The present invention relates to compositions-of-matter capable of specifically binding particular antigen-presenting molecule (APM):antigen complexes. More particularly, the present invention relates to compositions-of-matter capable of specifically binding a particular human APM:pathogen-derived antigen complex.
Diseases caused by pathogens, such as viruses, mycoplasmas, bacteria, fungi, and protozoans, account for a vast number of diseases, including highly debilitating/lethal diseases, affecting all human individuals at numerous instances during their lifetime. For example, diseases caused by retroviruses are associated with various immunological, neurological, and neoplastic disorders. For example, diseases caused by lymphotropic retroviruses, such as acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV), or, the closely related human T-cell lymphotropic virus (HTLV), a causative agent of various lethal pathologies (for general references, refer, for example to: Johnson J M. et al., 2001. Int J Exp Pathol. 82:135-47; and Bangham C R., 2000. J Clin Pathol. 53:581-6), account for lethal disease epidemics of devastating human and economic impact.
However, satisfactory methods of diagnosing, characterizing, and treating many kinds of pathogen-associated diseases such as diseases associated with lymphotropic viruses such as HIV or HTLV are unavailable.
HTLV-1 was the first human retrovirus identified (Poiesz B. J. et al., 1980. Proc Natl Acad Sci USA. 77:7415-7419). It infects both CD4+ and CD8+ T-lymphocytes and is associated with a variety of diseases, including adult T-lymphocyte leukemia/lymphoma (ATLL; Yoshida M. et al., 1982. Proc Natl Acad Sci USA. 79:2031-2035) and a non neoplasic inflammatory neurological syndrome called human T lymphotropic type I (HTLV-1)-associated myelopathy/tropical virus spastic paraparesis (HAM/TSP; Osame M. et al., 1986. Lancet 1:1031-1032; reviewed in Ribas J G. and Melo G C., 2002. Rev Soc Bras Med Trop. 35:377-84; and Plumelle Y., 1999. Med Hypotheses. 52:595-604). Other diseases linked to HTLV-1 infection on the basis of seroepidemiological studies include Sjogren's syndrome, inflammatory arthropathies, polymyositis, and pneumopathies (Coscoy L. et al., 1998. Virology 248: 332-341). The HTLV protein Tax seems to play a major role in the pathogenesis of HTLV-1 associated diseases. Tax protein is known to stimulate the transcription of viral and cellular genes such as the genes coding for interleukin-2 (IL-2) and other cytokines, interleukin-2 receptor (IL-2R), proto-oncogenes, c-jun and c-fos, and major histocompatibility complex (MHC) molecules (Yoshida M., 1993. Trends Microbiol. 1:131-135). The transforming potential of Tax has been demonstrated in different experimental systems. It has been shown that rodent fibroblastic cell lines expressing Tax form colonies in soft agar and tumors in nude mice (Tanaka A. et al., 1990. Proc Natl Acad Sci USA. 87:1071-1075). Also, Tax transforms primary fibroblasts in cooperation with the Ras protein (Pozzatti R. et al., 1990. Mol Cell Biol. 10:413-417), and immortalizes primary T-lymphocytes in the presence of IL-2 (Grassmann R. et al., 1989. Proc Natl Acad Sci USA. 86:3351-3355). Transgenic mice carrying the tax gene develop different types of tumors (Grossman W. J. et al., 1995. Proc Natl Acad Sci USA. 92:1057-1061). Tax binds directly to DNA but acts in cooperation with several cellular transcription factors, but the role of these different interactions in the cell transformation mediated by Tax is still unclear (Coscoy L. et al., 1998. Virology 248: 332-341).
HAM/TSP is a progressive chronic demyelinating disorder affecting the white matter of the central nervous system (CNS) and the spinal cord. The disease affects approximately twice as many females as males, and typically the time of disease onset occurs during the fourth decade of life. The disease causes numerous highly debilitating symptoms, with common early symptoms and signs including gait disturbance and weakness and stiffness of the lower limbs. The disease affects the lower extremities to a much greater degree than upper extremities, spasticity may be moderate to severe, and lower back pain commonly occurs. Disease progression is associated with bowel and bladder dysfunction, and sensory loss and dysesthesia. Patients examined via magnetic resonance imaging may exhibit nonspecific lesions in the brain as well as spinal cord atrophy. Immune manifestations associated with HAM/TSP include inflammatory infiltrates in the central nervous system consisting predominantly of monocytes, and large numbers of CD8+ T-cells which are primarily reactive with peptides of the HTLV-1 Tax protein. The frequency of such T-cells in the peripheral blood and cerebrospinal fluid (CSF) has been shown to be proportional to the amount of HTLV-1 proviral load and the levels of HTLV-1 tax mRNA expression. It has further been shown that in patients carrying the HLA-A2 allele, the immune response is dominated by CD8+ T-lymphocytes that recognize the Tax11-19 peptide (Bieganowska K. et al., 1999. J Immunol. 162:1765-1771; Nagai, M. et al., 2001. J Inf Dis. 183:197-205). Thus, immunological determinants, such as the Tax11-19 peptide and antigenic mimics thereof, shared by thymus, brain and HTLV-1 are thought to direct lymphocytic neurotropism and demyelinization in nervous tissues. It is thought that the specificity of thoracic spinal cord involvement could be linked to shared thymic and thoracic spinal cord determinants, genetically peculiar to HAM/TSP patients. In a first stage, disease onset may be dependent on CD4+ T-lymphocytes specific for such determinants, reactivated in response to HTLV-1 infection, and that demyelinization during this stage could potentially be initiated as a result of stoppage in the synthesis of myelin following alteration of expression of oligodendrocytic and neuronal adhesion molecules. The second stage of the disease, involving chronic inflammatory manifestations, may depend on CD8+ T-lymphocytes specific for viral peptides, but also on CD8+ T-lymphocytes specific for peptides generated as a result of proteolysis of myelin layer, and other central nervous system proteins.
