Compositions and methods for modulation of immune responses

专利摘要:
The present invention relates to a novel mechanism for modulating the immune response. More specifically, the present invention relates to the modularization of said receptor by HLA-E + binding peptides, leading to the inhibition or absence of inhibition of the 5CD94 / NKG2 receptor. In a preferred embodiment, the present invention relates to a HLA-E binding hsp (heat shock protein) 60 peptide.
公开号:KR20040041575A
申请号:KR10-2004-7001590
申请日:2002-07-31
公开日:2004-05-17
发明作者:칼 페터 쇠더스트룀
申请人:칼 페터 쇠더스트룀；
IPC主号:

专利说明:

Modular composition of immune response and method of modularization {COMPOSITIONS AND METHODS FOR MODULATION OF IMMUNE RESPONSES}
[4] Natural killer (NK) cells are lymphocytes associated with an innate immune response to certain microbial and parasitic infections. Recent studies have also suggested an important role for NK cells in experimental autoimmune models, but little is known about the function of NK cells in human autoimmune diseases. Herein we describe MHC class I molecules for NK cells as well as ab T cells and gd T cells derived from synovial fluid (SF) and peripheral blood (PB) in patients with arthritis, mostly rheumatoid arthritis (RA) The expression of killer cell immunoglobulin (Ig) -like (KIR) and C-type lectin-like (CD94 / NKG2) receptors specific for is studied. We found that SF in arthritis patients contained a greater proportion of NK cells as compared to PB paired with them. In contrast to PB-NK cells, the SF-NK cell population expressed almost uniformly the CD94 / NKG2A cell surface receptor and contained a much smaller proportion of KIR + NK cells. Functional analysis has shown that both in vitro cultured polyclonal SF-NK cells and PB-NK cells derived from patients can completely kill a range of target cells. However, SF-NK cytolysis was inhibited by the presence of HLA-E on the transfected target cells. By blocking CD94 on SF-NK cells or blocking HLA on autologous cells, SF-NK cells were able to perform self-directed lysis. Thus, HLA-E may play a fundamental role in the regulation of major NK cell populations in inflammatory joints.
[5] MHC class I molecules regulate the function of natural killer (NK) cells, such as their ability to mediate lysis of target cells (Ljunggern et al. , Immunol. Today 11 : 237-244, 1990, referenced herein). This regulation is controlled by the complex repertoire of MHC class I specific receptors displayed on the surface of NK cells. These receptors monitor the expression of MHC class I on neighboring cells and transmit inhibitory signals that block NK cell-mediated cytotoxicity of MHC class I-expressing normal cells (Lainer et al., Immunity 6; 371-378, 1997 , Referenced in the text).
[6] HLA-E is a widely distributed, non-traditional MHC class I molecule expressed on the cell surface in association with beta 2-microglobulin. HLA-E, along with b2-microglobulin and peptides, are widely expressed at low concentrations on the cell surface (see Wei et al. , Hum. Immunol. 29: 131, 1990). Peptide loading of HLA-E is believed to be TAP-dependent, although TAP-independent signs have been reported. In contrast to traditional MHC class I molecules, HLA-E exhibits a somewhat restrictive polymorphism, whose peptide binding cleft is derived from the signal sequence of specific HLA-A, -B, -C and G-molecules. Occupied predominantly by nonamers (Lazetic et al. , J. Immunol. 157: 4741-4745, 1996, referenced herein). Such peptides generally share a common motif, such as: common motifs such as methionine at position 2 and leucine or isoleucine at position 9 (Arnett et al . , Arthritis Rheum. 31: 315-324, 1988, referenced herein). ). Crystal structure analysis of HLA-E revealed peptide selectivity of this molecule (Soderstrom et al. , J. Immunol. 159: 1072-1075, 1997, referenced herein). The murine HLA-E homologue, named Qa-1b, also shows peptides derived from the signal sequence of several mouse MHC class I molecules with anchor residues similarly conserved at positions 2 and 9 (Miller et al . , Proc. Natl.Acad . Sci. USA 70 : 190-194, 1973; Hendrich et al., Arthritis Rheum . 34 : 423-431, 1991, referenced herein). Recently, however, both HLA-E and Qa-1b have been shown to be able to bind to various arrays of peptides derived from random peptide libraries (Fort et al. , J. Immunol. 161 : 3256-3261, 1998; Phillips Et al., Immunity 5 , 163-172, 1996, respectively). In addition, Qa-1b can represent peptides derived from mouse and bacterial heat shock protein 60 (hsp60), and these complexes are directed to T cells via their antigen-specific T cell receptor (TCR). Has been reported (Litwin et al . , J. Exp. Med. 180 : 537-543, 1994, referenced herein).
[7] Conservative anchor motifs found between the signal sequences of several MHC class I molecules are believed to be important for binding to pockets within the HLA-E peptide binding cleft. HLA-E molecules, when loaded with these HLA class I signal peptides, are C-type lectins, termed CD94 / NKG2A, -B, -C, -E, expressed on a subset of NK cells and T cells. It is believed to form functional ligands for analogous receptor dimers.
[8] In humans at least two types of inhibitory receptors have been described, one is a killer cell immunoglobulin (Ig) -like receptor (KIR) and the other is a C-type lectin-like receptor. KIR has several KIRs characterized by two (2D) or three (3D) extracellular Ig-like domains with short (S) long (L) cytoplasmic tails. Depending on their structure, the KIRs are further divided into several family subgroups, with members with three Ig-domains (KIR 3DL) specifically recognizing the HLA-B molecular group, while other KIRs with two Ig-domains (KIR 2DLs) recognize HLA-C molecules. In addition, homodimers of two KIR3D molecules have been reported to recognize HLA-A molecules (Long et al., Http://www.ncbi.nlm.nih.gov/prow/guide/679664748_g.htm , 1999, Referenced in the text). NKG2 family (NKG2A, -B, and C) (Chang et al. , Eur. J. Immunol. 25 : 2433-2437, 1995; Lazetic et al. , J. Immunol. 157 : 4741-4745, 1996, referenced herein) C-type lectin-like receptors, including CD94 covalently bound to them, specifically recognize relatively non-polymorphic HLA-E molecules (Braud et al., Nature 391: 795-799, 1991). , Referenced in the text). The CD94 / NKG2A receptor is believed to mediate an inhibitory signal for NK cells when recognized by cells of HLA-E loaded with appropriate peptides expressed on the bystander target cells. This CD94 / NKG2A mediated signal is believed to prevent activation of NK cells (eg, cytotoxicity and cytokine release) while encountering normal autologous cells. NK cells producing CD94 / NKG2A receptors that regulate their own self-tolerance can kill cells that have lost the expression of protective HLA-E molecules. Protective HLA-E molecules are those loaded with peptides derived from the signal sequence of certain other MHC class I molecules.
[9] Initially, it was understood that hybrid constructs consisting of HLA-G leader sequences implanted on HLA-B * 5801 transfected into 721.221 cells significantly upregulated protective endogenous HLA-E levels in this cell line (Braud Et al., 1991, supra. These experiments suggested that in order to generate and migrate to the cell surface detected by the CD94 / NKG2A inhibitory receptor, an HLA class I leader must be present for stable and mature HLA-E proteins.
[10] CD94 / NKG2 receptors are expressed mostly by NK cells in both humans and mice and interact with the non-traditional MHC class I molecule HLA-E and its mouse analog, Qa-1b, respectively. (Vance et al., J. Exp. Med. 188 : 1841, 1998; Braud et al., Nature 391: 6669: 795, 1998, respectively referenced in the text). NKG2A contains an intracellular immunoreceptor tyrosine-based activating motif (ITIM) that mediates inhibition signals (Brooks et al . , J. Exp. Med. 185 : 795, 1997, referenced herein). NKG2C mediates positive signaling in association with an immunoreceptor tyrosine-based activating motif (ITAM) that produces the adapter molecule DAP-12 (Lanier et al., Immunity 8 : 693, 1998, text). ). CD94 / NKG2A / C receptors have been reported to be able to distinguish between different HLA-E and Qa-1b binding peptides (Kraft et al . , J. Exp. Med . 192: 613, 2000; Llano et al . , Eur. J. Immunol . 28: 2854, 1998; Vales-Gomez et al., Embo J. 18: 4250, 1999; Brooks et al ., J. Immunol . 162: 305, 1999, respectively, referenced in the text). It is not.
[11] In order to avoid autoimmune attack mediated by NK cells, it has been proposed that at least one MHC class I-specific inhibitory receptor for one self-MHC class I molecule should be expressed by each single NK cell. (Lanier et al., Immunity 6: 371-378, 1997, referenced in the text). Most normal cells usually express all MHC class I molecules at sufficient levels, so they are protected from NK cell-mediated attacks. However, if one or several MHC class I molecule (s) are lost or down-regulated (this often occurs during certain viral infections and neoplastic transformations), these cells can become susceptible to destruction by NK cells. (Homologous). In addition, lymphocytes from patients with autoimmune diseases, including rheumatoid arthritis (RA), have been shown to have abnormalities in the expression of MHC class I (Fu et al. , J. Clin. Invest. 91: 2301-2307, 1993, Referenced in the text). It is not known whether this affects NK cell tolerance, and the role for NK cells in RA is also generally ambiguous.
[12] Recent studies in various experimental models of autoimmune disease, however, have gathered a regulatory role for NK cells that have been shown to be pathologically important. For example, NK cells appear to play an important role in down-regulating TH1-mediated colitis by modulating the response of effector T cells in a perforin dependent manner (Foert et al. , J. Immunol. 161: 3256-3261, 1998, see text). In experimental autoimmune encephalomyelitis (EAE), a model for multiple osteomyelitis (MS) in humans, administration of NK cell stimulating compound linomide prevented the mouse from developing the disease and lacked the same model of NK cells. In the city, the production of TH1 cytokines was increased to aggravate the disease (Matsumoto et al. , Eur. J. Immunol. 28: 1681-1688, 1998; Zhang et al. , J. Exp. Med. 186: 1677-1687, 1997, respectively. Referenced in the text). These reports suggest that the presence of NK cells is beneficial in preventing prototypie TH1-mediated diseases. In contrast, a pathological role of NK cells has also been suggested in the asthma model of rats with prototype TH2-mediated disease, where the deficiency of NK cells protects the mice from allergen-induced inflammation in the airway epithelium ( Korgren et al., J. Exp. Med. 189: 553-562, 1999, referenced herein).
[13] RA is an autoimmune disease characterized by chronic inflammation of the joints causing progressive destruction of cartilage and bone. After the onset of RA, the synovial compartment contains not only activated T cells but also granzyme-positive NK cells (Tak et al . , Arthritis Rheum. 37: 1735-1743, 1994, referenced herein). Although potent NK cell stimulating cytokines such as IL-15 are found in the joints (Thurkow et al., J. Pathol. 181: 444-450, 1997, referenced in the text), newly isolated synovial NK cells produce peripheral blood (PB). It is less cytotoxic than NK cells derived from and appears to produce less IFN-γ (Lipsky Clin. Exp. Rheumatol. 4: 303-305, 1986; Berg et al., Clin. Exp. Immunol. 1: 174 -182, 1999, respectively referenced in the text). Because signaling through KIR- as well as CD94 / NKG2 molecules has been known to regulate both NK cell-mediated cytotoxicity and cytokine production, it is important to study receptor availability to NK cells at the site of inflammation.
[14] Heat shock proteins (hsps), exemplified by hsp60, are also highly conserved through evolution between humans and bacteria. hsp60 is present in all living cellular organisms (Lindquist et al . , Annu. Rev. Genet. 22: 631, 1988; Bukau et al., Cell 92: 351, 1998, referenced herein). In eukaryotic cells it plays an important role as a mitochondrial chaperone and in bacteria it acts as an intracellular protein involved in the assembly and disassembly of multi-subunit protein complexes (Fink, Physiol. Rev. 79: 425, 1999, see text). Given the various stress stimuli such as temperature rise, nutrient depletion, exposure to toxic chemicals, inflammatory responses and allograft rejection, levels of hsp60 are elevated (Lindquist, 1988, supra; Anderton et al. , Eur. J. Immunol. 23:33, 1993; Birk et al . , Proc. Natl. Acad. Sci. USA 96: 5159, 1999). Hsp60 is believed to play an important role in protecting cells from the consequences of these harmful stimuli. At the same time, this can make these cells more susceptible to attack by hsp60-directed innate and acquired immune responses, and it is known that hsp60 is highly immunogenic. For example, an immune response directed against bacterial-hsp60 at infection may cross react with self-hsp60.
[15] hsp60 is a major self antigen in mammalian autoimmunity. The large increase in endogenous hsp60 expression in chronic inflammatory tissues (eg, as in rheumatoid joints) has generated great interest among research groups studying autoimmune mechanisms and autoimmune diseases. Increased levels of hsp60 are also found during cellular stress, such as abnormal hyperthermia, for example. Abnormal high fever throughout the body is also used as a therapy for cancer.
[16] Based on these and other reports, it modulates HLA-E / CD94 / NKG2 cellular receptor interactions, or HLA-E / CD94 / NKG2 cellular receptor interactions and / or cellular stressors, inflammation and There is no clear teaching or suggestion in the art of using common agents to modulate abnormal immune responses associated with changes in the state of related diseases, including autoimmune symptoms. Likewise, the role of stress-induced proteins and peptides, which may be associated with HLA-E / CD94 / NKG2 cellular receptor interactions and abnormal regulation of immune responses that participate in symptoms such as chronic inflammation and autoimmunity, are still It is not revealed.
[17] In view of the above circumstances, the technical field includes HLA-E / CD94 / NKG2 modularizing HLA-E / CD94 / NKG2 cellular receptor interactions and potentially mediated by abnormal immune responses, in particular cellular stressors. There is an urgent need for additional tools and methods for controlling abnormal immune responses associated with changes in cellular receptor interactions. There is also a need for effective compositions and methods for alleviating the symptoms of related disease states including inflammation, autoimmune diseases and cancer. Surprisingly, the present invention achieves this object and also satisfies additional objects and advantages that will be apparent from the following description.
[18] Summary of the Invention
[19] The present invention provides methods and compositions using proinflammatory or anti-inflammatory binding peptides to modulate an immune response in a mammalian subject. Typically, these binding peptides are composed of a major histocompatibility complex class I (MHC class I) molecule, such as an HLA-E MHC class I molecule, on an antigen presenting cell (APC) and a proinflammatory or anti-inflammatory binding peptide. Binding complexes allow HLA-E to interact with MHC class I-specific inhibitory receptors. MHC class I-specific inhibitory receptors will generally be CD94 / NKG2 cellular receptors. Interactions between pro- or anti-inflammatory binding peptides and HLA-E binding peptides modulate the interactions between these binding peptides / HLA-E complexes and receptors, resulting in new regulation of immune responses in cell populations expressing inhibitory receptors. .
[20] In a more specific aspect of the invention, the interaction between the pro-inflammatory or anti-inflammatory binding peptides and the HLA-E binding peptides is characterized by a population of cells or other subjects, such as autoimmune diseases, inflammatory diseases or symptoms (eg, chronic inflammation, or surgery). Inflammation leading to trauma), transplant rejection, viral infections, cancer, or any other disease or condition that can be treated by modulating the immune response according to the present invention to facilitate a proinflammatory or anti-inflammatory response in a mammalian subject. Do it.
[21] In certain embodiments of the invention, the anti-inflammatory binding peptides interact with HLA-E molecules on the cell surface producing peptides bound to HLA-E, and the resulting peptide-HLA-E complexes are MHC class I-specific. Recognized by inhibitory receptors. This recognition results in a protective immune response characterized by reduced cytotoxic activity and / or induction of expression of one or anti-inflammatory cytokine (s) by cells producing CD94 / NKG2 cellular receptors.
[22] In another aspect, the pro-inflammatory or anti-inflammatory binding peptides of the invention may exhibit activity to upregulate the expression of HLA-E molecules on cells exposed to the peptide in vitro or in vivo .
[23] In another embodiment of the invention, the proinflammatory binding peptide binds to the HLA-E molecule at the cell surface producing the peptide bound to HLA-E, and the resulting peptide-HLA-E complex is MHC class I-specific. Interfere with protective recognition by inhibitory receptors. In other words, the binding of this peptide inhibits the protective immune response mediated by the CD94 / NKG2 cellular receptor. This inhibition of CD94 / NKG2 receptor-mediated protection HLA-E between the pro-inflammatory binding peptide and one or more protective (ie anti-inflammatory) peptides that become inefficient or obsolete by binding competition with the pro-inflammatory binding peptides. It is related to competition over the combination with. In this context, the proinflammatory binding peptides competitively occupy HLA-E binding cleft and the complex between the proinflammatory binding peptide and HLA-E is not recognized by the CD94 / NKG2 cellular receptor. Inhibition of the CD94 / NKG2 cellular receptor-mediated protein induces and / or increases the expression of one or more proinflammatory cytokine (s) by cells producing CD94 / NKG2 cellular receptors (such as NK or T cells). Reflected by toxic activity.
[24] For proinflammatory binding peptides, these peptides may compete with anti-inflammatory binding peptides for binding to MHC class I molecules and / or stimulate cytotoxic or proinflammatory cytokine inducing responses in cells expressing CD94 / NKG2 cellular receptors. It will have biological activity.
[25] "Antigen producing cells" refers to a cell class capable of providing an antigen to cells of the immune system that can recognize the antigen when the antigen is associated with a major histocompatibility complex molecule. Antigen producing cells generally mediate an immune response to a particular antigen by processing the specific antigen into a form that can bind to the major histocompatibility complex molecule on the surface of the antigen producing cell. Antigen producing cells include a variety of cell types such as, for example, macrophages, T cells and synthetic (“artificial”) cells.
[26] In general, immune responses to be modularized by the methods and compositions of the present invention include cytotoxic and proinflammatory and anti-inflammatory cytokine induction in cells expressing MHC class I-specific inhibitory receptors. In an exemplary embodiment, these cells are selected from natural killer (NK) cells and cytotoxic T lymphocytes (CTL: cytotoxyc T lymphocytes). The elicited immune response may be to suppress or enhance one or more activities of NK or T cells, including suppression or enhancement of cytotoxic activity, cytokine production, proliferation, chemotaxis, and the like.
[27] The methods of the present invention generally involve a subject of the present invention that binds to a HLA-E molecule at the subject's surface and at that surface raises or inhibits binding of the CD94 / NKG2 cellular receptor to the peptide / HLA-E complex. Exposure to an effective amount of an inflammatory or anti-inflammatory binding peptide. In certain methods of the invention, subjects are isolated or bound CD94 / NKG2 cellular receptors, membrane or cell preparations containing the receptors, cell populations, tissues or organs expressing the receptors, or mammalian patients. In more detailed embodiments, the subject includes a cell population, tissue or organ selected for in vivo or ex vivo treatment or diagnostic processing. On the other hand, the subject may be a mammalian patient susceptible to an inflammatory or autoimmune disease or condition, viral infection, rejection of transplant or cancer. In such cases, proinflammatory or anti-inflammatory binding peptides may be administered in a prophylactic or therapeutically effective amount to prevent or inhibit the associated disease symptoms or signs.
[28] In a further embodiment of the invention, the pro-inflammatory or anti-inflammatory binding peptide is (a) binding an HLA-E molecule or HLA-E / peptide complex to the cell surface by CD94 / NKG2 cellular receptor (b) APC Cytotoxic or cytokine-inducing activity of (e.g., NK cells or CTLs), or (c) inhibiting or increasing a biological activity selected from one or more of the disease symptoms or signs associated with inflammatory or autoimmune diseases, viral infections, transplant rejection or cancer It is administered to the subject in an amount.
[29] Pro- or anti-inflammatory binding peptides may be naturally occurring or synthetic. Often these peptides may be peptide analogs or mimetic or allelic variants found among native pro- or anti-inflammatory binding peptide sequences. Peptides, peptide analogs or mimetic peptides may, for example, add, mix, or conjugate additional amino acids, peptides, proteins, chemical reagents or moieties that do not substantially alter the biological activity (eg, HLA-E binding activity) of the peptide. It can be modified in various ways.
[30] In a further aspect, the present invention relates to assays for HLA-E binding peptides or analogs, the assay comprising the following steps: a) providing a peptide library; b) forms an HLA-E / peptide complex; c) selecting an appropriate complex capable of inhibiting or activating the CD94 / NKG2 receptor on NK or T cells; d) Isolate stable peptide / peptide analogs from the complex.
[31] In another aspect, the present invention relates to a pharmaceutical composition containing any peptide according to the present invention together with a pharmaceutically acceptable carrier. In the methods and compositions of the invention, the pro-inflammatory or anti-inflammatory binding peptides are combined in various combinations with pharmaceutically acceptable carriers, diluents, excipients, adjuvants or other active or inactive agents, among the disease symptoms or signs identified below. It may be formulated in an amount or dosage form sufficient to prevent or alleviate one or more of the agents.
[32] In another aspect of the invention, the pro-inflammatory or anti-inflammatory binding peptides are combined combinations or active agent (s) for one or more additional antiviral, anti-inflammatory, anti-cancer, or anti-transplant rejection treatments or in accordance with a cooperative treatment protocol. It can be administered by the method described above. Within the scope of the methods and compositions, proinflammatory or anti-inflammatory binding peptides can be mixed or co-administered with one or more therapeutic adjuvants (simultaneously or sequentially) to prevent or alleviate one or more selected disease signs or symptoms described below.
[33] The invention also contains proinflammatory or anti-inflammatory binding peptides optionally with other active or inactive ingredients and / or means for administering them to diagnose, manage and / or prevent and treat selected disease symptoms or signs described below. It also relates to kits, packages and multicontainer units. In general, these kits comprise a diagnostic or pharmaceutical preparation of a pro-inflammatory or anti-inflammatory binding peptide, formulated with a biologically suitable carrier and optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional packaging materials may include labels or instructions for use, indicating the desired use of the kit, as described below.
[34] Additional aspects of the invention include vector constructs and polynucleotide molecules encoding the pro-inflammatory or anti-inflammatory binding peptides of the invention, including mimic peptides or peptide analogs.
[35] Vaccines and other immunogenic compositions also elicit an immune response associated with the production of antibodies that target one or more pro- or anti-inflammatory binding peptides of the invention, which may be useful for diagnostic and / or therapeutic purposes, as described below. Is provided. Various additional diagnostic and therapeutic tools and reagents are also provided, as set forth in the description below, in accordance with the present invention.
[1] The present invention relates to novel compositions and methods for modulating the immune response in mammals. More specifically, the present invention relates to the modularization of CD94 / NKG2 receptor function by HLA-E + binding peptides, wherein the peptide results in the inhibition or absence of inhibition of the receptor.
[2] Related reference
[3] This application claims the benefit of US Provisional Application No. 60 / 308,598, filed July 31, 2002, which is incorporated herein by reference.
[226] 1 provides the protein sequence of human hsp60. Mitochondrial targeting signals are grayed out. The box portion is four peptide sequences representing methionine accompanying the C-terminus of the seven amino acids of leucine or isoleucine, two residues important for binding to the HLA-E pocket. hsp60 corresponds to residues 10-18 (QMRPVSRVL) of this sequence.
[227] FIG. 2 shows stabilization of HLA-E by hsp60sp and B7sp in K562 cells transfected with HLA-E * 0101 or HLA-E * 01033. Cell surface expression of HLA-E * 0101 (top panel) and HLA-E * 01033 (bottom panel) after overnight incubation at 26 ° C. with hsp60sp (left panel, thick line) or B7sp (right panel thick line) at 300 ° C. overnight. Dotted line shows HLA-E expression after incubation with 300 mM of control peptide (P18I10). Cells were stained with anti-MHC class I mAb DX17 and then treated with RPE-conjugated goat-anti-mouse IgG. HLA-E expression was confirmed by staining with anti-HLA-E mA3D12. Staining with isota-leaf matched control antibody is indicated by hatched gray. One representative example of the ten experiments is shown.
[228] 3 shows that upregulation of HLA-E by overexpression of full length hsp60 signal peptide was increased by cellular stress. HLA-E surface expression was monitored for cells with increasing cell density. The cells were pooled and analyzed for HLA-E expression between Day 1 and Day 5 (indicated on the histogram). The numbers in the upper right corner of each histogram are the cell density (cell / ml) and the percentage of viability at the time of analysis, respectively. The numbers in the lower right corner of each histogram in (a) represent the MFI (top, black) of HLA-E expression and the MFI (bottom, gray) of GFP. The numbers in the lower right corner of each histogram of (b) represent the MFI of HLA-E expression. All cells were stained with HLA specific antibody (DX17, dashed line) or control Ig (grey histogram) followed by RPE-conjugated goat anti-mouse IgG. (a) Co-trans with HLA-E * 01033 and a full length (residual 1-26) wild type hsp60 signal peptide-GFP construct (wild type hsp60L, top panel) or variant hsp60 signal peptide GFP (mutated hsp60L, bottom panel) Specified K562 cells cultured with increasing cell density. The gate was set on GFP positive cells and 10 000 events were acquired within this gate. (b) K562 cells (top panel) and K562 transfected with HLA-E * 01033 (K562 E * 01033 lower panel), incubated with increasing cell density. The K562-E * 01033 cell line of (b) and the co-transfected cell line shown in FIG. (a) were independently generated and screened and had higher values than the HLA-E background levels observed on Day 1. Can be represented. Thus, the absolute level of HLLA-E cannot be directly compared between FIGS. 3A and 3B.
[229] 4 shows binding of soluble HLA-E tetramer (tetramer) molecules to the CD94 / NKG2 receptor. (a) Ba / F3 cells transfected with CD94 and NKG2A were HLA-E / B7sp tetramer- (bold line), HLA-E / hsp60sp-tetramer (thin line), or control H-2Db / gp33- Incubation with tetramer (dotted line). (b) Ba / F3 cells transfected with CD94, NKG2C and DAP-12 were treated with HLA-E / B7sp-tetramer (bold line), HLA-E / hsp60sp-tetramer (thin line), or control H- Incubation with 2Db / gp33-tetramer (dashed line). (c) NK cell line NKL was incubated with HLA-E / B7sp-tetramer (bold line), HLA-E / hsp60sp-tetramer (thin line), or control H-2Db / gp33-tetramer (dotted line) . (d) HB-120 B-cell hybridomas (anti-MHC class I) were replaced with HLA-E / B7sp-tetramer (bold line), HLA-E / hsp60sp-tetramer (thin line), or control H- Incubation with 2Db / gp33-tetramer (dashed line). All incubations were performed for 45 min at 4 ° C. in PBS supplemented with 1% FCS. HLA-E / hsp60sp-tetramer did not bind to both CD94 / NKG2A + and CD94 / NKG2C + cells over the entire HLA-E / hsp60sp-tetramer concentration range. This is the representative of one of five experiments performed.
[230] 5 shows that hsp60sp did not prevent death of K562-E * 01033 cells by NK cells. K562-E * 01033 cells were incubated with various peptides at 26 ° C. for 15-20 hours and then tested by 2h 51 Cr release assay. To clearly demonstrate that HLA-E levels with protective peptides, rather than HLA-E levels alone, provided protection, we maintained non-protective peptides in assays, but omitted B7sp. (a) Killing K562-E * 01033 cells by NKL (left) or Nishi (right) after incubation with 300 mM P18I10 control peptide, 300 mM hsp60sp, or 30 mM B7sp. 50 mM P18I10 control peptide and hsp60sp were also included in the analysis. Data with an E: T ratio of 30: 1 are shown. This figure shows the average of the results of at least three experiments. Error bars represent standard error of the mean. (b) K562-E * 01033 cells by NKL (left panel) or Nishi (right panel) incubated overnight with 30 mM B7sp, 300 mM P18 (10 (pCtrl), 300 mM B7R5V, 300 mM hsp60sp, or 300 mM hsp60, V5R) 50 mM of all peptides except B7sp were included in this analysis Peptide concentrations were selected according to Figure 5c This figure shows the mean of three or more experimental values The error bars represent the standard error of the mean. (c) Surface expression of HLA-E cells by K562-E * 01033 after analysis, cold target preparations were prepared in parallel as in (a) and (b) and then DX17 mAb (anti-HLA class I), RPE- Stained with conjugated goat-anti-mouse IgG A representative experiment of five experiments was shown: As in (a) and (b), 50 mM of all peptides except B7sp were present in this assay and this was indicated by Hsp6-sp. , Hsp60 V5R and B7 R5V showed more HLA-E expression for B7sp (D) increasing the amount of 0.1 mM B7sp and the competing peptides (hsp60sp, hsp60.4, B7 R5V and P18I10) and incubating with them for 30 minutes at room temperature followed by the induction of K562-E * 01033 cells by Nishi. Killed All peptides were maintained throughout the assay.
[231] 6 demonstrates that increasing HLA-E cell surface levels for K562-E * 01033 after cellular stress does not prevent NK cell mediated killing. (a) Killing K562-E * 01033 cells by NKL in 2h 51Cr release assay (incubation with increasing cell density as in FIG. 3B). (b) Same experimental settings as above in the presence of 100 mM B7sp. Colored circles-high density; Uncolored square-medium density; Colored triangles-low density. (c) HLA-E expression on K562-E * 01033 cells after culture with increasing cell density.
[232] FIG. 7 demonstrates the increased proportion of NK cells present in synovial fluid (SF) in patients suffering from rheumatoid arthritis (RA). Mononuclear cells freshly isolated from SF and PB of RA patients and PB of healthy control group were stained with mAbs for CD56 (PE-conjugated) and CD3 (Cydhrome-conjugated). About 10,000 events were obtained by setting the gate to the lymphocyte population and analyzed by flow cytometry. Results are shown as mean ± SEM of the percentage of CD56 ⁺ CD3 ⁻ cells in lymphocyte gates. The proportion of CD56 ⁺ CD3 ^- NK cells was increased in lymphocytes in SF (14.1 ± 2.2%) compared to patient PB (9.4 ± 1.3%; p <0.05).
[233] 8A-8C demonstrate that SF-NK cells are CD94 ^bright and NKG2A ⁺ , similar in phenotype to the minor subset of CD56 ^bright PB-NK cells. 8A: CD94 (DX22; thick, mid-row histogram) freshly isolated from SF (median and lower row histograms) and PB (top histogram, respectively) derived from the right and left knees of a representative RA patient, Stained three times with antibody to NKG2A (Z199; dark line, histogram in right column) or cIg (dotted line), followed by anti-mouse Ig and anti-CD3 (Cychrome conjugated) and anti-CD56 of FITC-conjugated goat (PE conjugated, dark, histogram in left column). Gates were set for the CD56 ⁺ CD3 ^- NK cell populations within the lymphocyte gate. CD94 staining is significantly biphasic among PB-NK cells (divided into CD94 ^faint and CD94 ^bright subsets) and most SF-NK cells belong to the CD94 ^bright NKG2A ⁺ subset, while some fraction of PB-NK cells Only NKG2A ⁺ can be seen. Figure 8B: The percentage of CD94 ^faint _, CD94 ^bright and NKG2A expressing cells in the CD56 ⁺ CD3 ^- NK gated lymphocyte population was calculated (5000-10000 events were obtained at this NK cell gate). Compared to PB-NK cells (black bars), which are mostly CD94 ^faint (69.2 ± 4.9%, n = 15), the significantly increased fraction of SF-NK cells (white bars) was CD94 ^bright (78.5 ± 3.0%, n = 17, p <0.001). Increased fraction of CD94 ^bright SF-NK cells involved an increase in NKG2A ⁺ cells (93.6%, n = 6, p <0.001). 8C: Small subsets of CD56 ^bright PB-NK cells are phenotypically similar to major SF-NK cell subsets. Freshly isolated PB mononuclear cells derived from 7 healthy blood donors were collected in the same cell number and immediately triple-stained with the following antibodies: control Ig (Y-axis, upper left), anti-KIR mAbs (Y-axis). Of DX9, DX27 and DX31 on the upper right) and anti-CD94 (DX22 on the Y-axis, lower left) and anti-NKG2A (Z199 on the Y-axis, lower right), and the term of PE-conjugated chlorine Mouse antibody with anti-CD3 (Cychrome conjugated) and anti-CD56 (FITC conjugated, X-axis). About 10 ⁵ events in the lymphocyte gate were obtained, resulting in at least 10 ³ events in the CD56 ^bright cell population and an assay gate was set for CD3 ^- cells. KIR expression was confined ^{to the} CD56 ^faint NK cell population, whereas the CD56 ^bright NK cell population was KIR ⁻ and expressed high levels of CD94 and NKG2A.
[234] 9A-9C demonstrate that SF-NK cells functionally recognize HLA-E. 9A: Untransfected 721.221 cells (HLA class I ⁻ , black bars), G _L -B * 5801 transfected cells (HLA-G leader peptide transplanted onto HLA-B * 5801 protein, FIG. In vitro cultured polyclonal from two patients in an Alamar-blue cytotoxicity assay on 721.221 cells expressing chimeric protein, hatched bars) and wild-type HLA-B * 5801 transfected 721.221 cells (white bars) Raw SF-Nk cell lines were used as effectors. E / T ratio was 1: 1. FIG. 9B: Two polyclonal SF-NK cell lines identical to those used in FIG. 8A, were untransfected 721.221 cells (HLA class I ⁻ , white bars) and G _L -B * 5801 transfected cells (FIG. Alamar-blue cytotoxicity assay (black bars) (E / T ratio was 1: 1) was tested as effectors. Blocking MHC class I or CD94 with specific mAbs reverses the protection conferred by HLA-E expression on G _L -B * 5801 transfected cells. Anti-CD94 (DX22), anti-HLA class I (w6 / 32) or cIg were present in cytotoxicity assays at a concentration of 1 μg / ml. 9C: Tetramer HLA-E molecules brightly stain most SF-Nk cells. Freshly isolated cells from PB (left) and SF (right) of representative RA patients were treated with control tetramers (mouse H2-K ^b molecules conjugated to streptavidin-PE, Y-axis on top contour plot) and HLA- E tetramer molecule (refolded in the presence of HLA-B * 0701 nonamer (quarmer) peptide conjugated to streptavidin-PE on the Y axis, lower contour plot) and CD56-FITC (X-axis) Stained. CD3-Cycrome ^- set the gate on lymphocytes.
[235] 10 demonstrates that CD94 / NKG2A binding of self-HLA class I is a major receptor / ligand interaction that protects autologous cells from lysis by SF-NK cells. Polyclonal SF-NK and PB-NK cell lines (Patient 2) analyzed in FIGS. 9A and 9B were 4-hour ⁵¹ Cr-releasing cells for EBV-transformed autologous cells at an E / T ratio 4: 1. Used for toxicity analysis. Blocking MHC class I (hatched bars) or CD94 / NKG2A (black bars) with specific mAbs induced apoptosis of autologous cells, but cIg (white bars) had no effect. When using the SF-NK cell line as an effector, similar levels of killing were observed in the presence of anti-MHC class I or anti-CD94 mAbs, with most self-protection interacting with HLA-class I on autologous cells. This is due to the CD94 / NKG2A. Anti-CD94 (DX22) or anti-HLA class I (w6 / 32) or cIg were present in cytotoxicity assays at a concentration of 1 μg / ml.
