JP2008524245A - Antibodies against antigen-presenting cells and uses thereof - Google Patents

Abstract

Antibodies against antigen-presenting cells can be used to prevent interaction between antigen-presenting cells and immune cells including T cells. Peptides can be linked to the antibody, thereby generating an immune response against such peptides. Preferably, the peptide linked to the antibody is associated with autoimmunity. One embodiment of the present invention provides an antibody that recognizes the L-SIGN receptor and shows at least 40 percent internalization into isolated human liver non-parenchymal cells in 2 hours. The

Description

(Citation of related application)
This application is a continuation-in-part of US Application No. 11/016647 (filed December 17, 2004). This US application is a partial continuation application of PCT / US04 / 06570 (filed on March 4, 2004), and this PCT application is filed with US Provisional Patent Application No. 60 / 548,385 (filed February 28, 2004). ); Claims priority to US Provisional Patent Application No. 60 / 529,500 (filed on Dec. 15, 2003); and US Provisional Patent Application No. 60 / 451,816 (filed on Mar. 4, 2003) . The complete disclosures of all these applications are hereby incorporated by reference.
(Technical field)
Develop and repair innate immune tolerance against self antigens to treat or prevent autoimmune diseases.

(Background of related technology)
The T cell mediated disease, insulin dependent diabetes mellitus ("T1DM") is a major health problem affecting more than 1.5 million Americans. This autoimmune disease results from T cell-mediated destruction of insulin-producing β-cells of islets of Langerhans in the pancreas. Despite treatment with insulin, mortality due to T1DM has increased over the past 20 years, but mortality in cancer, cardiovascular disease and stroke has decreased (Hurlbert et al., 2001). Also, the complications of treatment with exogenous insulin, including nephropathy, neuropathy and retinopathy, are very debilitating.

  T1DM is believed to be a Th1-mediated disease, and early intervention that converts the immune response to the Th2 type, for example by systemic administration of IL-4, can prevent the onset of the disease (Cameron et al., 1997). Year). The balance of effector T cells, Th1 and Th2, can be important in maintaining immune tolerance, and changes in balance can result in autoimmunity. However, protection from autoimmune disease is not an intrinsic property of Th2 cells since the Th2 cell line from NOD mice has also been shown to metastasize disease (Pakkala et al., 1997).

  The immune system has evolved in a complex manner to maintain self tolerance. The thymus provides an important initial selection of T cells. This selection results in the peripheral transport of T cells that are tolerant to self antigens present in the thymus. However, many tissue specific proteins are not expressed at levels sufficient to induce tolerance. For example, islets of Langerhans reactive T cells have been found in healthy subjects but probably have a low affinity (Lohman et al., 1996). Several mechanisms of peripheral tolerance complement the central tolerance mechanism of the thymus to keep autoreactive T cells under control. One important mediator of peripheral tolerance is the antigen-presenting cell (“APC”). APCs such as dendritic cells ("DCs") and macrophages capture autoantigens from other cells and present them to autoreactive T cells, resulting in deletions, anergy, and / or regulatory T cell Development induces T cell tolerance (Non-Patent Document 1). The current hypothesis is that immature APCs, such as APCs in the steady state immune system, tolerate rather than activate T cells, presumably due to lack of costimulatory molecules. Hawiger et al. Targeted the antigen to the major histocompatibility class II ("MHC II") pathway of DC using an antibody against the DC-restricted endosite receptor DEC-205 (Hawiger et al., 2001). As indicated by the lack of response to subsequent peptide immunity, antigen presentation by these DCs stimulated a short burst of CD4 + T cell proliferation, after which deletion occurred and the recipient became tolerant to the antigen. In contrast, immunity was induced when antigen targeting was achieved by strong DC maturation stimuli such as anti-CD40.

  Dendritic cells can also induce peripheral tolerance by generating regulatory T cells that affect the function of effector T cells through inhibitory cytokines or contact-dependent mechanisms (Jonuleit et al. 2000, Dhodapkar and Steinman, 2001). Many different protocols have been developed for the induction of regulatory T cells, generally with "suboptimal" T cell stimulation. Suboptimal stimulation of T cells can be achieved by antigen presentation without costimulation, or by inflammation, or by partial blocking of the T cell receptor or its co-receptors CD4 and CD8. Regulatory T cell phenotypes and mechanisms of action are heterogeneous. Many suppressor cells are CD4 + CD25 +, but it is becoming increasingly clear that in many situations CD4 + CD25 + cells are equally effective. Other markers identified in regulatory T cell populations include CD62L, GITR and CD103 (Lafaille and Lafile, 2002), and CD8 + regulatory T cells have also been reported (Dhodapkar and Steinman, 2002). Year). Several regulatory T cells have been shown to produce the immunosuppressive cytokine, interleukin (“IL”)-10 (Wakach et al., 2001, Barrat et al., 2002), although oral immunization has been demonstrated. Regulatory T cells induced by tolerance are characterized by the production of transforming growth factor-β (“TGF-β”) in addition to the Th2-type cytokines IL-4 and IL-10 (Weiner). , 2001). Contact-dependent suppressor cells are generated by activating CD4 + CD45RA + human peripheral T cells in the presence of TGF-β (Yamigawa et al., 2001). Induction of regulatory T cells requires stimulation through the T cell receptor, but their suppressive effects appear to be non-antigen specific (Thorton and Shevach, 2000).

  Immunoregulatory T cells have been shown to play a role in downregulating pathogenic autoreactive T cells in NOD mice. There is evidence that pre-diabetic mice contain immunoregulatory T cells and a decrease in their number or their functional capacity is a major contributory event in disease progression (3). Co-metastasis experiments have shown that CD4 + T spleen cells from prediabetic mice completely prevent disease metastasis to immune-deficient recipients by diabetes-inducing cells (Boitard et al., 1989, Hutchings and Cooke, 1990). ). In addition, in the NOD mouse model, the induction of regulatory T cells by immature DCs correlated with disease prevention (Huges et al., 2002).

  Responds to insulin, glutamate decarboxylase (“GAD”), heat shock protein (“HSP”) 60, or protein tyrosine phosphatase-like molecule (“IA-2”), and other undefined β-cell antigens in humans Autoreactive T cells have been described (Roep et al., 1990, Atkinson et al., 1992, Honeyman et al., 1993, Reijonen et al., 2002).

  GAD is a biosynthetic enzyme for γ-aminobutyric acid, an inhibitory neurotransmitter (Baekkeskov et al., 1990). Two different isoforms of 65% homology, GAD65 and GAD67 have been cloned. While GAD65 is the predominant isoform in humans, GAD67 is the major form in NOD mice, and antibodies against both isoforms are detected in humans (Kaufman et al., 1992). In NOD mice, anti-GAD antibodies were previously detected before or at the onset of isletitis and before the development of antibodies against other β-cell antigens. This timing implies that GAD is the major antigen that initiates β-cell autoimmunity in this model (Tisch et al., 1993). Further evidence of the important role of GAD in diabetes arises from the observations by many laboratories that GAD-specific T cells isolated from the spleen or pancreas of diabetic mice can metastasize disease to naïve animals ( Non-Patent Document 4, Wen et al., 1998, Zekzer et al., 1998). Although there is still controversy about the central role of GAD in the pathogenesis of T1DM, evidence from animal experiments suggests at least an important role for this protein.

  Immunization with purified GAD65, either thymus or intravenously, at an early age can tolerate T cells against pancreatic β-cells in NOD mice, thereby preventing islet inflammation and diabetes (Tian et al., 1996, Ma et al., 1997). Tolerization to GAD could also prevent the development of immune responses against other antigens such as HSP65. Further studies examined which GAD peptides can induce tolerance (Tisch et al., 2001, Tisch et al., 1999, Zechel et al., 1998). Protection from the onset of diabetes can also be achieved by either insulin or HSP65 treatment by the intravenous, subcutaneous, oral or nasal route (Elias et al., 1991, Elias and Cohen, 1994, Elias et al., 1997, Atkinson et al., 1990). Antigen-specific therapies are highly effective in preventing disease onset when administered early, but few attempts have been successful in controlling ongoing disease (Elias and Cohen, 1994, Tian et al., (1996).

  General peptide immunity cannot control whether antigen-presenting cells present peptides at the stage of inducing immunity or by antigen-presenting cells that can alter the immune response to tolerance, and thus immune stimulation or Either immunosuppression can occur.

  The occurrence of diabetes can be prevented by weakening the immune system. Any and all common agents that suppress T cell function, such as FK506, anti-CD4, anti-CD8, anti-CTLA-4 and others have been shown to prevent or delay the onset of diabetes in NOD mice (Reviewed in Atkinson and Leiter, 1999). However, none of these reagents are specific for diabetes-inducing T cells, and most of these can prevent the onset of the disease, but once the disease is established, they are ineffective. Common immunosuppressants such as cyclosporine tested in clinical trials were effective in the short term (Feutren et al., 1988, Skyler and Rabinovitch, 1992). However, withdrawal of immunosuppression leads to immediate recurrence, and side effects such as nephrotoxicity prevent long-term treatment (Parving et al., 1999).