While, at best, therapy of HAM/TSP with corticosteroids, and IFN-gamma may result transient responses, similarly to numerous diseases associated with lymphotropic viruses there is currently no effective treatment for HAM/TSP, nor does the state of the art currently enable optimal prediction, diagnosis, staging, monitoring, and prognosis of the disease in patients.
The immune system employs two types of immune responses to provide antigen specific protection from pathogens; humoral immune responses, and cellular immune responses, which involve specific recognition of pathogen antigens via antibodies and T-lymphocytes, respectively.
T-lymphocytes, by virtue of being the antigen specific effectors of cellular immunity, play a central and direct role in the body's defense against diseases mediated by intracellular pathogens, such as viruses, intracellular bacteria, mycoplasmas, and intracellular parasites, by directly cytolysing cells infected by such pathogens. However, helper T-lymphocytes also play a critical role in humoral immune responses against non intracellular pathogens by providing T-cell help to B lymphocytes in the form of interleukin secretion to stimulate production of antibodies specific for antigens of such pathogens.
The specificity of T-lymphocyte responses is conferred by, and activated through T-cell receptors (TCRs). T-cell receptors are antigen specific receptors clonally distributed on individual T-lymphocytes whose repertoire of antigenic specificity is generated via somatic gene rearrangement mechanisms analogously to those involved in generating the antibody gene repertoire. T-cell receptors are composed of a heterodimer of transmembrane molecules, the main type being composed of an alpha-beta dimer and a smaller subset of a gamma-delta dimer. T-lymphocyte receptor subunits comprise a transmembrane constant region and a variable region in the extracellular domain, similarly to immunoglobulins, and signal transduction triggered by TCRs is indirectly mediated via CD3/zeta, an associated multi-subunit complex comprising signal transducing subunits.
The two main classes of T-lymphocytes, helper T-lymphocytes and cytotoxic T-lymphocytes (CTLs), are distinguished by expression of the surface markers CD4 and CD8, respectively. As described hereinabove, the main function of helper T-lymphocytes is to secrete cytokines, such as IL-2, promoting activation and proliferation of CTLs and B lymphocytes, and the function of CTLs is to induce apoptotic death of cells displaying immunogenic antigens.
T-lymphocyte receptors, unlike antibodies, do not recognize native antigens but rather recognize cell-surface displayed complexes comprising an intracellularly processed fragment of a protein or lipid antigen in association with a specialized antigen-presenting molecule (APM): major histocompatibility complex (MHC) for presentation of peptide antigens; and CD1 for presentation of lipid antigens, and to a lesser extent, peptide antigens. Peptide antigens displayed by MHC molecules and lipid antigens displayed by CD1 molecules have characteristic chemical structures are referred to as MHC-restricted peptides and CD1 restricted lipids, respectively. Major histocompatibility complex molecules are highly polymorphic, comprising more than 40 common alleles for each individual gene. “Classical” MHC molecules are divided into two main types, class I and class II, having distinct functions in immunity.
Major histocompatibility complex class I molecules are expressed on the surface of virtually all cells in the body and are dimeric molecules composed of a transmembrane heavy chain, comprising the peptide antigen binding cleft, and a smaller extracellular chain termed β2-microglobulin. MHC class I molecules present 9- to 11-amino acid residue peptides derived from the degradation of cytosolic proteins by the proteasome a multi-unit structure in the cytoplasm, (Niedermann G., 2002. Curr Top Microbiol Immunol. 268:91-136; for processing of bacterial antigens, refer to Wick M J, and Ljunggren H G., 1999. Immunol Rev. 172:153-62). Cleaved peptides are transported into the lumen of the endoplasmic reticulum (ER) by TAP where they are bound to the groove of the assembled class I molecule, and the resultant MHC:antigen complex is transported to the cell membrane to enable antigen presentation to T-lymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7; Yewdell J W. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8).