[236] 11 demonstrates that SF-NK cells bind HLA-E in combination with eg the VMAPRTVLL peptide. Tetramer HLA-E / B7sp molecules brightly stain most SF-Nk cells. Freshly isolated cells from PB (left) and SF (right) of representative RA patients were treated with control tetramers (mouse H2-Kb molecule conjugated to streptavidin-PE, Y-axis on top contour plot) and HLA-E. Stained with tetramer molecules (refolded in the presence of VMAPRTVLL peptide, conjugated to streptavidin-PE on the Y-axis, bottom contour plot) and CD56-FITC (X-axis). Gates were set on CD3-Cycrome-negative lymphocytes.
[237] 12 shows that SF-NK cells bind to HLA-E in combination with the VMAPRTVLL (B7sp) peptide but not to HLA-E in combination with the QMRPVRSVL (hsp60sp) peptide. Tetramer HLA-E / B7sp molecules stain most brightly most SF-NK cells (upper row, middle contour plot) and fractions of SF-T cells (lower row, middle contour plot). HLA-E / hsp60sp was observed not to stain SF-NK cells or SF-T cells (top row, right contour plot and bottom row, right contour plot, respectively). Control tetramer staining (mouse H2-Kb molecule conjugated to streptavidin-PE) is shown on the left.
[238] Figure 13 shows that SF-NK cells produce IFN-gamma and TNF-alpha better when stimulated with LPS compared to PB-NK cells in RA patients or healthy subjects. PB and SF mononuclear cells (MC) were stimulated with LPS (10 mg / ml) overnight or with K562 (1: 1 cell ratio) for 4 hours in the presence of GolgiStop ^™ . Cells were surface stained for CD3 and CD56 and then stained for IFN-gamma or TNF-alpha. Analysis was performed by flow cytometry.
[239] FIG. 14 shows that SF-Nk cells produce IFN-gamma better after stimulation with IL-2 compared to PB-NK cells. PB and SF mononuclear cells (MC) were stimulated with IL-2 (200 U / ml) overnight. Cells were surface stained for CD3 and CD56 and then stained intracellularly for IFN-gamma or TNF-alpha. Analyze by flow geometry.
[240] Figure 15 demonstrates that HLA-E produced B7 signal peptide (VMAPRTVLL) is sufficient to inhibit NK cell IFN-gamma and TNF-alpha cytokine production. Incubation with various doses of HLA-B7 signal sequence derived peptide (VMAPRTVLL) stabilizes HLA-E expression on K562 cells transfected with HLA-E * 01033. PB and SF mononuclear cells (MC) of RA patients were then incubated with peptide stabilized K562 cells (1: 1 cell ratio) for 4 hours in the presence of GolgiStop ^™ . Cells were surface stained for CD3 and CD56 and then intracellularly stained for IFN-gamma or TNF-alpha. Analyze by flow geometry.
[36] The present invention satisfies the aforementioned needs and provides additional objects and advantages by providing novel methods and compositions for the diagnosis and treatment of inflammatory diseases and symptoms, autoimmune diseases, viral infections, transplant rejections and cancers, among other diseases and symptoms. To achieve. Such compositions and methods use proinflammatory or anti-inflammatory binding peptides to modulate the immune response in a subject, typically a mammalian subject, having a disease or condition that may be treated in accordance with the methods and compositions of the present invention.
[37] Peptides used in the present invention exhibit specific binding interactions with major histocompatibility complex class I (MHC class I) molecules, such as HLA-E MHC class I molecules. In general, these MHC I molecules will be present on antigen producing cells (APCs). The complex between the peptide bound to the MHC I molecule and the MHC I molecule is formed when the cell is exposed to the peptide. The resulting binding complex interacts with an MHC class I-specific inhibitory receptor, generally a CD94 / NKG2 cellular receptor, consisting of CD94 and its paired NKG2A or splice variant NKG2B. The interaction of pro- or anti-inflammatory binding peptides with HLA-E binding peptides (including optional additional binding peptides) modulates the interaction between the binding peptide / HLA-E complex and the receptor to express inhibitory receptors. New regulation of immune responses.
[38] The term "major histocompatibility complex molecule" refers to a molecule on antigen producing cells that has the ability to form antigen-associated antigen producing cells in association with the antigen. Recognition of antigen-associated production cells by NK and T cells is mediated by the CD94 / NKG2 cellular receptor.
[39] Class I molecules consisting of beta-2-microglobulin molecules linked covalently with heavy chains include clifts or crevices for receiving pro- or anti-inflammatory binding peptides. Thus, the peptide is sized and dimensioned enough to allow the peptide to enter the crevasses. The size and dimensions of crevasses are known to those skilled in the art (F. Latron Science 257: 964; 967, 1992, referenced herein). Preferably, the peptide fits substantially within the crevasses, but when associated with a class I molecule can access NK or T cells capable of recognizing antigen. In general, the peptide will consist of about 4-24 amino acids in length, often about 6-15 amino acids in length, more often about 8 to about 10 amino acids in length. In an embodiment, the peptide is nonamer (a hexamer). Frequently, the two amino acids of the peptide are hydrophobic residues to trap the peptide in the crevasses. This peptide can be derived from a tumor, tissue, viral protein or viral protein, for example.
[40] In related embodiments of the invention, the proinflammatory or anti-inflammatory binding peptides induce or prevent NK cell activation (such as cytotoxicity and cytokine release) during encountering normal and abnormal (eg cancer or virus infected) cells. NK cells producing CD94 / NKG2A receptors that regulate their own self-tolerance can kill cells that have lost the expression of protective HLA-E molecules.
[41] In a more detailed aspect of the invention, the proinflammatory binding peptide is a peptide derived from the signal sequence of another MHC class I molecule. Anti-inflammatory peptides are typically peptides derived from stress-induced or stress-related proteins, or heat shock proteins (hsp60), such as hsp60. In contrast to traditional MHC class I molecules, HLA-E exhibits a somewhat restrictive polymorphism, whose peptide binding cleft is mainly a nonmeric peptide derived from the signal sequence of specific HLA-A, -B, -C and -G molecules. (Lazetic et al. , J. Immunol. 157: 4741-4745, 1996, referenced in the text). These peptides generally share a common motif: methionine at position 2, leucine or isoleucine at position 9 (Arnett et al . , Arthritis Rheum. 31: 315-324, 1998, referenced herein). In addition, the peptide also shows a third common motif element that is a proline residue at position 4. Peptides that share this motif or similar structures bind operable pro-inflammatory and anti-inflammatory binding peptides that can bind to HLA-E and modulate interactions with CD94 / NKG2 cellular receptors to mediate the regulation of immune responses. It is useful as a candidate peptide for screening in the present invention to identify.
[42] In certain embodiments of the invention, the HLA-E binding peptide is derived from the signal sequence of a stress-inducing protein. For example, exemplary peptides can be selected from the stress inducing peptide hsp60. In one embodiment of the invention, the hsp60 peptide is nonamer. Examples of preferred peptides can be given (standard one letter code) VMAPVTVLL and QMRPRSRVL.
[43] To identify peptides derived from human hsp60 that have the potential to bind HLA-E, HLA-E permissive motifs (methionine at position 2 at the C terminus and leucine or isoleucine at position 9) The full length amino acid sequence of hsp60 was scanned for the peptides indicated. Of these four peptides identified (FIG. 1: Table 1), one was initially selected based on its position in the hsp60 leader sequence (denoted QMRPVSRVL, hsp60sp). In addition, hsp60sp not only has methionine at position 2 and leucine at position 9, but also shares amino acids common at positions 4 and 8 with some peptides known to bind HLA-E effectively (Table 1). In particular, four of the nine amino acids of hsp60sp share some peptides found in the HLA class I leader sequence (eg, HLA-A * 0201, and -A * 3401, Table 1).
[44]
[45] Diseases and symptoms treatable and diagnosed according to the methods and compositions of the present invention include rheumatoid arthritis, juvenile arthritis, Crohn's disease, ulcerative colitis, acute myeloid leukemia, multiple osteomyelitis, insulin-dependent diabetes mellitus, systemic lupus erythema, SjUgren syndrome, Vase Dow's disease, Hashimoto's disease, autoimmune hemolytic anemia, cancer (eg, ovarian cancer), cardiomyopathy, early cardiovascular disease, atherosclerosis, hypertension, Hodgkin's disease, and transplant rejection. In an exemplary report supporting this broad applicability of the present invention, NK cells have been shown to play an important role in down-regulating TH1-mediated colitis by controlling the response of effector T cells in a perforin dependent manner ( Fort et al. , J. Immunol. 161: 3256-3261, 1998, incorporated herein by reference. In laboratory autoimmune encephalomyelitis (EAE), a model of human multiple osteomyelitis (MS), administration of the Nk cell stimulating compound linamide has prevented the mouse from causing the disease, and the lack of NK cells in the same model led to TH1. Cytokine production increased and disease worsened (Matsumoto et al. , Eur. J. Immunol. 28: 1681-1688; Zhang et al. , J. Exp. Med. 186: 1677-1687, 1997, respectively referenced in the text). These reports suggest that the presence of NK cells is beneficial for protection against prototype TH1-mediated diseases. In contrast, the pathologic role of NK cells has been suggested in the asthma model of rats with prototype TH2-mediated disease, where the deficiency of NK cells protects the mice from allergen-induced inflammation in the airway epithelium.
[46] In order to identify additional peptides derived from heat shock protein (hsp) and other proteins, a speculative design and screening method similar to that used above for hsp60 was used. Candidate hsps with the potential to bind HLA-E can be identified based on the structural considerations described herein and their known activity that mediates the onset or exacerbation of a disease state. As shown in the following Tables 2 and 3, many hsps are included in serious diseases and symptoms that can be treated according to the methods and compositions of the present invention. To identify candidate pro-inflammatory or anti-inflammatory binding peptides from these subject proteins with known detrimental activity associated with the disease, the full-length amino acid sequence of the protein is HLA-E replicable motif (eg, C-terminus). Was scanned for peptides representing methionine at position 2 and leucine or isoleucine at position 9). Candidate peptides so identified were evaluated and screened according to the methods presented below.
[47]
[48]
[49] References cited above
[50] Albani S. et al. Nat Med. May; 1 (5): 448-52.1995
[51] 2. de Graeff-Meeder, E. R. et al. Clin Exp Rheumatol 11 Suppl 9, S25-28.1993
[52] Xu, Q. et al Arterioscler Thromb 13: 1763-1769.1993
[53] 4. Yokota, S. et al. Clin Immunol Immunpathol. 67: 163-170.1993
[54] 5. Rambukkana, A. et al. J Invest Dermatol 100,87-92. 1993
[55] 6. Raz I. et al Lancet. Nov 24; 358 (9295): 1749-53.2001
[56] 7. Salvetti M et al J Neuroimmunol. Apr; 65 (2): 143-53.1996
[57] 8. Jenkins SC et al Tissue Antigens. Ju1; 56 (1): 38-44.2000
[58] 9. Hayem G. et al. Ann Rheum Dis. May; 58 (5): 291-6.1999.
[59] 10. Blass S et al Arthritis Rheum. Apr; 44 (4): 761-71.2001
[60] 11. Somersan S. et al. J Immunol. Nov 1; 167 (9): 4844-52.2001
[61] The peptides identified in Table 3 above are considered pro- or anti-inflammatory binding peptides that are useful candidates for use in the diagnostic and therapeutic methods of the present invention, respectively.
[62] In addition, several other proteins were analyzed to determine if candidate pro- or anti-inflammatory binding peptides could be used in the present invention. In one such illustrative analysis, a BLAST study was performed to identify nucleotide stretches in Homo sapiens beta defensin 2 (HBD2: Homo sapiens beta defensin 2) showing 85% amino acid homology with human hsp60 leader sequences. The reading frame was inverted (-2) starting at position HBD2: 718 to 659, showing 85% homology with the hsp60-leader peptide.
[63] BLAST Survey Results:
[64] gi | 3818536 | gb | AF071216. AF071216, complete cds
[65] Score = 37.0 bits (84), expected = 0.34
[66] Identity = 17/20 (85%), Positive = 18/20 (90%)
[67] Frame = -2
[68] Question human hsp60sp: 1 MLRLPTVFRQMRPVSRVLAP 20
[69] Identity ML LPTVF QMRPVSR + LAP
[70] HBD2: 718 MLPLPTVFHQMRPVSRLLAP 659
[71] From these and other subject proteins and peptide sequences, amino acid sequences are scanned for peptides that exhibit the HLA-E copying motif. Candidate peptides so identified are assessed and screened according to the methods presented herein. Table 4 shows the large assemblies of candidate HLA-E binding peptides for use in the present invention.
[72] POS. 2: M, POS. 4. P and POS. 9: Putative HLA-binding peptides derived from non-MHC human protein with I or L protein ACC.NO.SWISS PROT. order location Inter-alpha Trypsin Inhibitory Heavy Chain H1 Precursor P19827 AMGPRGLLL 4-12 (Signal Peptides) Plasminogen Activator Inhibitor-1 P05121 QMSPALTCLGMAPALRHL 2-10 (Signal Peptide) Cell surface A33 antigen precursor Q99795 KMWPVLWTL 4-12 (Signal Peptides) Acrosine precursor P10323 EMLPTAILL 3-11 (Signal Peptides) Class II Histocompatibility Antigens P28067 QMLPLLWLL 13-21 (Signal Peptides) Membrane-Anchor Protein Precursors Specific to the Brain Q9UK28 LMPPPLLLL 6-14 (Signal Peptides) GC-rich Sequence DNA Binding Factors P16383 AMAPRSRLL 60-68 T box warrior P57082 TMMPRLPTL 450-458 factorRetinoblastoma Related Protein P06400 KMTPRSRIL 824-832 Fatty acid synthase 049327 TMDPQLRLL 74-82 Transient Internal Quality Semang P55072 GMTPSKGVL 507-515 Carboxeptidase M Precursor P14384 PMIPLYRNL 411-419
[73] Thromboxane-A Synthase P24557 IMVPLARIL 235-243 Interferon Consensus Sequence Binding Protein Q02556 DMAPLRSKL 356-364 Lymphocyte Cytosol Protein P13796 PMNPNTNDL 145-153 Ryanodine receptor P21817 QMGPQEENLEMCPDIPVLYMEPALRCL 2169-21773238-32464639-4647 Proteasome Subunit Alpha Type 2 P25787 GMGPDYRVL 77-85 60S Ribosome Protein P08526 FMKPGKVVL 3-11 Heteronuclear Ribotucleoprotein P52272 RMGPGIDRL 403-411 Seryl-TRNA Synthetase P49591 FMPPGLQEL 458-466 Telomerase reverse transcriptase O14746 QMRPLFLEL 388-396 Protein Disulfide Isomerase P13667 VMDPKKDVL 539-547 Vinculin P18206 MMGPYRQDL 532-540 Wilm Seed Tumor Protein P19544 RMFPNAPYL 126-134 Zinc finger protein O43670 GMPPGIPPL 155-163 RNA helicase O43738 IMNPSYYNL 828-836 CPSB Q9P2I0 QMKPRQLII 352-360
[74] Tight Junction Protein ZO-3 O95049 QMKPVKSVL 189-197 Phosphoenolpyruvate Carboxinase Q16822 SMGPVGSPL 163-171 ATP-binding cassette, subfamily A, member 3 Q99758 GMDPVARRL 1544-1552 Actin crosslinked family protein 7 Q9UPN3 TMPPVGTDL 4180-4188 Potential Phospholipid-delivery ATPase O60312 LMTPVAALL 943-951 Chromodomain-helicase-DNA-binding protein 1 O14646 RMRPVKAAL 1420-1428 Chromodomain-helicase-DNA-binding protein 2 O14647 RMRPVKKAL 1475-1483 Cytochrome P450 XXIB P08686 SMEPVVEQL 134-142 DNA ligase III P49916 LMTPVQPML 390-398 Hyperlipolipid Receptor Q99500 AMNPVIYTL 292-300 GC-rich sequence DNA-binding factor Q9Y5B6 EMTPVTIDL 382-390 HomologueRAP1 GTPASE-GDP Dissociation Stimulant 1 P52306 EMPPVQFKL 414-422 Host Cell Factor C1 (HCF) P51610 RMAPVCESL 1253-1261 Leukotriene A-4 Hydrolase P09960 SMHPVTAML 594-602
[75] Neuropeptide Y Receptor Type 2 P49146 KMGPVLCHL 117-125 All Factory Receptor 3A2 047893 EMQPVVFVL 25-33 PAX-7 P23759 HMNPVSNGL 374-382 Protocadherin 15 precursor Q96QU1 LMDPVKQML 86-94 Perilipine (PERI) O60240 SMEPVVRRL 72-80 26S proteasome non-ATPase regulatory subunit 13 Q9UNM6 LMHPVLESL 217-225 Melanosite-specific Transporter Proteins Q04671 TMIPVLLNL 747-755 Chromosome Condensation Regulator P18754 SMVPVQVQL 162-170 Ring Finger Protein 11 Q9Y3C5 CMEPVDAAL 139-147 Sideeroplexin 2 Q96NB2 FMVPVACGL 277-285 SGT1 Protein O95905 VMAPVDVDL 590-598 Hypothetical Q14139 VMIPVFDIL 275-283 Protein KIAA0126Hypothetical Zinc Finger Protein Q9Y2H8 QMAPVQKNL 62-70 Zinc Finger Protein ZNF287 Q9HBT7 LMRPVQKEL 179-187
[76] Human tetracyclinetransporter-like protein Q14728 EMAPWFALL 201-209 Human KU80 Autoantigen Homolog Q9W627 LMLPDFDLL 82-90 Adapter-Related Protein Complex 3beta 1 Subunit O00203 TMDPDHRLL 292-300 Adapter-Related Protein Complex 3beta 2 Subunit Q13367 VMDPDHRLL 292-300 Cyclin A1 P78396 LMEPPAVLL 455-463 Complement C5 precursor P01031 NMVPSSRLL 533-541 Cytochrome P450 4F2 P78329 WMGPISPLL 91-99 1 cytochrome P450 4F12 Q9HCS2 AMSPWLLLL 15-23 G protein-coupled receptor kinase GRK7 Q8WTQ7 DMKPENVLL 316-324 Glutathione S-Transferase A3-3 Q16772 RMEPIRWLL 15-23 Solute Carrier Family 2 P14672 AMGPYVFLL 444-452 ATP-dependent DNA helicase II P13010 LMLPDFDLL 82-90 Mitogen-Activated Protein Kinase Kinase Kinase 5 Q99683 LMQPNFELL 237-245 Celerian Inhibitor Precursor P03971 RMTPALLLL 244-252
[77] Coronary Multispecific Organ Anion Transporters O15438 EMGPYPALL 831-839 Transition-Related Protein MTA1 Q13330 HMGPSRNLL 614-622 Sodium / Hydrogen Exchanger 6 Q92581 LMRPLWLLL 24-32 PYD-Containing Protein 2 Q9NX02 VMLPKAALL 325-333 PYD-Containing Protein 4 Q96MN2 KMLPEASLL 268-276 Panexin 3 Q96QZ0 EMLPAFDLL 306-314 Peroxysome Biogenesis Factor 1 O43933 WMQPSVVLL 653-661 Long Transient Receptor Potential Channel 2 O94759 TMDPIRDLL 618-626 Uteroglobin-associated protein 1 precursor Q96PL1 FMDPLKLLL 45-53 Williams-Beyren Syndrome Chromosome Band 14 Q9NP71 PMAPPTALL 417-425 Hypothetical Q15053 AMCPIAMLL 41-49 Protein KIAA0040
[78] Reference
[79] Braud, V. M., D. S. Allan, C. A. O'Callaghan, K. Soderstrom, A. D'Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J.1. Bell, I H. Phillips, L. L. Lanier, and A. I McMichael. 1998. HLA-E binds to natural killer cell receptors CD94 / NKG2A, B and C (see comments). Nature 391: 795.
[80] 2. Kleinau, S., K. Soderstrom. , R.Kiessling, and L. Klareskog. 1991. A monoclonal antibody to the mycobacterial 65 kDa heat shock protein (ML 30) binds to cells in normal and arthritic joints of rats. Scand J Immunol 33: 195.
[81] Karlsson-Parra, A., K. Soderstrom, M. Ferm, I Ivanyi, R. Kiessling, and L. Klareskog. Presence of human 65 kD heat shock protein (hsp) in inflamed joints and subcutaneous nodules of RA patients [corrected and republished with original paging, article originally printed in Scand J Immunol 1990 Mar; 31 (3)-. 283-8]. Scand J Immunol 31: 283.
[82] 4. Boog, C. J. P., E. R. de Graeff-Meeder, M. A. Lucassen, R. R. van der Zee, M. M. Voorhorst-Ogink, P. I S. van Kooten, H. J. Geutz, and W. van Eden. 1992. Two monoclonal antibodies generated against human hsp60 show reactivity with synovial membranes of patients with juvenile chronic arthritis. J Exp. Med 175: 1805.
[83] 5. Lo, W.-F. et al. Molecular mimicry mediated by N4HC class Ib molecules after infection with Gram-negative pathogens. Nature Med 6,215-218 (2000)
[84] 6. Kraft, J. R. et al. Analysis of Qa-I b peptide binding specificity and the capacity of CD94 / NKG2A to discriminate between Qa-I-peptide complexes.J Exp. Med 192, 613-623 (2000).
[85] hsp60 signal peptide or other pro-inflammatory HLA-E binding proteins (such as stress proteins, heat shock proteins) that can potentially compete with MHC class I-peptides in the strong and protective HLA-E cleavage of HLA-E Or derived from other proteins disclosed herein, and analogs thereof, to induce activation of NK cells and to express CTLs against tumor cells outside the immune detection network based on maintained protective HLAE expression. New therapeutic tools can be developed to lower the activation titers of -NKG2A. Such compositions and methods include preventing or inhibiting the growth of tumor cells or cancer tissues by exposing the patient's tumor cells or cancer tissues to a therapeutically effective amount of proinflammatory binding peptides.
[86] The method also applies to the treatment of virally infected cells. Exposure of such infected cells to therapeutically effective amounts of proinflammatory binding peptides would result in peptides competing with protective MHC class I-peptides at the cleft of HLA-E, activating NK cells and otherwise leaving the immune detection network. It is possible to lower the activation titer of CD94-NKG2A expressing CTL against virus infected cells.
[87] Peptide Analogs and Mimetics
[88] In the present invention, the definition of the term biologically active peptide includes chemically modified natural, synthetic, therapeutically or prophylactically active peptides (where two or more amino acids are covalently linked), peptide analogs and active peptides. Derivatives or salts are included. Often these peptides are muteins readily achievable by partial substitution, addition or deletion of amino acids in naturally occurring or natural (eg wild type, naturally occurring variant, or allelic variant) peptide sequences. Also included are biologically active fragments of natural peptides. These mutants and fragments substantially retain the desired biological activity of natural peptides. In the case of peptides with carbohydrate chains, biologically active variants marked by alterations in these carbohydrate species are also within the scope of the present invention.
[89] In additional embodiments, the peptides to be used in the present invention are prepared by addition or conjugation of polyethylene glycol, natural polymers such as hyaluronic acid, or synthetic polymers such as any sugars (such as galactose, mannose), sugar chains or non-peptide compounds. It can be modified. Substances added to the peptide by this modification will either specify or enhance binding to a specific receptor or antibody or otherwise enhance the mucosal delivery, activity, half-life, cell- or tissue-specific targeting or other beneficial properties of the peptide. For example, such modifications make the peptide more lipophilic. That is, for example, by addition or conjugation of phospholipids or fatty acids. Methods and compositions of the invention include linkage (eg, chemical) to two or more peptides, protein fragments or functional domains (eg, extracellular, transmembrane and cytoplasmic domains, ligand-binding zones, active site domains, immunogenic epitopes, etc.) Peptides obtained by binding), such as fusion peptides produced recombinantly for incorporating functional elements of a plurality of peptides in a single coding molecule.
[90] Biologically active peptides used in the methods and compositions of the present invention therefore include natural or "wild-type" peptides of such molecules and naturally occurring variants such as naturally occurring allelic variants and mutant proteins. Also included are synthetic or chemically recombinantly processed peptides and polymers of peptides and protein “analogs” and chemically modified variants, fragments, conjugates, and naturally occurring peptides. The term peptide “analog” herein includes modified peptides involving one or more amino acid substitutions, insertions, rearrangements or deletions, when compared to the natural amino acid sequence of the selected peptide. Thus modified peptides and protein analogs actually preserve the biological activity comparable to that of the corresponding natural peptide, which is at least 50%, generally at least 75%, often 85% -95 relative to the activity level of the corresponding natural protein or peptide. It means having an activity level of% or more.
[91] Fusion polypeptides between pro- or anti-inflammatory binding peptides and other homologous or heterologous peptides are also provided. Many growth factors and cytokines are homodimeric, and the repetitive structure of the linked peptides to form a "cluster peptide" will provide various advantages, such as reduced sensitivity to proteolytic degradation. Various alternative multimeric constructs are also provided comprising the peptides of the invention. In one embodiment, US Pat. Nos. 6,018,026 and 5,843,725 by linking one or more pro- or anti-inflammatory binding peptides of the invention with heterologous multimerized polypeptides such as immunoglobulin heavy chain constants, or immunoglobulin light chain constants Various polypeptide fusions described in the call can be accomplished. The biologically active, multimerized polypeptide fusions thus constructed may be hetero- or homo-multimers, such as heterodimers or homodimers, each of which comprises one or more different pro-inflammatory or anti-inflammatory binding peptides of the invention ( May be included). Other heterologous polypeptides can also bind to this peptide to produce fusions, including hybrid proteins that exhibit, for example, heterologous (such as CD4) receptor binding specificities. Similarly, heterologous fusions may be prepared that exhibit a combination of properties or activities of derivative proteins. Another typical example is the ease of localization of a fused protein as a fusion of a reporter polypeptide, such as CAT or luciferase with a peptide of the invention (see, eg, Dull et al., US Pat. No. 4,859,609, herein). Other gene / protein fusion partners in this context include bacterial beta-galactosidase, trpE, protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor (eg, Godowski et al. , Science 241: 812-816, 1988, referenced herein).
[92] The invention also provides for the use of pro- or anti-inflammatory binding peptides modified by covalent or aggregate binding with chemical moieties. Such derivatives generally fall into three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes with, for example, cell membranes. Such covalent or aggregated derivatives are useful for various purposes, such as immunogens, as reagents in immunoassays or purification methods such as affinity purification of ligands or other binding ligands. For example, the pro-inflammatory or anti-inflammatory binding peptides are covalently immobilized on a solid carrier, such as cyanogen bromide-activated Sepharose, or specifically bound to pro- or anti-inflammatory binding peptides by methods known in the art. It may be adsorbed on the polyolefin surface in the presence or absence of glutaraldehyde crosslinking for use in the analysis or purification of the antibody. Proinflammatory or anti-inflammatory binding peptides may also be labeled with a detectable group, such as radioiodized by a chloramine T process, covalently bound to rare earth chelates, or conjugated to other fluorescent moieties used in diagnostic assays.
[93] For the purposes of the present invention, the term biologically active peptide “analog” refers to derivatives of natural peptides, such as amino and / or carboxyl terminal deletions and fusions, insertions, substitutions or deletions of single or multiple amino acids in sequence. Or synthetic variants. Insertive amino acid sequence variants are those in which one or more amino acid residues are introduced into a position in a protein. Random insertion is also possible by appropriate screening of the resulting product. Deletion variants are characterized by the removal of one or more amino acids from the sequence. Substitutable amino acid variants are those wherein at least one residue in the sequence has been removed and another residue has been inserted in its place.
[94] When a natural peptide is modified by the substitution of an amino acid, the amino acid is generally caused by other amalic acids with conservatively related similar chemical properties, such as hydrophobicity, hydrophilicity, electronegativity, small or bulky side chains, and the like. Replaced. Residue positions that are not identical to the native peptide sequence are thus replaced by amino acids having similar chemical properties, such as charge or polarity, where such changes will not substantially affect the properties of the peptide analogs. Such and other minor modifications generally affect the biological properties of the corresponding natural peptide (eg, binding molecules, or binding to other ligands or receptors), immunohomology (eg, one or more monoclonal antibodies that recognize the natural peptide). Perception) and other biological properties substantially.
[95] As used herein, the term "conservative amino acid substitutions" refers to the general interchangeability of amino acid residues having similar side chains. For example, common compatibility groups of amino acids with aliphatic side chains include alanine, valine, leucine and isoleucine; Amino acid groups having aliphatic hydroxyl side chains include serine and threonine; Amino acid groups having amide-containing side chains include asparagine and glutamine; Amino acid groups having aromatic side chains include phenylalanine, tyrosine and tryptophan; Amino acid groups having basic side chains include lysine, arginine and histidine; Amino acid groups with sulfur-containing side chains are cysteine and methionine. Examples of conservative substitutions are the intersubstitutions between nonpolar (hydrophobic) residues such as isoleucine, valine, leucine or methionine. Likewise, the present invention also encompasses the substitution of polar (hydrophilic) residues such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine. In addition, the substitution of basic residues such as lysine, arginine or histidine or the substitution of acidic residues such as aspartic acid or glutamic acid is included. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.
[96] The term biologically active peptide analog also refers to modifications of natural peptides, including twenty general amino acids, or stereoisomers of non-natural amino acids such as, for example, α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid (eg, D-amino acids). Included forms are also included. These and other unusual amino acids may also be substituted or inserted in natural peptides useful in the present invention. Examples of unusual amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine , 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine and other similar amino acids and imino acids (eg, 4-hydroxyproline). In addition, biologically active peptide analogs include single or multiple substitutions, deletions and / or additions of carbohydrate, lipid and / or protein moieties that occur naturally or artificially as a structural component of the peptide, or are linked to or otherwise linked to the peptide. .
[97] In order to facilitate the production and use of peptide and protein analogs in the present invention, homologous proteins found in different species, such as species, genus, family or other taxonomic groups (eg, humans, rats, rats, and / or cattle). Molecular lineages that characterize conservative and diverse protein structures and functional elements among various members of various stress induced or thermal shock proteins, between family members, allelic variants, and / or naturally occurring variants, including See developmental studies. In this regard, the available findings provide a detailed assessment of the relationship between structure-function at the microscopic molecular level for modifying the majority of peptides disclosed herein to facilitate the production and selection of functional peptides and protein analogs. Will provide. Such studies include, for example, detailed sequence comparisons that identify various structural elements that are conserved among multiple isoforms or species or allelic variants of the subject's pro- or anti-inflammatory binding peptides. These various conserved structural elements facilitate the practice of the present invention by pointing to targets useful for modifying natural peptides to impart desired structural and / or functional changes.
[98] In this context, analyze sequence sequences and compare sequences in an assay to identify corresponding protein regions and amino acid positions between species and protein family members between species variants of the protein of interest using conventional sequence alignment methods. May be used. Such comparisons are useful for identifying the various conserved structural elements of interest, where the latter will often be useful for incorporation in biologically active peptides, thereby producing functional analogs thereof. In general, one or more amino acid residues that mark various structural elements within different reference peptide sequences are incorporated in functional peptide analogs. For example, a cDNA encoding a natural pro-inflammatory or anti-inflammatory binding peptide may contain one or more corresponding amino acid position (s) (ie, isoforms, species of the subject's pro-inflammatory or anti-inflammatory binding peptides according to the applied alignment method). Or a heterologous reference peptide sequence, such as an allelic variant, or a synthetic mutant, with residues representing the structural element of interest, corresponding positions that match or occupy similarly aligned sequence elements). May be modified to encode an amino acid deletion, substitution or insertion that alters the corresponding residue (s) in the natural peptide to produce operable peptide analogs of the invention having similar structural and / or functional elements as reference peptides. .
[99] In this rational design to address biodegradably active peptide analogs, the natural or wild type identity of the residue (s) at the amino acid position corresponding to the desired contributing element of the heterologous reference peptide, the corresponding in the reference peptide. May be modified with residue identity identical or conservatively related to the amino acid residue (s). However, it is often possible to non-conservatively alter native amino acid residues with respect to the corresponding reference protein residue (s). In particular, many non-conservative amino acid substitutions, especially non-conservative amino acid substitutions at various positions known to be easier to modify, may have moderate damage or neutral effects as compared to the function of the natural peptide, or It can even enhance selected biological activity.
[100] Sequence alignment and comparison to find useful peptide and protein analogs and mimics is associated with known computer modeling techniques in the art to determine crystal structure analysis of natural biologically active peptides (eg, Loebermann et al . , J. Molec. Biol. 177; 531-556, 1984; Huber et al., Biochemistry 28: 8951-8966, 1989; Stein et al., Nature 347: 99-102, 1990; Wei et al., Structural Biology 1: 251-255, 1994, respectively referenced in the text ) Will be more precise. This assay provides a detailed structure-function map for identifying desired structural elements and modifications for incorporation into peptide and protein analogs and mimetics that will exhibit substantial activity comparable to that of natural peptides for use in the methods and compositions of the present invention. It makes writing possible.
[101] Biologically active peptides and protein analogs of the present invention generally exhibit substantially sequence homology with the corresponding native peptide sequence. "Substantial sequence identity" means at least 65% sequence homology, often when two target amino acid sequences are optimally aligned, such as when aligned by program GAP or BESTFIT using a default gap penalty. Refers to 80-85% sequence homology, often at least 90-95% sequence homology or more. "Percentagle amino acid identity" refers to a comparison of the amino acid sequence of two peptides having a predetermined percentage of approximately identical amino acids when optimally aligned. Sequence comparisons are generally made to a reference sequence through a comparison window of at least 10 residue positions, often at least 15-20 amino acids, where the percentage of sequence homology is associated with the second sequence. Computed by comparison (where this second sequence is a peptide analogue sequence comprising one or more deletions, substitutions or additions of a total of 20%, typically less than 5-10%, relative to the reference sequence, eg, via a comparison window). The reference sequence may be a subset of larger sequences, such as for example a subset of hsp60 leader sequence residues. The optimal alignment of sequences to align the comparison window is based on the local homology algorithm of Smith and Watermn (Adv. Appl. Math.2: 482, 1981), homology alignment algorithm of Needleman and WUnsch (J. Mol. BIol.48: 443, 1970), to study the similarity method of Pearson and Limpman (Proc. Natl. Acad. Sci. USA85: 2444, 1988) or a computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and / or TFASTA eg provided by Wisconsin Genetics Software package Release 7.0 of Genetic Computer Group, 575 Science Dr., Madison, WI). Such as that).