  Clinical trials have been initiated to evaluate the efficacy of antigen-specific therapy for diabetes. In early-onset diabetes, the HSP6O p277 peptide (DiaPep277) was tested (Raz et al., 2001). Multiple immunizations with peptides have slowed disease progression, and extensive studies have been initiated to confirm and extend the results. Clinical trials are underway using the beta chain of human insulin in combination with Freund's incomplete adjuvant, a modified peptide ligand of insulin B9-23 and GAD. However, trials to treat recently diagnosed diabetes with oral insulin have failed (Pozzili et al., 2000, Chaillous et al., 2000), and parenteral insulin administration does not prevent the prevention of high-risk pre-diabetes. It was a success (Diabetes Prevention Trial-Type 1 (DPT) Study Group, 2002). Failure may be due to a number of factors, including antigen selection, antigen dose (Kurts et al., 1999), timing and route of administration. Also, antigen therapy cannot control which type of immune cells absorb antigen. Although mice are under controlled, pathogen-free conditions, this is not the case for human studies. If there is a concurrent bacterial or viral infection, priming occurs rather than tolerance. In animals, diabetes could be induced by antigen immunization under certain conditions (Blana et al., 1996, Bellmann et al., 1998).

Since an understanding of how the immune system maintains tolerance to self-antigens has developed considerably over the past decade, current therapeutic strategies for preventing or treating T1DM restore immune tolerance to beta cell antigens Intended to let you. Current immunotherapy strategies are intended to induce tolerance to β-cell antigens either by directly inactivating self-reactive T cells and / or inducing T cells with regulatory capacity. . Induction of regulatory T cells appears to be a promising approach for the treatment of many autoimmune diseases.
Heath WR and Carbon FR, “Cross-presentation, dendritic cells, tolerance and immunity.”, Annu Rev Immunol, 2001, Vol. 19, p. 47-49 Roncarolo MG, Leavings MK, Traversi C, “Differencement of Tregularity cells by immediate dendritic cells.”, J Exp Med, 2001, Vol. 193, F5-F9. Sempe P, Richard MF, Bach JF, Boitard C, “Evidence of CD4 + regulatory T cells in the non-obesity diabetic male mouse,” Vol. 37, Diabetolologica. 337-343 Rohan PW, Shimada A, Kim DT, Edwards CT, Charles B, Shultz LD, Fastman CG, Islet-infiltrating lymphocytes from predibetic NOD. Diabetes, 1995, 44, p. 550-554

(Summary)
The present disclosure relates to a method of treating autoimmune diseases by inducing immune tolerance. By presenting self-antigens on antigen-presenting cells, immune tolerance is induced. The self antigen is linked to an antibody that recognizes the antigen internalization receptor. Antigen presenting cells internalize self antigens on antigen presenting cells and cause inhibition of self-reactive T cells.

  In particularly useful embodiments, the methods and compounds described herein are used to provide tolerance to self-antigens, which can be, inter alia, beta cell antigen, GAD or epitope thereof, insulin or epitope thereof, HSP or epitope thereof. By inducing diabetes mellitus is treated. Autoantigens are linked to antibodies that recognize DC-SIGNR or DC-SIGNR mutations that are antigen internalization receptors. Autoantigens are internalized in target hepatic sinusoidal endothelial cells or other tolerized APCs expressing DC-SIGNR on the surface. Autoantigens are presented to target hepatic sinusoidal endothelial cells and inhibit the proliferation of self-reactive T cells or activate the suppressive effect of regulatory T cells.

  In another aspect, an antibody / peptide construct comprising an antibody to a receptor on an antigen presenting cell linked to a peptide is described. Preferably, the peptide is an antigen, more preferably an autoantigen. In particularly useful embodiments, the antibody / self-antigen construct or portion thereof is internalized by the antigen-presenting cell to achieve immune tolerance to the self-antigen. In some cases, the toxin can be combined with an antigen of the present disclosure and administered to the patient. Where the toxin is directed against, for example, tumor cells, the antibodies of the present disclosure can be utilized to direct the toxin to tumor cells, thereby concentrating the administration of the toxin on the tumor cells.

  In another aspect, a method for recombinantly producing engineered antibodies, including antibodies to receptors or antigen presenting cells, linked to self antigens is described.

  The present disclosure also relates to antibodies to DC-SIGNR that interfere with the interaction between DC-SIGNR expressing cells and ICAM expressing cells such as T cells. Such blocking of interactions can result in immune stimulation. Furthermore, antibodies that are antagonistic to L-SIGN can alter the antigen presentation properties of the target cells, which can result in either activation or suppression of immunity.

  In another embodiment, antibodies against DC-SIGNR prevent viral entry into cells containing hepatocytes such as hepatic sinusoidal endothelial cells and their infection into other cells. Antibodies against DC-SIGNR can also be used to prevent viral entry into T cells and their infection into other cells. In some embodiments, the disclosure includes the use of antibodies against DC-SIGNR in a vaccine.

  In other embodiments, the antibodies of this disclosure are utilized to bind to DC-SIGNR and / or L-SIGN, thereby including viruses such as HIV, HCV, Ebola, SARS, CMV and Sindbis. Non-limiting infectious agent binding, infection, and transmission can be blocked.

  In yet another embodiment, an antibody against DC-SIGNR is used to bind, infect, or infect Mycobacterium bacteria including M. tuberculosis and M. bovis. And can block propagation. In other embodiments, antibodies against DC-SIGNR can be utilized to block the binding, infection, and transmission of parasites such as Schistosoma mansoni.

  In yet another embodiment, an antibody or antibody / peptide construct of the present disclosure can be labeled with a toxin against DC-SIGNR expressing cells. Administration of a toxin-labeled anti-DC-SIGNR antibody or anti-DC-SIGNR antibody / peptide construct can then be used to reduce the level of DC-SIGNR expressing cells, such as treatment of autoimmune diseases, etc. In some instances, it may be beneficial.

  The antibodies against DC-SIGNR of the present disclosure can also be used as a conventional diagnostic method for tumor types associated with DC-SIGNR expression, and in some embodiments, provided as part of a diagnostic kit You can also

  The antibodies against DC-SIGNR of the present disclosure can also be used as therapeutics for the treatment of cancer and tumor types associated with DC-SIGNR expression.

  DC-SIGNR expressing cells can also be isolated from cells that do not express DC-SIGNR using the antibodies of the present disclosure.

  In some embodiments, an antibody against DC-SIGNR of the present disclosure can be a humanized antibody. In other embodiments, an antibody against DC-SIGNR of the present disclosure can be a scFv.

  In some embodiments, the disclosure provides an antibody that recognizes L-SIGN receptor and blocks HIV gp120 binding to L-SIGN, an L-SIGN receptor that recognizes L-SIGN receptor, and an Ebola envelope protein to L-SIGN Recognizing L-SIGN receptor and blocking HIVgp120 binding to DC-SIGN Recognizing L-SIGN receptor and blocking Ebola envelope protein binding to DC-SIGN Antibodies that recognize both L-SIGN and DC-SIGN receptors and block the binding of HIV gp120 to DC-SIGN, and / or both L-SIGN and DC-SIGN receptors Recognize and block Ebola envelope protein binding to DC-SIGN. To an antibody. This antibody may bind to the same epitope to which Ebola envelope protein or HIV gp120 binds.

  In yet other embodiments, the present disclosure relates to chimeric antibodies comprising a non-human variable domain and a human IgG constant region, wherein the non-human variable domain binds to a receptor on an antigen presenting cell, optionally a DC-SIGN receptor. In some embodiments, the non-human variable domain recognizes the L-SIGN receptor and the human constant region is an IgG1 region. Such antibodies can block the binding of HIV gp120 or Ebola envelope protein to L-SIGN and / or DC-SIGN. Chimeric antibodies can bind to L-SIGN and / or DC-SIGN more advantageously than with non-human variable domains alone.

  Further embodiments of the present disclosure relate to prophylactic and diagnostic techniques using the composition and / or including the methods described above. Also provided is a composition comprising an antibody against DC-SIGNR of the present disclosure in a pharmaceutically acceptable carrier.

(Detailed explanation)
The method induces immune tolerance to self antigens or self peptides involved in autoimmune diseases.

  By administering an antibody / autoantigen construct (sometimes referred to herein as an “engineered antibody”) to a subject, the present disclosure induces immune tolerance. Antibody / self-antigen constructs include a self-antigen linked to an antibody.

  The antibody component can be an antibody that binds to any receptor on any antigen presenting cell. As will be appreciated by those skilled in the art, antigen presenting cell types include dendritic cells, macrophages, endothelial cells, Kupffer cells and B cells. Currently known receptors or antigen presenting cells include DEC-205, mannose receptor, DC-SIGN, DC-SIGNR, MHC, toll receptor, Langerin, asialoglycoprotein receptor, β-glucan receptor, type C There are lectin receptors and dendritic cell immune receptors. In particularly useful embodiments, the receptor is one that internalizes an STT antibody. Whether internalization occurs at a particular receptor can be determined experimentally using techniques known to those skilled in the art. Receptors or antigen presenting cells currently known to provide antibody internalization include DEC-205, mannose receptor, DC-SIGN and DC-SIGNR.

  The antibody component may be a natural antibody (isolated using conventional techniques) or may be an antibody prepared synthetically by recombinant methods within the purview of those skilled in the art. The antibody can be, for example, a fully human antibody, a non-human antibody, a humanized antibody, a chimeric antibody or an antibody of the type described above engineered in any way (eg, site-specific modification or deimmunization). Antibodies can be advantageously selected from a library of antibodies using techniques known to those skilled in the art, such as phage display and panning.