Major histocompatibility complex class II molecules are expressed on a restricted subset of specialized antigen-presenting cells (APCs) involved in T-lymphocyte maturation and priming. Such APCs in particular include dendritic cells and macrophages, cell types which internalize, process and display antigens sampled from the extracellular environment. Unlike MHC class I molecules, MHC class II molecules are composed of an alpha-beta transmembrane dimer whose antigen binding cleft can accommodate peptides of about 10 to 30, or more, amino acid residues.
The antigen presenting molecule CD1, whose main function, as described hereinabove, is presentation of lipid antigens, is a heterodimer comprising a transmembrane heavy chain paired with beta2-microglobulin, similarly to MHC class I, and is mainly expressed on professional APCs, similarly to MHC class II (Sugita M. and Brenner M B., 2000. Semin Immunol. 12:511). CD1:antigen complexes are specifically recognized by TCRs expressed on CD4−CD8− T-lymphocytes and NKT lymphocytes and play a significant role in microbial immunity, tumor immunology, and autoimmunity.
The cells of the body are thus scanned by T-lymphocytes during immune surveillance or during maturation of T-lymphocytes for their intracellular protein or lipid content in the form of such APM:antigen complexes.
One strategy which has been proposed to enable optimal diagnosis, characterization, and treatment of diseases, such as HAM/TSP, associated with an infection by a pathogen involves using molecules capable of specifically binding APM:antigen complexes composed of a particular combination of APM and an antigen derived from such a pathogen. Such molecules, for example, could be conjugated to functional moieties, such as detectable moieties or toxins, and the resultant conjugates could be used to detect such complexes or cells displaying such complexes, or to kill cells displaying such complexes. Hence, such conjugates could be used to diagnose/characterize and treat a pathogen infection in an individual, respectively. Alternately, molecules capable of specifically binding such complexes could be used to bind such complexes on cells so as to block activation of T-lymphocytes bearing TCRs specific for such complexes. Such molecules could further be used, for example, to isolate such complexes, or cells displaying such complexes, such as cells infected with a pathogen, or APCs exposed to a pathogen-derived antigen.
Several prior art approaches have been described involving molecules capable of binding particular APM:antigen complexes.
One approach involves using TCRs or derivatives thereof specific for particular MHC:peptide complexes in attempts to provide reagents capable of specifically binding such complexes.
Another approach involves using antibodies or derivatives thereof specific for particular mouse MHC:peptide complexes in attempts to provide reagents capable of specifically binding such complexes (Aharoni, R. et al., 1991. Nature 351:147-150; Andersen, P. S. et al., 1996. Proc. Natl. Acad. Sci. U.S.A 93:1820-1824; Dadaglio, G. et al., 1997. Immunity 6:727-738; Day, P. M. et al., 1997. Proc. Natl. Acad. Sci. U.S.A. 94:8064-8069; Krogsgaard, M. et al., 2000. J. Exp. Med. 191:1395-1412; Murphy, D. B. et al., 1989. Nature 338:765-768; Porgador, A. et al., 1997. Immunity 6:715-726; Reiter, Y. et al., Proc. Natl. Acad. Sci. U.S.A. 94:4631-4636; Zhong, G. et al., 1997. Proc. Natl. Acad. Sci. U.S.A. 94:13856-13861; Zhong, G. et al., 1997. J. Exp. Med. 186:673-682).
A further approach involves utilizing antibodies or derivatives thereof specific for the human MHC class I molecule HLA-A1 in complex with an HLA-A1 restricted peptide derived from the melanoma specific tumor associated antigen melanoma associated antigen (MAGE)-A1 in attempts to provide reagents capable of specifically binding such a complex (Chames, P. et al., 2000. Proc. Natl. Acad. Sci. U.S.A. 97:7969-7974).
An additional approach involves employing antibodies or derivatives thereof specific for the human MHC class I molecule HLA-A2 in complex with an HLA-A2 restricted peptide derived from the melanoma specific tumor associated antigen gp 100 in attempts to provide reagents capable of specifically binding such a complex (Denkberg, G. et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426).
Yet another approach involves using antibodies or derivatives thereof specific for human MHC class I molecule HLA-A2 in complex with an HLA-A2 restricted peptide derived from human telomerase catalytic subunit (hTERT) in attempts to provide reagents capable of specifically binding such a complex (Lev, A. et al., 2002. Cancer Res. 62:3184-3194).
However, all of the aforementioned prior art approaches suffer from significant disadvantages: (i) approaches involving the use TCRs or portions thereof as compounds capable of specifically binding particular MHC:peptide complexes are suboptimal due to the relatively low intrinsic binding affinity of TCRs for such complexes; (ii) approaches involving the use of antibodies or portions thereof specific for MHC:peptide complexes comprising non-human MHC are not suitable for human application; and (iii) approaches involving antibodies or portions thereof specific for MHC:non-pathogen-derived antigen complexes are not suitable for specifically binding complexes comprising pathogen-derived antigens.
Thus, all prior art approaches have failed to provide an adequate solution for providing molecules capable of specifically binding with high specificity and affinity a particular human APM:pathogen-derived antigen complex.
There is thus a widely recognized need for, and it would be highly advantageous to have, molecules devoid of the above limitation.
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