[102] By optimizing the corresponding natural peptides and peptide analogs, and by using appropriate assays such as adsorption proteins or receptor binding assays to determine the selected biological activity, the operable peptides and proteins to be used in the methods and compositions of the invention It is easy to identify. Operable peptide and protein analogs generally specifically immunoreact with antibodies raised against the corresponding natural peptide. Likewise, operable peptides and nucleic acids encoding proteins will share substantial sequence homology as described above for the corresponding native peptide-encoding nucleic acids and are generally under acceptable, mild or very stringent hybridization conditions. It will optionally hybridize with a portion of the nucleic acid sequence encoding the corresponding natural peptide or fragment thereof or with the entire nucleic acid sequence (Sambrook et al., Molecular Cloning: A Laboratory Manual , 3rd Edition, Cold Spring Harbor Laboratories, Cld Spring Harbor, NY). , 2001, text). The term " selectively hybridizing to " hybridizes preferentially to specific target DNA or RNA sequences, for example when the target sequence is present in a heterologous preparation such as total cellular DNA or RNA. It refers to selective interactions between nucleic acid probes that are dosed or multiplied. Generally, nucleic acid sequences encoding biologically active peptides and protein analogs or fragments thereof are selected under stringent conditions (such as about 5 ° C. below the melting point (Tm) for the subject sequence at a given ionic strength and pH, where Tm Will hybridize with the nucleic acid sequence encoding the corresponding natural peptide under a predetermined ionic strength and temperature under pH where 50% of the complementary or target sequence is perfectly matched to the probe. For nucleic acid probe design and annealing conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual , 3rd Edition, Cold Spring Harbor Laboratories, Cld Spring Harbor, NY, 2001, or Current Protocols in Molecular Biology , F. Ausubel et al., Greene. See Publishing and Wiley-Interscience, New York, 1987 (each referenced in the text). Generally, stringent or optional conditions will be at pH 7 with a salt concentration of at least about 0.02 moles and a temperature of at least about 60 ° C. Less stringent selective hybridization conditions may also be selected. Combination of variables is more important than any particular means, as other factors, such as base composition and complementary strand size, presence of organic solvents and degree of base mismatching can greatly affect the precision of hybridization.
[103] In another aspect of the present invention, there is also provided a mimetic peptide comprising a peptide or non-peptide molecule that mimics the tertiary binding structure and activity of a selected natural feldtide functional domain (such as a binding motif or active site). do. These mimetic peptides include peptides that are recombinantly or chemically modified as well as non-peptide preparations, such as called molecular drug mimetics as described below.
[104] In one aspect, peptides (including polypeptides) useful in the present invention may comprise one or more naturally occurring one or more side chains of twenty genetically encoded amino acids (or D amino acids) in other side chains such as alkyl, lower alkyl, cyclic 4-, 5-, 6- or 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and lower ester derivatives thereof, and 4-, 5-, 6-7 membered hetero By replacing the cycle, mimic peptides are made. For example, proline analogs can be made in which the ring size of the proline residue is changed from 5 to 4, 6 or 7 members. The cyclic group may be saturated or unsaturated and, if unsaturated, may be aromatic or non-aromatic. Heterocyclic groups may contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazinyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (such as morpholino), oxazolyl, piperazinyl (such as , 1-piperazinyl), piperidyl (eg 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyri Midinyl, pyrrolidinyl (such as 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (such as thiomorpholino), and triazolyl. have. Such heterocyclic groups may be substituted or unsubstituted. When substituted, the substituents can be alkyl, alkoxy, halogen, oxygen or substituted or unsubstituted phenyl.
[105] Peptides, peptide and protein analogs and mimetics are also described in US Pat. No. 4,640,835; U.S. 4,496,689; US Patent 4,301,144; US Patent 4,670,417; US Patent 4,791,192; Or covalently bind to one or more various nonproteinaceous polymers, such as, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkenes according to the methods set forth in US Pat. No. 4,179,337.
[106] Other peptide and protein analogs and mimetics of the present invention include glycosylated variants and covalent or aggregated conjugates with other chemical moieties. Covalent derivatives can be prepared by linking functional groups to groups found at the N- or C-terminal or amino acid side chains by means well known in the art. Such derivatives include amide or aliphatic esters of residues containing a carboxyl terminus, or carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives or amino group containing residues of amino-terminal amino acids, such as lysine. Or arginine, including but not limited to. The acyl group is selected from the group of alkyl-moieties including C3 to C18 normal alkyl and thus alkanoyl aroyl species are formed. Covalent linkages to carrier proteins such as, for example, immunogenic moieties may also be used.
[107] In addition to these modifications, glycosylation modifications of biologically active peptides can be achieved during the activity and processing of the peptide, or by further processing steps, such as by modifying the glycosylation pattern of the peptide. A particularly preferred means for this is to expose this peptide to glycosylase, such as, for example, mammalian glycosylase, which is derived from cells that generally provide such processing. Deglycosylation enzymes can also be used successfully to produce modified peptides useful within the scope of the present invention. Natural major amino acid sequence versions with other minor modifications are also included, including ribosyl groups or crosslinking agents, such as phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, or phosphorus threonine or other moieties.
[108] Peptidomimetics may also have amino acid residues that are chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, especially those having a molecular shape similar to phosphate groups. In some embodiments, such modifications may serve as useful labeling reagents, or as purification targets such as, for example, affinity ligands.
[109] Major mimetic peptide groups within the scope of the present invention include covalent conjugates of natural peptides, or fragments thereof, with other proteins or peptides. These derivatives can be synthesized using agents known in the art for their usefulness in crosslinking to proteins via reactive side groups or in recombinant culture such as N- or C-terminal fusions. Preferred peptide and protein induction sites for targeting by crosslinkers are free amino groups, carbohydrate moieties and cysteine residues.
[110] Fusion polypeptides between biologically active peptides and other homologous or heterologous peptides are also provided. Many growth factors and cytokines are homodimers and repeat structures of these molecules or active fragments thereof have several advantages, such as reduced sensitivity to proteolytic degradation. Repeating and other fusion constructs of pro- or anti-inflammatory binding peptides show similar advantages in the methods and compositions of the present invention. Various other hairy structures are also provided, including peptides useful within the scope of the present invention. In certain embodiments, biologically active polypeptide fusions are described, for example, in US Pat. Nos. 6,018,026, 5,843,725, 6291,646, 6,300,099 and 6,323,323, each of which is incorporated herein by reference, for example. The active peptide is provided by linking with a heterologous multimeric polypeptide such as an immunoglobulin heavy chain constant region, or an immunoglobulin light chain constant region. The biologically active, multimerized polypeptide fusions so constructed may be hetero- or homo-multimers, such as heterodimers or homodimers, which may contain one or more pro-inflammatory or anti-inflammatory binding peptide element (s). Other heterologous polypeptides combine with active peptides to produce fusions that represent a combination of properties or activity of a derivative protein. Other common examples include fusions with peptides described herein to facilitate localization of a peptide fused with a reporter polypeptide, such as CAT or luciferase (see, eg, Dull et al., US Pat. No. 4,859,609, referenced herein). ). Other fusion partners useful in this context include bacterial beta-galactosidase, trpE, protein A, beta-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factors (eg, Godowski et al., Science 241). : 812-816, 1998, referenced in the text).
[111] The invention also provides for the use of pro- or anti-inflammatory binding peptides modified by covalent or aggregate binding with chemical moieties. Such derivatives generally fall into three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes with, for example, cell membranes. Such covalent or aggregated derivatives are useful for various purposes, such as immunogens, as reagents in immunoassays or purification methods such as affinity purification of ligands or other binding ligands. For example, the active peptide may be glued for use in the analysis or purification of an antibody which is covalently immobilized on a solid carrier, such as cyanogen bromide-activated Sepharose, or which specifically binds to the active peptide by known methods in the art. It may be adsorbed on the polyolefin surface with or without taraldehyde crosslinking. The active peptide may also be labeled with a detectable group, such as radioiodinated by a chloramine T process, covalently bound to rare earth chelates, or conjugated to other fluorescent moieties used in diagnostic assays.
[112] Those skilled in the art will prepare mimic peptides and mimic proteins having the same or similar desired biological activity as the corresponding natural peptides, but with further improved solubility, stability and / or sensitivity to hydrolysis or proteolysis than the peptides. A variety of techniques are known that can be used (eg, Morgan and Gainor, Ann. Rep. Med. Chem. 24: 243-252, 1989, referenced herein). Certain mimetic peptide compounds are based on the amino acid sequences of the proteins and peptides described for use in the present invention. In general, mimetic peptide compounds are the primary, secondary of a selected peptide or structural domain, active site or binding zone thereof (such as a homotape or heterotape binding site, catalytically active site or domain, receptor or ligand binding interface or domain, etc.). And / or tertiary structures, and / or synthetic compounds having a three-dimensional structure (at least part of the mimicking compound) that mimics electrochemical properties. Peptide-mimetic structures or partial structures (also referred to as peptide mimetic “motifs” of mimetic peptide compounds) may block, for example, recognition or protective HLA-E binding by the CD94 / NKG2 cellular receptor of the MHC leader sequence peptide / HLA-E complex. Or desired biological activity, such as binding activity for HLA-E, will be shared with the natural peptide. In general, the biological activity of the subject of a mimic compound is not substantially less than that, often the same or better, compared to the activity of the natural peptide modeled for that mimic. In addition, mimetic peptide compounds may have other desired properties that enhance their therapeutic application, such as increased cell permeability, high affinity and / or acid group, and extended biological half-life. The mimetic peptides of the present invention are partially or completely non-peptide, but will have a "backbone" having the same side groups as those of the amino acid residues occurring in the peptide modeled for the mimetic peptide. Several types of chemical bonds, such as, for example, ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are generally useful substituents of peptide bonds in the manufacture of protease-resistant mimetic peptides. Known.
[113] Next, a method for producing a peptide and protein mimetic modified at an N-terminal amino group, a C-terminal carboxyl group and / or a method for changing one or more amido bonds in the peptide to non-amido bonds is described. It is known that two or more such modifications can be coupled in one peptide mimetic structure (eg, including --CH ₂ -carbamate bonds between modifications at the C-terminal carboxyl group and two amino acids in the peptide) Letting). For N-terminal modifications, as described above, peptides are generally synthesized as free acids, but can be readily prepared as amides or esters. The amino and / or carboxy terminus of the peptide compound may also be modified to produce other compounds useful in the present invention. Amino terminal modifications include methylation (ie --NHCH ₃ or --NH (CH ₃ ) ₂ ), acetylation, carbobenzoyl group addition or amino terminus RCOO-- (where R is naphthyl, acridinyl, And blocking with a blocking group containing a carboxylate functional group, which is selected from steroididyl and similar groups). Carboxy terminal modifications include forming cyclic lactans at the carboxy terminus or replacing the free acid with a carboxamide group to introduce structural tension. The amino terminal modifications are as described above and are the addition of alkylation, acetylation, addition of carbobenzoyl groups, succinimide groups and the like. N-terminal amino groups can be reacted as follows:
[114] (a) An amide group of the formula RC (O) NH--, where R is as defined above, is formed by reacting with an acid halide [eg, RC (O) Cl] or an acid anhydride. In general, this reaction is carried out by contacting the peptide with about equimolar or excess (eg about 5 equivalents) of the acid halide, preferably in contact with the peptide in an inert diluent (eg dichloromethane) containing an excess of tertiary amine such as diisopropylethylamine. This can be done by capturing the acid generated during the reaction. Other reaction conditions are customary (such as 30 minutes at room temperature). The alkylation of terminal amino to provide lower alkyl N-substitution and subsequent reaction with an acid halide followed by N- of formula RC (O) NR-- Alkyl amide groups will be provided;
[115] (b) A succinimide group is formed by reaction with succinic anhydride. As above, using an equivalent (such as about 10 equivalents) of tertiary amine, such as diisopropylethylamine, in an appropriate inert solvent (such as dichloromethane), using about equimolar amount or succinic anhydride (such as about 5 equivalents) Amino groups are converted to succinimides by known methods including those described in, eg, Wollenberg et al., US Pat. No. 4,612,132, referenced herein. It is known that succinimides can be substituted, for example at the N-terminus of the peptide, by substituting with a SR substituent or C ₂ -C ₆ alkyl, e. Such alkyl substituents are prepared by reacting maleic anhydride with lower olefins (C _2-6 ) in the method of Wollenberg et al. (US Pat. No. 4,612,132) and the --SR substituents are substituted with RSH (where R is as defined above) with maleic anhydride. It makes it by reaction.
[116] (c) Equivalent or excess CBZ-Cl (such as benzyloxycarbonyl chloride) or substituted CBZ-Cl, preferably in an appropriate inert diluent containing tertiary amines (such as dichloromethane) to capture the acid produced in the reaction By reaction with benzoyloxycarbonyl--NH-- or substituted benzyloxycarbonyl--NH-- groups;
[117] (d) reacting sulfonamides by reacting the same or excess (eg 5 equivalents) RS (O) ₂ Cl, where R is as defined above, in a suitable inert diluent (dichloromethane) to convert terminal amines to sulfonamides. To form. Preferably, the inert diluent contains an excess of tertiary amine (such as 10 equivalents), such as diisopropylethylamine, to capture the acid produced during the reaction. Other reaction conditions are typical (such as 30 minutes at room temperature);
[118] (e) Equivalent or excess (such as 5 equivalents) of R-OC (O) Cl or R-OC (O) OC ₆ H ₄ -p in a suitable inert diluent (such as dichloromethane) to convert terminal amines to carbamate Carbamate groups are formed by reacting with —NO ₂ , where R is as defined above. Preferably, the inert diluent contains an excess (eg about 10 equivalents) of tertiary amine, such as diisopropylethylamine, in order to capture any acid produced during the reaction. Other reaction conditions are common (such as 30 minutes at room temperature).
[119] (f) equivalent or excess (such as 5 equivalents) in an appropriate inert diluent (such as dichloromethane) to convert terminal amines to urea (ie, RNHC (O) NH--) groups where R is as described above) A urea group is formed by reacting with R-N = C = O. Preferably, the inert diluent contains an excess (eg about 10 equivalents) of tertiary amine, such as diisopropylethylamine. Other reaction conditions are typical (eg about 30 minutes at room temperature).
[120] In preparing mimic peptides in which the C-terminal carboxyl group has been replaced by an ester (ie, --C (O) OR where R is as defined above), the resins used to produce the peptide acid are generally used and the acid The side chain protected peptide is cleaved with a base and a suitable alcohol such as methanol. The side chain protecting groups are then removed in a conventional manner by treatment with hydrogen fluoride to give the desired ester.
[121] In preparing mimetic peptides in which the C-terminal carboxyl group has been replaced by amide-C (O) NR ₃ R ₄ , benzhydrylamine resin is used as a solid support for peptide synthesis. Upon completion of the synthesis, the free peptide amide (ie, C-terminus --C (O) NH ₂ ) is produced directly by hydrogen fluoride treatment for peptide release from the support. On the other hand, free peptide amides are produced by the use of chloromethylated resins in the synthesis of peptides coupled with the reaction with ammonia to cleave the side chain protected peptides from the support and side chain protected by reaction with alkylamines or dialkylamines. Alkylamides or dialkylamides (ie, the C-terminus is —C (O) NRR ₁ where R and R ₁ are as defined above) are produced. The side chain protecting groups are then removed in a conventional manner to produce free amides, alkylamides or dialkylamides by hydrogen fluoride treatment.
[122] In another embodiment of the invention, the C-terminal carboxyl group or C-terminal ester of the biologically active peptide is an internal displacement of --OH or ester (--OR) or ester of the carboxyl group, respectively, with an N-terminal amino group. By cyclization to form a cyclic peptide. For example, peptide acids are produced by synthesis and cleavage, for example by means of suitable carboxyl activators such as dicyclohexylcarbodiimide (DCC) in solutions such as methylene chloride (CH ₂ Cl ₂ ), dimethyl formamide (DMF) mixtures. The free acid is converted to an activated ester. The cyclic peptide is then formed by internal replacement of the activated ester with the N-terminal amine. Internal cyclization as opposed to polymerisation can be facilitated by using very dilute solutions. Such methods are well known in the art.
[123] Active peptides for use in the present invention can be cyclized or desamino or descarboxyl residues incorporated at the end of the peptide to eliminate terminal amino or carboxyl groups, thereby reducing sensitivity to proteases or limiting the arrangement of the peptides. have. C-terminal functional groups among the peptide analogs and mimic peptides of the present invention include amides, amide lower alkyls, amide di (lower alkyls), lower alkoxy, hydroxy and carboxy, and lower ester derivatives thereof. Acceptable salts are also included.
[124] Peptide and protein derivatives and other methods of making mimetics for use in the methods and compositions of the present invention are disclosed in Biochem J. 268 (2): 249-262, 1990, incorporated herein by Hruby et al . According to this method, biologically active peptides serve as structural models of non-peptide mimetic compounds with similar biological activity as natural peptides. Those skilled in the art can use a variety of techniques to prepare compounds that have the same or similar desired biological activity as the lead peptide, or that the desired properties such as solubility, stability and hydrolysis and proteolysis are better than the lead peptide. Will be recognized (see, eg, Morgan and Ganior, Ann. Rep. Med. Chem. 24: 243-252, 1989, text). Such techniques include, for example, replacing the backbone of the peptide with a backbone consisting of phosphonates, amidates, carbamates, sulfonamides, secondary amines and / or N-methylamino acids.
[125] For example, one or more peptidyl bonds [--C (O) NH--] may be substituted for --CH ₂ -carbamate bonds in conventional pefanid synthesis by merely replacing amino acid reagents with appropriately protected amino acid analogs at appropriate positions in the synthesis. Phosphonate bonds, --CH ₂ -sulfonamide bonds, urea bonds, secondary amine (--CH ₂ NH--) bonds, and alkylated peptidyl bonds [--C (O) NR ₆ -wherein R ₆ Is lower alkyl] to produce mimetic peptides and proteins that are replaced by a bond. Suitable reagents include, for example, amino acid analogues in which the carboxyl groups of the amino acids are replaced by moieties suitable for forming any of these bonds. For example, replacing a --C (O) NR-- bond in a peptide with a --CH ₂ -carbamate bond (--CH ₂ OC (O) NR--) followed by a carboxyl (-COOH) of appropriately protected amino acid The group is first reduced to a --CH ₂ OH group and then converted to the --OC (O) Cl functional group or para-nitrocarbonate --OC (O) OC ₆ H ₄ -p-NO ₂ functional group according to the conventional method. At the N-terminus of the partially assembled peptides found in the solid support, the reaction of a free or alkylated amine with these functional groups forms a --CH ₂ OC (O) NR-- bond. A more detailed description of such --CH ₂ -carbamate bond formation can be found in Cho et al. (Science 261: 1303-1305, 1993, referenced herein).
[126] Replacing amido bonds in active peptides with --CH ₂ -sulfonamide bonds reduces the carboxyl (--COOH) group of an appropriately protected amino acid to a --CH ₂ OH group followed by a hydroxyl group according to the conventional method. Achievement by conversion to a suitable leaving group such as By reacting this derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination, the --CH ₂ --S (O) ₂ Cl functional group is provided, which otherwise replaces the carboxyl group of an appropriately protected amino acid. The use of such properly protected amino acids in peptide synthesis results in the production of mimetic peptides by providing an inclusion of —CH ₂ S (O) ₂ NR − bonds that replaces the amino bonds in the pentide. A more complete description of converting carboxyl groups of amino acids to --CH ₂ S (O) ₂ Cl groups is described, for example, in Weinstein and Boris (Chemistry & Biochemistry of Amino Acids, Peptides, Vol. 7, pp. 267-357, Marcel Dekker, Inc., New York, 1983, referenced herein). Replacing amido bonds in peptides with urea bonds can be performed, for example, in the manner set forth in US Patent Application Serial No. 08.147,805 (incorporated herein).
[127] Secondary amine bonds in which --CH ₂ NH-- bonds replace the amido bonds in the peptide, for example, employ appropriately protected dipeptide analogues in which the carbonyl bonds of the amido bonds are reduced to CH ₂ groups by conventional methods. It can be manufactured by. For example, in the case of diglycine, H ₂ NCH ₂ CH ₂ NHCH ₂ COOH is produced by reducing the amide to amine and then deprotecting and using it in N-protective form in the next coupling reaction. The preparation of such analogues by reduction of carbonyl groups of amido bonds in dipeptides is well known in the art.
[128] Biologically active peptides and protein preparations of the invention may be present in monomeric form without disulfide bonds formed by thiol groups of cysteine residue (s) that may be present in the subject peptide. Alternatively, intermolecular disulfide bonds between thiol groups of cysteine on two or more peptides can be made to produce multimeric (eg, dimer, tetramer or larger oligomer) compounds. Some of these peptides can be cyclized or dimerized by replacing leaving groups by sulfur of cysteine or homocysteine residues (Barker et al. , J. Med. Chem. 35: 2040-2048, 1992; and Or et al., J. Org. Chem. 56: 3146-3149, 1991). Thus, one or more natural cysteine residues can be replaced with homocysteine. Intramolecular or intermolecular disulfide derivatives of the active peptide provide analogs in which one of several sulfurs is replaced by a CH ₂ group or another isostere of sulfur. Such analogs can be carried out through intramolecular or intermolecular replacement by using known methods.
[129] All naturally occurring peptides, recombinant peptides and synthetic peptides identified as agents useful in the present invention, and peptides and protein analogs, act as inhibitors of isotype or heterozygous binding between membrane adsorption proteins for enhancing epithelial permeability. Additional compounds, other peptides, proteins, analogs and mimetics, which will play a variety of roles in the compositions, may be used for screening (such as in kits and / or screening assays).
[130] Several automated assays have been developed in recent years that can screen tens of thousands of compounds in a short period of time (eg, Fodor et al., Science 251: 767-773, 1991 and U.S. Patents 5,677,195; 5885,837; 5,902,723; 6,027,880; 6,040,193 and 6,124,102, respectively). Referenced in the text). Encoded synthetic libraries (ESL), as described, for example, in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503, and WO 95/30642, each referenced herein Large combinatorial libraries of compounds can be constructed. Peptide libraries can also be produced by phage display (eg, Devln, WO 91/18980, referenced herein). Many other publications describing chemical diversity libraries and screening methods are also considered to be a reflection of the current state of the art in support of this aspect of the invention and are incorporated herein by reference in their entirety.
[131] One method (such as small molecule drug peptide mimetics) for screening new biologically active agents for use in the present invention is to use eukaryotic or prokaryotic host cells stably transformed with recombinant DNA molecules expressing the active peptide. Such cells may be used in standard assays such as ligand / receptor binding assays in live or fixed form (eg, Parce et al., Science 246: 243-247, 1989; and Owicki et al . , Proc. Natl. Acad. Sci. USA 87: 4007-4011, 1990, respectively referenced in the text). Particularly useful in competitive assays such as assays in which cells are incubated in contact with an antibody having a known binding affinity for a labeled receptor or peptide ligand and a test compound or sample to be measured for binding affinity. Subsequently, labeled or bound free binding components are separated to assess the degree of ligand binding. The amount of bound test compound is inversely proportional to the amount of labeled receptor bound to a known source. Numerous techniques can be used to separate the bound ligand from the free ligand to assess the degree of ligand binding. Such separation steps may include known procedures such as adsorption on filters and subsequent washing, adsorption and washing on plastics, or cell membrane centrifugation.
[132] Another technique for screening drugs within the scope of the present invention is suitable binding affinity for target molecules such as, for example, HLA-E molecules, HLA-E peptide complexes, or HLA-E / peptides / CD94 / NKG2 cellular receptor complexes. Approaches to provide high speed screening for compounds having have are described in detail in Gysen, European Patent Application 84/03564, September 13, 1984 (incorporated herein). First, a number of different test compounds, such as small peptides, are synthesized on a solid substrate such as, for example, plastic fins or other suitable surfaces (see, eg, Fodor et al., Science 251: 767-773, 1991 and Fodor et al., US Pat. Nos. 5,677,195; 5,885,837; 5,902,723; 6,027,880; 6,040,193; and 6,124,102. All referenced herein). All pins are then reacted with solubilized peptide formulations of the invention and then washed. The next step is to detect the bound peptide.
[133] Inferential drug design may also be based on structural studies of the molecular shape of biologically active peptides determined to work in the methods of the present invention. A variety of methods are available, including, for example, x-ray crystallography and two-dimensional NMR techniques, to characterize, map, translate, and reproduce the structural properties of the peptides, which will guide the production and selection of new mimetic peptides. Methods are well known in the art. These and other methods will allow for rational prediction of the amino acid residues present in the molecular contact zone of the selected peptide form, eg required for specificity and activity (eg Blundell and Johnson, Protein Crystallography, Academic Press, NY, 1976, Referenced in the text).
[134] Activatable analogs and mimetics of the pro-inflammatory or anti-inflammatory binding peptides disclosed herein retain partial, complete, or more increased activity than natural peptides. In this regard, the operable analogs and mimetics used in the present invention are at least 50%, often 75%, 95-100% or more when compared to the same activity as observed in selected natural phetides or unmodified compounds. One or more selected activities will be maintained. Such biological activity of an altered peptide or non-peptide mimetic can be measured according to any suitable assay disclosed or referenced herein.
[135] Compounds of the invention according to the present disclosure are useful in vitro as unique tools to analyze the properties and functions of pro- or anti-inflammatory binding peptides, HLA-E molecules, and CD94 / NKG2 cell receptors, thus enhancing mucosal epithelial permeability. It will also serve as a leader in various programs for designing additional peptide and non-peptide (such as small molecule drug) formulations to facilitate and facilitate drug delivery through mucous membranes.
[136] In addition, the proinflammatory or anti-inflammatory binding peptides, analogs and mimetics disclosed herein are useful as immunogens, immunogen components, such as to reduce autoimmune symptoms or inflammation by blocking HLA-E binding by, for example, proinflammatory binding peptides, or It is useful for producing antibodies and related agents that would be useful for targeting or initiating NK and CTL responses against tumor cells or virus infected cells. In the latter case, localizing the antibody to a tumor or virus infected cell or tissue may be facilitated by coupling the antibody to a tumor or viral targeting factor, such as an antibody or antibody fragment that binds to a tumor-associated or virus-associated antigen. Can be.
[137] Thus, the peptides of the present invention typically bind to an immunized peptide (s) or peptide conjugate (s) with high affinity and strength, but do not similarly recognize unrelated peptides, typically in conjugate form. (Eg, a multimeric peptide, or peptide / carrier or peptide / hapten conjugate) as an immunogen.
[138] In this context, the present invention provides diagnostic and therapeutic antibodies, including monoclonal antibodies directed against proinflammatory or anti-inflammatory binding peptides. Such antibodies can be used to determine whether a peptide is associated with an interaction between the peptide and, for example, an HLA-E molecule. The functional part may be specifically recognized. Such immunotherapy reagents include humanized antibodies and are combined or optionally with additional active or inactive ingredients such as those described herein for therapeutic use, such as conventionally pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants. It can be used in combination with an adjuvant or bindingly active agent, such as an antiretroviral drug. Methods of preparing functional antibodies, including humanized antibodies, antibody fragments, and other related agents, are known in the art (eg, Harlow & Lane, Antibodies, A Laboratory Manual, CSHP, NY, 1988; Queen et al., Proc. Natl. Acad. Sci USA 86: 10029-10033, 1989 and WO90 / 07861, respectively referenced in the text).
[139] Humanized forms of mouse antibodies can be generated by binding the CDR bands of non-human antibodies to human constant regions by recombinant DNA techniques (eg, Queen et al . , Proc. Natl. Acad. Sci. USA 86: 10029-10033, 19889). And WO 90/07861, respectively referenced in the text). Human antibodies are obtainable by phage-display (eg Dower et al., WO 91/17271; MaCafferty et al., WO 92/01047, respectively referenced in the text). In these methods phage libraries are prepared in which members exhibit various antibodies on their outer surface. Antibodies are usually represented as Fv or Fab fragments. Phages representing antibodies with the desired properties are selected by affinity enrichment for human cytochrome P450 or fragments thereof. Human antibodies are selected by competitive binding test or with the same epitope specificity as specific mouse antibodies.
[140] The present invention also provides fragments of the native antibodies described above. In general, these fragments compete with the original antibody from which they are derived for specific binding to HLA. Antibody fragments include separate heavy chain, light chain Fab, Fab'F (ab ') 2, Fv and single chain antibodies. Fragments can be produced by enzymatic or chemical separation of native immunoglobulins. For example, F (ab ′) 2 fragments can be obtained from IgG molecules by proteolysis with pepsin at pH 3.0-3.5 using standard methods as described by Harlow and Lane, supra. Fab fragments can be obtained from F (ab ') 2 fragments by restrictive reduction or from whole antibodies by cleavage with papain in the presence of a reducing agent. Fragments can be produced by recombinant DNA technology. Nucleic acid segments encoding selected fragments are prepared by cleavage of the full length coding sequence using restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. This anti-glare mode is advantageous for affinity-sharpening of antibodies.
[141] To recombinantly produce the antibodies of the invention, nucleic acids encoding light chain and heavy chain variable regions optionally linked to the constant region are inserted into an expression vector. Light and heavy chains can be cloned into the same or different expression vectors. DNA segments encoding the antibody chains are operatively linked to regulatory sequences in the expression vector (s) to ensure antibody chain expression. Such regulatory sequences include signal sequences, promoters, enhancers and transcription termination sequences. Expression vectors can generally be replicated in the host organism either as an integral part of the host chromosome or as an episome. E. coli is a prokaryotic host that is particularly useful for expressing the antibodies of the invention. Other microbial hosts suitable for use include Bacillus, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonela, Serratia, and various Pseudomonas species. have. In these prokaryotic hosts, expression control sequences and regulatory sequences, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems from phage lambda, which can coexist with host cells (such as the origin of replication), are generally Expression vectors containing can also be made. Other microorganisms, such as yeast, can also be used for expression. Saccharomyces is a preferred host having suitable vectors with expression control sequences such as the desired 3-phosphoglycerate kinase or other glycolitic enzymes and promoters including replication origins, termination sequences and the like.