  As used herein, “antibody” and “immunoglobulin” are used interchangeably and refer to an entire immunoglobulin molecule or a molecule comprising an immunologically active portion of an entire immunoglobulin molecule; Includes Fab, F (ab ′) 2, scFv, Fv, heavy chain variable region and light chain variable region.

  The nucleic acid encoding the antibody, when selected, is, for example, US patent application Ser. Nos. 10 / 251,085 and 2001 filed Sep. 19, 2002, the disclosure of which is incorporated herein by reference. Amplification can be performed using techniques known to those skilled in the art, such as conventional PCR or amplification techniques, each described in US Ser. No. 10/014012 filed Dec. 10.

  An autoantigen is linked to an antibody to prepare an antibody / self antigen construct according to the present disclosure. For the purposes of this disclosure, the terms “antibody / autoantigen construct” and “antibody / peptide” are used interchangeably.

  Any autoantigen can be used. Autoantigens may be naturally occurring and may be isolated using techniques known to those skilled in the art. Alternatively, if the amino acid sequence of the autoantigen is known, it can be prepared synthetically using known techniques. Suitable self-antigens include insulin, GAD, Hsp, nuclear antigen, acetylcholine receptor, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, myelin-related glycoprotein, glomerular basement membrane protein and thyroid gland Examples include stimulating hormone receptors. In particularly useful embodiments, the self-antigen is one that induces immune tolerance upon presentation by tolerized antigen-presenting cells.

  Autoantigens can be linked to antibodies by any suitable method. Certain specific methods are described in the Examples below, but the present disclosure is not limited to any specific method of producing antibody / autoantigen constructs.

  The present methods for inducing immune tolerance against self-antigens target antigen-presenting cells (“APC”) and direct the self-antigen to these cells by antibodies. FIG. 1 schematically illustrates the interaction of an antibody / autoantigen construct according to the present disclosure with antigen presenting cells (APCs) and T cells. The antibody recognizes a receptor on the target cell. These two are linked to guide the delivery of the autoantigen by the antibody. Although this disclosure depicts the use of vector cloning, this ligation can be accomplished by any method. The antibody targets and binds only the unique antigen internalization receptor, thereby ensuring delivery of the autoantigen to the desired cell type.

  After the antibody binds to the target antigen internalization receptor, the linked autoantigen and antibody are internalized in the antigen-presenting cell. The autoantigen is presented on the surface of the APC, possibly through the interaction of the autoantigen with the intracellular major histocompatibility complex (“MHC”). When autoantigens are expressed on the surface of APCs by costimulatory capacity, naïve autoreactive T cells become activated and can target and react with that specific autoantigen. The absence of costimulatory molecules on the surface of APC is most likely related to limiting the T cell response. Autoreactive effector T cells can only kill a limited number of antigen-expressing tissue cells. After killing a small number of target cells, the effector cells die. The self antigen presenting cells are then tolerized. Presentation of antigen to naive T cells by tolerizing antigen presenting cells induces regulatory T cells. Subsequently, regulatory T cells prevent activation of other potential autoreactive T cells by stimulating antigen presenting cells.

  Thus, in some embodiments, an antibody of the present disclosure can be utilized to form an antibody / self-antigen construct that can bind to hepatic sinusoidal endothelial cells, thereby stimulating the growth of regulatory cells. The antibodies of the present disclosure can or can be utilized to form antibody / self-antigen constructs that can bind to hepatic sinusoidal endothelial cells, thereby inhibiting the activity of autoreactive T cells. The antibody / autoantigen constructs of the present disclosure can also be used to deliver vaccine antigens to sinusoidal endothelial cells, including those found in lymph nodes, thereby stimulating the proliferation of antigen-specific T cells. it can.

  The antibody / autoantigen construct can be obtained by known methods such as intravenous or intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional injection or infusion, topical, Alternatively, it can be administered according to the sustained release system as described below. The antibody / autoantigen construct is preferably administered continuously by infusion or by bolus injection. The antibody / self-antigen construct can be administered in a local or systemic manner.

  The antibody / autoantigen construct can be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulating and administering the compounds of the present application are described in "Ramington's Pharmaceutical Sciences", Mack Publishing Co. Can be seen in the latest edition, Easton, Pennsylvania. The therapeutic composition can be administered intravenously or through the nose or lung, preferably as a liquid or powder aerosol (lyophilized). The composition can also be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic composition should be sterile, free of heat source materials, and in a parenterally acceptable solution with due consideration of pH, isotonicity, and stability. These conditions are known to those skilled in the art.

  Briefly, a dosage formulation of the antibody / autoantigen construct can be stored or administered by mixing a compound having the desired degree of purity with a physiologically acceptable carrier, excipient, or stabilizer. Be prepared for. Such substances are non-toxic to recipients at the dosages and concentrations used, buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; Low molecular weight (less than about 10 residues) peptides such as polyarginine, and proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; Sugars, disaccharides, and other hydrocarbons including cellulose or derivatives thereof, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or TWEEN, PLURO It may include non-ionic surfactants such as ICS or polyethylene glycol.

  When used for in vitro administration, the antibody / autoantigen construct formulation must be sterile and can be formulated according to conventional pharmaceutical practice. This is easily accomplished by filtration through sterile filtration membranes before or after lyophilization and reconstitution. Antibodies are usually stored in lyophilized form or in solution. Other vehicles such as naturally occurring vegetable oils such as sesame, peanut or cottonseed oil or synthetic fat vehicles such as ethyl oleate may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to generally accepted pharmaceutical practice.

  Pharmaceutical compositions suitable for use include compositions wherein one or more antibody / autoantigen constructs are included in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of antibody effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. A therapeutically effective amount can be determined by using in vitro and in vivo methods.

  The effective amount of antibody / self-antigen construct used in therapy will depend, for example, on the therapeutic objectives, the route of administration, and the condition of the patient. The attending physician may also alter the action of the drug, including severity and disease type, weight, gender, diet, time and route of administration, other drug applications and other relevant clinical factors. Various known factors are taken into account. Therefore, the therapist will need to titrate the dosage and change the route of administration as necessary to obtain the optimal therapeutic effect. Typically, the clinician will administer antibody / autoantigen constructs until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

For any antibody / autoantigen construct, the therapeutically effective dose can be estimated initially from cell culture assays. For example, it prescribes a dose in animal models, it is possible to achieve a circulating concentration range that includes the EC ₅₀ s are measured in cell culture (e.g., the concentration of the test molecule which promotes or inhibits cellular proliferation or differentiation). Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the antibody / autoantigen constructs described herein are, for example, LD ₅₀ (a dose that is lethal to 50% of the population) and ED ₅₀ (a dose that is therapeutically effective in 50% of the population). To do so, it can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Molecules that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such molecules is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the respective physician in view of the patient's condition. (See, for example, Chapter 1, page 1 of “Finger et al., 1975,“ The Pharmacological Basis of Therapeutics ”.)
Dosage amount and interval can be adjusted individually to provide plasma levels or minimal effective concentrations (MECs) of the antibody / self-antigen construct that are sufficient to promote or inhibit cell proliferation or differentiation. The MEC will vary for each antibody / autoantigen construct, but can be estimated from in vitro data using the described assays. The dosage required to achieve MEC will depend on individual characteristics and route of administration. However, plasma concentrations can be determined using HPLC assays or bioassays.

  Dosage intervals can also be determined using the MEC value. The antibody / autoantigen construct molecule is administered using a regimen that maintains plasma levels above the MEC for between 10-90% of the time, preferably between 30-90%, most preferably between 50-90%. Should be.

  In cases of local administration or selective uptake, the effective local concentration of the antibody / autoantigen construct may not be related to plasma concentration.

  A typical daily dosage may range from about 1 μg / kg to 1000 mg / kg or more, depending on the factors described above. Typically, the clinician will administer the antibody / autoantigen construct until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

  Depending on the type and severity of the disease, for example, from about 0.001 mg / kg to about 1000 mg / kg, more preferably about 0, whether by one or more separate administrations or by continuous infusion. An antibody / autoantigen construct of .01 mg to 100 mg / kg, more preferably about 0.010 to 20 mg / kg may be the first candidate dosage for administration to a patient. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs or a desired improvement in the patient's condition is achieved. However, other dosing regimes may also be useful.

  In particularly useful embodiments, the disclosed methods can be used to treat diabetes mellitus by inducing immune tolerance to the insulin-producing β cells of the islets of Langerhans in the pancreas. The autoantigens of these cells are linked to an antibody that recognizes the desired antigen internalization receptor. Suitable autoantigens for use in the present disclosure are beta cell antigens as well as epitopes, or peptides presenting epitopes, of insulin, glutamate decarboxylase (“GAD”) and heat shock protein (“HSP”). Linking a series of peptides covering epitopes from insulin, GAD and hsp to anti-DC-SIGNR antibodies has the ability to induce tolerance to all major antigens involved in T1DM. Other autoantigens are known and can be used or discovered and used by those skilled in the art.

