgcacccaaa gcctcagacc tgctgggggc ccggggccca gggggaacct tccagggccg 2100agatcttcga ggcggggccc atgcctcctc ttcttccttg aaccccatgc caccatcgca 2160gctgcagctg cccacactgc ccctagtcat ggtggcaccc tccggggcac ggctgggccc 2220cttgccccac ttacaggcac tcctccagga caggccacat ttcatgcacc agctctcaac 2280ggtggatgcc cacgcccgga cccctgtgct gcaggtgcac cccctggaga gcccagccat 2340gatcagcctc acaccaccca ccaccgccac tggggtcttc tccctcaagg cccggcctgg 2400cctcccacct gggatcaacg tggccagcct ggaatgggtg tccagggagc cggcactgct 2460ctgcaccttc ccaaatccca gtgcacccag gaaggacagc accctttcgg ctgtgcccca 2520gagctcctac ccactgctgg caaatggtgt ctgcaagtgg cccggatgtg agaaggtctt 2580cgaagagcca gaggacttcc tcaagcactg ccaggcggac catcttctgg atgagaaggg 2640cagggcacaa tgtctcctcc agagagagat ggtacagtct ctggagcagc agctggtgct 2700ggagaaggag aagctgagtg ccatgcaggc ccacctggct gggaaaatgg cactgaccaa 2760ggcttcatct gtggcatcat ccgacaaggg ctcctgctgc atcgtagctg ctggcagcca 2820aggccctgtc gtcccagcct ggtctggccc ccgggaggcc cctgacagcc tgtttgctgt 2880ccggaggcac ctgtggggta gccatggaaa cagcacattc ccagagttcc tccacaacat 2940ggactacttc aagttccaca acatgcgacc ccctttcacc tacgccacgc tcatccgctg 3000ggccatcctg gaggctccag agaagcagcg gacactcaat gagatctacc actggttcac 3060acgcatgttt gccttcttca gaaaccatcc tgccacctgg aagaacgcca tccgccacaa 3120cctgagtctg cacaagtgct ttgtgcgggt ggagagcgag aagggggctg tgtggaccgt 3180ggatgagctg gagttccgca agaaacggag ccagaggccc agcaggtgtt ccaaccctac 3240acctggcccc tgaggatcc 32591214PRTArtificial Sequencesynthetic 12Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly1 5 1013927DNAArtificial Sequencesynthetic 13atg gat ttt cag gtg cag att ttc agc ttc ctg cta atc agt gcc tca 48Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15gtc ata atg tct aga atg tta cca ttt ctg ttt ttt tcc acc ctg ttt 96Val Ile Met Ser Arg Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe 20 25 30tct tcc ata ttt act gaa gct cag aag cag tat tgg gtc tgc aac tca 144Ser Ser Ile Phe Thr Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser 35 40 45tcc gat gca agt att tca tac acc tac tgt gat aaa atg caa tac cca 192Ser Asp Ala Ser Ile Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro 50 55 60att tca att aat gtt aac ccc tgt ata gaa ttg aaa gga tcc aaa gga 240Ile Ser Ile Asn Val Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly65 70 75 80tta ttg cac att ttc tac att cca agg aga gat tta aag caa tta tat 288Leu Leu His Ile Phe Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr 85 90 95ttc aat ctc tat ata act gtc aac acc atg aat ctt cca aag cgc aaa 336Phe Asn Leu Tyr Ile Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys 100 105 110gaa gtt att tgc cga gga tct gat gac gat tac tct ttt tgc aga gct 384Glu Val Ile Cys Arg Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala 115 120 125ctg aag gga gag act gtg aat aca aca ata tca ttc tcc ttc aag gga 432Leu Lys Gly Glu Thr Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly 130 135 140ata aaa ttt tct aag gga aaa tac aaa tgt gtt gtt gaa gct att tct 480Ile Lys Phe Ser Lys Gly Lys Tyr Lys Cys Val Val Glu Ala Ile Ser145 150 155 160ggg agc cca gaa gaa atg ctc ttt tgc ttg gag ttt gtc atc cta cac 528Gly Ser Pro Glu Glu Met Leu Phe Cys Leu Glu Phe Val Ile Leu His 165 170 175caa cct aat tca aat ggt cac ctc gtg aaa ggg aaa cac ctt tgt cca 576Gln Pro Asn Ser Asn Gly His Leu Val Lys Gly Lys His Leu Cys Pro 180 185 190agt ccc cta ttt ccc gga cct tct aag ccc ttt tgg gtg ctg gtg gtg 624Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val 195 200 205gtt ggt gga gtc ctg gct tgc tat agc ttg cta gta aca gtg gcc ttt 672Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 210 215 220att att ttc tgg gtg agg agt aag agg agc agg ctc ctg cac agt gac 720Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp225 230 235 240tac atg aac atg act ccc cgc cgc ccc ggg ccc acc cgc aag cat tac 768Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 245 250 255cag ccc tat gcc cca cca cgc gac ttc gca gcc tat aga tct caa gtg 816Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val 260 265 270cga aag gca gct ata acc agc tat gag aaa tca gat ggt gtt tac acg 864Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr 275 280 285ggc ctg agc acc agg aac cag gag act tac gag act ctg aag cat gag 912Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 290 295 300aaa cca cca cag tag 927Lys Pro Pro Gln305144408DNAArtificial Sequencesynthetic 14atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtct 60agaatgttac catttctgtt tttttccacc ctgttttctt ccatatttac tgaagctcag 120aagcagtatt gggtctgcaa ctcatccgat gcaagtattt catacaccta ctgtgataaa 180atgcaatacc caatttcaat taatgttaac ccctgtatag aattgaaagg atccaaagga 240ttattgcaca ttttctacat tccaaggaga gatttaaagc aattatattt caatctctat 300ataactgtca acaccatgaa tcttccaaag cgcaaagaag ttatttgccg aggatctgat 360gacgattact ctttttgcag agctctgaag ggagagactg tgaatacaac aatatcattc 420tccttcaagg gaataaaatt ttctaaggga aaatacaaat gtgttgttga agctatttct 480gggagcccag aagaaatgct cttttgcttg gagtttgtca tcctacacca acctaattca 540aatggtcacc tcgtgaaagg gaaacacctt tgtccaagtc ccctatttcc cggaccttct 600aagccctttt gggtgctggt ggtggttggt ggagtcctgg cttgctatag cttgctagta 660acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 720tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 780ccaccacgcg acttcgcagc ctatagatct caagtgcgaa aggcagctat aaccagctat 840gagaaatcag atggtgttta cacgggcctg agcaccagga accaggagac ttacgagact 900ctgaagcatg agaaaccacc acagtagctt tagactcgag cgggatcaat tccgcccccc 960ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt 1020attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt 1080cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa 1140tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac 1200cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg 1260tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt 1320tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca 1380gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt 1440ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg 1500aaaaacacga taatagcatg cagcgctacc ggtcgccacc atggtgagca agggcgagga 1560gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 1620gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 1680catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 1740cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 1800cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 1860caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1920gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 1980cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 2040gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 2100ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 2160cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 2220cgccgggatc actctcggca tggacgagct gtacaagtcc ggccggactc agatctcgag 2280ctcaagcttc gaattcatgc ccaaccccag gcctggcaag ccctcggccc cttccttggc 2340ccttggccca tccccaggag cctcgcccag ctggagggct gcacccaaag cctcagacct 2400gctgggggcc cggggcccag ggggaacctt ccagggccga gatcttcgag gcggggccca 2460tgcctcctct tcttccttga accccatgcc accatcgcag ctgcagctgc ccacactgcc 2520cctagtcatg gtggcaccct ccggggcacg gctgggcccc ttgccccact tacaggcact 2580cctccaggac aggccacatt tcatgcacca gctctcaacg gtggatgccc acgcccggac 2640ccctgtgctg caggtgcacc ccctggagag cccagccatg atcagcctca caccacccac 2700caccgccact ggggtcttct ccctcaaggc ccggcctggc ctcccacctg ggatcaacgt 2760ggccagcctg gaatgggtgt ccagggagcc ggcactgctc tgcaccttcc caaatcccag 2820tgcacccagg aaggacagca ccctttcggc tgtgccccag agctcctacc cactgctggc 2880aaatggtgtc tgcaagtggc ccggatgtga gaaggtcttc gaagagccag aggacttcct 2940caagcactgc caggcggacc atcttctgga tgagaagggc agggcacaat gtctcctcca 3000gagagagatg gtacagtctc tggagcagca gctggtgctg gagaaggaga agctgagtgc 3060catgcaggcc cacctggctg ggaaaatggc actgaccaag gcttcatctg tggcatcatc 3120cgacaagggc tcctgctgca tcgtagctgc tggcagccaa ggccctgtcg tcccagcctg 3180gtctggcccc cgggaggccc ctgacagcct gtttgctgtc cggaggcacc tgtggggtag 3240ccatggaaac agcacattcc cagagttcct ccacaacatg gactacttca agttccacaa 3300catgcgaccc cctttcacct acgccacgct catccgctgg gccatcctgg aggctccaga 3360gaagcagcgg acactcaatg agatctacca ctggttcaca cgcatgtttg ccttcttcag 3420aaaccatcct gccacctgga agaacgccat ccgccacaac ctgagtctgc acaagtgctt 3480tgtgcgggtg gagagcgaga agggggctgt gtggaccgtg gatgagctgg agttccgcaa 3540gaaacggagc cagaggccca gcaggtgttc caaccctaca cctggcccct gaggatccgc 3600gccgctcgcg actcgagaga tccggattag tccaatttgt taaagacagg atatcagtgg 3660tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat 3720agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct 3780gtaggtttgg caagctagct taagtaacgc cattttgcag gcatggaaaa atacataact 3840gagaatagag aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa 3900caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc 3960tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag 4020aacagatggt ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt 4080tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 4140cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 4200cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat 4260aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct 4320gagtgattga ctacccgtca gcgggggtct ttcacatgca gcatgtatca aaattaattt 4380ggtttttttt cttaagtatt tacattaa 44081520DNAArtificial Sequencesynthetic 15agtccgggca ggtctacttt 201620DNAArtificial Sequencesynthetic 16gaggcaacct gaccactctc 201719DNAArtificial Sequencesynthetic 17tctggaggaa ctggcaaaa 191820DNAArtificial Sequencesynthetic 18tgagctcatt gaatgcttgg 201920DNAArtificial Sequencesynthetic 19tacagggctt tcgattcagc 202020DNAArtificial Sequencesynthetic 20cgcacacagc agttcttctc 202120DNAArtificial Sequencesynthetic 21tccttgggaa gcaattgaag 202220DNAArtificial Sequencesynthetic 22aactggccac agttttcagg 202321DNAArtificial Sequencesynthetic 23ctaagcaagg acggcgaatg t 212420DNAArtificial Sequencesynthetic 24ggctgggaac aggatactgg 202520DNAArtificial Sequencesynthetic 25gcctgcggac tctaccataa 202620DNAArtificial Sequencesynthetic 26cagggatgac atgtgtctgg 202720DNAArtificial Sequencesynthetic 27gtgttcctac ccccaatgtg 202820DNAArtificial Sequencesynthetic 28cttgctcagt gtccttgctg 2029388PRTArtificial Sequencesynthetic 29Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile
Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ser Ser 20 25 30Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser 35 40 45Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr 50 55 60Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser65 70 75 80Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 85 90 95Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 100 105 110Val Tyr Tyr Cys Lys Gln Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly 115 120 125Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser 130 135 140Glu Gly Lys Gly Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val145 150 155 160Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp 165 170 175Phe Ser Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 180 185 190Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr 195 200 205Thr Pro Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys 210 215 220Asn Thr Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala225 230 235 240Leu Tyr Tyr Cys Ala Arg Arg Tyr Gly Asn Tyr Trp Tyr Phe Asp Val 245 250 255Trp Gly Ala Gly Thr Thr Val Thr Val Lys Gly Lys His Leu Cys Pro 260 265 270Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val 275 280 285Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 290 295 300Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp305 310 315 320Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 325 330 335Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val 340 345 350Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr 355 360 365Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 370 375 380Lys Pro Pro Gln38530167PRTArtificial Sequencesynthetic 30Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu 20 25 30Arg Val Leu Ala Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His 35 40 45Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val 50 55 60Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr65 70 75 80Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu 85 90 95His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 100 105 110Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 115 120 125Ser Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly 130 135 140Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu145 150 155 160Lys His Glu Lys Pro Pro Gln 16531162PRTArtificial Sequencesynthetic 31Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30Lys Tyr Lys Gly His Leu Val Lys Gly Lys His Leu Cys Pro Ser Pro 35 40 45Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 50 55 60Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile65 70 75 80Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 85 90 95Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 100 105 110Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg Lys 115 120 125Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu 130 135 140Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro145 150 155 160Pro Gln32195PRTArtificial Sequencesynthetic 32Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys Gly 20 25 30Lys Tyr Lys Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys 35 40 45Gly Gln Val Pro Ala Gln Leu Leu Val Gly Ala Leu Arg Val Leu Ala 50 55 60Tyr Ser Arg Leu Lys Val Thr Val Lys Gly Lys His Leu Cys Pro Ser65 70 75 80Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val 85 90 95Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile 100 105 110Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 115 120 125Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 130 135 140Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val Arg145 150 155 160Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly 165 170 175Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys 180 185 190Pro Pro Gln 19533308PRTArtificial Sequencesynthetic 33Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe 20 25 30Ser Ser Ile Phe Thr Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser 35 40 45Ser Asp Ala Ser Ile Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro 50 55 60Ile Ser Ile Asn Val Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly65 70 75 80Leu Leu His Ile Phe Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr 85 90 95Phe Asn Leu Tyr Ile Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys 100 105 110Glu Val Ile Cys Arg Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala 115 120 125Leu Lys Gly Glu Thr Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly 130 135 140Ile Lys Phe Ser Lys Gly Lys Tyr Lys Cys Val Val Glu Ala Ile Ser145 150 155 160Gly Ser Pro Glu Glu Met Leu Phe Cys Leu Glu Phe Val Ile Leu His 165 170 175Gln Pro Asn Ser Asn Gly His Leu Val Lys Gly Lys His Leu Cys Pro 180 185 190Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val 195 200 205Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe 210 215 220Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp225 230 235 240Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr 245 250 255Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gln Val 260 265 270Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr 275 280 285Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu 290 295 300Lys Pro Pro Gln305
Patent applications by Zelig Eshhar, Rehovot IL
Patent applications by YEDA RESEARCH AND DEVELOPMENT CO. LTD.
Patent applications in class Leukocyte
Patent applications in all subclasses Leukocyte