[142] Mammalian tissue cell cultures can also be used to express and produce the antibodies of the invention (see, eg, Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987, text). Eukaryotic cells are preferred because several eukaryotic cells have been developed that can secrete native native antibodies. Preferred host cells suitable for expressing nucleic acids for encoding immunoglobulins of the invention include the following: Monkey kidney CV1 cells (COS-7, ATCC CRL 1651) transformed by SV40; Human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36:59, 1977, referenced herein); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cell-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216, 1980, incorporated herein by reference); Mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251, 280, referenced herein); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); Human cervical carcinoma cells (HELA, ATCC CCL2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (HEp G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CCl51); And TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-46, 1982, referenced herein): and baculovirus cells.
[143] Vectors containing the desired polynucleotide sequences (such as heavy and light chain coding sequences and expression control sequences) can be delivered to a host cell. Calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation is used for other cellular hosts (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2nd). ed., 1989, referenced herein). When the heavy and light chains are cloned on separate expression vectors, these vectors are co-transformed to obtain the expression and assembly of the original unique immunoglobulin. After introduction of the recombinant DNA, cell lines expressing the immunoglobulin product are selected. Cell lines capable of stable expression are preferred (ie, the expression level does not decrease even after passage of the cell line 50).
[144] Once expressed, all of the antibodies of the invention, their dimers, individual light and heavy chains, or other immunoglobulin forms, are known in the art, such as ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, and the like. Purification can be done according to techniques (see, eg, Scopes, Protein Purifications, Springer-Verlag, NY, 1982, text). Substantially pure immunoglobulins having at least about 90-95% homology are preferred, most preferably having 98-99% homology.
[145] The pro-inflammatory or anti-inflammatory binding peptides of the present invention are commonly used in drug screening compositions and methods as described above to be used for HLA-E, HLA-E / peptide complexes, or HLA-E / peptides / CD94 / NKG2 cellular receptor complexes. As described herein, it can be used to identify additional compounds having binding affinity for and / or act as agonists or antagonists for HLA-E mediated protective interactions with CD94 / NKG2 cellular receptors. It functions as an immune modulatory agents. Many different screening methods and formats are available and are well known in the art. Biological assays can subsequently be used to determine whether the screened compounds have binding activity or other activities useful in the present invention. In such assays, the compounds of the invention may be used without modification or in a variety of different ways; Modifications can be made in a variety of ways, including labeling such as for example, covalently or non-covalently binding to a portion that provides a detectable signal directly or indirectly. Direct labeling possibilities include radioactive labels, enzymes such as peroxidase and alkaline phosphatase (eg, US Pat. No. 3,645,090; and US Pat. No. 3,940,475, respectively, referenced in the text), and fluorescent labels. Indirect labeling possibilities include biotinylation of one component and subsequent binding to avidin coupled to one of the labeling groups. These compounds may also include spacers or linkers when the compound is attached to a solid support.
[146] The pro-inflammatory or anti-inflammatory binding peptides of the present invention are based on their ability to bind to complexes with HLA-E and CD94 / NKG2 cellular receptors, and thus are alive in biological fluids, tissue homogenates, purified, natural biological materials, and the like. It can be used as a reagent for detecting and / or quantifying HLA-E molecules on cells, immobilized cells. For example, by labeling such peptides, cells having HLA-E molecules on their surface can be identified and / or quantified. In addition, based on its more detailed activity, proinflammatory or anti-inflammatory binding peptides may be used to quantify the presence and activity of other HLA-E binding peptides and CD94 / NKG2 cellular receptors. Peptides of the invention can be used in in situ staining, FACS (fluorescence-activated cell sorting), western blotting, ELISA and the like. In addition, the peptides of the present invention can be used to purify HLA-E and CD94 / NKG2 cellular receptors or to purify cells expressing HLA-E.
[147] The pro-inflammatory or anti-inflammatory binding penendoids of the present invention may also be used as commercial reagents for various medical research or diagnostic applications. Such uses include, but are not limited to: (1) Use as a calibration standard for quantifying the presence or activity of HLA-E, other HLA-E binding peptides, and / or CD94 / NKG2 cellular receptors. ; (2) use in structural analysis of HLA-E and CD94 / NKG2 cellular receptors via co-crystallization; And (3) use to investigate HLA-E / peptide / CD94 / NKG2 cellular binding and activation mechanisms.
[148] In another aspect of the invention, the immunomodulatory activity of the peptide of the subject may be enhanced by a link to a sequence containing at least one epitope capable of inducing an NK, CTL or T helper cell response. For example, in this context certain conjugates may define CTL epitopes that are one or more different or overlapping with the proinflammatory binding peptide. On the other hand, such combinatorially active peptides / epitopes can be combined into "cocktails" to provide enhanced immunogenicity in NK or CTL responses. Peptides may be combined with a peptide having different MHC restriction elements. Such compositions can be used to effectively extend the immunological coverage provided by the therapeutic, vaccine or diagnostic methods and compositions of the present invention in a variety of different populations.
[149] Peptides of the invention can be formulated in the composition as a mixture with or without a link to a polymer (multimer) via a link. The same peptide can be linked to itself to form a homopolymer of plural repeating epitope units. Couplings to carriers or links for homo- or hetero-polymers can be provided in various ways. For example, controlled oxidation of cysteine residues can add cysteine residues at both the amino- and carboxy-terminus to which the peptides are covalently bound. Many heterobifunctional agents are also useful, including N-succidimidyl-3- (2-pyridyldithio) propionate (SPDP), which generates disulfide links in one functional group and peptide links in another functional group. These reagents make disulfide bonds between themselves and cysteine residues in one protein and amide bonds through amino on lysine or other amine groups in another protein. Many such disulfide / amide forming reagents are known. For example, Immune. Rev. 62: 185 (1982). Other bifunctional coupling agents form thioethers better than disulfide bonds. Many of these thioether-forming reagents are commercially available and include 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimido-methyl) cyclohexane-1-carboxylic acid, and the like. This is an example. Carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. Particularly preferred coupling agents are succinimidyl 4-N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Of course, this combination should not substantially interfere with the linked group function.
[150] In a preferred embodiment the pro- or anti-inflammatory binding peptides of the invention are conjugated to other peptides by spacer molecules. Spacers typically comprise relatively small, neutral molecules, such as amino acids or amino acid mimetics, which may actually have straight or branched side chains without being charged under physiological conditions. The spacer is generally selected from, for example, neutral spacers of Ala, Gly or neutral polar amino acids or nonpolar amino acids. In certain preferred embodiments herein the neutral spacer is Ala. It will be appreciated that the spacer, optionally present, need not consist of identical residues and can therefore be hetero- or homo-oligomer. Examples of preferred spacers include Ala's homo-oligomers. This spacer, if present, will consist of at least one or two residues, more generally three to six residues.
[151] Delivery vehicle and method
[152] In certain aspects of the invention, pro- or anti-inflammatory binding peptides are administered in a formulation comprising a biocompatible polymer that functions as a carrier or base. Such polymer carriers include polymer powders, matrices or particulate delivery vehicles, among other polymer forms. The polymer may be of plant, animal or synthetic origin. Often polymers are crosslinked. In addition, the peptides in these delivery systems can be functionalized in such a way that it can be covalently attached to the polymer and cannot be separated from the polymer by simple washing. In another embodiment, the polymer is chemically modified with other agents or enzyme inhibitors that can degrade or inactivate the biologically active agent (s) and / or delivery promoter (s).
[153] Drug delivery systems based on biodegradable polymers are desirable in many biomedical applications because such systems can be degraded into non-toxic molecules by hydrolysis or enzymatic reactions. The rate of degradation is controlled by manipulating the composition of the biodegradable polymer matrix. This kind of system can thus be used in certain settings for long term release of biologically active agents. Biodegradable polymers such as poly (glycolic acid) (PGA), poly- (lactic acid) (PLA), and poly (D, L-lactic-co-glycolic acid) (PLGA) have low toxicity of the degradation products of these polymers. As it turns out, it is attracting attention as a possible drug delivery carrier. Under normal metabolic functions of the body, these polymers are broken down into carbon dioxide and water (Mehta et al . , J. Control. Rel. 29: 375-384, 1994). These polymers also exhibit excellent biocompatibility.
[154] In order to extend the biological activity of pro- or anti-inflammatory binding peptides, they can be incorporated into polymeric matrices such as polyorthoesters, polyanhydrides or polyesters. This sustains the release and activity of the active agent (s) as measured, for example, by polymer matrix degradation (Heller, Formulation and Delivery of Proteins and Peptides , pp. 292-305, Cleland et al., Edited by ACS Symposium Series 567). , Washington DC, 1994; Tabata et al., Pharm. Res. 10: 487-496, 1993; and Cohen et al . , Pharm. Res. 8: 713-720, 1991, respectively,). Although encapsulating biotherapeutic molecules inside synthetic polymers will stabilize them during storage and delivery, the greatest challenge of polymer-based release technology is that these therapeutic molecules during formulation processes often associated with thermal, ultrasonic or organic solvents. (Tabata et al . , Pharm. Res. 10: 487-496, 1993; and Jones et al., Drug Targeting and Delivery Series, New Delivery Systems for Recombinant Proteins-Practical Issues from Proof of Concept to Clinic , Vol. 4, pp. 57-67, Lee et al., Eds., Harwood Academic Publishers, 1995).
[155] Polymers suitable for use in the present invention should generally be stable on their own and when combined with the selected biologically active agent (s) and additional ingredients of the mucosal composition and are stable in the pH condition range of about pH 1 to pH 10. Gels should be formed. More generally, they should be stable in the range of about 3-9 pH and form polymers without additional protective coating. However, the desired stability can be adjusted to the characteristic physiological parameters of the target site for delivery (eg, secondary delivery sites such as nasal mucosa or systemic circulation). Thus, in certain compositions, higher or lower stability may be more desirable at certain pH and selected chemical and biological environments.
[156] Absorption-promoting polymers of the present invention can include polymers from homo- and copolymer groups based on various combinations of the following vinyl monomers: acrylic acid and methacrylic acid, acrylamide, methacrylamide, hydroxyethylacrylate Or methacrylates, vinylpyrrolidone, and polyvinyl alcohols and their copolymers and terpolymers, polyvinylacetates, monomers and 2-acrylamido-2-methyl-propanesulfonic acids (AMPS) Copolymers and terpolymers). Particularly very useful are the monomers specified above and for example acrylic or methacryl amide acrylate or methacrylate esters, wherein the ester groups are four or more which may contain straight or branched chain alkyl, alkyl substituents of from 1 to 6 carbons. Copolymers with copolymerizable functional monomers such as derived from aryl having an aromatic ring; Steroidal, sulfate, phosphate or cationic monomers such as N, N-dimethylaminoalkyl (meth) acrylamide, dimethylaminoalkyl (meth) acrylate, (meth) acrylicoxytrimethylammonium chloride, (meth) acryloxyalkyl Dimethylbenzyl ammonium chloride.
[157] Additional absorption-promoting polymers usable in the present invention are dextran, those classified as dextrins, and substances classified as natural gums and resins, or processed collagen, chitin, chitosan, pullulan, juglan, alginate and " Kelcoloid "(polypropylene glycol modified alginate) gellan gums such as" Kelocogel ", xanthan gums such as" Keltrol ", statin, alpha hydroxy butyrate and copolymers thereof, hyaluronic acid and derivatives thereof, polylactic acid and glycolic acid.
[158] Very useful polymer classes applicable in the present invention include olefinically-unsaturated carboxylic acids containing at least one activated carbon to carbon olefin double bond and at least one carboxyl; That is, an acid or functional group that is readily converted to an acid containing an olefinic double bond that readily functions during polymerization, due to its presence in the monomer molecule, whether at the alpha-beta position or as part of a terminal methylene group, with respect to the carboxyl group. . Olefinically unsaturated acids of this class include acrylic acid such as acrylic acid itself, alpha-cyano acrylic acid, beta methacrylic acid (crotonic acid), alpha-phenyl acrylic acid, beta-acryloxy propionic acid, cinnamic acid, p-chlorocinnamic acid, 1-carboxy-4-phenyl butadiene-1,3, itaconic acid, citric acid, mesaconic acid, glutamic acid, aconitic acid, maleic acid, fumaric acid and tricarboxy ethylene. The term "carboxylic acid" herein includes polycarboxylic acids and their acid anhydrides, such as maleic anhydride, where the anhydride is formed by removing a molecule of water from two carboxyl groups located on the same carboxylic acid molecule.
[159] Representative acrylates useful as the absorbent-promoting agent in the present invention include methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, methyl methacrylate, Methyl acrylate, ethyl methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate, isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate, hexyl acrylate, n-hexyl methacrylate The rate etc. are mentioned. Higher alkyl acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl acrylate, stearyl acrylate, behenyl acrylate and mesyl acrylate and methacrylate versions thereof. Mixtures of two or three or more long chain acrylic esters can be successfully polymerized with one carboxylic monomer. Other comonomers include olefins including alpha olefins, vinyl ethers, vinyl esters, and mixtures thereof.
[160] Still other useful absorbent promoting substances include alpha-olefins containing 2 to 18 carbon atoms, more preferably 2 to 8 carbon atoms; Dienes containing 4 to 10 carbon atoms; Allyl esters and vinyl esters such as vinyl acetate; Vinyl aromatics such as styrene, methyl styrene and chloro-styrene; Vinyl and allyl ethers and ketones such as vinyl methyl ether and methyl vinyl ketone; Chloroacrylate; Cyanoacrylates such as alpha-cyanomethyl acrylate, and alpha-, beta-, and gamma-cyanopropyl acrylate; Alkoxyacrylates such as methoxy ethyl acrylate; Haloacrylates such as chloroethyl acrylate; Vinyl halides and vinyl chlorides such as vinyl chloride, vinylidene chloride and the like; Divinyl, diacrylate and other multifunctional monomers such as divinyl ether, diethylene glycol diacrylate, ethylene glycol dimethacrylate, methylene-bis-acrylamide, allylpentaerythritol, and the like; And bis (beta-haloalkyl) alkenyl phosphonates such as bis (beta-chloroethyl) vinyl phosphonate and the like are well known to those skilled in the art. Copolymers in which the carboxy-containing monomer is a minor member and other vinylidene monomers are present as the major component are readily prepared according to the methods described herein.
[161] In another related aspect, a pro- or anti-inflammatory binding peptide covalently coupled with a triglyceride backbone moiety via a polyalkylene glycol spacer group bonded to a carbon atom of the triglyceride backbone moiety, and a carbon atom of the triglyceride backbone moiety Multiligand conjugated peptide complexes comprising one or more fatty acid moieties covalently attached directly or covalently linked via a polyalkylene glycol spacer moiety are provided (eg US Pat. No. 5,681,811. Referenced). In such multiligand conjugated therapeutic agent complexes, the alpha 'and beta carbon atoms of the triglyceride bioactive moiety may have fatty acid moieties attached by covalent or indirect covalent attachment through the polyalkylene glycol spacer moiety. . On the other hand, the fatty acid moiety may be covalently attached to the alpha and alpha carbons of the triglyceride backbone moiety through or directly through the polyalkylene glycol spacer moiety, wherein the bioactive agent is attached to the gamma-carbon of the triglyceride backbone moiety. Directly covalently or indirectly via a polyalkylene glycol spacer moiety. It will be appreciated that a wide variety of structural, collateral and sequence forms are possible as multiligand conjugated therapeutic complexes comprising triglyceride backbone moieties within the scope of the present invention. In such multiligand conjugated therapeutic agent complexes, the biologically active agent (s) can be covalently coupled with the triglyceride modified backbone moiety through an alkyl spacer group or through other acceptable spacer groups within the scope of the present invention. Will also be understood. In such a burial, the acceptability of spacer groups refers to the three-dimensional, compositional and end-use applications of a particular acceptable characteristic.
[162] In another aspect of the invention, (i) a fatty acid group; And (ii) functional groups covalently coupled to their alpha, alpha 'and beta carbon atoms, including polyethylene glycol groups having proinflammatory or anti-inflammatory binding peptides covalently bound, such as those bound to appropriate functional groups of polyethylene glycol groups Conjugation-stabilization complexes are provided that include a polysorbate complex that includes a polysorbate moiety that includes a triglyceride backbone having a structure (see, eg, US Pat. No. 5,681,811, referred to herein). Such covalent bonds may be directly to the hydroxy end functional groups of the polyethylene glycol group or on the other hand, the covalent bonds may be indirect by, for example, reactively mapping the hydroxy end of the polyethylene glycol group by a terminal carboxy functional spacer group. As a result, the capped polyethylene glycol group has a terminal carboxyl functional group to which a proinflammatory or anti-inflammatory binding peptide can be covalently bound.
[163] Liposomes and micelle delivery vehicles
[164] Formulations in combination with the harmonized methods of administration of the present invention may optionally include a carrier, processing agent, or delivery vehicle based on a lipid or fatty acid that is effective for providing an improved formulation for delivery of pro- or anti-inflammatory binding peptides. For example, by mixing or encapsulating one or more pro-inflammatory or anti-inflammatory dainpop peptides together or in concert with a mixed micelle carrier liposome, or emulsion, to enhance the chemical and physical stability of the biologically active agent and reach its half-life to the mucosa. Various combinations and methods are provided for mucosal delivery, such as by increasing until (eg, by decreasing sensitivity to proteolysis, chemical modification and / or denaturation).
[165] In certain aspects of the invention, particular delivery systems for pro- or anti-inflammatory binding peptides include small lipid pockets known as liposomes (eg, Chonn et al . , Curr. Opin. BIotechnol. 6: 698-708, 1995; Lasic , Trends Biotechnol. 16: 307-321, 198; and Gregoriadis, Trends Biotechnol. 13: 527-537, 1995, respectively referenced). They are generally made from natural, biodegradable, nontoxic and non-immunogenic lipid molecules and effectively capture or bind drug molecules, including peptides and proteins, onto their membranes. The advantages of liposomes as peptide and protein delivery systems in the present invention are further highlighted by the fact that the encapsulated proteins remain in the preferred aqueous environment in their pockets while the liposome membranes protect them from proteolysis and other destabilizing factors. Although all known liposome preparations may not be suitable for encapsulating them due to the unique physical and chemical properties of proteins and peptides, some methods encapsulate these macromolecules without substantially inactivating them (eg, Weiner, See Immunomethods 4: 201-209, 1994. See text).
[166] Methods of preparing liposomes for use in the present invention are varied (eg, Ann. Rev. Biophys. Bioeng. 9: 467, 1980; Szoka et al., And methods described in US Pat . Nos. 4,235,871, 4,505,728 and 4,837,028, respectively.) Referenced). To be used for liposome delivery, the biologically active agent is generally confined within the liposome or lipid bag or bound to the outside of the bag. Several strategies have been devised to increase the effectiveness of liposome-mediated delivery by targeting liposomes to specific tissues and specific cell types. Liposomal combinations, including those containing cationic lipids, have been shown to be safe and well tolerated in human patients (Treat et al . , J. Natl. Cancer Instit. 82: 1706-1710, 1990, referenced herein).
[167] Like liposomes, unsaturated long-chain fatty acids, which enhance mucosal absorption activity, can also form closed pockets with structures similar to bilayers (so-called “ufasomes”). These include, for example, biologically active peptides and oleic acids. Proteins can be used in the present invention to be confined for mucosal delivery, such as for example in the nose.
[168] Another delivery system for use in the present invention is to use them together to take advantage of both the polymer and liposome vehicles at once. As an example of this type of hybrid delivery system, liposomes containing model protein horseradish peroxidase (HRP) have been effectively encapsulated inside the natural polymer fibrin (Henschen et al., Blood Coagulation , pp. 171-241, Zwaal et al., Elsevier, Amsterdam, 1986, referenced in the text). Because of its biocompatibility and biodegradability, fibrin is a useful polymer matrix for drug delivery systems in this respect (eg, Senderoff et al . , J. Parenter. Sci. Technol. 45: 2-6, 1991; and Jackson, Nat. Med 2: 637-638, 1996, referenced in the text). In addition, the release of biotherapeutic compounds from this delivery system can be controlled by the use of covalent crosslinks and by adding antifibrin degradants to the fibrin polymers (Uchino et al., Fibrinolysis 5: 93-98, 1991, referenced herein). ).
[169] A more convenient delivery system for use in the present invention is the use of cationic lipids as the delivery vehicle or carrier, which provides an electrostatic interaction between the lipid carrier and the charged biologically active agent as the protein and the polyanionic nucleic acid. Can be used effectively (eg, Hope et al., Molecular Membrane Biology 15: 1-14, 1998, referenced herein). This allows the effective packaging of the drug in a form suitable for mucosal administration and / or subsequent delivery to the systemic compartment. Such systems and related systems are particularly suitable for delivering polymeric nucleic acids, such as in the form of gene constructs, antisense oligonucleotides and ribozymes. These drugs are large, usually negatively charged molecules with molecular weights of about 106 for genes and about 103 oligonucleotides. Although the targets of these drugs are intracellular, because of their physical properties, they do not cross cell membranes by passive diffusion like conventional drugs. In addition, unprotected DNA is degraded in minutes by nucleases present in the normal cytoplasm. To prevent inactivation by endogenous nucleases, antisensor oligonucleotides and ribozymes can be chemically modified to be resistant to enzymes by a variety of known methods, but plasmid DNA can be modified to viral or non-viral envelopes. It must be protected by encapsulation or condensation in the form of particles tightly packed by polycations such as proteins or cationic lipid bags. Recently, small unilamellar vesicles (SUVs) made of cationic lipids and dioleoylphosphatidylethanolamine (DOPE) as polynucleic acid pouches, such as plasmid DNA, are activated through a plasma membrane into the cytoplasm of a wide range of cells. It has been successful to form particles capable of delivering polynucleotides. This process (called lipofection or cytofection) is widely used as a means of delivering plasmid constructs intracellularly to study the effects of transient gene expression. Delivery vehicles of this type for use in the present invention include cationic lipids (eg, N- (2,3- (dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA)), quaternary Ammonium salts (e.g., N, N-dioleyl-N, N-dimethylammonium chloride (DODAC)), cationic derivatives of cholesterol (e.g., 3 (N- (N'N-dimethylaminoethane-carbamoyl-cholesterol) (DC-chol)) and lipids characterized by multivalent headgroups (eg, dioctadecyldimethylammonium chloride (DOGS), Transfectam Commercially available).
[170] Additional delivery vehicles for use in the present invention include long- and medium-chain fatty acids, micelles mixed with fatty acids and surfactants (eg, Muranshi, Crit. Rev. Ther. Drug Carrier Syst. 7: 1-33, 1990 , Referenced in the text). Most naturally occurring lipids in ester form have important means with respect to shifting the mucosal surface itself. Free fatty acids and their monoglycerides with polar groups attached have been demonstrated to act as penetration enhancers against the intestinal barrier in the form of mixed micelles. The discovery of this barrier modification function of free fatty acids (chain lengths of 12 to 20 carbon atoms) and their polar derivatives has actively stimulated the study of applying these agents as mucosal absorption enhancers.
[171] For use in the methods of the invention, long-chain fatty acids, in particular fusogenic lipids (monoglycerides and unsaturated fatty acids such as oleic acid, linoleic acid, linoleic acid, monoolein, etc.), are proinflammatory or anti-inflammatory binding peptides, as described herein. Provided are useful carriers that enhance the delivery of analogs and mimetics thereof. Medium chain fatty acids (C6 to C12) and monoglycerides have also been shown to aid drug absorption in the small intestine and to be suitable for use in the delivery formulations and methods of the present invention. In addition, the sodium salts of heavy and long chain fatty acids are effective delivery vehicles and absorption promoters for the delivery of pro- or anti-inflammatory binding peptides in the present invention. Thus, non-toxic surfactants can be added, such as polyoxyethylated hydrogenated castor oil, sodium taurocholate, or the fatty acids can be used in the soluble form of sodium salts. Mixed micelles of naturally occurring unsaturated long chain fatty acids (oleic acid or linoleic acid) and their monoglycerides with bile salts have been demonstrated to exhibit essentially harmless absorption promoting capacity in the small intestine mucosa (eg, Muranishi, Pharm. Res. 2: 108-118, 1985; and Crit. Rev. Ther.drug carrier carrier Syst. 7: 1-33, 1990, respectively). Other fatty acids and mixed micelle preparations useful in the present invention include, but are not limited to, Na caprylate (C8), Na caprate (C10), Na laurate (C12) or Na oleate (C18). And, optionally, bile salts such as glycolate and taurocholate.
[172] PEGylation
[173] Additional methods and compositions provided herein include chemical modifications of pro-inflammatory or anti-inflammatory binding peptides, eg by covalent attachment of polymeric materials such as dextran, polyvinyl pyrrolidone, glycopeptides, polyethylene glycols and polyamino acids. Related to As a result, the conjugated peptides retain biological activity and solubility for clinical administration. In another embodiment, pro- or anti-inflammatory binding peptides are conjugated to polyalkylene oxide polymers, in particular polyethylene glycol (PEG) (see, eg, US Pat. No. 4,179,337, referenced herein). Numerous reports in the literature discuss the potential advantages of PEGylated peptides and proteins that exhibit increased resistance to proteolysis, extended plasma half-life, increased solubility and reduced antigenicity and immunogenicity (Nucci et al., Advanced Drug Deliver Reviews 6). : 133-155, 1991; Lu et al. , Int. J. Peptide Protein Res. 43: 127-138, 1994, respectively referenced). L-asparaginase, streptokinase, insulin, interleukin-2, adenosine deamidase, L-asparaginase, interferon alpha 2b, superoxide dismutase, streptokinase, tissue plasminogen activator (tPA), euro Many proteins, including kinases, freecases, hemoglobin, TGF-beta, EGF, and other growth factors, are bound to PEG and evaluated for their altered biochemical properties as drugs (eg, Ho et al., Drug Metabolism and Disposition 14: 349- 352, 1986; Abuchowski et al. , Prep. Biochem. 9: 205-211, 1979; and Rajagopaian et al. , J. Clin. Invest. 75: 413-419, 1985, Nucci et al . , Adv. Drug Delivery Rev. 4; 133- 151, 1991, respectively, referenced in the text). Although the in vitro biological activity of PEGylated proteins may be reduced, this loss of activity is usually offset by increased in vivo half-life in the bloodstream (Nucci et al., Advanced Drug Deliver Reviews 6: 133-155, 1991, text). ). Thus, these and other polymer-coupled peptides and proteins exhibit improved properties such as extended half-life and reduced immunogenicity when administered mucosally according to the methods and compositions of the present invention.
[174] Several methods have been reported for attaching PEG to proteins and peptides and then purifying them (Abuchowski et al. , J. Biol. Chem. 252: 3582-3586, 1977; Beauchamp et al. , Anal. Biochem. 131: 25-33 , 1983, respectively). In addition, Lu et al. Int. J. Peptide Protein Res. 43; 127-138, 1994 (referenced herein) compared the PEGylation process of protein to peptide, explaining various technical considerations (Katre et al . , Proc. Natl. Acad. Sci. USA 84 : 1487-1491, 1987; Becker et al. , Makromol. Chem. Rapid Commun. 3: 217-223,1982; Mutter et al. , Makromol. Chem. Rapid Commun. 13: 151-157, 1992; Merrifield, RB, J. Am. Chem. Soc. 85: 2149-2154, 1993; Lu et al . , Peptide Res. 6: 142-146,1993; Lee et al. , Bioconjugate Chem. 10 : 973981,1999, Nucci et al. , Adv. Drug Deliv. Rev. 6: 133-151, 1991; Francis et al., J. Drug Targeting 3: 321-340, 1996; Zalipsky, S., Bioconjugate Chem. 6: 150-165, 1995; Clark et al. , J. Biol. Chem. 271: 21969-21977,1996; Pettit et al. , J. Biol. Chem. 272: 2312-2318, 1997; Delgado et al . , Br. J. Cancer 73: 175-182,1996; Benhar et al. , Bioconjugate Chem. 5: 321-326,1994; Benhar et al. , J. Biol. Chem. 269: 13398-13404,1994; Wang et al. , Cancer Res. 53: 4588-4594,1993; Kinstler et al . , Pharm. Res. 13: 996-1002, 1996, Filpula et al . , Exp. Opin. Ther.Patents 9: 231-245, 1999; Pelegrin et al . , Hum. Gene Ther. 9: 2165-2175,1998, respectively, referenced in the text).
[175] According to these and other guidelines in the art, proinflammatory or anti-inflammatory binding peptides, with the expected results of prolonging the cycle life of PEGylated actives and / or reducing immunogenicity while maintaining acceptable levels of activity. Can be easily conjugated to the polyethyleneglycol polymer. Amine-reactive PEG polymers for use in the present invention include SC-PEG having molecular weights of 2000, 5000, 10000, and 20,000; U-PEG-10000; NHS-PEG-3400-Biotin; T-PEG-5000; T-PEG-12000 and TPC-PEG-5000 are mentioned. Chemical conjugation chemistry of these polymers is already published (eg, Zalipsky, S., Bioconjugate Chem. 6: 150-165, 1995; Greenwald et al. , Bioconjugate Chem. 7: 638-641, 1996; Martinez et al . , Macromol. Chem. Phys. 198: 2489-2498, 1997; Hermanson, GT, Bioconjugate Techniques , pp. 605-618,1996; Whitlow et al . , Protein Eng. 6: 989-995,1993; Habeeb, AFSA, Anal.Biochem . 14 : 328-336, 1966; Zalipsky et al., Poly (ethyleneglycol) Chemistry and Biological Applications , pp. 318-341, 1997; Harlow et al., Antibodies: a Laboratory Manual , pp. 553-612, Cold Spring Harbor Laboratory, Plainview, NY , 1988; Milenic et al . , Cancer Res. 51: 6363-6371, 1991; Friguet et al. , J. Immunol.Methods 77: 305-319,1985, each of which is incorporated herein by reference). PEGylation of biologically active peptides and proteins can be accomplished by modifying carboxyl moieties (eg, aspartic acid or glutamic acid groups in addition to the carboxyl terminus). The use of PEG-hybridase in the selective modification of carbodiimide-activated protein carboxyl groups under acidic conditions has been described (Zalipsky, S., Bioconjugate Chem. 6: 150-165,1995; Zalipsky et al. , Poly (ethyleneglycol) (See Chemistry and Biological Applications , pp. 318-341, American Chemical Society, Washington, DC, 1997, text). On the other hand, bifunctional PEG modifications of biologically active peptides and proteins can be used. In some processes, charged amino acid residues, including lysine, aspartic acid, and glutamic acid, have a prominent tendency to be solvent accissible at the protein surface. Conjugating a protein to a carboxylic acid group is a less tried approach in making protein bioconjugates. However, Zalipsky and his colleagues (Zalipsky, S., Bioconjugate Chem. 6: 150-165,1995; Zalipsky et al. , Poly (ethyleneglycol) Chemistry and Biological Applications , pp. 318-341, American Chemical Society, Washington, DC, 1997 Hybridase / EDC chemistry, described by the present disclosure, provides a realistic way to link PEG polymers to protein carboxyl sites. For example, such modified conjugation chemistry has been shown to be effective in amine binding for PEGylation of brain-derived neurotrophic factor (BDNF), while maintaining biological activity (Wu et al . , Proc. Natl.Acad.Sci . USA 96: 254-259, 1999, incorporated herein by reference). Maeda and his colleagues also found that carboxy-targeted PEGylation is a preferred approach for bilirubin oxidase conjugation (Maeda et al., Poly (ethylene glycol) Chemistry.Biotechnical and Biomedical Applications, JM Harris, ed., Pp. 153- 169, Plenum Press, New York, 1992, referenced herein).
[176] Often, PEGylation of peptides for use in the present invention involves activating PEG with a functional group that will react with lysine residues on the peptide or protein surface. In some alternatives of the present invention, biologically active peptides and proteins are modified without substantial loss of activity by PEGylation of other residues such as His, Trp, Cys, Aps, Glu and the like. When PEG modification of a selected peptide or protein is completed, the activity of the peptide or protein is often reduced. Thus, the PEG modification process herein results in a loss of activity of PEGylated active agents to less than about 50%, more often less than about 25%, while substantially increasing half-life (such as serum half-life) and effective administration of PEGylated actives. The amount is generally limited to partial PEGylation of the peptide or protein to a substantial extent.
[177] Other Stabilization Modifications of Activators
[178] Proinflammatory or anti-conjugation via conjugation to other known protective or stabilizing compounds, such as to make fusion proteins with active peptides, proteins, analogs or mimetics linked to one or more carrier proteins, such as one or more immunoglobulin chains. By protecting inflammatory binding peptides, in addition to PEGylation, proinflammatory or anti-inflammatory binding peptides can be modified to increase circulating half-life (see, eg, US Pat. Nos. 5,750,375; 5,843,725; 5,567,584 and 6,018,026, referenced herein). Such modifications result in reduced degradation, sequestration or removal of the peptide and prolong half-life in the physiological environment (eg, in the circulatory system or on the mucosal surface). Active agents modified by these and other stabilizing conjugation methods are therefore useful ones with improved efficacy in the methods of the present invention. In particular, these modified peptides retain activity for longer periods of time at the delivery or action target compared to the unmodified active agent. Even when the active agent is thus modified, it substantially retains its biological activity comparable to that of the unmodified compound.