Antigen internalizing receptors are presented on specialized APCs. For this method, the antigen-internalizing receptor selected is DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-capturing non-integrin-related receptor). DC-SIGNR is expressed by hepatic sinusoidal endothelial cells (“LSEC”), which are liver-resident antigen presenting cells. ( ¹ Pohlmann et al., 2001). DC-SIGNR belongs to a family of pathogen internalization receptors that internalize receptor binding proteins and facilitate antigen presentation. ( ¹ Geijtenbeek et al., 2002). It has been shown that presentation of antigens by LSEC results in antigen-specific tolerance (Limmer et al., 2000). In contrast to other dendritic cell types that can mature from an immature tolerogenic state to an activated state, hepatic sinusoidal endothelial cells cannot be induced to develop into activated antigen presenting cells ( ² Knoll et al., 1999). Human DC-SIGNNR (also referred to as L-SIGN) homologue to human DC-SIGN shows 77% identity to DC-SIGN at the amino acid level and has a typical domain of internalization receptors (Bashirova Et al., 2001, Soleilux et al., 2000). DC-SIGNR is highly expressed in LSEC and is also found in a subpopulation of lymph node macrophage-like cells, but is not expressed by DC.

  For the purposes of this disclosure, the terms “DC-SIGNR” and “L-SIGN” are used interchangeably.

C-type lectin mouse DC-SIGN (DC209) has recently been identified as a DC-specific receptor. DC-SIGN mediates the transendothelial migration of DC, allowing an initial immune response by initiating transient DC-T cell interactions ( ³ Geijtenbeek et al., 2000, ² Geijtenbeek et al., 2000) . DC-SIGN also serves as an internalizing antigen receptor that recognizes pathogens through carbohydrate structures. In addition to its prominent role in the initiation of DC-T cell clustering and T cell responses, DC-SIGN is also associated with HIV-1, HIV-2, SIV-1, hepatitis C virus (HCV), Ebola virus, site Viruses such as megalovirus (CMV) and dengue fever virus; bacteria such as Helicobacter pylori, Klebsiella pneumoniae and Mycobacterium tuberculosis; yeasts such as Candida albicans; pifanoi), a major receptor involved in the infection of DCs and subsequent transmission to T cells of parasites such as Schistosoma mansoni. MSIGNR1, a mouse homologue of DC-SIGNR, captures antigens that are rapidly internalized and targeted to lysosomes for processing ( ¹ Geijtenbeek et al., 2002). Based on the amino acid sequence, mouse mSIGNR1 is as homologous to human DC-SIGNR as it is to human DC-SIGN and is therefore useful for animal modeling studies.

  Accordingly, in another aspect, the present disclosure relates to anti-DC-SIGNR (ie, anti-L-SIGN) antibodies. As mentioned above, the term “antibody” as used herein refers to a whole immunoglobulin molecule or a molecule comprising an immunologically active part of a whole immunoglobulin molecule, Fab, F (ab ′) 2, scFv, Fv, heavy chain variable region and light chain variable region.

  In certain embodiments, the antibodies of this disclosure comprise a light chain. As used herein, “light chain” means the smaller polypeptide, or fragment thereof, of an antibody molecule composed of one variable domain (VL) and one constant domain (CL). To do. In another embodiment, the portion of the antibody comprises a heavy chain. As used herein, a “heavy chain” is the large of an antibody molecule composed of one variable domain (VH) and three or four constant domains (CH1, CH2, CH3, and CH4) Of the other polypeptide, or a fragment thereof.

In another embodiment, the antibody comprises the Fab portion of the antibody. As used herein, “Fab” means a monovalent antigen-binding fragment of an immunoglobulin that consists of one light chain and part of a heavy chain. This can be obtained by brief papain digestion or by recombinant methods. In another embodiment, the portion of the antibody comprises the F (ab ′) ₂ portion of the antibody. As used herein, “F (ab ′) ₂ fragment” means a divalent antigen-binding fragment of an immunoglobulin that consists of both light chains and part of both heavy chains. This can be obtained by brief pepsin digestion or by recombinant methods. In other embodiments, the antibody can be a Fab ′ fragment. A Fab expression library can be obtained, for example, by the method of Huse et al., Science 245: 1275 (1989).

  In addition, “humanized” antibodies that bind to receptors on antigen-presenting cells may be used. Methods of humanization are disclosed, for example, in WO 98/49306, US Pat. Nos. 5585089, 5225539, and 5697661 and WO 90/07861, the disclosure of which Incorporated herein by reference in its entirety. As used herein, a “humanized” antibody is an antibody in which amino acids outside the CDRs are replaced with the corresponding amino acids from a human immunoglobulin molecule. “CDR” or “complementarity determining region” refers to a highly variable sequence of amino acids in the variable domain of an antibody. Using recombinant DNA technology, the immunoglobulins of one immunoglobulin variable region have different specificities so that the humanized antibody recognizes the desired target but is not significantly recognized by the immune system of the human subject Humanized antibodies can be generated that are substituted with the CDRs of

  In other embodiments, chimeric antibodies comprising non-human variable domains that bind to any receptor on any antigen presenting cell linked to a human constant region are contemplated. The receptor to which the variable domain binds can be, for example, L-SIGN or DC-SIGN, and the constant region can be a human IgG1 constant region. Surprisingly, in certain embodiments described in more detail below, such chimeric antibodies may exhibit advantageous binding to the receptor on any antigen presenting cell over the variable domain alone. know.

  For conversion of antibody clones to complete IgG, the coding regions of both the light and heavy chains, or fragments thereof, can be cloned separately from the bacterial vector into the mammalian vector (s). it can. Using a single vector system such as pDR1 or its derivatives, both light and heavy chain cassettes can be cloned into the same plasmid. Alternatively, dual expression vectors can be used in which the heavy and light chains are generated by separate plasmids. The mammalian signal sequence must either be already present in the final vector or be added to the 5 'end of the light and heavy chain DNA inserts. This can be accomplished by first strand transfer to a shuttle vector (s) containing the appropriate mammalian leader sequence. Following restriction enzyme digestion, the light and heavy chain regions, or fragments thereof, are introduced into the final vector (s) where the remaining constant regions of IgG are provided with or without introns.

  The antibodies according to the present disclosure can bind to both L-SIGN and DC-SIGN and bind preferentially to L-SIGN. That is, the binding to L-SIGN can be stronger than the binding to DC-SIGN. The binding to L-SIGN can be, for example, 1.1 to 200 times stronger than the binding to DC-SIGN. Alternatively, the binding to -SIGN can be 1.2-100 times stronger than the binding to DC-SIGN.

  In some embodiments, the antibody against DC-SIGNR modulates, ie inhibits or enhances the interaction of DC-SIGNR expressing cells with ICAM expressing cells. In one embodiment, the anti-DC-SIGNR antibody binds to a DC-SIGNR receptor site on the surface of an antigen presenting cell such as LSEC and prevents interaction (s) between LSEC and T cells. More specifically, antibodies against DC-SIGNR reduce adhesion between LSECs and T cells by interfering with adhesion between DC-SIGNRs on the surface of T cells and ICAM receptors. By blocking this interaction, the immune response can be modulated. For example, antibodies to DC-SIGNR can modulate immune function by blocking the interaction of L-SIGN with T cells, thereby increasing the proliferation of regulatory T cells, including those found in the liver. Can cause. In another embodiment, antibodies against DC-SIGNR can modulate immune function by blocking the interaction of L-SIGN with T cells, thereby causing self-relevance, including those found in lymph nodes. Reactive T cell proliferation can be suppressed.

  In some further embodiments, the antibodies of the present disclosure can be utilized to form antibody-peptide constructs that can bind to hepatic sinusoidal endothelial cells, thereby stimulating the growth of regulatory cells. Or can utilize the antibodies of the present disclosure to form antibody-peptide constructs that can bind to hepatic sinusoidal endothelial cells, thereby inhibiting the activity of autoreactive T cells can do. The antibody-peptide constructs of the present disclosure can also be used to deliver vaccine antigens to sinusoidal endothelial cells, including those found in lymph nodes, thereby stimulating the proliferation of antigen-specific T cells. .

  As used herein, “ICAM receptor (s)” refers to both ICAM-2 and ICAM-3 receptors, particularly ICAM-3.

  In some embodiments, an antibody against DC-SIGNR of the present disclosure does not bind to DC-SIGN. In other embodiments, the antibodies of the present disclosure have a high affinity for DC-SIGNR and a low affinity for DC-SIGN. The antibodies of the present disclosure may be capable of binding to both linear and steric epitopes on DC-SIGNR.

  It is also contemplated that the antibody or antibody / peptide construct can be labeled with a toxin against DC-SIGNR presenting cells. Administration of a toxin-labeled anti-DC-SIGNR antibody or anti-DC-SIGNR antibody / peptide construct can then be used to reduce the level of DC-SIGNR expressing cells, which may result in autoimmune disease, cancer Or it may be advantageous in some cases, such as the treatment of inflammatory diseases. In this way, DC-SIGNR expressing cells can also be killed or removed in vivo using the present antibodies or antibody / peptide constructs. This includes administering an antibody or antibody / peptide construct conjugated to a cytotoxic drug (eg, a toxin or radiation emitting compound) to a subject in need of such treatment. Since the antibody or antibody / peptide construct recognizes DC-SIGNR expressing cells (eg, cancer cells or hepatic sinusoidal endothelial cells), any such cells to which the antibody or antibody / peptide construct is bound are destroyed. In one embodiment, a method of treating cancer according to the present disclosure comprises an effective, killing amount of a cancer cell with a toxin-conjugated anti-DC-SIGNR antibody or anti-DC-SIGNR antibody / peptide construct. Administration to cancer patients. In another embodiment, a method of treating an inflammatory disease according to the present disclosure comprises an effective, killing DC-SIGNR expressing cell in an amount of a toxin-bound anti-DC-SIGNR antibody or anti-DC-SIGNR. Administration of the antibody / peptide construct to a patient suffering from an inflammatory disease.

  In other embodiments, antibodies against DC-SIGNR of the present disclosure block viral entry into hepatocytes, such as hepatic sinusoidal cells, and their infection into other cells. The antibodies against DC-SIGNR of the present disclosure can also block viral entry into T cells and their infection into other cells.

  In some embodiments, antibodies to DC-SIGNR of the present disclosure can be utilized to block infection by Mycobacterium spp. Infections that can be blocked include those caused by M. tuberculosis and M. bovis.

  By interfering with T-cell adhesion to antigen-presenting cells, the use of antibodies against DC-SIGNR has made antigen-presenting cell-T cell clustering, T cell activation, and contact between antigen presenting cells and T cells. Affects other interactions that depend on These other interactions include direct cell-cell contact or the close proximity of antigen presenting cells and T cells.

  In other embodiments, an anti-DC-SIGNR antibody of the present disclosure is linked to a peptide, such as a self antigen, or a peptide of a self antigen. These peptides can be linked to the anti-DC-SIGNR antibody by any suitable method, including transplanting the vector into an antibody fragment and cloning the linked vector / antibody or chemically linking. Can do. Methods of vector ligation, cloning or chemical ligation are well known to those skilled in the art.

A peptide, preferably a self-antigen, along with the linked antibody is then internalized in the LSEC. LSEC has surface molecules necessary for antigen presentation, such as MHC II, CD80 and CD86 (Lohse et al., 1996, Rubinstein et al., 1986). In addition to inducing a regulatory phenotype in naïve CD4 + T cells (Knolle et al., 1999), LSEC can induce tolerance in CD8 + T cells by cross-presenting exogenous antigens. (Limmer et al., 2000). LSEC responds to stimulation as TNF-α and endotoxin by preventing MHC down-regulation and endosomal processing ( ² Knoll et al., 1999). Furthermore, LSEC does not migrate from the liver to the lymphoid organs.

  This internalization facilitates the presentation of self-antigen peptides to the surface of LSECs mediated by MHC interactions. Once the autoantigen is expressed on the surface of LSECs that have costimulatory capabilities, untreated self-reactive T cells can become activated. T cells target and react with linked autoantigens. Effector T cells kill a few LSECs and die without costimulatory molecules. This presentation of autoantigens by LSEC results in self-antigen specific tolerance.

The liver has a unique microenvironment with abundant immune tolerance-inducing mediators such as IL-10 and TGF-β, and specialized APCs that favor the development of immunological tolerance ( ¹ Knoll and Gerken, 2000). The tolerogenic properties of the liver are supported by the discovery that allogeneic liver transplantation can be tolerated across the MHC barrier (Calne, 1969). Furthermore, application of antigen by portal vein is more likely to lead to tolerance than systemic application of antigen (Kamei et al., 1990). Excretion through the liver has been reported to be a requirement for oral tolerance induction (Yang et al., 1994). Blood passing through the hepatic blood vessels first comes into contact with Kupffer cells and LSECs. Blood flow through the hepatic sinusoids is slow, allowing contact between hepatic sinusoidal cell populations and passing white blood cells. LSEC has surface molecules necessary for antigen presentation, such as MHCII, CD80 and CD86 (Lohse et al., 1996, Rubinstein et al., 1986). In addition to inducing a regulatory phenotype in naïve CD4 + T cells (Knolle et al., 1999), LSEC can induce tolerance in CD8 + T cells by cross-presenting exogenous antigens. (Limmer et al., 2000). Klugewitz et al. (Klugewitz et al., 2002) found that injection of Th1, IFN-γ producing TCR transgenic cells into mice resulted in suppression of IFN-γ production by these cells in the liver after intravenous protein immunization, and Th2 It was shown to cause cell promotion. In contrast to professional myeloid APC, which can differentiate from an immature tolerogenic stage to a mature stage that initiates immunity, LSEC prevents THC-α by preventing MHC down-regulation and endosomal processing. And responds to stimuli as endotoxins ( ² Knoll et al., 1999). Furthermore, LSEC does not migrate from the liver to the lymphoid organs. LSEC may not be the only APC that is specialized when inducing tolerance. Recently identified a small subset of splenic DCs that induced tolerance by presenting endogenously expressed self-antigens (Pugliese et al., 2001). Overall, LSEC appears to be a desirable cell type for presenting β-cell antigens for the purpose of tolerance induction.

Also, both DC-SIGN and L-SIGN have many viruses such as HIV (Bashilova et al., 2001, ² Pohlmann et al., 2001), HCV (Gardner et al., 2003), Ebola (Alvarez et al., 2002). , Simons et al., 2003), SARS (Jeffers et al., 2004), CMV (Harry et al., 2002), and Sindbis (Klimstra et al., 2003). These receptors act as HIV and HCV attachment points, spreading them prematurely to other cells, such as T cells (Bashilova et al., 2001, ² Pohlmann et al., 2001, Cormier et al., 2004). Year), they also bind to Ebola, SARS, CMV and Sindbis virus, allowing them to enter and infect receptor-expressing cells. Also, both DC-SIGN and L-SIGN are bacterial pathogens of the genus Mycobacterium including Mycobacterium tuberculosis ( ⁴ Geijtenbeek et al., 2003, Koppel et al., 2004), and parasites such as Schistosoma mansoni ( Serves as a receptor for infection by Van Liempt et al., 2004).

  Accordingly, in another embodiment, the antibodies of the present disclosure are utilized to bind to DC-SIGN and / or L-SIGN, thereby causing viruses such as HIV, HCV, Ebola, SARS, CMV and Sindbis, human tuberculosis It can block the binding, infection and transmission of infectious agents including but not limited to bacterial pathogens of the genus Mycobacterium, including fungi and Mycobacterium bovis, and parasites such as Schistosoma mansoni.

  The antibodies against DC-SIGNR of the present disclosure can also be used as a common diagnostic method for tumor types associated with DC-SIGNR expression. For example, because up-regulation of immune receptors is expected to occur only under pressure by the immune system, up-regulation of DC-SIGNR in cancer samples assesses whether the cancer has become exposed to the immune system Could be used as the basis of diagnostic tools for Using methods known to those skilled in the art, including immunohistochemistry and / or FACS analysis, a tumor biopsy is exposed to an antibody against DC-SIGNR, and then binding is an indicator of cancer associated with DC-SIGNR expression The presence of the antibody may be analyzed. In some embodiments, the antibody may be provided as part of a diagnostic kit for determining the presence of a cancer that expresses DC-SIGNR.

  The antibodies against DC-SIGNR of the present disclosure can also be used as therapeutic techniques for cancer treatment. In one embodiment, an antibody of the present disclosure induces ADCC (antibody dependent cytotoxicity) or CDC (complement dependent cytotoxicity) of tumor cells, thereby killing the cells.

  The antibody or antibody / peptide construct can also be utilized to kill or remove cancerous cells in vivo. This includes administering an antibody or antibody / peptide construct conjugated to a cytotoxic drug to a subject in need of such treatment. Since the antibody or antibody / peptide construct recognizes cancer cells, any such cells to which the antibody or antibody / peptide construct is bound are destroyed.

  Various cytotoxic compounds can be delivered using the antibodies or antibody / peptide constructs of the present disclosure. Any cytotoxic compound can be fused to the antibody or antibody / peptide construct. Fusion can be accomplished chemically or genetically (eg, by expression as a single fusion molecule). A cytotoxic compound can be a biological or small molecule such as a polypeptide. As one skilled in the art will appreciate, chemical fusion is used for small molecules, but for biological compounds, either chemical or genetic fusion can be used.

  Using the antibodies or antibody / peptide constructs of the present disclosure, a variety of cytotoxic drugs, including therapeutic agents; compounds that emit radiation; molecules derived from plants, fungi, or bacteria; biological proteins; and mixtures thereof. Can be delivered. The cytotoxic drug may be a cytotoxic drug that acts intracellularly, such as a short-range radiation emitter, including, for example, a short-range high-energy α-emitter. Enzymatically active toxins and fragments thereof include, for example, diphtheria toxin A fragment, non-binding active fragment of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modesin A chain, alpha-sacrine, certain Aureites fordii proteins, certain diantine proteins, Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantia ) Inhibitors, Kursin, Crotin, Saponaria officinalis inhibitors, gelonin, mitodiline, restrictocin, phenomycin, and et Exemplified by puromycin. Procedures for the preparation of immunotoxins, enzymatically active polypeptides, are described in WO 84/03508 and WO 85/03508, which are incorporated herein by reference. Yes. Certain cytotoxic moieties are derived from, for example, adriamycin, chlorambucil, daunomycin, methotrexate, neocalcinostatin, and platinum.

  Procedures for conjugating an antibody or antibody / peptide construct with a cytotoxic agent have been described previously.

Alternatively, an antibody or antibody / peptide construct of the present disclosure binds to a radioisotope such as ¹³¹ I which is a gamma emitter that kills several cell diameters when localized at the tumor site May be. See, for example, S., which is incorporated herein by reference. E. Order, “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies in Cancer Threat, Monoclonal Antibiotic.” W. See Baldwin et al. (Ed.), Pages 303-316 (Academic Press, 1985). Other suitable radioisotopes include α-emitters such as ²¹² Bi, ²¹³ Bi, and ²¹¹ At, and β-emitters such as ¹⁸⁶ Re and ⁹⁰ Y. Because prostate cancer is a relatively radiosensitive tumor, radiation therapy is expected to be particularly effective in connection with prostate cancer.