[179] According to another aspect of the invention, pro- or anti-inflammatory binding peptides are conjugated with relatively low molecular weight compounds such as aminolecithin, fatty acids, vitamin B ₁₂ , and glycosides to increase stability (eg, Igarishi et al. , Proc. Int. Symp. Control.Rel.Bioact.Materials , 17, 366, (1990). Another exemplary modified peptide for use in the compositions and methods of the present invention is advantageously modified in vivo by Will be:
[180] (a) a mammalian signal peptide (such as Lin et al., J. Biol. Chem. 270: 14255, 1995, referenced herein) or bacterial peptide (such as Joliot et al., Proc. Natl. Acad. Sci. USA 88: 1864 , 1991, incorporated herein by reference. It is directed to the active peptide or protein across cytoplasmic and organelle membranes and / or to the desired intracellular compartment (eg, endogenous reticulum (ER) of antigen producing cells (APCs), such as increased CTL induction). To serve as traffic to;
[181] (b) adding a biotin moiety to the active peptide, which serves to direct the active conjugate, particularly its binding capacity, through the cell membrane to the translocator present on the cell surface (ie, binding affinity is about 10 ⁶ , 10 ⁷ , 10 ⁸ or 10 ⁹ or 10 ¹⁰ M ^-1 ) (Chen et al., Analytical Biochem. 227: 168, 1995, referenced herein);
[182] (c) adding either or both of the amino- and carboxy-terminus of the active peptide of the blocking agent to increase stability in vivo. This may be useful in situations where the ends of the active peptide or protein tend to be degraded by proteases prior to cell uptake or during intracellular trafficking. Such blocking agents include, but are not limited to, additional related or unrelated peptide sequences that may be attached to the amino and / or carboxy terminal residues of the therapeutic polypeptide or peptide to be administered. This can be done chemically in peptide synthesis or by recombinant DNA techniques. Blocking agents, such as pyroglutamic acid or other molecules known in the art, may also be attached to amino and / or carboxy terminus residues or the amino group at the amino terminus or the carboxyl group at the carboxy terminus may be replaced by other moieties.
[183] Variants of prodrugs
[184] Another processing and formulation strategy useful within the scope of the present invention is to modify prodrugs. By temporarily inducing groups such as carboxyl, hydroxyl and amino groups in small organic molecules, i.e., bioreversibly, undesirable physicochemical properties of these molecules (e.g., charge, hydrogen bond potential to reduce mucosal penetration) And the like may be "masked" without permanently changing the pharmacological properties of the molecule. Bioreversible prodrug derivatives of therapeutic small molecule drugs have been shown to improve the physicochemical (eg, solubility, affinity) properties of many typical therapeutic agents, particularly those containing hydroxyl and carboxylic acid groups.
[185] The preparation of prodrugs of amine containing actives such as peptides of the present invention can be accessed through the acylation of amino groups. Optionally, acyloxyalkoxycarbamate derivatives of amines are discussed as prodrugs. 3- (2'-hydroxy-4 ', 6'-dimethylphenyl) -3,3-dimethylpropionic acid prepares linear, esterase-sensitive, phosphatase-sensitive and dehydrogenase-sensitive prodrugs of amines (Amsberry et al., Pharm. Res. 8: 455-461, 1991; Wolfe et al., J. Org. Chem. 57: 6138, 1992, each incorporated herein by reference). These systems include the first stage, which is a slow rate-determining enzyme-catalyst (esterase, phosphatase, or dehydrogenase) stage, and the second stage, which is a fast (t _1/2 = 100 sec, pH 7.4, 37) chemical stage. It is known to degrade through a two-step mechanism. (Amsberry et al., J. Org. Chem. 55: 5867-5877, 1990, incorporated herein by reference). Recently, phosphatase-sensitive systems have been used to prepare prodrugs with very high water-soluble (> 10 mg / ml) water in TAXOL, which exhibits distinct anti-tumor activity in vivo . It's interesting. These and other prodrug modification systems and the resulting therapeutics are useful in the scope of the methods and compositions according to the invention.
[186] To prepare prodrugs of peptides useful within the scope of the present invention, US Pat. No. 5,672,584 (incorporated herein by reference) discloses cyclic prodrugs of biologically active peptides and peptide nucleic acids (PNAs). The preparation and use of is further described. To prepare these cyclic prodrugs, the N-terminal amino group and C-terminal carboxyl group of the biologically active peptide or PNA are linked via a linker, or the C-terminal carboxyl group of the peptide is linked to a side chain amino group via a linker. Or a side chain hydroxyl group, or the N-terminal amino group of the peptide is linked to a side chain carboxyl group via a linker, or the side chain carboxyl group of the peptide is linked to a side chain amino group or a side chain hydroxyl group through a linker. Linkers useful herein include 3- (2'-hydroxy-4 ', 6'-dimethyl phenyl) -3,3-dimethylpropionic acid linkers and derivatives thereof, and acyloxyalkoxy derivatives. The disclosures included herein are opioids that exhibit advantageous physicochemical characteristics (eg, reduced size, intramolecular hydrogen bonding, and amphophilic properties) to improve linear peptides such as cell membrane permeability and metabolic stability. Methods useful for the characterization and preparation of cyclic prodrugs synthesized from peptides are provided. Methods of modifying such peptide prodrugs are also useful for preparing modified peptide therapeutic derivatives within the scope of the methods and compositions of the present invention.
[187] Purification and Manufacturing
[188] Peptides of the invention can be prepared in a wide variety of ways. Because these peptides are relatively short, they can be synthesized according to conventional techniques in solution or on a solid support. Various types of automatic synthesizers are commercially available and can be used according to known protocols. See, eg, Stewart and Young, Solid Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341-347 (1986); And Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. See 1-284 (1979).
[189] Alternatively, a nucleotide sequence encoding a proinflammatory or anti-inflammatory binding peptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and then cultured under conditions suitable for expression. DNA technology can be used. These procedures are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, cold Spring Harbor Press, Cold Spring Harbor, New York (1982), and Ausubel et al. (Ed.) Current Protocols in Molecular Biology, which is incorporated herein by reference. , John Wiley and Sons, Inc., New York (1987), and US Pat. Nos. 4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428, 941, generally known in the art. Thus, fusion proteins comprising one or more peptides of the invention can be used to provide proinflammatory or anti-inflammatory binding peptides.
[190] Coding sequences of peptides with the lengths envisioned herein are described in chemical techniques such as Matteucci et al., J. Am. Chem. Soc. Since it can be synthesized according to the phosphoester ester method of 103: 3185 (1981), it can be modified by simple substitution of appropriate base (s) in bases encoding natural peptide sequences. The appropriate linker is then provided to the coding sequence to ligate to an expression vector generally available in the art, and the vector is used to transform the appropriate host to produce the desired fusion protein. Numerous vectors and suitable host systems are currently available. To express the fusion protein, coding sequences with operably linked start codons and end codons, promoter regions and terminator regions, and usually replication systems, will provide an expression vector for expression in the desired cellular host. For example, promoter sequences suitable for bacterial hosts are provided in plasmids with restriction sites suitable for insertion of the desired coding sequence. The resulting expression vector is transformed into a suitable bacterial host. Of course, yeast or mammalian cell hosts may be used, using appropriate vectors and control sequences.
[191] Peptides according to the invention and pharmaceutical products and vaccine compositions thereof are useful for the treatment and / or prevention of various diseases and symptoms by administration to mammals, especially humans. It is common to administer the pro-inflammatory or anti-inflammatory binding peptides directly to the subject in substantially purified form. As used herein, the term "substantially purified" refers to a peptide, protein, nucleic acid or other compound in which the peptide, protein, nucleic acid or other active compound is separated in whole or in part from naturally bound proteins and other contaminants. It means purified to a measurable degree in proportion to the naturally-occurring state, for example in proportion to the purity in the cell extract.
[192] In some embodiments, the term "substantially purified" is isolated from cells, cell culture media, or other crude preparations and then fractionated to yield various components of the initial preparation, such as proteins, cellular debris. (debris), and peptide composition with other components removed. The purified preparation may be a substance covalently bound to an active agent, such as a glycoside moiety, or a substance mixed or conjugated with an active agent which is desired to obtain modified derivatives or analogs of the active agent or to prepare combination therapeutic formulations, conjugates, fusion proteins, and the like. Of course, it may include. Thus, the term "purified" refers to a peptide wherein an additional compound or moiety such as polyethylene glycol, biotin or other moiety is bound to the active agent for the provision of a combination useful for the attachment and / or treatment or diagnosis of other compounds. And preferred products such as protein analogs or mimetics or other biologically active compounds.
[193] When applied to a polynucleotide, the term "substantially purified" does not contain materials normally accompanied by that polynucleotide, but the noncoding portion of the message, for example when DNA is derived from the cDNA library, is reverse transcribed. It means that the additional sequence may be included at the 5 'and / or 3' end of the coding sequence, which may occur or may include reverse transcript for the mature protein coding sequence and the signal sequence.
[194] When applied to peptides, proteins and peptide analogs (including other peptides and / or peptide fusions with proteins) according to the invention, the term "substantially purified" means that the peptide, protein or analog is normally non-purified, such as By means of a composition in which the other cellular component in its natural state or environment is partially or not present at all. Purified peptides and proteins are generally homogenous or nearly homogeneous, whether in the dry state or in aqueous solution. Purity and homogeneity are generally measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
[195] In general, substantially purified peptides, proteins and other active compounds for use within the scope of the present invention include pharmaceutical carriers, excipients, buffers, absorption enhancers, stabilizers, preservatives in a complete pharmaceutical combination for therapeutic administration. 80% of all macromolecular species present in preparations prior to admixtures or formulations of adjuvants or other co-ingredients with peptides, proteins or other active agents It includes the above. More generally, peptides or other active agents are purified to represent at least 90% and often at least 95% of all macromolecular species present in the purified preparations prior to admixture with other combination ingredients. In other cases the purified preparation of the active agent is essentially homogeneous, in which case other macromolecular species cannot be detected by conventional techniques.
[196] Various techniques suitable for use in peptide and protein purification are well known to those skilled in the art. Such techniques include, for example, centrifugation after precipitation with heat denaturation or precipitation with ammonium sulfate, PEG, antibodies, and the like; Chromatography steps such as ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxylapatite chromatography and / or affinity chromatography; Isoelectric focusing; Gel electrophoresis; And merging the above and other techniques. Particularly useful purification methods include selective precipitation with materials such as ammonium sulfate; Column chromatography; Affinity methods such as immunopurification; And others (see, eg, R. Scopes, Protein Purification: Principles and Practice , Springer-Verlag: New York, 1982, incorporated herein by reference). Generally, biologically active peptides and proteins can be extracted from cell cultures or tissues expressing the peptides, and then the peptides and proteins can be further purified by standard protein chemistry / chromatography followed by immunoprecipitation.
[197] Peptides and proteins used in the methods and compositions according to the invention can be obtained in various ways. Many peptides and proteins are readily available in purified form from commercial sources. Smaller peptides (less than 100 amino acids in length) can be readily synthesized by standard chemistry familiar to those skilled in the art (see, eg, Creighton, Proteins: Structures and Molecular Principles , WH Freeman and Co., NY, 1983). ). Larger peptides (greater than 100 amino acids) can be prepared by a variety of methods including recombinant DNA techniques (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, NY, 1989, each incorporated herein by reference). And techniques described in Ausubel et al., Eds., Current Protocols in Molecular Biology , Green Publishing Associates, Inc., and John Wiley & Sons, Inc., NY, 1989). Alternatively, RNA encoding the protein can be chemically synthesized. See, eg, the technology described in Oligonucleotide Synthesis , Gait, MJ, ed., IRLPress, Oxford, 1984 (incorporated herein by reference).
[198] In some embodiments of the present invention, biologically active peptides or proteins will be constructed using peptide synthesis techniques such as solid peptide synthesis (Merrifleld synthesis), or the like, or recombinant DNA techniques well known in the art. Peptide and protein analogs and mimetics can also be prepared according to the above methods. Techniques for making substitution mutations in predetermined sites of DNA include, for example, M13 mutagenesis. Manipulation of DNA sequences to make substitutions, insertions, or deletion variants is described in other documents, such as, for example, Sambrook et al. ( Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). . In accordance with these and related teachings, defined mutations can be introduced into biologically active peptides or proteins to site-specific cDNA copies of a variety of conventional techniques, such as portions of genes encoding selected peptide fragments, domains or motifs. Site-directed mutagenesis can produce the desired analogs and mimetics. This is MUTA-gen from Bio-Rad Laboratories (Richmond, Calif.) Chameleon from Strategene (La Jolla, CA) as a template or through a single stranded intermediate, such as the use of a kit This can be accomplished through direct use of a double stranded plasmid such as a mutagenesis kit, or by polymerase chain reaction using oligonucleotide primers or a template comprising the desired mutant (s). The mutated subfragments can then be assembled into cDNAs encoding complete peptide analogs. Many other mutagenesis techniques are known and can be routinely used for making mutations in the desired biologically active peptides and proteins for use within the scope of the present invention.
[199] Formulations and Administration
[200] Pro-inflammatory or anti-inflammatory binding peptides are typically combined with one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients. The carrier (s) should be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not exhibiting a deleterious effect that is unacceptable to the patient. Such carriers are already described above, otherwise they are known to those skilled in the art of pharmacology. Preferably, the combination should not contain substances such as oxidants or enzymes known to be incompatible with the biologically active agent administered. The combination may be prepared by any of the methods known in the pharmaceutical art.
[201] For disease prevention and treatment purposes, the proinflammatory or anti-inflammatory binding peptides disclosed herein may be administered to a patient via a suitable route of administration, including intravenous, subcutaneous, intratumoral, intrapulmonary, perfusion, and the like. May be administered. For therapeutic purposes, the peptides of the present invention may also be expressed by an attenuated viral vector or other gene therapy delivery constructs. Vectors such as vaccinia or fowlpox are typical examples of these well known means. Such an approach includes, for example, using vaccinia virus as a vector expressing a nucleotide sequence encoding a peptide (or conjugate) of the invention. When introduced into a target region (eg, an intratumoral region, an inflammatory region, or a viral infection region), the recombinant vaccinia virus modulates the proinflammatory or antiinflammatory immune response by expressing proinflammatory or antiinflammatory binding peptides. . Methods useful for vaccinia vectors and immune protocols are described in US Pat. No. 4,722,848 (incorporated herein by reference). Another useful vector is BCG (bacille Calmetter Guerin). BCG vectors are described in Nature 351 : 456-460, 1991 to Stover et al. (Incorporated herein by reference). Various other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as, for example, Salmonella typhi vectors, are described from the description herein to those skilled in the art to which this invention pertains. Will be obvious.
[202] Proinflammatory or anti-inflammatory binding peptides can be administered over long periods of continuous delivery (eg, continuous transdermal, mucosal or intravenous delivery), or by repeated dosing protocols (eg, over time, days or weeks). Repeated administration protocols), in the form of one large bolus delivery. In this regard, therapeutically effective dosages of pro- or anti-inflammatory binding peptides may include repeated administrations within the long-term prophylactic or therapeutic regime, which are associated with the target disease or conditions as described above. Will have clinically important consequences of alleviating one or more symptoms or conditions that can be detected. In this regard, the determination of the effective dose is usually based on animal model studies and subsequent clinical trials in humans to determine an effective dosage or administration protocol that significantly reduces the incidence or severity of the patient's target disease symptom or condition. Is guided by one. Suitable models in this regard include, for example, murine, rats, pigs, cats, primates other than humans, and other acceptable animal model patients known in the art. . Alternatively, the effective dose can be determined using an in vitro model (eg, immunological and histopathological analysis). When using this model, only normal calculations and adjustments are usually required to determine the concentrations and dosages suitable for administering a therapeutically effective amount of proinflammatory or anti-inflammatory binding peptide (s). In an alternative embodiment, an "effective amount" or "effective dose" of a biologically active agent may simply inhibit or enhance one or more selected biological activities associated with a disease or condition as described above, for therapeutic or diagnostic purposes. .
[203] Proinflammatory or anti-inflammatory binding peptides can be used to describe the specific pharmacology of a biologically active agent to exhibit the required active or biological response, as well as the patient's disease indications and specific conditions (e.g., the patient's age, height, health ( fitness), the severity of symptoms, susceptibility factors, etc.), time and route of administration, and other drugs or treatments administered together. Dosage regimens may be adjusted to provide the optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which a therapeutically useful effect outweighs any of the toxic or deleterious side effects of pro- or anti-inflammatory binding peptides in a clinical sense.
[204] A therapeutically effective amount of a non-limiting range of biologically active agent in the methods and combinations of the invention is 0.01 μg / kg-10 mg / kg, more typically between 0.05 and 5 mg / kg, in certain embodiments about Between 0.2 and 2 mg / kg. Dosages within this range can be achieved by single or multiple doses, including daily or weekly doses. For administration, an amount of at least 1 microgram of pro-inflammatory or anti-inflammatory binding peptide, more typically between 10 μg and 5 mg, in certain embodiments between about 100 μg and 1.0 or 2.0 mg per average human patient It is preferable to administer. For each particular patient, the specific dosage regimen is assessed over time based on individual judgment and professional judgment by the person managing or supervising the administration of the permeabilizing peptide and other biologically active agent (s). It is further noted that it should be adjusted.
[205] The dose of pro- or anti-inflammatory binding peptides can be varied to maintain the desired concentration at the target site by the clinician participating in the procedure. For example, selected local concentrations of a biologically active agent in the CNS or blood stream may vary from about 1-50 nanomoles per liter, often 1.0 nanomoles per liter and 10, 15 or 25 per liter, depending on the condition of the patient and the projected or measured response. It is between nanomoles. The concentration may be selected to be higher or lower depending on the mode of delivery, such as mucosal versus intravenous or subcutaneous delivery. The dosage should be adjusted according to the release rate of the administered formulation, such as for example nasal sprays versus powders, sustained release oral formulations versus injected granules or transdermal delivery formulations. To achieve the same serum concentration levels, for example, sustained-release particles with a release rate of 5 nanomoles (under standard conditions) will need to be administered about twice as much as those with particles having a release rate of 10 nanomoles.
[206] Additional guides for specific dosages of selected biologically active agents for use in the present invention will be found extensively in the literature.
[207] The invention is further illustrated by the following specific examples without intention of limiting the scope of the invention.
[208] The following examples document the discovery that HLA-E molecules can also bind to human heat-shock protein 60 (hsp60), a protein derived from exemplary shock-induced proteins. In accordance with the foregoing description, the hsp60 peptide, which is a HLA-E binding candidate, was identified in the signal sequence of the hsp60 protein (corresponding to the target sequence of the mitochondria). During cellular stress, these peptides access the nascent HLA-E molecules, causing upregulation of HLA-E on the cell surface. Notably, HLA-E molecules that bind these hsp60 peptides are no longer recognized by inhibitory CD94 / NKG2A receptor pairs.
[209] Thus, during normal cell growth, HLA-E binds to signal peptides derived from MHC class I signal sequences and these cells are protected from NK-cell mediated attacks. During cellular stress, HLA-E molecules will be able to bind mainly to peptides derived from other endogenous proteins, such as hsp60 signal peptides.
[210] When HLA-E was transfected into K562 cells (red leukemia cell line K562 lacks HLA class I cell surface expression) with a full length hsp60 leader sequence, a slight increase in cell surface HLA-E expression was observed. . In contrast, large scale upregulation was observed when these cells were placed under cell culture stress, such as high density cell growth conditions. Only a small amount of HLA-E upregulation was observed in K562 cells transfected by the same HLA-E construct (and also by point mutations in the hsp60 leader sequence), indicating that the important peptide sequence found in the hsp60 leader This suggests that HLA-E can be accessed. This observation indicates a novel peptide-presenting effect of HLA-E on cellular distress.
[211] CD94 / NKG2A uncoupling on NK cells by HLA-E mediated presentation of stress-induced peptides allows NK cells (and CD94 / NKG2A expressing T cells) to undergo autologous cells that are stressed, for example, in sustained chronic inflammation. It indicates that it can be detected and removed.
[212] These findings presented herein demonstrate that an exemplary HLA-E that binds to a pedeide from a stress-induced protein can no longer be engaged with CD94 / NKG2A inhibitory receptors, which is peptide-loaded. In addition to NK cellular cytotoxicity against HLA-E transfected cells, tetramerine upon binding to CD94 / NKG2A expressed by cellular transfectants or endogenously and functionally expressed on NK cells It is also measured by using HLA-E / beta2 microglobulin / hsp60 peptide-complex. These results form a complex in which the representative hsp60 peptide and other related peptides presented by HLA-E uncouple the CD94 / NKG2A inhibitory receptor, which would result in cellular activation of cells producing the CD94 / NKG2A receptor. It suggests that you can.
[213] Cell culture
[214] K562 (human HLA-class I negative red leukemia), and 721.221 (human HLA-class I low B-lymphoid cells) were treated with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin supplemented was maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD). Two human CD94 / NKG2A ⁺ (but KIR ⁻ ) cytotoxic NK cell lines (NKL, provided by Dr. M. Robertson, Indiana University School of Medicine, Indianapolis, IN) and Nishi (Dr. H. Wakiguchi, Dept of Pediatrics, Provided by Kochi Medical School, Japan), 7% pooled heat-inactivated human AB + serum, 200 U IL-2 / ml (Pepro Tech Inc., Rocky Hill NJ), 2 mM L-glutamine, 100 U / ml penicillin , And 100 □ g / ml streptomycin (Life Tecnologies) incubated in IMDM supplemented. Ba / F3 cells co-transfected with CD94 and NKG2A, CD94, DAP-12 and NKG2C-GFP or CD94 and DAp-12 have been described previously (Lanier et al., Immunity 6: 371-378, 1997, Referenced in the text). HB-120 (pan-HLA class I specific hybridoma) from American Type Culture Collection, Rockville, MD, 10% FCS, 2 mM L-glutamine, sodium pyruvate, HAT, 100 U / ml penicillin, 100 g / ml Streptomycin (Life Technologies) incubated in DMEM supplemented.
[215] Peptide, HLA-E Stabilization and Cell Culture Stress Analysis
[216] Synthetic peptides purchased from Research Genetics were dissolved in PBS. Peptides used were B7sp (VMAPRTVLL), hsp60sp (QMRPVSRVL), B7 R5V (VMAPVTVLL), hsp60 V5R (QMRPRSRVL) and P18I10 (RGPGRAFVTI) (all purchased from Research Genetics, Huntsville, AL). Cells and their HLA-E transfected derivatives were incubated with synthetic peptides (3-300 μM) in serum-free AIM-V medium at a temperature of 26 ° C. for 15-20 hours. Cells were harvested and washed in PBS, stained with mAbs and analyzed by flow cytometry. Cells were stressed by culturing the cells to increase cell density.
[217] Briefly, cell culture was performed at different time points for a period of up to 6 days at a cell concentration of 0.2 10 ⁶ cells / ml. At the end point, cell concentration and viability were measured by trypan blue exclusion. Expression of cell-surface HLA class I molecules was analyzed by flow cytometry. Cell cultures with> 90% survival at three densities were selected as targets for cytotoxicity assays.
[218] HLA-E tetramer production
[219] HLA-E tetramer complexes were prepared according to the methods described above (Michaelsson et al., Eur. J. Immunol. 30: 300, 2000; Braud et al., Nature 391: 795-799, 1991, respectively referenced in the text). In summary, HLA-E and □₂Microglobulin (□₂m) was highly expressed in E. coli BL21 pLysS and then purified from inclusion bodies and dissolved in 8M urea solution followed by synthetic peptides (B7sp, hsp60sp, B7 R5V or hsp60 V5R).In vitroRefold by dilution. HLA-E heavy chain, □₂m And the complex of peptides was purified by size exclusion chromatography on a Superose 12 column (Amersham-Pharmacia Biotech), then biotinylated with BirA enzyme (Avidity, Denver CO) according to the manufacturer's instructions, followed by rapid freezing. Store at ° C. Tetramer HLA-E complexes were made by mixing biotinylated monomers with streptavidin-phycoerythrin (Molecular Probes, Leiden, Neterlands) in a 4: 1 molar ratio. Other tetramers of similar quality were identified by gel-shift analysis as well as pan-HLA specific hybridoma (HB-120) staining.
[220] Antibodies and Flow Cytometry
[221] Monoclonal antibodies (Mabs) used: DX22 (anti-CD94, DNAX, Palo Alto, CA), anti-NKG2A (Z199, Dr. Lorenzo Moretta, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy) , CD56 (B159, BD Pharmingen), anti-MHC class I mabs (DX17, DNAX) and W6 / 32 (American Type Culture Collection). 3H5 (anti-MICA) and 3D12 (anti-HLA-E) mAbs, respectively, were determined by Drs. T. Spies and D. Geraghty (Fred Hutchinson Cancer Center, Seattle, USA). Anti-hsp60 (ML30) was determined by Dr. Provided by J. Ivanyi (University of London, England). Anti-MICB (7C5) in vitro was immunized by immunizing mice with pCDNA3 expression vectors comprising the N-terminal CD8 leader peptide followed by stable P815 cells stably transfected with FLAG epitopes and extracellular, transmembrane and cytoplasmic MICB cDNA. Generated in. Hybridoma 7C5 (anti-MICB) was selected and shown to bind to 721.221 and P815 cells transfected with MICB * 002cDNA expression vector, while MICA * 005 as well as untransfected or control transfected cells Transfected cells were negative. FITC- and PE-conjugated goat anti-mouse IgG (both from Dakopatts, Glostrup, Denmark) was used as the second stage reagent. DAK-GO1 was used as negative control mAbs for triple-colors (Dakopatts). Cells were analyzed on FACScan ^™ (Becton Dickinson, San Jose, Calif.). Immunofluorescence staining was performed following standard protocols. Briefly, K562 cells transfected with wild type (wt) or variant full-length hsp60 signal peptide-GFP were stained with nuclear stain Hoechst 33342 at 37 ° C. for 30 minutes and mitochondria at 37 ° C. for 15 minutes. After staining with dye tetramethylrhodamine ethyl ester (MRE), a three step wash was performed. Cells were analyzed using a Nikon Eclipse E400 Universal Microscope connected to a Hamamatsu C4742-98 digital camera. Filters suitable for immunofluorescence analysis of labeled cells were used and images were acquired using Jasc Paint Shop Pro 6.0 and imported into Adobe Photoshop ^™ .
[222] Generation of Expression Vectors and Transfected Cells
[223] The synthetic sense and anti-sense DNA encoding the full length hsp60 signal peptide adjacent to the 5'EcoRI / 3'BamHI site (5'CGGAATTCATGCTTCGGTTACCCACAGTCTTTCGCCAGATGAGACCG GTGTCCAGGGTACTGGCTCCTCATCTCACTCGGGCTTATGGATCCGC3 ') was purchased from Interactiva (Ulm, Germany). Annealing and degradation products were linked to pEGFP-N3 expression vector (Clontech, Palo Alto, USA). Triplets encoding the Met-residue of position 11 of the hsp60 signal peptide were prepared using the following oligonucleotide primers: 5'CAGTCTTTCGCCAGGGGAGACCGGTGTCCAG-3 ', site-directed mutagenesis kit (QuikChange) ^TM , Stratagene, La Jolla, Calif.), Were used to transform into triplets encoding gly-residues and confirmed by sequencing. HLA-E * 0101 and HLA-E * 01033 cDNA encoding plasmid (pCDNA3) were obtained from Drs. M. Ullbrecht and E. Weiss (Institut fuer Anthropologie und Humangenetik, Munich, Germany). 721.221 and K562 cells were transfected by electroporation (Gene pulser, BioRad, Hercules CA) according to standard protocols. HLA-E and GFP were used in a ratio of 10: 1 (HLA-E: GFP) to perform transient co-transfection experiments using chimeric GFP and HLA-E encoding plasmids. Transfected cells were selected in complete medium supplemented with 1 mg / ml G418 (BioRad). Stable transfected cells were isolated by flow cytometry (FACScan) based on their green fluorescence properties.
[224] NK cell-mediated cytotoxicity assay
[225] NK cell-mediated cytotoxicity was measured using a 2-hour standard ⁵¹ Cr radioisotope release assay. In brief, target cells were incubated for 15-20 hours at 26 ° C. with various peptides of varying concentrations in the range 1-300 μM and then labeled with ⁵¹ Cr. Non-protective hsp60sp, B7 R5V and hsp60 V5R analyzed peptides except for some experiments that resulted in higher levels of HLA-E expression throughout the assay compared to targets incubated with protective B7sp. Wash before setup. For mAb blocking experiments, cells were preincubated with mouse serum or irrelevant isotaleaf matched antibodies to block Fc-receptors. Blocking target or effector cells with mAb was performed at 4 ° C. and antibodies were included in the analysis.
[241] Example I
[242] Hsp60sp stabilizes HLA-E cell surface expression
[243] To identify peptides obtained from human hsp60 having the potential to bind HLA-E, HLA-E permissive motif (with methionine at site 2 and then leucine or isoleucine at site 9 at C-terminus) The full-length amino acid sequence of hsp60 is scanned for a peptide. Of the four peptides identified (FIG. 1; Table 3), one was chosen first (QMRPVSRVL, referred to as hsp60sp) based on the position in the hsp60 leader sequence. In addition, hsp60sp not only contains methionine at site 2 and leucine at site 9, but also shares amino acids at sites 4 and 8 in common with the same peptide known to bind HLA-E efficiently (Table 1). In particular, four of the nine amino acids of hsp60sp are shared with the same peptide found in the HLA class I leader sequence (eg, HLA-A * 0201, and A * 3401, Table 1).
[244] By study of peptide and HLA-E interactions, the presence of HLA-E binding peptides was determined by flow cytometry, either in transfected cDNA expressing plasmids or by extraneous addition of synthetic peptides. It is shown to be sufficient to stabilize and up-regulate HLA-E cell surface expression to a level that is acceptable (Braud et al., Nature 391 : 795-799, 1991; Lee et al., Proc. Natl. Acad. Sci. USA 95 : 5199, 1998; Borrego et al., J. Exp. Med. 187 : 813,1998, each of which is incorporated herein by reference). To test whether the hsp60-derived peptides can bind to HLA-E, incubated overnight at 26 ° C. with different synthetic peptides to stabilize stabilized HLA-E cell surface expression. To this end, it lacks 721.221 (HLA-A, -B, -C, and -G, as well as K562 cells transfected with HLA-E * 01033 (K562-E * 01033) or HLA-E * 0101). MHC class I deficient cell lines such as expressing HLA-E and -F intracellularly) were used. H562-transfected K562 with 721.221 cells expresses low levels of HLA-E at the cell surface during normal cell growth but does not express K562 that is not HLA-E transfected. The basal level of their HLA-E expression suggests that there is a small amount of intracellular peptides sufficient to stabilize nascent HLA-E molecules.
[245] As shown in FIG. 2, using hsp60sp, a substantial increase in HLA-E expression in both HLA-E * 01033 and HLA-E * 0101 transfected K562 cells was observed. HLA-E expression levels after loading hsp60sp were compared with the levels of cells loaded with peptides derived from the leader sequence of HLB * 0701 (B7sp, VMAPRTVLL) (FIG. 2). However, at 37 ° C., the HLA-E / hsp60sp complex degraded faster than HLA-E / B7sp, reaching basal levels almost immediately after. Along with hsp60sp, hsp60.4 peptide (GMKFDRGYI) was also able to stabilize HLA-E molecules on 721.221 cells as well as on transfected K562 cells. The peptide has already been shown to bind to mouse Qa-l ^b molecules (Lo et al., Nature Med. 6: 215, 2000, incorporated herein by reference). HLA-E stabilization was not observed when using two other hsp60 derived peptides ((hsp60.2 and hsp60.3; Table I), possibly due to the low solubility of the assay medium.
[246] Example II
[247] Hsp60 signal peptide achieves intracellular access to HLA-E and HLA-E / hsp60sp levels are up-regulated during cellular stress