  In another embodiment, an antibody to L-SIGN of the present disclosure is used to treat a tumor by binding to L-SIGN and preventing negative regulation of the immune system through L-SIGN expressing cancer cells. It can be used as a therapeutic technique. By preventing this negative regulation, the immune system can progress and eradicate cancer cells.

  The efficacy of the treatment described above can be confirmed using methods known to those skilled in the art, including xenograft models.

  An antibody against L-SIGN according to the present disclosure utilized as a cancer therapy is also a cancer treatment in combination with any other immunomodulatory therapy such as cancer vaccine, anti-CTLA-4, anti-CD25 or cyclophosphamide. An increase in therapeutic efficacy can be achieved.

  Antibodies to L-SIGN according to the present disclosure may be used in some embodiments to isolate L-SIGN expressing cells found in a mixture of cells including cells that do not express L-SIGN. . For example, human non-parenchymal hepatocytes comprising a mixture of sinusoidal endothelial cells, red blood cells, Kupffer cells and a small number of other cell populations can be readily obtained from commercial sources. To isolate L-SIGN expressing cells, i.e., hepatic sinusoidal cells, from human non-parenchymal hepatocytes, erythrocytes can be exposed to agents such as ammonium chloride that cause lysis of erythrocytes. Dead cells can be removed using commercially available dead cell removal kits, including those sold by Miltenyi Biotech (Germany). The remaining cells can be counted and labeled with anti-L-SIGN antibody. After washing the cells, they are resuspended in an appropriate buffer and the remaining cells are exposed to anti-L-SIGN antibody, which in some cases may be conjugated to beads or similar separation media. Cells, that is, L-SIGN expressing cells can be isolated. Commercial separation media and methods of their use are known to those skilled in the art and include commercially available beads from Milteny. In some particularly useful embodiments, L-SIGN expressing cells are transformed into L-SIGN by using anti-L-SIGN composite beads, such as anti-mouse IgG composite beads, according to the manufacturer's instructions (Milteny). It can be isolated from cells that are not expressed. Cells that bind to the anti-L-SIGN antibody are L-SIGN expressing cells, and are thereby isolated from the remaining cells that do not express L-SIGN. Once isolated from the remaining cells, L-SIGN expressing cells can be removed from the anti-L-SIGN antibody using methods readily known to those skilled in the art. Isolation quality should be monitored by methods known to those skilled in the art, including FACS analysis using a panel of antibodies against molecules such as CD54, mannose receptor, LYVE-1, CD40, asialoglycoprotein and others. Can do.

  The practice of this method, including its further preferred aspects and embodiments, will be better understood and should not be construed as limiting in any way from the following examples, which are presented for purposes of illustration only.

Example 1
Obtaining anti-mSIGNR1 antibodies Using phage display technology, a panel of single chain antibodies (scFv) that recognize mSIGNR1 were identified. The scFv includes a variable light chain region and a variable heavy chain region connected by a linker. Their short length makes these antibody fragments very suitable for antigen binding and preserves the ability to bind to the receptor. The phage display vector pRL4, described in International Application Publication No. 02 / 46436A2, published June 13, 2002, immunized with recombinant mSIGNR1, the disclosure of which is incorporated herein by reference. Was used to construct a scFv antibody library. Antibody fragments in this system are displayed on the gene III coat protein of the phage. Antibodies recognizing mSIGNR1 were isolated by four solid phase pannings of recombinant mSIGNR1. Six different antibodies were identified. The amino acid sequences of these six antibodies are shown in FIGS. 2A and B (SEQ ID NOs: 1-6 and 7-12, respectively). All antibodies recognized mSIGNR1 in solid phase ELISA and no cross-reactivity with mDC-SIGN, a mouse homologue of human DC-SIGN, was observed. The antibody was epitope tagged with HA and HIS ₆ . Both mSIGNR1 and DC-SIGNHIS were generated by 3T3 EBNA cells and purified on a nickel column.

(Example 2)
Identifying anti-mSIGNR1 antibodies that are internalized upon binding to cell surface receptors Screening for cell lines expressing mSIGNR1 A panel of mouse macrophage cell lines (P388D1, 1-13.35, WEHI-3 and J774) is screened for expression of mSIGNR1 by RT-PCR by standard methods. Primers were designed based on the mSIGNR1 GenBank sequence and used in mouse organ RT-PCR. Cell lines expressing mSIGNR1 at the mRNA level are identified and surface expression is confirmed by FACS analysis. 5 × 10 ⁵ cells were incubated with 1 μg of anti-mSIGNR1 antibody in PBS containing 1% BSA and 0.1% NaN ₃ for 15 minutes on ice in conditions that did not allow antibody internalization. After two washes with PBS containing 1% BSA and 0.1% NaN ₃ , bound anti-mSIGNR1 was detected with biotinylated anti-HA (Roche) followed by PE-conjugated streptavidin (Becton Dickinson) Cells are analyzed using a FACSCalibur (Becton Dickinson, Mountain View, CA). Alternatively, internalization is determined in primary cells known to express mSIGNR1, such as hepatic sinusoidal endothelial cells. The expression of mSIGNR1 in LSEC can also be confirmed by FACS as described above, but only 1 × 10 ⁵ cells are added per reaction.

Internalization Measurement Once an mSIGNR1-expressing cell line or primary cell type is identified, internalization of a panel of antibodies is assessed by FACS analysis. In order to show that internalization is based on mSIGNR1 binding, cell lines that do not express mSIGNR1, such as JAWS1 mouse dendritic cells, are included. Anti-mSIGNR1 detection using biotinylated anti-HA antibody in intact and permeabilized cells followed by PE-conjugated streptavidin is compared as described for anti-DEC-205 antibody (Mahnke et al., 2000). Incubate 1.5 × 10 ⁶ cells for cell lines or 3 × 10 ⁵ cells for primary cells with 3 μg mSIGNR1 in PBS containing 1% bovine serum albumin (BSA) for 20 minutes at 4 ° C. The antibody is bound to the surface without internalization. Unbound antibody is removed by two washes with PBS containing 1% BSA at 4 ° C. Divide each sample into three. One third is fixed with 4% paraformaldehyde and surface antibodies are detected as described above. The other two-thirds are further incubated at 37 ° C. for 30 minutes to allow internalization before fixation. One-half is detected directly with anti-HA and streptavidin and the other half is permeabilized by incubating the cells with PBS containing 0.1% (volume / weight) saponin (Sigma-Aldrich). The amount of internalized antibody is calculated by subtracting the average fluorescence in the fixed cells from that recorded with the fixed and permeabilized cells. The antibody with the highest percentage of internalization within 30 minutes is selected for further studies to link the peptide to the antibody. An existing, unrelated rabbit scFv is used as a negative control, and a commercially available ER-TR9 antibody recently shown to bind to mSIGNR1 ( ¹ Geijtenbee et al., 2002) is used as a positive control. Also, Fab fragments of ER-TR9 are generated by papain digestion and tested for internalization to demonstrate that dimerization is not a requirement for internalization. If desired, the scFv may be converted to Fab′2 or IgG.

  In an alternative embodiment, the mSIGNR1 library is Panning for internalized antibodies as described by Marks group (Poul et al., 2000). A process suitable for this embodiment is outlined below.

Selection of Internalized Antibody from mSIGNR1 Phage Library As described above, from the mSIGNR1 library displaying 5 × 10 ⁶ cells identified to express mSIGNR1 on the surface and fused to gene 3 protein. Incubate with 1 × 10 ¹² colony-forming units of 1 phage for 1.5 hours at 4 ° C. to allow phage binding without internalization. After phage binding, the cells are washed 5 times with phosphate buffered saline to remove non-specifically or weakly bound phage. The cells are then incubated at 37 ° C. for 15 minutes to allow surface-bound phage endocytosis but avoid intracellular phage degradation. To remove phage bound to the cell surface, the cells are removed by washing 3 times with low pH glycine buffer. The cells are then trypsinized, washed with PBS and lysed with high pH triethylamine. The cell lysate containing the phage is used to infect E. coli and prepare the phage for the next round of selection. Make a total of 3 selections. The titer of phage bound to the cell surface (as seen in the first low pH glycine wash) and the number of phage recovered from within the cell are monitored for each round. The increase in the number of endocytosed phage indicates a successful selection of internalized phage antibodies.

To determine if any of the internalized scFv antibody fragments bound to mSIGNR1, the third 500 clones were selected using the Robot Qpix (Genetics) system and in HiGrow Shaker (Gene Machines) Grow overnight in 96 well dishes in SB medium. The next day, the dish is slowed down and the supernatant is tested in a solid phase mSIGNR1 ELISA using the Robot Genesis Freedom 200 (Tecan) system. Coat 96-well ELISA plates with 1 μg mSIGNR1 / ml PBS overnight at 4 ° C. The next day, the plate is blocked with 1% BSA, followed by 3 washes with PBS containing 0.05% Tween. Coat control plates with 1% BSA. The supernatant containing the antibody is added to wells of mSIGNR1 or BSA only at a concentration between 0.05-5 μg / ml in PBS containing 1% BSA. After 2 hours at room temperature on a shaker, the plate is washed 3 times with PBS containing 0.05% Tween. For detection of bound scFv, anti-HA antibody (12CA5 mouse ascites, Strategic Biosolutions, Del.) Is added at a 1: 1,000 dilution in PBS containing 1% BSA. After 2 hours on a shaker at room temperature, the plates are washed again and alkaline phosphatase conjugated anti-mouse IgG (Sigma) is added over 2 hours. After three additional washes, bound antibody is detected using Sigma 104® substrate. The plate is read at various times using an ELSA plate reader (Molecular Device) at OD ₄₀₅ .

Clones that show a positive signal in the ELISA are characterized by a restriction enzyme digestion pattern. DNA is isolated using a Qiagen miniprep kit. 2 μg of DNA is digested with 5 U EcoRII for 2 hours at 37 ° C., then the sample is run on a 4% NuSieve agarose gel. Compare the patterns and purify the sequence in small amounts (about 100-300 μg). ScFv is properly assembled and secreted in the periplasmic space of bacteria. The scFv may be isolated from either the supernatant or the periplasmic space. The clones were grown in 4 liters of SB to an OD _{600 of} 0.8 and induced with 1 mM isopropyl-pD-thiogalactopyranoside (IPTG) for 3-4 hours at 30 ° C. ScFv is generated. To isolate single chain antibodies from the periplasmic space, cell pellets are resuspended in cold PBS supplemented with Complete Mini (Roche) protease inhibitors and sonicated with a Sonics Vibra-cell VC750. Cell debris is pelleted and the supernatant is applied to a Qiagen Ni-NTA column using Akta FPLC (Pharmacia). The antibody is eluted with imidazole. This method generally yields about 100-300 μg of purified antibody / liter. Endotoxin is removed by filtration through a Sartorius QI5 filter, generally resulting in an antibody preparation containing less than 10 U / ml endotoxin as measured by the LAL test (an assay commercially available from BioWhittaker). Antibodies are analyzed again for internalization as described above and analyzed for binding to recombinant mSIGNR1 in a solid phase ELISA. Select the antibody with the highest percentage of internalization within 30 minutes and a good signal in solid phase ELISA (> 10D after 1 hour at 1 μg / ml) to make a peptide-antibody construct.

(Example 3)
Vectors and Cloning Strates that Bind GAD Peptides to Antibodies Following the identification of the best bacterially produced scFv, conversion to a mammalian expression system is performed. Mammalian expression allows appropriate secondary modification of the peptide and production without endotoxin. A vector with compatible restriction enzyme recognition sites as shown in FIG. 3 is used (eg, the disclosure of which is described in US Pat. No. 6,355,245, incorporated herein by reference). DNA from the antibody of interest in pRL4 (described above) is cut with Sfi and inserted into an Apex 3P vector containing a CMV promoter and a mammalian antibody leader sequence. Available sites (MCS = NaeI, FseI, XbaI, EcoRI, PstI, EcoRV, BSABI, BstXI, NotI, BsrBI, Xho, not included in the antibody sequence to insert nucleotides encoding the peptide of interest. Restriction enzyme recognition sites are selected from PbvIOI, SphI, NsiI, and XbaI). An oligonucleotide encoding a peptide is synthesized by an operon having appropriate restriction enzyme recognition sites at both ends and inserted using T4 DNA ligase. The resulting construct contains an scFv followed by a spacer determined by the selected restriction enzyme followed by a peptide and a HIS-tag (FIG. 3). After sequence confirmation using standard techniques, DNA is transfected into 393 EBNA cells for antibody production.

Peptide Selection It should be understood that any of the known diabetic autoantigen peptides insulin, hsp and GAD 65 and 67 can be used in the methods described herein, Therefore, GAD is selected as the peptide. GAD-reactive T cells are the first autoreactive T cells detected in NOD mice (Tisch et al., 1993, Kaufman et al., 1993) and have been shown to be important in the disease process. Furthermore, human and mouse GAD are 95% homologous. Epitopes recognized by splenic NOD T cells have been extensively characterized (Kaufman et al., 1993, Tisch et al., 1999, Zechel et al., 1998), and many immunodominant peptides are found in NOD mice and T1DM patients. Similar and used interchangeably in in vitro T cell assays (Kaufman et al., 1993). The initial immune response in NOD mice is directed against a defined region (peptide 509-528, peptide 524-543) in the carboxy terminal region of GAD65 (Kaufman et al., 1993). Later, T cell responses are also directed against other regions between 200-300 as well as other autoantigens. Early CD4 GAD65 T cell epitope, peptide 524-543 (SRLSKVAPVIKARMMEYGGT (SEQ ID NO: 13), same sequence in mouse and human) and late mouse GAD65 epitope, peptide 247-266 (NMYAMLLIARYK MFPEVKEKG (SEQ ID NO: 14), underlined 2 amino acids between human and mouse) and peptide 290-309 (ALGIGTDSVILIKCDGERGK (SEQ ID NO: 15), same sequence in mouse and human) were selected for use as peptides in this experiment To do. All three epitopes can induce a spontaneous proliferative response in NOD splenocytes. Furthermore, peptide immunization with peptides 247-266 and 290-309 has been shown to delay the onset of diabetes in NOD mice (Ma et al., 1997, Tisch et al., 1999, Zechel et al., 1998). In addition to CD4 T cell epitopes, tolerance to CD8 T cell epitopes has also been reported to be important (Quinn et al., 2001, Bercovici et al., 2000). As a negative control, antibody constructs are made with hen egg lysozyme peptide 116-124 (KGTDVQAWI (SEQ ID NO: 16). The most effective peptides from these in vitro studies are then ligated in various combinations with antibody constructs and NOD Test in a diabetes model.

Example 4
Antibody-Peptide Construct Production and Purification For T cell experiments, approximately 300 μg of each antibody construct is produced in EBNA293 human embryonic kidney cells. Cells are grown in DMEM containing 10% FCS, 2 mM glutamine and 250 U / ml G418 (Sigma). Cells in TI75 flasks are transfected with DNA using Qiagen Effectin reagent according to manufacturer's instructions. After 3 days, the medium is replaced with serum-free medium. Supernatants are collected on days 4 and 8, cell debris is removed by centrifugation, and the clarified supernatant is loaded onto a Ni column using Akta-FPLC. The antibody is eluted with imidazole, dialyzed into PBS, and the correct size is demonstrated by running 1 μg on an SDS gel.

(Example 5)
Internalization of antibody-peptide constructs by LSEC resulting in peptide presentation and the effect of this presentation on T cells With peptide-antibody constructs, hepatic sinusoidal cells are targeted in vitro and these cells are derived from young NOD or Balb lc mice Determine whether phenotypic changes can be induced in the T cells.

Isolation of mouse sinusoidal endothelial cells Liver sinusoidal endothelial cells are isolated from 3 week old NOD or 4-6 week old Balb / c mice. As described by Kretz-Rommel, the calcium is first chelated by portal perfusion with EGTA, loosening cell-cell contact, followed by perfusion with 0.05% collagenase A in Hanks buffer. Cells are obtained by degrading the interstitial matrix (Kretz-Rommel and Boelsterli, 1995). Remove the perfused liver from the mouse and gently work with bending forceps. The resulting crude cell suspension is filtered through a series of metal sieves (30, 50, 80 mesh) to remove larger tissue fragments. Sinusoidal cells are separated from parenchymal cells by density gradient centrifugation on a metrizamide gradient (1.089 g / cm 3), and cell debris is removed by two subsequent washing steps (3 Knoll et al., 1999). At this point, a mixture of Kupffer cells and hepatic sinusoidal cells is obtained. This is sufficient for FACS experiments because F4 / 80 antibodies that recognize Kupffer cells but not hepatic sinusoids can be used to distinguish Kupffer cells from hepatic sinusoid endothelial cells. However, for co-culture experiments with T cells and peptides, cells were labeled with PE-conjugated F4 / 80 (BD Pharmingen) followed by Milteny's anti-PE microbeads and a MACS column according to the manufacturer's instructions. And Kupffer cells are removed by magnetic separation of labeled cells using a separator. The remaining cell population is seeded in 96 well tissue plates in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 2% glutamine. The purity of the cell population is examined after 3 days of isolation by FACS staining for surface markers with anti-mSIGNR1 and anti-F4 / 80. There is no mSIGNR1 on Kupffer cells ( ¹ Geijtenbeek et al., 2002). A purity of 90% is considered sufficient to continue the experiment. Using two mouse livers per experiment, the expected yield is approximately 2 × 10 ⁷ cells (Knook and Sleyster, 1976, estimated for mice).

T cell phenotype assay The T cell assay indicates whether the peptide-antibody construct results in the presentation of the peptide by hepatic sinusoidal endothelial cells and whether the peptide presentation can induce a phenotypic change in the T cell. . Hepatic sinusoidal endothelial cells are cultured at a density of 1 × 10 ⁵ cells / well in flat bottom microtiter plates. After maintaining the sinusoidal cells for 3 days, CD4 + T cells were purified as described above from 3 and 8 week old NOD mice or 4 to 6 week old Balb / c mice, and 10 ⁴ or 10 ⁵ Add in cells / well. Also, add each antibody-peptide construct at a concentration of 0.1-5 pg / well. As a positive control, only each GAD and control peptide is included. Peptides are synthesized by SynPep (Dublin, CA). Negative control wells contain T cells alone or hepatic sinusoid cells alone.