[248] Hsp60 is a mitochondrial matrix protein and is encoded in genomic DNA (Bukau et al., Cells 923 : 351, 1998; Itoh et al., J. Biol. Chem. 270 : 13429, 1995, each of which is herein incorporated by reference. Included). It is synthesized as a precursor protein with an N-terminal mitochondrial targeting sequence consisting of 26 amino acids (hsp60L, see FIG. 1). Biochemical studies suggest that cleavage of hsp60L requires the import of precursor proteins into the mitochondrial substrate, and that mitochondrial import of hsp60 is not observed in the absence of hsp60L, so this cleavage does not appear to occur in the cytoplasm (Singh et al., Biochem. Biophys. Res. Commun. 1692 : 391, 1990, incorporated herein by reference). The final purpose of hsp60L after cleavage is unknown. During stress, hsp60 is regulated by post-transcriptional processes that affect increased transcription as well as its intracellular concentration and distribution (Belles et al., Infect. Immun. 67 : 4191, 1999; Samali et al., Embo J. 18 : 2040, 1999; Feng et al., Blood 97 : 3505, 2001; Soltys et al., Exp. Cell.Res . 222 : 16, 1996, each of which is incorporated herein by reference).
[249] For localization of hsp60L, in particular, to determine whether hsp60sp can achieve access to HLA-E molecules, one of the wild type hsp60L or a mutant variant in which methionine at position 11 is substituted with glycine A model system based on K562 cells transfected with the containing chimeric construct was developed. The methionine residue of interest corresponds to position 2 in the hsp60sp nonamer and is required for stable binding with HLA-E. Wild type and mutated hsp60L were implanted in frame onto the N-terminus of the green fluorescent protein (GFP) to ensure that green fluorescent cells also transform each hsp60 leader sequence during transfection. do. After transfection, GFP was located inside the mitochondria in both hsp60L-GFP transfected cell lines, demonstrating that substitution of methionine at position 11 does not alter transport to mitochondria. GFP showed no apparent subcellular localization when the GFP gene was transfected alone.
[250] Already, cell surface concentrations of mouse Qa-1 ^b molecules have been reported to be substantially up-regulated during cellular stress (Imani et al., Proc. Natl. Acad. Sci. USA 88 : 10475, 1991, herein incorporated by reference. Included). In view of the homology between Qa-1 ^b and HLA-E in all aspects of sequence, biological function and peptide binding specificity, the nonamer peptide located inside the mitochondrial targeting sequence of hsp60, in particular under cellular stress conditions, The experiment was designed to test whether the approach to HLA-E can be achieved. For this purpose, K562 cells are HLA-E * 01033-encoded plasmid loco-transfected with either wild type hsp60L-GFP construct or one of its variants. The HLA-E cell surface expression of these transfectants was then monitored by stressing the culture with increased cell density proliferation.
[251] Cells transfected with wild-type hsp60L-GFP constructs consistently expressed HLA-E at higher levels than cells co-transfected with variant constructs (FIG. 3A). This difference depends on growth conditions; At day 1, it should be noted that the difference in HLA-E cell surface concentrations between cells expressing wild type and mutated hsp60sp is appropriate, while at day 5 it becomes large. In addition, a certain increase in HLA-E concentration was observed in cells transfected with the mutated hsp60L-GFP constructs upon growth under stress as compared to normal conditions (FIG. 3A, day vs. day 1). This may be due to either the residual capacity to bind the HLA-E of the mutated peptide, or the approach to the HLA-E of the endogenously derived hsp60 peptide. Consistent with the latter possibility, HLA-E concentrations increased as a result of culture-induced stress in K562 cells transfected with the HLA-E gene alone (FIG. 3B, bottom panel), while untransfected K562 cells Is still HLA-E negative (FIG. 3B, top panel). In addition to the regulation of post-transcription peptide-independent HLA-E in stressed cells, the possibility remains to be affected by other HLA-E binding peptides. In addition, the co-transfected cell line and K562-E * 01033 cell line shown in FIG. 4A were independently generated and selected, which could explain the high HLA-E background concentration observed on day 1. Thus, the absolute concentration of HLA-E is not necessarily directly comparable between FIGS. 3A and 3B.
[252] The transfectant studies above show that the stress response results in increased intracellular access of mitochondrial hsp60sp to HLA-E and ultimately results in up-regulation of HLA-E / hsp60sp cell surface concentrations. This is at least in part due to post-transcriptional regulation of hsp60sp during the stress response, since the GFP expression levels did not change with increasing cell density and the hsp60L-GFP and HLA-E constructs used were under the control of the same CMV promoter. Seems to be (FIG. 3A). Eventually, although K562 constitutively expresses the active NKG2D ligands MIC-A and MIC-B, additional cell surface up-regulation of these stress-induced MHC class I-like molecules during this assay Not observed. However, upregulation of other active stress-inducing ligands such as, for example, UL16 binding protein (ULBP) could not be ruled out.
[253] Example III
[254] HLA-E mediated presentation of hsp60sp abolishes recognition by CD94 / NKG2A and CD94 / NKG2C receptors
[255] Inhibitory lectin-like receptor heterodimer CD94 / NKG2A is present on about 50% of all NK cells in the peripheral blood of both humans and mice. The HLA-E specific receptor mediates negative signals for binding to HLA-E expressing various protective HLA-class I signal peptides, which leads to inactivation of NK cell effector action. In a similar fashion, Qa-l ^b in complex with the permeable MHC class I leader peptide can be effectively recognized by the CD94 / NKG2A receptor in mice, which suggests that evolutionary conservation of humans and mice at both receptor and ligand levels Indicates. To characterize possible NK cell receptors that interact with HLA-E as complexes with hsp60sp or MHC class I signal peptides, whether the MHC tetramer complex can bind to CD94 / NKG2 receptors expressed on transfectants and NK cells The study was designed to measure. In vitro recombinant soluble HLA-E molecules were refolded in the presence of human β ₂ -microglobulin and B7sp (VMAPRTVLL) or hsp60sp (QMRPVSRVL). The refolded MHC complex was used to generate tetrameric HLA-E molecules, which enabled analysis of HLA-E binding receptors. All peptides allow effective refolding of in vitro HLA-E and are effectively biotinylated as analyzed by gel-shift analysis. This study showed that HLA-E / B7sp tetramers effectively bind to CD94 and NKG2A or mouse Ba / F3 pro-B cells co-transfected with CD94, NKG2C and DAP12 (FIG. 4, panels a and b). . These results were confirmed by staining NK-cell lines expressing inhibitory receptor CD94 / NKG2A (FIG. 4, panel c), or newly isolated NK cells predominantly expressing CD94 / NKG2A receptor. In contrast, HLA-E / hsp60sp tetramer did not bind to either CD94 / NKG2A or CD94 / NKG2C / DAP12, and to Ba / F3 pro-B cells co-transfected with all NK cells tested ( 5, panel ac). However, both HLA-E / B7sp and HLA-E / hsp60sp bound to a similar extent as regulatory B cell hybridomas specific for HLA class I molecules (FIG. 5, panel d). Thus, despite hsp60sp being able to access HLA-E physiologically effectively, these complexes are no longer recognized by the CD94 / NKG2A and CD94 / NKG2C receptors, indicating that they are peptide selective.
[256] Example IV
[257] HLA-E / hsp60sp does not inhibit CD94 / NKG2A + NK cells in cytotoxicity assays; An important role for position 5 within the peptide
[258] To examine the functional significance of increasing HLA-E / hsp60sp cell surface concentration, the study was designed to determine whether cells expressing these MHC complexes could be protected from death by CD94 / NKG2A ⁺ NK cells. . K562-E * 01033 cells incubated overnight at 26 ° C with either the hsp60sp or B7sp peptide were tested as targets in the 2 hrs chromium release assay with CD94 / NKG2A ⁺ NK cell lines Nishi and NKL as effectors. Clear protection from death was observed when other sensitive K562-E * 01033 cells were incubated with B7sp, whereas no significant protection was seen when incubated with hsp60sp (FIG. 5, panel a). As can be seen above, hsp60sp and B7sp have different rates of separation from HLA-E, which may explain the difference in target sensitivity. Therefore, HLA-E surface expression was monitored before and after the cytotoxicity test to confirm the relative concentration of HLA-E on the target through the test.
[259] Targeted mutations were introduced into B7sp and hsp60sp to pinpoint residues that cause loss of HLA-E recognition by CD94 / NKG2A. It has already been shown that changes in p5R in the Qa-l ^b binding peptide Qdm abolish recognition by CD94 / NKG2A in mice (Kraft et al., J. Exp. Med. 192 : 613, 2000, herein Included by reference). To test the degree of functional conservation of position 5 in both peptides, experimental peptides B7 R5V (VMAPVTVLL) and hsp60 V5R (QMRPRSRVL) were prepared. The ability of these peptides to protect K562-E * 01033 cells was tested in the cytotoxicity test as described above. K562-E * 01033 cells cultured at 26 ° C. with B7 R5V expressed high levels of HLA-E (FIG. 6, panel c), but they were effectively killed by CD94 / NKG2A ⁺ NK cells (FIG. 5, panel). b). Thus, this variation is sufficient to extinguish the protective capacity of B7sp. However, V5R mutations introduced into hsp60sp were not sufficient to restore protection from death using the same effector cells (FIG. 5, panel b).
[260] Recently, it has been reported that hsp60.4 peptide (GMKFDRGYI) can bind Qa-l ^b but does not induce protection from CD94 / NKG2A ⁺ NK cells (Gays et al., J. Immunol. 166 : 1601-). 1610, 2001, incorporated herein by reference). However, the peptide is not comparable to the protective Qdm-peptide for binding to Qa-l ^b even when mixed with 1 nM Qdm (Id.) In a 100,000-fold excess. In contrast, the present disclosure shows that hsp60sp can interfere with HLA-E mediated protection by competing with MHC class I signal peptides.
[261] In brief, K562-E * 01033 cells were incubated with 0.1 □ M B7sp with increasing concentrations of competing peptides and tested in cytotoxicity assays. Cells incubated with 0.1 □ M B7sp and cells incubated with the control peptide remain protected from killing at all test concentrations, whereas cells incubated with 0.1 □ M B7sp and hsp60sp die as the concentration of hsp60sp increases. More sensitive to (FIG. 6D). The B7R5V peptide was a much stronger competitor than hsp60sp (FIG. 6D). In line with the results on Qa-lb peptide binding competition as reported by Gays et al. Consistent with the results for Qa- ^1b peptide binding competition as reported by (36), hsp60.4 did not compete with B7sp for binding to HLA-E (FIG. 5D).
[262] Further studies were conducted to determine whether the stress-induced HLA-E cell surface up-regulation observed in K562-E * 01033 cells caused protection from NK cell mediated cytolysis. K562-E * 01033 cells grown at different densities were tested as targets in the 2 hrs chromium release assay using NKL and Nishi as effectors. Despite showing an increase in HLA-E concentrations, killing increased rather than decreased, indicating that the HLA-E molecules induced on these cells are not protective. All target cells had a survival rate higher than 90% as determined by AnnexinV staining and trypan blue (data not included). In addition, importantly, cells grown at high density could be rescued from death by the addition of B7sp peptide (FIG. 6, panel b). This shows that the increase in killing is not finally determined by cell culture conditions and that the HLA-E concentration is sufficient for protection in the presence of the appropriate peptide. In addition, the data show that stress induced HLA-E expression is not sufficient to protect against NK cell mediated killing. It should be noted that although K562 constitutively expresses the MIC-A and MIC-B ligands for the active receptor NKG2D, they are not further up-regulated by the cellular stress imposed on this test. Thus, the increase in death after cellular stress observed in some of the experiments herein does not appear to be due to increased expression of MIC-A or MIC-B. Up-regulation of other active ligands, such as, for example, ULBP's, can also lead to increased killing.
[263] Summarizing the above examples, HLA-E has been shown to bind to novel stress-related peptides derived from the signal sequence of hsp60. The complex thus obtained cannot be effectively recognized by the inhibitory CD94 / NKG2A receptor. This was manifested by lack of binding of HLA-E / hsp60sp tetramer to CD94 / NKG2A expressing cells and NK cell mediated killing of cells expressing such as HLA-E / peptide complexes. In addition, studies based on transfected cells show that hsp60sp can achieve access to HLA-E molecules in vivo, particularly during cellular stress conditions. Thus, it is shown that the proportion of HLA-E in the complex with these peptides increases during stress, which causes a gradual transition from NK cell protectors of the HLA-E peptide list to non-protected complexes.
[264] According to this model, NK cells can detect stressed cells during infection and inflammatory responses, despite monitoring of HLA-E / peptide complexes in peptide selective methods. This may be particularly important for a subset of NK cells that uniformly express CD94 / NKG2A as a major inhibitory receptor and also a subset of activated T-cells expressing such receptors.
[265] Already, in the sense that normal magnetic peptides in complex with MHC class I are tolerant to binding of inhibitory receptors, viral peptides or other non-magnetic peptides are not tolerated, missing-self recognition is It has been discussed whether it can be based on peptide specific recognition. There is ample evidence that some receptors are strongly affected by the bound peptide. It is suitable for immunoglobulin-like receptors as well as C-type lectin-like receptors, including CD94 / NKG2A. However, protective capacity is not correlated with the origin of the peptide, ie, whether the peptide is self- or non-self, or healthy or pathological. However, the balance between different HLA-E complexes can indicate the state of whether the cell signals "normal" or "abnormal" through a peptide that is competitive for MHC dependent presentation. Thus, HLA-E mediated protection will depend not only on whether sufficient transmission signal peptides (mainly derived from various MHC class I molecules) are produced, but also on how they are balanced by non-permeable, stress induced peptides. .
[266] Although KIR recognition of MHC class I may be affected by binding peptides, a mechanism based on peptide selective monitoring of stressed cells may preferentially be associated with CD94 / NKG2 receptors, which may be involved in the protection peptide. This is because it is specifically designed to recognize oligomorphic HLA-E molecules as complexes with a limited set. KIRs, on the other hand, have evolved preferentially to recognize a very diverse list of polymorphic HLA-A, -B, and -C molecules. Similar monitoring mechanisms of stressed cells require that there is a large array of stress-induced peptides that can be loaded onto each HLA class I allele when manipulated through KIRs.
[267] The first focus of this study on stress induced peptide interference (SPI) on inhibitory receptors is related to the structural aspects of different HLA-E peptide complexes. The crystal structure of HLA-E / B7sp shows that five peptide residues are located in well-defined pockets of the HLA-E molecule (O'Callaghan et al., Mol. Cell. 1: 531, 1998, herein Included as a reference), which compresses the conformation of the peptide through the binding groove. The comparison between hsp60sp and the MHC class I signal peptide sequence (TableI) shows the differences in five positions: p1, p3, p5, p6, and p7. Among them, p3, p6 and p7 are buried in pockets D, C and E, respectively, and pl and p5 are exposed on the surface.
[268] Based on the HLA-E / B7sp structure, O'Callaghan et al. Suggested that p5R in B7sp acts as an HLA-E contact residue for the HLA-E binding receptor. In fact, the substitution of arginine to valine (corresponding residue of hsp60sp) at position 5 of B7sp was sufficient to completely abolish HLA-E mediated protection from killing by CD94 / NKG2A expressing NK cells. However, the intersubstitution at hsp60sp (valine of p5 to arginine) was not sufficient to achieve protection, meaning that additional amino acids were important in the peptide. Special attention is paid to arginine at positions 3 and 7, which appear to be difficult to fit in shallow, hydrophobic D- and E-pockets. Alteration of the localization and identity of these side chains will be useful within some aspects of the present invention, designed to disrupt receptor binding either directly or indirectly by changing the overall conformation of the peptides in the HLA-E grooves.
[269] Another important focus for further expansion within the scope of the present invention relates to the biological relevance of this HLA-E / hsp60sp complex. The evidence presented above shows that the increase in HLA-E concentration observed during stress is due to the influx of hsp60 derived peptides into the HLA-E presentation pathway. For rigorous studies these K562 cells were co-transfected with full length hsp60 signal sequence (hsp60L-GFP) bound to HLA-E * 01033 and GFP. This resulted in the expression of GFP in mitochondria and HLA-E expressed at high levels intracellularly but at low levels at the cell surface. Cell surface HLA-E concentrations were compared with the mutated hsp60L-GFP constructs substituted with important HLA-E anchor residues and the control cells transfected with HLA-E * 01033, the cells cultured in stress-induced dogs. Increased in In addition, upregulation of HLA-E is planned as a result of altered endogenous hsp60sp distribution and high concentrations during stress. Accordingly, K562 cells transfected with HLA-E * 01033 alone also show an increase in cell surface HLA-E during stress. In addition, as a result of stress, up-regulation of HLA-E at the cell surface did not prevent NK cell mediated killing in any of these experiments.
[270] However, by adding a protective peptide such as, for example, B7sp, one may be able to restore HLA-E mediated protection. In fact, stressed cells were protected by simply adding protective B7sp peptides at the time of testing. This shows that endogenous hsp60sp can be presented by HLA-E during stress. HLA-E is therefore considered important as a presenter of stress-induced peptides against NK cells and T cells during infection, autoimmunity and inflammation. Peptide elution and sequencing from isolated HLA-E molecules of cells grown under normal conditions and cells exposed to various stress stimuli indicate whether hsp60sp is actually predominantly provided by the stressed cells of this disease and other disease states. Will be judged.
[271] The results further show that at least a portion of the stress-induced increased access of Hsp60sp to HLA-E must be due to post-transcriptional factors. Such factors include changes in protease activity, more effective peptide transport from mitochondria to ER, altered hsp60 distribution, or changes in the permeability of the mitochondrial membrane. Most of the hsp60 pool (80-90%) is located in the mitochondrial matrix of healthy cells (Soltys et al., Exp. Cell. Res. 222 : 16, 1996, incorporated herein by reference). However, not only after bacterial infection (Belles et al., Infect. Immun. 67 : 4191,1999, incorporated herein by reference), but also after proapopto processes and cellular stress (Feng et al., Blood 97 : 3505, 2001; Samali et al., Embo J. 18 : 2040, 1999, each of which is incorporated herein by reference), also reports an increase in the concentration of hsp60 other than mitochondria. This observation was made using mature hsp60 and at least the effect on mitochondrial permeability could also be applied to the cleaved signal peptide.
[272] In addition to increased hsp60sp expression and altered mechanisms of peptide loading, other peptides capable of binding HLA-E can be up-regulated during stress. Reportedly, simple heat treatment of L-cells also increases the cell surface concentration of Qa-l ^b (Imani et al., Proc. Natl. Acad. Sci. USA 88 : 10475, 1991, incorporated herein by reference. ). Recently, it has been reported that hsp60-derived peptides (GMQFDRGYL in Salmonella and GMKFDRGYI in mice) bind to Qa-lb (Lo et al., Nature Med. 6: 215,2 0, incorporated herein by reference) . In addition, the studies performed herein confirmed that the peptide GMKFDRGYI (hsp60.4 in Table I) can also bind to HLA-E. In contrast to hsp60sp, this peptide could not compete with B7sp for binding to HLA-E (Figure 5, panel d), or reportedly could not compete for binding with Qa-1 ^b (Gays et al. , J. Immunol. 166 : 1601-1610, 2001, incorporated herein by reference). It has also been reported that Qa-l ^b , which provides hsp60.4, does not protect cells from NK cell mediated cytolysis (Id.). Thus, these ligands cannot interlock with CD94 / NKG2A, but instead appear to be detectable by clonal T cells upon Salmonella infection in mice (Lo et al., 2000, supra). Thus, it appears that heat shock proteins, including peptides derived from other stresses induced and additional hsp60-derived peptides, may be HLA-E accessible during cellular stress caused by intracellular infection. It is believed that these peptides convert the functional role of HLA-E molecules as ligands for the CD94 / NKG2 receptor into complexes that can be recognized by specific T cells via antigen-specific TCR during infection.
[273] T cells also express CD94 / NKG2A inhibitory receptors, and therefore it is also believed that the balance between MHC class I signal peptides and HLA-E molecules with hsp60sp regulates T cells in the inflammatory response. In this regard, the findings are supported by a recently published report that effector cytotoxic T-lymphocytes induced against viral antigens can be inhibited through the expression of CD94 / NKG2A (Moser, JM et al., Nature Immunol. 3 : 189-196, 2002, incorporated herein by reference). Recognition of Qa- ^1b through these receptors has a dramatic effect on acute infection as well as oncogenesis by polyoma virus, and inhibits the effector effect and proliferation of T-cells. It was speculated that the peptide loading of Qa-l ^b could be affected under pathological conditions and could affect the interaction with suppressed T-cells.
[274] The present disclosure shows loading HLA-E with peptides that are not only induced in stressed cells but also interfere with protection against CD94 / NKG2A ⁺ NK cells normally provided by HLA-E. These findings clearly explain the role of CD94 / NKG2A expression during the regulation of T-cell responses. Co-expression of these receptors can complement the TCR pathway in distinguishing healthy from diseased cells by sensing the decrease in MHC class I molecule production as well as the increased accessibility of stress-induced peptides to HLA-E. To make these mechanisms more clear, studies are planned to determine whether CD94 / NKG2 expressing CD94 / NKG2 expressing human T-cells can be affected by stress-induced changes in target cells. In this relationship, analysis of peripheral blood from healthy donors demonstrates that a subset of CD94 / NKG2A ⁺ T-cells also binds to HLA-E / B7sp tetramers. In addition, further studies presented herein showed that HLA-E / hsp60sp tetramers were not detected to bind to either CD94 / NKG2A ⁺ or CD94 / NKG2A-T cells, which means that T cells are the same as NK cells. This means that there are not many T cells in healthy individuals that distinguish different HLA-E complexes and express TCR specific for HLA-E / hsp60sp.
[275] HLA-E molecules are recognized by CD94 / NKG2A inhibitory and CD94 / NKG2C activating complexes. The role of the activation form is not yet clear. The possibility of HLA-E / hsp60sp complexes being recognized by CD94 / NKG2C or other unknown activated NK has been raised. This may explain why stressed K562-E * 01033 cells are more effectively killed by NK cells despite increasing HLA-E concentrations. However, NKL cell lines do not express activating NKG2C receptors, HLA-E / hsp60sp tetramer did not bind to CD94 / NKG2C transfectant and all tested NK cells. Thus, other ligands that induce NK cell activating receptors may be involved. Neither MIC-A or MIC-B, ligands for NKG2D, are upregulated on stressed cultured K562 or K562-E * 01033 cells. However, additional ligands, or other activating receptors for NKG2D, may affect the sensitivity of K562 and K562-E * 01033 cells. Additional experiments using reagents that specifically block activating NK cell receptors may assist in clarifying the mechanism behind increased NK cell sensitivity during culture stress.
[276] NK cells can be classified into two major subpopulations based on CD56 cell surface expression levels (CD56 ^dim and CD56 ^bright ) (Sedlmayr et al., Int. Arch. Allergy. Immunol. 110 : 308, 1996, herein Included as a reference). Cells belonging to a small number of CD56 ^bright subpopulations all express high levels of CD94 / NKG2A and only part of them express KIRs. In contrast, most CD56 ^dim NK cells express KIRs and exhibit low cell surface concentrations of D94 / NKG2A (Jacobs et al., Eur. J. Immunol. 31 : 3121, 2001, incorporated herein by reference). Phenotypic classification of CD56 ^dim and CD56 ^bright NK cells is associated with different effector functions (Cooper et al., Blood 97 : 3146, 2001, incorporated herein by reference). Upon stimulation, CD56 ^bright NK cells have been suggested to be less cytotoxic and easier to produce cytokines, and therefore immunomodulatory (Chen et al., J. Immunol. 162: 3212, 1999, herein Included by reference). These cells are potentially responsive to proinflammatory signals (based on the expression patterns of adhesion molecules and chemokine receptors) and are over-expressed in large quantities at the site of inflammation (see below). In addition, macrophages have been reported to respond to human hsp60 by increasing the production of IL-12 and IL-15 (Id.), Which are important activators of this NK cell subpopulation. Based on the findings presented herein and the fact that hsp60 is upregulated during inflammation, the binding of hsp60sp by HLA-E is expected to result in cytokine production mainly by CD94 / NKG2A ⁺ , CD56 ^bright NK cells.
[277] In the following examples, additional findings are shown, including showing that CD56-bright natural killer cells expressing functional HLA-E specific inhibitory receptors preferentially accumulate in joints with arthritis. As can be seen from above, natural killer (NK) cells are lymphocytes involved in the innate immune response to certain microbial and parasitic infections. Recent reports suggest an additional important role of NK cells in autoimmune models, but little is known about the function of NK cells during human autoimmune disease. In the following examples, MHC class in αβ T cells and γδ T cells derived from peripheral blood (PB) and synovial fluid (SF) in arthritis, mainly rheumatoid arthritis (RA) patients, as well as in NK cells The expression of killer cell immunoglobulin (Ig) -like (KIR) and C-type lectin-like (CD94 / NKG2) receptors specific for I molecules is analyzed.
[278] From these studies, it was determined that SF of arthritic patients contained an increased proportion of NK cells compared to paired PBs. In contrast to PB-NK cells, the SF-NK cell population expressed CD94 / NKG2A cell surface receptors almost uniformly and contained significantly reduced proportions of KIR ⁺ NK cells. Functional analysis shows that both polyclonal SF-NK cells and PB NK cells extracted from patients and cultured in vitro can completely kill a series of target cells. However, SF-NK cell lysis is inhibited by the presence of HLA-E in the transfected target cells. By blocking CD94 in SF-NK cells, or by masking HLA in autologous cells, SF-NK cells were able to perform self-directed cytolysis. Thus, HLA-E is believed to play a fundamental role in regulating major NK cell populations in inflamed joints.
[279] Patient, Control, and Cell Separation
[280] All 17 patients had knee arthritis and were receiving SF aspiration treatment at the time of analysis. RA patients met American College's rheumatism classification criteria for RA (Arnett, Arthritis Rheum. 31 : 315324, 1988, incorporated herein by reference). All patients received disease-modifying antirheumatic drugs, except for a 44 year old woman diagnosed with early oligoarthritis. Extra-articular signs between RA patients include manifestations among the RA patients included diabetes (diabetes mellitus, 1 patient), Raynaud's phenomenon (2 patients), and secondary Sjogren's syndrome. , 1 patient). Samples paired with SF and PB from patients, and PB from 8 healthy female controls (mean age 52.7 years, ranging from 49 to 61 years) were collected into preservative-free heparin, and monocytes were collected from FICOLL-HYPAQUE (Amersham). Pharmacia Biotech, Uppsala, Sweden) using a density gradient centrifuge to separate according to standard protocols.
[281] NK cell culture
[282] Anti-mouse Ig-coated dynabeads (a ratio of beads to cells = 4: 1) and anti-CD3 mAb (OKT3, American Type Culture Collection, as recommended by the manufacturer (Dynal AS, Oslo, Norway) Rockville, MD) was used to reduce CD3 ⁺ cells to generate PB- and SF-NK cell lines. The remaining NK cell enriched population was maintained intact as described above with slight modifications (Soderstrom et al., J. Immunol. 159 : 1072-1075, 1997, incorporated herein by reference). In brief, CD3-cells were treated with 2% congested human AB ⁺ serum, 10% FCS, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 100 U / ml IMDM supplemented with human recombinant IL-2 (Life Technologies, Gaithersburg, MD) was mixed and plated in 24 well culture plates (Costar, Cambridge, Mass.) At a concentration of 1 × 10 ⁶ cells / ml. CD56 + CD3-PB- and SF-NK cell lines were tested in functional assays 2-3 weeks after initiation of culture.
[283] mAbs and flow cytometry
[284] Anti-KIRmAbs DX9 (anti-KIR3DLl), DX27 (anti-KIR2DL2, KIR2DL3 and KIR2DS2), DX31 (anti-KIR3DL2), and DX22 (anti-CD94 / NKG2A, -B, and -C) were Drs. Lewis L. Lanier and Joseph H. Phillips (UCSF, San Francisco and DNAX, Palo Alto, CA, respectively). Other antibodies include NKG2A (Z199, Dr Lorenzo Moretta, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy), CD3 (UCHT1, BD Pharmingen, San Diego, CA), CD56 (B 159, BD Pharmingen), CD16 (Leu-llc, Becton & Dickinson, San Jose, CA) TCRαβ (WT31, Becton & Dickinson) and TCRγδ (Immu 510, Coulter-Immunotech, Miami, Fol), MHC Class I (w6 / 32, American Type Culture Collection) I used things about. Using negative controls for FITC- and PE-conjugated rabbit anti-mouse Ig (both from Dakopatts, Glostrup, Denmark) and triple-colour (triple-colour, DAK-GO1, Dakopatts) as two-stage reagents It was. The cotton fluorescence staining was performed using standard protocols. Cells were analyzed on FACScan ^™ .
[285] cell
[286] K562 (human HLA-class I-leukemia), Daudi (human 2m ^- Burkitt's lymphoma), P815 (mastocytoma), 721.221 (human HLA-class I ^- B-limpoblastois) Lymphoblastoid cells) were a complete medium consisting of RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin I left it on. 721.221 cells transfected with a chimeric gene consisting of HLA-B * 5801 transfected 721.221 cells and HLA-G leader fused to HLA-B * 5801 were produced in DNAX (Palo Alto, USA). Briefly, PCR using wild-type HLA-G and HLA-B * 5801 cDNA as a template a leader fragment of HLA-G and a chimeric cDNA comprising the extracellular domain, transmembrane domain and cytoplasmic domain of HLA-B * 5801 (For details and primer sequences see "Braud et al., Nature 391 : 795-799, 1991, incorporated herein by reference"). The resulting product was confirmed by insertion into the pBJ-neo expression vector and sequencing. 721.221 cells were transfected by electroporation and selected in complete medium supplemented with 1 mg / ml G418. Transfected cells expressing high levels of cell surface HLA class I were isolated by flow cytometry. EBV transformed B-lymphoblastoid cell line (B-LCL) was established ex vivo by infecting patient B-cells with B95-8 EBV-strain. In brief, 10 ⁶ mononuclear cells obtained from patient PB were treated for 1 hour with supernatant of B95-8 EBV-producing cell line (Miller et al., Proc. Natl. Acad. Sci. USA 70 : 190-194, 1973, inches). Incubated at 37 ° C. under 5% CO ₂ . Infected B-cells were then incubated for about 2 weeks in complete medium supplemented with 5 μg / ml cyclosporin A (Sigma, St Louis, MO). Successful transformation was confirmed by typical cell clumping and proliferation of EBV-transformed B cells established ex vivo.
[287] NK cell-mediated cytotoxicity assay
[288] NK cell-mediated cytotoxicity was determined using a 4 hrs ⁵¹ Cr radioisotope or 18-hrs Alamar Blue Survival Assay (Alamar Biosciences, Sacramento, CA) as described above. (Soderstrom et al., J. Immunol. 159 : 1072-1075, 1997, incorporated herein by reference). In some experiments with blocking, mAbs were added at a final concentration of 1 μg / ml and were present during the analysis.
[289] HLA-E tetramer production
[290] HLA-E expression vectors for tetramer production were provided from Dr Veronique Braud (Oxford, UK). Tetramer HLA-E complexes were generated essentially as described above (Braud et al., Nature 391 : 795-799, 1991, incorporated herein by reference). Briefly, HLA-E heavy chain fused with BirA substrate peptide (bsp) at the C-terminus, and human beta-2 microblobulin (β2m) were overexpressed in E. coli BL21 pLysS, purified from cell inclusions, DTT It was dissolved in 8M urea solution containing. In vitro refolding of HLA-E-bsp, human β2m and peptides, complexes of HLA-E-bsp, human β2m and synthetic peptides (VMAPRTVLL, derived from HLA-B * 0701 leader sequence, Research Genetics, Huntsville, AL) Produced by The refolded complex is purified by size exclusion chromatography on a Superrose 12 column (Amersham Pharmacia Biotech) and then biotinyl according to the manufacturer's instructions using BirA enzyme (Avidity, Denver, CO) It is done. Free biotin was removed using a NAP-5 desalting column (Amersham Pharmacia Biotech). As measured in the gel-shift analysis, the degree of biotinylation was about 90%. Biotinylated HLA-E / β2m / peptide monomer and streptavidin-PE (Sigma) were mixed in a 4: 1 molar ratio to generate tetramers.
[291] *statistics
[292] The percentage of positive cells is expressed as mean ± SEM. Paired investigators' T-tests were used for comparison between SF and PB.
[293] Example V
[294] Expression of KIR and CD94 / NKG2 Molecules on NK Cells in Arthritis Patients
[295] Flow cytometry analysis was performed after triple staining using a panel of mAbs to determine the phenotype and subpopulation distribution of freshly isolated NK cells from patients suffering from arthritis. As shown in FIG. 7, a slight increase in the proportion of NK cells (CD3 ⁻ CD56 ⁺ ) in SF was observed as compared to patient PB. In addition, most PB-NK cells in both patients and healthy individuals expressed the NK cell marker CD 16 (FC □ RIII), whereas in SF a decrease in CD 16 ⁺ NK cell frequency was observed, confirming other reports. (Hendrich et al., Arthritis Rheum. 34 : 423431, 1991, incorporated herein by reference). In addition, the low T-cell ratios in both SF and PB of patients and healthy controls were double-positive for CD56 and CD3, but no significant difference was observed between SF and PB of patients and controls. .
[296] Clearly, all patients contained significantly lower fractions of KIR3DL1 ⁺ , KIR2DL2 / KIR2DL3 ⁺ and KIR3DL2 ⁺ NK cells in SF compared to paired PB samples (Table 6). The expression of these KIR molecules on PB-NK cells of patients was not significantly different from PB-NK cells of exogenous and healthy controls. A significant reduction in the percentage of NK cells expressing KIR molecules specific for certain typical HLA-A, -B, and -C molecules in SF distinguishes SF-NK cells from the KIR-type of the molecule to modulate effector function. Implying other MHC class I-specific inhibitory receptors. In light of these findings, a lectin-like MHC class I-specific receptor that forms an inhibitory unit in which the CD94 chain paired with NKG2A (or its splice variant NKG2B) specifically binds to atypical HLA-E molecules ( That is, expression analysis of the CD94 / NKG2 receptor complex) was performed (Braud et al., Nature 391 : 795-799, 1991, incorporated herein by reference).
[297] Expression of KIR Molecules on NK Cells patientRA 1NK cell expression rate: ^a)KIR3DL1KIR2DL2 / 3KIR3DL2 SFPBSFPBSFPB RA 13.218.73.517.53.713.4 RA 214.728.616.621.522.842.4 RA 37.715.48.625.711.918.6 RA 42.014.26.025.36.623.6 RA 52.02.85.622.012.821.7 RA 62.519.16.721.65.610.4 RA 75.0 / 4.820.910.6 / 10.338.215.6 / 14.230.9 RA 80.10.18.18.93.76.5 RA 90.8 / 1.89.45.4 / 9.227.04.1 / 5.913.0 Psor.A10.637.37.227.012.030.8 AS2.036.24.346.59.030.4 Mono.A1.16.47.227.23.02.2 Poly.A1.09.35.923.66.728.3 Oligo. A13.75.67.055.211.513.0 Oligo. A20.88.06.733.88.031.0 Mean ± SEM3.8 ± 0.915.5 ± 3.08.4 ± 1.127.1 ± 3.39.2 ± 1.321.1 ± 2.9 Control PB (n = 8) Mean ± SEM9.8 ± 2.330.1 ± 4.925.3 ± 4.6
[298] a) Newly isolated cells from SF and PB of patients and healthy subjects (control PB) using mAbs for various MHC class I specific receptors (KIR3DL1, KIR2DL2 / L3 and KIR3DL2), and as a second step, FITC- Conjugated goat anti-mouse antibodies were used followed by triple-staining using conjugated mAbs for CD56 (PE) and CD3 (Cychrome). These results are shown as paired data for individual patients. The SF values presented for RA patients 7 and 9 correspond to the right / left knees. The percentage of KIR expressing cells in the CD56 ⁺ CD3 ^- gated lymphocyte population was shown (5000-10000 events were obtained within the NK cell gate). Patient samples contained significantly lower proportions of NK cells expressing KIR in SF compared to paired PB samples (p <0.001 for KIR3DL1, p <0.001 for KIR2DL2 / L3, p for KIR3DL2). <0.001; paired Students T-test). KIR expression in PB-NK cells of patients was not different from PB-NK cells in healthy controls.
[299] While most SF-NK cells brightly stained with antibodies to CD94 have a single histogram peak, there has been a clear and consistent study that the anti-CD94 staining pattern on PB-NK cells is biphasic. These populations are classified into the CD94 ^dim and CD94 ^bight subgroups. FIG. 8A shows the histogram profile of a representative patient and FIG. 8B summarizes the data obtained from all study subjects. Interestingly, most SF-NK cells also clearly expressed NKG2A molecules, whereas only a portion of PB-NK cells were NKG2A ⁺ (FIGS. 8A and 8B). These findings, together with the results presented in FIG. 6, show that an absolute majority of SF-NK cells express inhibitory CD94 / NKG2A receptors and contain a significantly reduced proportion of KIR expression subpopulations. Expression levels of CD56 also showed that the majority of SF-NK cells belonged to the CD56 ^bright subpopulation (57.7 ± 5.3%, n = 16), whereas only a small percentage of PB-NK cells had bright CD56 levels (24.1 ± 5.0%, n = 14, p <0.001, compared to paired SF-NK cells), which is close to the proportion (17.5 ± 3.2%, n = 8) seen among PB-NK cells from healthy subjects.
[300] This analysis further showed that KIR expression on patient PB-NK cells was always limited to CD56 ^dim , while some CD56 ^bright PB-NK cells were CD94 ^bright and NKG2A ⁺ . Thus, based on the CD56, KIR, CD94 and NKG2A staining patterns, the predominant SF-NK cell subpopulation is similar to the minority subpopulation of CD56 ^bright NK cells present in the PB of both patients as well as healthy individuals (FIG. It expresses high levels of CD56, CD94 and NKG2A molecules similar to SF-NK cells but appears to be almost completely lacking at least KIR2DL2, KIR2DL3, KIR3DL1 and KIR3DL2 molecules. Taken together, inflamed SF appears to be enhanced for NK cell subpopulations with more defined MHC class I specific receptors compared to PBs of patients and healthy controls.
[301] Example VI
[302] Expression of KIR and CD94 / NKG2 on T-lymphocyte Subpopulations in Patients with Chronic Arthritis
[303] Expression of KIR and CD94 / NKG2 molecules was measured on αβ- and γδ-T cells for patients and healthy subjects. Regardless of whether the cells were from SF or PB, the fractions of KIR and CD94 / NKG2A, B, and C expressing cells were lower among αβ-T cells as compared to γδ-T cells (Table 6, respectively). And Table 7). However, this is not surprising since it is well documented in the literature that these receptors are more common in γδ-T cells compared to αβ-T cells. Interestingly, nevertheless, the fractions of αβ-T cells and γδ-T cells expressing some of these molecules were significantly different between the paired PB and SF samples of some patients (Tables 6 and 7, respectively). This suggests that T cells expressing specific MHC class I specific receptors are preferentially accumulating in some patients. The difference between SF and PB was more pronounced in γδ-T cells, although some patients included a substantially lower proportion (> 5 fold) of specific KIR + subpopulations in SF compared to mated PB (eg, patient RA1). , RA4, RA5, RA8 and Poly.A), the opposite pattern was also observed. Thus, the results do not show any clear trends suggesting that an increase or decrease in the specific KIR and / or CD94 / NKG2 expressing αβ-T cells or γδ-T cell rate is associated with either PB or SF in RA patients. Do not.
[304]
[305] a) mAbs for various MHC class I specific receptors (KIR3DL1, KIR2DL2 / L3, KIR3DL2 and CD94 / NKG2A, B, C) were newly isolated from SF and PB in arthritis patients and healthy subjects (control PB). And FITC-conjugated goat anti-mouse antibody as the second step, followed by triple-staining using conjugated mAbs for TCRαβ (PE) and CD3 (Cychrome). These results are shown as paired data for individual patients. The SF values presented for RA patient 7 correspond to the right / left knees. The percentage of KIR and CD94 / NKG2 expressing cells in the CD3 ⁺ TCR ⁺ gated lymphocyte populations was shown (5000-10000 events were obtained in the gate).
[306]
[307] Functional Analysis of NK Cell Lines Derived from RA Patients
[308] Paired polyclonal CD3 ^- CD56 ⁺ SF-NK and PB-NK cell lines were established by ex vivo culture in the presence of IL-2. After 1-2 weeks, the cytotoxic potential of these NK-cell lines was tested against a panel of target cells (721.221, K562, Daudi and P815). As shown in Table 5, both polyclonal SF- and PB-NK cell lines were able to lyse target cells between them. These NK-cell lines also produce similar levels of proinflammatory cytokines IFNγ, TNFα, and IL-6, as tested in Table 8, and also similar amounts of IL as measured translationally using ELISA assays. -2 and IL-10 were secreted and at least 90% of SF- and PB-NK cells were stained for intracellular IFNγ after stimulation with PMA as measured by flow cytometry. Thus, no clear differences in cytotoxic potential and cytokine production could be observed between the SF- and PB-NK cell lines established ex vivo.
[309]
[310] a) The lysis of target cells was measured using a 4-hrs ⁵¹ Cr-release assay, and ex vivo cultured PB-NK cells (PB-right value) and SF-NK cells (SF- Data on the E / T ratio are shown. While the phenotype of short-term PB-NK cell lines is exogenous with respect to KIR and CD94 / NKG2A expression, the SF-NK cell line expresses CD94 / NKG2A endogenously and essentially lacks KIR expression.
[311] Example VII
[312] Synovial NK cell line functionally recognizes HLA-E, which is proposed as a major ligand for protecting autologous cells from NK-cell attack
[313] Surface expression of HLA-E depends on its binding to nonamer peptides derived from the signal sequences of several other HLA-A, -B, -C and -G molecules. Thus, the interaction of CD94 / NKG2A with HLA-E can be considered as a way for certain NK cells to indirectly monitor the expression of certain polymorphic and non-polymorphic HLA class I molecules. In a transfection system, an amount of HLA-E sufficient to interact with CD94 / NKG2A expressed in NK cells by overexpression of some HLA molecules (eg, HLA-G comprising a permeable HLA-E binding signal sequence). Can be collected (Braud et al., Nature 391 : 795-799, 1991, incorporated herein by reference).
[314] To functionally test whether SF-NK cells recognize HLA-E through their CD94 / NKG2A receptors, the HLA-G leader sequence is added to the extracellular domain of HLA-B * 5801 (G _L -B * 5801). Cytotoxic assays were performed using 721.221 cells stably transfected with the fused chimeric gene. As a control, full-length HLA-B * 5801 molecules (HLA molecules not involved in recognition by the CD94 / NKG2A receptor; see Phillips et al., Immunity 5 : 163-172, 1996, incorporated herein by reference) A 721.221 transfectant was used. These transfected turn teuneun all been shown that receptors specific for the HLA-type of Bw4- alleles KIR3DL1 ^{+ -} the HLA-B * 5801 on the cell surface that are equally well recognized by NK-cell clones (and the CD94 / NKG2A) (Litwin et al., J. Exp. Med. 180 : 537-543, 1994; D'Andrea et al., J. Immunol. 155 : 2306-2310, 1995, each of which is incorporated herein by reference) . In addition to HLA-B * 5801, GL-B * 5801 transfected cells also surface express HLA-E, which can be functionally detected by CD94 / NKG2A ⁺ NK cell clones. As shown in FIG. 9A, the polyclonal SF-NK cell line effectively killed 721.221 cells transfected with wild type HLA-B * 5801 as well as untransfected 721.221 cells. However, expression of the chimeric G _L -B * 5801 molecule protects against NK cell-mediated cytolysis. Protection is reversed in the presence of antibodies to either CD94 or HLA class I, clearly showing that polyclonal SF-NK cells can recognize HLA-E evenly through the inhibitory CD94 / NKG2A receptor (FIG. 9B). In addition, when staining freshly isolated NK cells from patient PB and SF with refolded HLA-E tetramer molecules in the presence of HLA-B * 0701 nonamer leader peptide sequence, most SF-NK cells were HLA- Although effectively stained with E tetramers, fresh PB-NK cells did not stain well (although only some, including most CD56 ^bright PB-NK cells, were HLA-E tetramer-positive; FIG. 9C shows one representative patient) ). The reason why many NK cells in PB are not stained with HLA-E tetramers seems to be largely due to the low concentration of CD94 / NKG2 molecules on the CD94 ^dim subpopulation.
[315] To test whether the interaction of CD94 / NKG2A with HLA-E can also protect SF-NK cells from the death of autologous cells, B-LCL from one RA patient was evaluated for NK cell-mediated cytotoxicity. It was used as a target of. As shown in FIG. 10, neither PB-NK cells nor SF-NK cells could lyse autologous B-LCL. However, cytolysis of autologous cells was enhanced by anti-HLA class I mAbs or anti-CD94 mAbs using both polyclonal PB-NK cells and SF-NK cells as effectors. Using SF-NK cells as an effector, anti-CD94 mAb restored lysis to nearly the same level as observed in anti-HLA class I mAbs-most of the self-HLA protection mechanisms interact with HLA-E. It is accompanied by a functioning CD94 / NKG2A. On the other hand, polyclonal PB-NK cell lines also have other receptor-ligand interactions, as anti-HLA class I results in almost complete lysis of autologous target cells, whereas addition of anti-CD94 only partially increases lysis. Controlled by action. These findings are consistent with the fact that only a fraction of polyclonal PB-NK cells were CD94 / NKG2A ⁺ and most all SF-NK cells were CD94 / NKG2A ⁺ . In addition, the results obtained from the patients shown in FIG. 10 show that CD94 / NKG2A is the major self-specific receptor present on SF-NK cells.
[316] Further research further confirms the important use of the present invention to modulate HLA-E / CD94 / NKG2 cell receptors and related immune responses associated with other immune and inflammatory diseases, including RA and transplant rejection responses. As noted above, human chronic joint inflammation is permanently persisted by the cascade of inflammatory cytokines produced in the synovial membrane and synovial fluid. NK cells are potential producers of cytokines and present at these sites of inflammation, but their role in chronic human arthritis is largely unknown so far. The function of NK cells is regulated by inhibitory and activating cell surface receptors that interact with neighboring cells. Herein, expression of killer immunoglobulin-like receptors (KIRs) and C-type lectin-like receptor CD94 / NKG2A in synovial fluid (SF) NK cells has been studied in detail. The ability to produce proinflammatory cytokines IFN-gamma and TNF-alpha in NK cells was also studied in detail. Novel regulation of NK cell cytokine production (IFN-gamma and TNF-alpha) in the presence of target cells expressing inhibitory HLA-E + peptide complexes is reported.
[317] In addition to the above examples, FIG. 11 shows that SF-NK cells bind HLA-E as a complex with a representative VMAPRTVLL peptide. 12 shows that SF-NK cells bind to HLA-E as a complex with the VMAPRTVLL (B7sp) peptide but not to HLA-E as a complex with the QMRPVRSVL (hsp60sp) peptide. FIG. 13 shows that SF-NK cells are stimulated to produce IFN-gamma and TNF-alpha upon exposure to lipopolysaccharide (LPS) as compared to PB-NK cells of either RA patients or healthy individuals. 14 shows that SF-NK cells are stimulated to produce IFN-gamma after exposure to IL-2 as compared to PB-NK cells. FIG. 15 shows that HLA-E presenting the B7 signal peptide (VMAPRTVLL) is sufficient to inhibit NK cell IFN-gamma and TNF-alpha cytokine production in these accepted model studies.
[318] These additional results show that human NK cells found in articular fluid extracted from patients with chronic inflammatory arthritis belong to a phenotypic and functionally distinct subset of NK cells, similar to the CD56-bright peripheral blood NK cell subset described above. NK cells in joints with arthritis can add proinflammatory cascades by their potential production of FN-gamma and TNF-alpha that react with other cytokines produced in the joints, and these NK cell cytokine responses May be significantly down-regulated by cellular contact with cells expressing HLA-E together with protective peptides, complexes recognized by the CD94 / NKG2A inhibitory receptor. HLA-E complexed with non-protecting peptides that are not recognized by CD94 / NKG2A inhibitory receptors cannot inhibit NK cell cytokine production.
[319] In summary, the NK cells obtained from SF of arthritic patients are phenotypically belonging to a distinct subset of NK-cells that endogenously express inhibitory CD94 / NKG2A heterodimers and lack most KIR molecules. It turned out. The description herein is believed to be the first explanation for the unique disease-related accumulation of certain NK cell subpopulations in all human autoimmune diseases.
[320] It has been established by previous studies that KIR expression on normal PB-NK cells varies and expresses clonally between individuals (Lanier et al., Immunity 6 : 371-378, 1997, herein incorporated by reference). Included). In addition, both the frequency of at least some KIR + NK cells in PB and the surface concentration of KIR appear to be stable over time and independent of the individual HLA class I haplotype (Gumperz et al., J. Exp. Med. 183 : 1817-1827, 1996, incorporated herein by reference). Thus, NK cells expressing certain KIR isoforms may be present in and lack an appropriate self-HLA class I molecule (Id.). Thus, certain individuals appear to have either inhibitory KIRs or CD94 / NKG2A for self recognition of MHC class I (Valiante et al., Immunity 7: 739-751, 1997, incorporated herein by reference). Some individuals rely on more “broad” reactive CD94 / NKG2A systems, but there is no marked reduction in the expression of KIR on their PB-NK cells. Thus, the apparently normal expression patterns of KIR3DL1, KIR2DL2, / KIR2DL3 and KIR3DL2 molecules on PB-NK cells of RA patients, and SF-NK, which mostly lack the expression of these KIR molecules and instead mainly express the CD94 / NKG2A receptor Specific accumulation of cells suggests that the inflamed joint only provides an environment for some NK cell subpopulations.
[321] Phenotypic similarity between a small subset of CD56 ^brignt PB-NK cells and a large number of SF-NK cell subsets is known to be present in macrophage inflammatory protein-1α, which is known to be present in, eg, inflamed joints. Macrophage inflammatory protein-1α; MIP-1α), MIP-1β or RANTES, preferentially supplemented to inflamed joints upon reaction with locally produced chemotactic factors (Hosaka et al., Clin.Exp. Immunol. 97 : 451-457, 1994, incorporated herein by reference), such a mechanism will be appreciated to further explain the theories herein. CD56 ^bright PB-NK cell subpopulations are also important molecules for leukocytes rolling on the vessel wall (eg, CD62L) as well as molecules required for attachment and ejection of leukocytes to inflammatory sites (eg, CD2, CDllc, CD44, CD49e). , CD54) Based on studies showing that they express brighter levels of (26), CD56 ^bright CD94 / NKG2A ⁺ KIR ^- NK cell subsets appear to be selectively supplemented to inflamed joints. In addition, cytokines (eg, IL-15) present in joints promote preferential proliferation and may involve structures from apoptosis of this particular subpopulation.
[322] In addition to the above findings, the examples herein show that SF-NK cells can functionally recognize HLA-E. Evidence is also shown that this recognition is a major functional receptor-ligand interaction that protects SF-NK cells from attack of autologous cells.
[323] Since the CD94 / NKG2A receptor itself recognizes the presence of most of the HLA-class I molecules containing permeable leader peptides, the presence of a uniformly expressed and broadly reactive system would be important for NK cells in inflamed joints. (Braud et al., Nature 391 : 795-799, 1991, incorporated herein by reference). However, because self-tolerance by SF-NK cells is maintained alone by HLA-E expression, relying primarily on these receptor-ligand interactions can make these systems vulnerable. Thus, to prevent SF-NK cell mediated cytotoxicity, it will be necessary to maintain high levels of HLA-E expression on cells in the joints. As long as typical MHC class I molecules are produced at normal levels, it can be predicted that a sufficient amount of protective leader-peptide is generated to inhibit the SF-NK cell response by intracellular loading of HLA-E molecules followed by cell surface localization. have. However, it has been reported that ideally low expression levels of MHC class I are expressed on the cell surface of lymphocytes of patients with various autoimmune diseases, including RA (Fu et al., J. Clin. Invest. 91 : 2301-2307 , 1993, incorporated herein by reference). Upon proper SF-NK-cell stimulation, these HLA-E concentrations are low enough to induce NK cell responses after interaction with specific lymphocytes, which may play an important regulatory role in the synovial compartment. In this regard, patients who have a genetic lack of transporter associated to antigen processing (TAP), which, as a result, do not express or express low amounts of MHC class I cell surface molecules on all cells, It is noteworthy that it overexpresses functional CD94 / NKG2A receptors on NK cells, suggesting that low levels of adaptation to MHC class I are associated with the expression of these receptors (eg, Zimmer et al., Incorporated herein by reference). al., J. Exp. Med. 187: 117-122, 1998). In addition, ex vivo activated NK cells obtained from these patients effectively lysed autologous LCL cells and fibroblasts, suggesting that the tolerance of this subpopulation was disrupted, and these NK cells had low levels of MHC class I. Enable autoimmune response to cells expressing (Id.).
[324] Newly isolated NK cells from RA patients generally show reduced lytic activity (reviewed in Lipsky, Clin. Exp. Rheumatol. 4 : 303-305, 1982, incorporated herein by reference) and incomplete IFNγ and incomplete Although there are many reports showing that it responds well (Berg et al., Clin. Exp. Immunol. 1 : 174-182, 1999, incorporated herein by reference), the reduction of NK cells from SF mononuclear cell-cultures ( There are other reports showing that depletion results in enhanced production of certain Ig-isotypes in vitro (Tovar et al., Arthritis Rheum. 29 : 1435-1439, 1986, incorporated herein by reference). This suggests that SF-NK cells are involved in the regulation of antibody production, which is probably due to direct cytotoxicity of certain B cells or indirectly to cytokine production (e.g. TGFβ), which can alternately induce inhibitory T cell responses. (Horwitz et al., Immunol. Today 18 : 538-542, 1997, incorporated herein by reference).
[325] Overall, these results show that SF in autoimmune arthritis patients contains significantly increased fractions of NK cells expressing mostly CD94 / NKG2A receptors and only a small number of KIR3DL1, KIR3DL2 / 3 and KIR3DL2 molecules. will be. Evidence is also provided that the SF NK cells are also able to bind HLA-E and functionally recognize HLA-E on the transfected cells. In addition, the CD94 / NKG2A receptor expressed on polyclonal SF-NK cell lines appears to be a major receptor involved in the regulation of self-MHC class I reactivity, as shown by the use of autologous LCL cells in blocking experiments. .
[326] Example VIII
[327] Screening of Synthetic HLA-E Binding Peptides Having “on / off Switch” Ability to Engage CD94-NKG2 Receptor-Pairs
[328] HLA-E complexes with hsp60 leader peptides tend to degrade when the peptide loaded cells migrate to 37 ° C. during the NK cell cytotoxicity assay and may be somewhat unstable. By identifying peptide variants that may enhance or reduce such binding interactions, additional active agents will be provided for use within the scope of the methods and compositions of the present invention, including therapeutic use in vivo. To clearly illustrate this aspect of the invention, large scale screening of synthetic peptides or peptide analogs can be performed using peptide variants characterized by sensitive modifications of the hsp60 peptide back-bone. Such screening can be used to isolate stable HLA-E binding peptides, which can show enhanced functional interaction with activating CD94 / NKG2 receptor pairs. Isolation of such peptide analogs is of greater importance in the treatment of extensive tumors. The following is a general outline of a representative large scale screening program for identifying useful peptide variants within the scope of the present invention.
[329] material:
[330] K562 cells transfected with either HLA-E * 0101 or HLA-E * 01033.
[331] Description: These cell lines can also be transfected using GFP-encoding plasmids to accelerate large scale peptide-screening approaches (see below).
[332] RPMI 1640 badge
[333] Nonamer peptide peptides, modified peptides based on hsp60 leader peptide back-bones, other potential synthetic or natural structural analogs that bind to HLA-E peptide binding gaps.
[334] -26 ℃ incubator
[335] -37 ℃ incubator
[336] Cell centrifuge with 96 well plate-holders
[337] 96 well round bottom plate
[338] Initial Screening for HLA-E Stabilization
[339] Studies of the interaction of peptides with HLA-E have shown that the presence of HLA-E binding peptides provided by the addition of synthetic nonamer peptides in the culture medium is responsible for HLA-E cell surface expression levels as measured by flow cytometry. It will be sufficient to upregulate and stabilize. To test whether synthetic peptides / peptide analogs bind to HLA-E, HLAE * 01033 or HLA-E * 0101 transfected K562 cells can be used to stabilize HLA-E cell surface levels overnight at 26 ° C. will be.
[340] Screening for HLA-E * 0101 and HLA-E * 01033 Binding Peptides / Peptide Analogues
[341] HLA-E transfected K562 cells will be washed twice in RPMI medium without FCS and mixed in 200 μM RPMI medium containing 300 μM peptide and placed in 96 well round bottom plates at 2 × 10e5 cell counts / well. Plates are incubated overnight at 26 ° C. and then washed twice in RPMI 1640 medium without FCS. A portion will be stained with anti-class I mAb and analyzed for HLA class I expression levels by flow cytometry. The remaining cells will be placed back at 37 ° C. and stained after 1, 2, 3, or 4 hours to obtain stability estimates of the HLA-E peptide complex. This approach will screen for a panel of HLA-E binding peptides that will form more stable complexes.
[342] Method for Assessing Potential Function of HLA-E Peptide Complexes by the Potential to Engage One of a CD94 / NKG2A (Inhibition) or CD94 / NKG2C (Activation) Receptor Pair
[343] Effector NK Cells Used for Screening :
[344] First analyze the presence of NKG2C cDNA transcripts in NKL and Nishi NK cell lines containing both inhibitory CD94 / NKG2A receptor pairs by staining RT-PCR or NKG2C specific mAbs, staining cell surface, and then flow cells Measurement was made. If these cell lines lack an activating NKG2C receptor chain, the presence of activating NKG2C receptor chains in exogenous polyclonal NK-cell populations established from rheumatoid joints that predominantly express functional CD94 / NKG2A receptor pairs will also be analyzed.
[345] Process :
[346] Stable HLA-E transfected K562 cells co-transfected with GFP will be loaded with a selected panel of HLA-E stabilized peptides in 96 well plates, as described above. After washing, these target cell plate-wells will be incubated with effector NK cells for 2-4 hours at 37 ° C. and NK-cell mediated cytotoxicity will be analyzed directly by flow cytometry without prior washing. First of all, the epidemiological requirements and exact details of this analytical procedure were shown in the representative HLA-B * 0701 signal peptide (VMAPRTVLL) and hsp60 signal peptide (QMRPVRSVL) loaded HLA-E * 0101 and HLA-E * 01033 transfected GFP- Positive K562 cells will be used to measure experimentally. This assay shows that HLA-E transfected target cells protected from NK-cell mediated lysis remain GFP-positive (green fluorescence) and stay within viable gates, and the lysed cells lose green fluorescence and eventually survive Based on being outside The advantage of this assay is the ability to quickly screen very large peptide libraries at one time. A potential drawback is that the limit level for determining whether target cells are lysed to significantly higher levels compared to control peptide-treated cells may be difficult to access. Therefore, it may be desirable to preferentially use this method to screen for peptides or peptide analogs that exhibit good protection from cytolysis. The effect of the remaining selected peptides is then tested by conventional methods (ie, NK cell-mediated cytotoxicity using 51-Cr radioisotope labeled target cells). This experimental approach allows for screening for novel peptides and peptide analogs that act as "off-switches" for CD94 / NKG2A inhibitory receptors and potentially act as "on-switches" for CD94 / NKG2 activating receptors. In turn, this experimental approach will allow for the screening of novel peptides and peptide analogs that form stable protective HLA-E complexes (ie, "on-switches" for CD94 / NKG2A inhibitory receptors).
[347] Example IX
[348] Evidence that Qa-lb (mouse homologue of HLA-E) is involved in tumor avoidance and can achieve enhanced rejection of Qa-lb expressing tumors by administration of CD94-NKG2A uncoupling peptides.
[349] HLA-E / hsp60 complex occurs during cell distress. This complex is not recognized by the inhibitory CD94-NKG2A receptor. CD94-NKG2A is expressed not only on NK cells but also on gamma / delta T cells and CD8 + cytotoxic T cells (CTLs). Downregulation of typical MHC class I molecules occurs in many tumors, perhaps as a avoiding mechanism from immunodeficiency. Thus, for example, melanoma cells with downregulated typical MHC class I and sustained expression of HLA-E may be attractive targets for the immune system and inhibit both CTL and NK activity. hsp60 signal peptide or other pro-inflammatory HLA-E binding peptides (eg, from stress proteins, heat shock proteins or other representative proteins disclosed herein), and in HLA-E gaps with strong ability to bind HLA-E CD94-expressing CTLs for tumor cells that induce activation of NK cells and avoid immune deficiency based on sustained HLAE expression, by the use of analogues that could potentially compete with protective MHC class I-peptides. New therapeutic measures can be developed to lower the limit on the activation of NKG2A.
[350] First, Qa-lb expressing tumor cells were combined with selected peptides or peptide analogs and GMKFDRGYI-a known as hsp60 peptide (Qa-lb binding, CD94 / NKG2A uncoupling peptide (Lo et al., Incorporated herein by reference). Nature Med. 6 : 215-218, 2000)), as well as AMAPRTLLL (Qa-lb binding CD94 / NKG2A coupling peptide (Kraft, J. Exp. Med. 192 : 613-623, 2000)). To determine whether enhanced NK-cell dependent tumor rejection is observed using the -NKG2A-uncoupling peptide and whether enhanced tumor establishment is observed using the CD94-NKG2A-coupling peptide in this manner. Assays will be performed in NK-cell reduced (anti-NK1.I treated) and unreduced mice.
[351] Example XIII
[352] Study of Mouse NK Cells in Experimentally Induced Arthritis
[353] In this model, the potential role for NK cells in the establishment and maintenance of collagen-induced arthritis (CIA) was determined. By using the NK-cell reducing antibody (NK1.1) before and during the induction of the disease, the role of the presence and absence of NK cells in the pathogenesis of the disease will be further elucidated. Various tissues (eg spleen, lymph-node, blood, joint-tissue) of NK cell reduction and non-reduction mice were collected and analyzed for the presence of NK cells and the expression of their various cell-surface markers. The main purpose of this assay is to provide an NK inflammatory tissue, such as synovial fluid of human rheumatoid arthritis (RA), that predominantly expresses a CD94-NKG2A receptor pair specific for the HLA-E homologue of a mouse called Qa-lb. To assess whether one accumulates only a specific subset of cells. These studies more clearly demonstrate that non-typical HLA-E / Qa-lb acts on NK cell regulation at the site of inflammation.
[354] Immunization with collagen type II has been shown to cause collagen-induced arthritis in C57B1 / 6 mice with joint histopathological changes similar to human RA (Cambell, et al. Eur. J. Immunol. 30 : 1568- 1575, 2000). Importantly, C57B1 / 6 mice carry an H-2b monomorphism carrying a Qalb-binding protective nonamer signal-peptide called qdm (AMAPRTLLL) and detect NK cells that can be detected by anti-NK1.1 antibodies. Have This CIA model allows further explanation of the role of Qalb + peptide and its interaction with mouse CD94 / NKG2 receptors expressed on NK cells and NK1.1-positive T cells.
[355] mouse
[356] At the start of the experiment, C57BL / 6 (H-2 ^b ) mice were 6-8 weeks old. All experiments were performed within the guidelines according to the Karolinska Institute's accreditation criteria.
[357] Induction of Collagen-induced Arthritis
[358] Complete Freund's adjuvant was prepared by mixing 100 mg of heat killed M. tuberculosis H37Ra (Difco, Detroit, MI) with 20 ml of incomplete Freund's adjuvant (IFA) (Difco). Chick CII (Sigma, St. Louis, MO) was solubilized in 10 mM acetic acid (Sigma) at a concentration of 2 mg / ml overnight at 4 ° C. The chick CII was then emulsified 1: 1 to CFA. 100 μl of the emulsion was injected intradermally into the base of the mouse tail.
[359] NK Cell Reduction in Arthritis Induction
[360] Mice were injected intraperitoneally with anti-NK1.1 (PK136, BD Biosciences) depleting mAb (200 μg / mouse dissolved in PBS) one day prior to immunization with CII of CFA. The reduction efficiency was monitored by FACS analysis of blood 2 days after NK1.1 injection and stained with plate-NK cell antibody (DX5, BD Biosciences) to confirm the reduction efficiency. A second reduction was performed 10 days after the first reduction (ie 9 days after immunization with CII). As a control, with NK1.1, 200 μl / mouse of PBS or the same volume (200 μl / mouse) of mouse IgG (Sigma) was injected intraperitoneally of the animal.
[361] Clinical Evaluation of Arthritis
[362] Clinical scores were assessed using visual scales, with one point corresponding to redness and swollenness of one joint (usually the toes), and two points to redness or swollenness of more than one joint. 3 points correspond to the whole foot affected state. Each animal was given a maximum of 12 points.
[363] Incidence of CIA
[364] 16 shows disease incidence in mice treated with anti-NK1.1 antibody (NK1.1), IgG control (IgG1) and PBS alone (CII / CFA). Because no additional immune collagen II injections were made, only a few mice in the control group (ie CII / CFA) established CIA (one of 10 mice with CIA on day 28 healed naturally on day 42 ). In contrast, 8 out of 10 mice injected with anti-NK1.1 antibody established CIA at day 42, whereas only 5 out of 10 mice treated with IgG control showed signs of disease at day 42.
[365] Disease total score
[366] FIG. 17 shows total arthritis scores in animals treated with anti-NK1.1 antibody (NK1.1), IgG control (IgG1) and PBS alone (CII / CFA). Mice treated with anti-NK1.1 antibody (NK1.1) show severe CIA in contrast to IgG-treated and PBS-treated control mice.
[367] These results show that the presence of cells expressing NK1.1 markers is necessary to prevent CIA induction.
[368] Peptide Treatment to Control Arthritis
[369] Qalb binding protective qdm-peptide (AMAPRTLLL), nonamer derived from mouse hsp60 (Accession ID: P19226) (positions 10-18; QMRPVSRAL) hsp60 signal-peptide and synthetic qdmRSV-peptide (AMAPVTLLL) before and after injection of collagen type II Will be administered. These experiments will explain the potential regulatory role of the interaction of Qalb with CD94 / NKG2A in the regulation of collagen-induced arthritis in this model.
[370] Experimental Therapeutic Approach Using Qa-lb Binding Peptides
[371] Like HLA-E, Qa-lb predominantly binds to nonamer peptides derived from MHC class I signal peptides. This Qa-lb / peptide complex forms a functional ligand for the CD94-NKG2A inhibitory receptor. There is evidence that HLA-E presents nonamer peptides from the heat-shock protein 60 (hsp60) signal peptide during cell distress. This presentation appears to be independent of the carriers associated with antigen presentations 1 and 2 (TAP) 1/2, otherwise it is necessary to load the MHC class I signal peptide on the initial HLA-E / Qa-lb molecule. . In particular, HLA-E / hsp60 signal peptide complexes cannot be recognized by CD94-NKG2A inhibitory receptor-pairs that recognize HLA-E complexes with appropriate MHC class I-signal peptides (Braud et al., Nature 391 : 795 -799, 1991, incorporated herein by reference). Hsp60 is known to be highly expressed in arthritis tissue in both human and experimental arthritis models (Kleinau et al., Scand. J. Immunol. 33 : 195, 1991; Karlsson-Parra et al., Scand. J. Immunol. 31 : 283, 1991; Boog et al., J. Exp. Med. 175 : 1805, 1992, each incorporated herein by reference). Potentially, inflammatory lesions predominantly comprise the HLA E / hsp60 signal peptide complex, which may be one important trigger for local NK cells. As a first step in an experimental arthritis model (ie CIA), administered Qa-lb binding MHC class known to form a relatively stable Qa-lb / peptide complex that can be recognized by inhibitory CD94-NKG2A receptor pairs. The potential therapeutic effect of the I signal peptide (ie AMAPRTLLL) will be assessed. Another mouse will receive an irrelevant control peptide and another group will receive a nonamer hsp60 peptide. These peptides will be administered preferentially during established CIA to assess the therapeutic potential. Such peptides will be administered prior to collagen II injection. Thereafter, clinical and histological evaluation of arthritis will be performed. Based on the results of peptide-treatment in experimental arthritis models, these findings can be applied to human clinical trials to specifically target the ability to form functional ligands for HLA-E and its human CD94-NKG2 receptor pairs. One treatment method can be developed.
[372] Restoring HLA-E molecules with appropriate protective peptides recognized by the CD94 / NKG2A receptor will be useful for the therapeutic regulation of ongoing chronic immune responses. In light of the results of studies showing that NK cells predominantly containing the CD94 / NKG2A receptor accumulate in the inflamed synovial fluid of patients with arthritis, it is now possible to treat the appropriate HLA-E binding peptide in accordance with the method of the present invention to inflamed It is possible to restore sufficient CD94 / NKG2A mediated responses in the joints. In addition, local administration of HLA-E binding peptides that do not couple to CD94 / NKG2A binding is believed to be of therapeutic value during cancer treatments that enhance NK cell (and T cell) mediated anti-tumor responses. Thus, HLA-E binding peptides are proposed that constitute a switch capable of turning on or off NK-cell mediated recognition of widely expressed HLA-E ligands.
[373] Although the invention has been described in detail above by way of examples in order to clarify the understanding of the invention, it is intended that certain modifications and changes can be made without undue experimentation within the scope of the appended claims as set forth herein. It is apparent to those skilled in the art to which the present invention pertains, and is given by way of example, but not limitation.

权利要求:
Claims (12)
[1" claim-type="Currently amended] Medical use of HLA-E binding peptides to modulate the effects of CD94 / NKG2 cell receptors.
[2" claim-type="Currently amended] The use according to claim 1, wherein the HLA-E binding peptide is derived from the signal sequence of the stress inducing protein.
[3" claim-type="Currently amended] The use of claim 2, wherein the stress inducing peptide is a peptide from hsp (heat shock protein) 60.
[4" claim-type="Currently amended] 4. Use according to claims 1 to 3, wherein the peptide is recombinant or synthetic and optionally derivatized or peptide analog.
[5" claim-type="Currently amended] The use according to claim 1 to 4, wherein the peptide is a stable peptide.
[6" claim-type="Currently amended] The use according to claims 1 to 5, wherein the CD94 / NKG2 receptor is a CD94 / NKG2A inhibitory receptor on NK and T cells.
[7" claim-type="Currently amended] Use according to claim 6 for the treatment of tumors.
[8" claim-type="Currently amended] 8. Use according to claims 3 to 7, wherein the hsp 60 peptide is a nonamer.
[9" claim-type="Currently amended] Peptide selected from VMAPVTVLLA and QMRPRSRVL.
[10" claim-type="Currently amended] The peptide according to any one of the preceding claims, which is in complex form with HLA-E.
[11" claim-type="Currently amended] A pharmaceutical composition comprising the peptide of claim 10 and a pharmaceutically acceptable carrier.
[12" claim-type="Currently amended] a) construction of peptide libraries;
b) HLA-E / peptide complex formation;
c) selection of stable complexes capable of inhibiting or activating the CD94 / NKG2 receptor on NK and T cells; And
d) separating stable peptides / peptide analogs from the complex
Comprising the step of, HLA-E binding peptide or method of analysis.

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同族专利:

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公开号 | 公开日

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US20070081991A1|2007-04-12|

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WO2003011895A2|2003-02-13|

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WO2003011895A3|2003-07-24|

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CN1555272A|2004-12-15|

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WO2003011895A8|2003-12-11|

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JP2005523236A|2005-08-04|

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EP1423140A2|2004-06-02|

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CA2456196A1|2003-02-13|

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US20030171280A1|2003-09-11|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-07-31|Priority to US30859801P

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2001-07-31|Priority to US60/308,598

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2002-07-31|Application filed by 칼 페터 쇠더스트룀

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2002-07-31|Priority to PCT/US2002/024311

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2004-05-17|Publication of KR20040041575A

优先权:

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申请号 | 申请日 | 专利标题

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US30859801P| true| 2001-07-31|2001-07-31|

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US60/308,598|2001-07-31|

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PCT/US2002/024311|WO2003011895A2|2001-07-31|2002-07-31|Compositions and methods for modulation of immune responses|