  There are four possible outcomes of peptide presentation to T cells by LSEC. 1) induction of regulatory T cells, characterized by production of TGF-P and / or IL-10 and IL-4 or expression of CD4 + CD25 + CD62L, 2) deletion of T cells or 3) complete absence or response. 4) Instead of inducing tolerance, it is also conceivable that peptide presentation results in stimulation of Th1 cells producing IL-2. To distinguish between these possibilities, culture supernatants (100 pl each) are collected at 24 and 48 hours and assayed for cytokine production as described below. The 3 week old T cell response is compared to that of 8 week old NOD mice as well as T cells from Balb / c mice that do not show a spontaneous response to GAD. The assay is assembled in triplicate and repeated twice. Since a mixture of T cells is used, the cytokine response in the supernatant will not be readily visible. However, the mixture of cells reflects the in vivo state, and a GAD-specific T cell response is seen using whole splenocytes (Tisch et al., 1993).

  As a more sensitive measure for the induction of regulatory T cells, expression of typical surface markers such as CD25, CD4 and CD62L (Lafile and Lafile, 2002) by FACS analysis after 3 days in culture Evaluate the cells. All reagents are available from BD-Pharmingen. In addition, IL-4 production is analyzed by FACS. A functional test of the potential immunomodulatory properties of LSEC / peptide-exposed T cells as described below is the final test for tolerance induction in the system. The potential of the peptides presented by LSEC to induce cell death is assessed in the culture supernatant using a Roche cell death ELISA kit according to the manufacturer's instructions.

Isolation of Spleen Cells and CD4 + T Cells Using techniques within the purview of those skilled in the art, the spleen from the NOD of Balb / c mice is removed in a sterile environment and placed in PBS. The cells are separated using an 18-21 standard needle and the larger piece is allowed to settle. Remove the supernatant and centrifuge at 200 g for 7 minutes. Erythrocytes are lysed using 5 ml 0.83% NH ₄ Cl per spleen. Cells are washed twice in PBS and then resuspended in medium. For certain experiments, whole splenocytes are used. For other experiments, CD4 + T cells are isolated using the Milteny (Auburn, Calif.) CD4 + T cell isolation kit according to the manufacturer's instructions. Magnetic isolation of various cell populations is within the purview of those skilled in the art. In some methods, isolation is based on depletion of non-CD4 + T cells using a cocktail of biotin-conjugated monoclonal antibodies against CD8a, CDI 1b, CD45R, DX5 and Ter-1 19. The purity of the isolated population is assessed by staining of a mixture of FITC conjugated anti-CD4, PE conjugated anti-CD8, APC conjugated anti-CD11b and cy-5 conjugated CD45R (all eBioscience, San Diego, Calif.). The expected purity is 90-95% with a yield of 70%. At least 1 × 10 ⁸ cells can be obtained from the mouse spleen, about 1.75 × 10 ⁷ cells, about 25% splenocytes are CD4 +, sufficient for 175 96-well microtiter plate wells Cells can be obtained.

Measurement of cytokine production The presence of IL-10, TGF-P, IFN-γ, IL-4 and IL-2 in the supernatant of the T cell / LSEC co-culture was determined as described in a standard sandwich. Measured by ELISA (Kretz-Romel and Rubin, 1997). All antibody pairs are available from BD Pharmingen. The cytokine capture antibody is coated on the plate overnight at 4 ° C. in PBS. After three washes with PBS / 0.05% Tween, culture supernatant and standard curve mouse recombinant cytokines are added and incubated on a shaker for 2 hours at room temperature. The plate is washed again and bound cytokine is detected with alkaline phosphatase conjugated anti-cytokine antibody. After three washes, Sigma 104 ™ substrate is added and the plate is read at OD ₄₀₅ at various times using an ELISA plate reader (Molecular Device).

(Example 6)
To assess whether T cells exposed to GAD peptides presented on LSEC can subsequently be prevented from activating autoreactive T cells by professional APCs that present GAD. It is tested whether T cells exposed to peptides on sinus endothelial cells for 3 days can negatively regulate autoreactive T cell activation by peptides presented on spleen professional APCs. The feasibility of inducing T cells with regulatory properties in vitro has been shown by many laboratories (Wakach et al., 2001, Barrat et al., 2002, Thorton and Shevach, 1998). Immunosuppressive properties can be tested by adding regulatory T cells to a culture system in which immune stimulation is normally observed. The addition of GAD peptide to splenocytes from 7 week old NOD mice containing both APCs and T cells provides such an immune stimulatory system as seen by a strong proliferative response. The addition of regulatory T cells eliminates this response. 10 ⁵ or 10 ⁶ splenocytes are added with 0.1, 1 or 10 μm of peptide per well containing T cells exposed to LSEC and peptide-conjugated antibody. Control wells contain splenocytes with peptides alone and LSEC + T cells alone. In addition, to exclude a significant contribution of putative T cell proliferation, control wells were also irradiated with splenocytes (600 RAD, performed at the UCSD radiation service facility by Joe Aguilera) and LSEC and peptide-antibody constructs. Includes previously exposed T cells. Irradiated splenocytes can present antigen but do not proliferate. During the last 16 hours of the 72 hour culture period, 1 μCi ³ H-thymidine is added to each well to label the newly synthesized DNA as a growth readout. Cells are harvested using a Packard Universal Cell Harvester and the incorporated ³ H-thymidine is evaluated using a Topcount. If ³ H-thymidine uptake in cultures containing splenocytes, and T cells previously exposed to LSEC + peptide, is reduced compared to splenocyte cultures, the T cells are successfully induced with regulatory properties. Yes. It is also tested whether peptide-conjugated anti-mSIGNR1 antibodies can induce regulatory T cells and stop disease in NOD mice.

(Example 7)
Mouse anti-human L-SIGN antibody was identified using recombinant phage technology. A mouse library (IgG1k and IgG2ak) derived from a combination of heavy and light chains of mice immunized with recombinant human L-SIGN is disclosed in WO 03/025202, the contents of which are incorporated herein by reference. Prepared by the disclosed method. Once prepared, the library was first panned for human DC-SIGN to remove antibodies that were cross-reactive with DC-SIGN. Unbound supernatant was used to select clones that were uniquely reactive with L-SIGN. A total of 95 colonies (36 / times) were induced for each of the two libraries (IgG1 and IgG2a), and antibody production and their reactivity with L-SIGN were determined by capture ELISA. Briefly, anti-human Fc (Caltag) was coated on ELISA plates overnight at 500 ng / ml. Plates were blocked with PBS containing 1% BSA followed by addition of recombinant L-SIGN at 2 μg / ml. After the plate was washed with PBS, the supernatant was added. After 12 hours incubation at room temperature, the plates were washed 3 times and alkaline-phosphatase conjugated anti-Fab antibody was added for 2 hours. Signal after addition of SigmaS substrate was evaluated using an ELISA reader (Molecular Device).

  Most of the clones showed good binding of the antibody on the phage (OD405> 1.0). Some clones from both IgG1 and IgG2a libraries showed positive reactivity with human L-SIGN. The results are shown in FIGS. FIG. 4 shows an IgG1 clone with human L-SIGN. FIG. 5 shows the reactivity of IgG2a clones with human L-SIGN.

(Example 8)
In order to identify clones that are uniquely reactive with human L-SIGN, all clones of Example 7 with an OD value 5 times greater than the background were selected and those by ELISA with human DC-SIGN were selected. Was tested for reactivity. Anti-human Fc (Caltag) was coated on ELISA plates overnight at 500 ng / ml. Plates were blocked with PBS containing 1% BSA, followed by the addition of recombinant DC-SIGN at 2 μg / ml. After the plate was washed with PBS, the supernatant was added. After 12 hours incubation at room temperature, the plates were washed 3 times and alkaline-phosphatase conjugated anti-Fab antibody was added for 2 hours. Signal after addition of SigmaS substrate was evaluated using an ELISA reader (Molecular Device).

  Ten clones from the IgG1 library and three clones from the IgG2a library were found to be uniquely reactive with human L-SIGN (OD values 5-10 times higher than DC-SIGN) . The results are shown in FIGS. FIG. 6 shows the reactivity of IgG1 positive clones with L-SIGN and DC-SIGN. FIG. 7 shows the reactivity of IgG2a clones with L-SIGN and DC-SIGN.

Example 9
Thirteen clones identified as reactive only with human L-SIGN, and nine with strong reactivity with both L-SIGN and DC-SIGN identified as above in Example 8. Clones were sequenced to determine the number of unique clones. Sequencing was determined by techniques known to those skilled in the art. The sequences of these clones are shown in FIGS. The sequences of heavy chain clones are shown in FIGS. 8A-8C (SEQ ID NOs: 17-36). The sequences of the light chain clones are shown in FIGS. 9A-9B (SEQ ID NOs: 37-55).

  Sequencing results showed a more diverse group of heavy chains compared to light chains. The diversity of antibody clones was also enhanced by cross-pairing of different light chains with the same heavy chain. A total of 5 unique clones reactive with L-SIGN were identified and the clones were classified based on their amino acid sequence similarity (see Table 1 below).

(Table 1)
Classification of antibody clones reactive with human L-SIGN based on amino acid sequence similarity

^ａＬ−ＳＩＧＮとのみ反応性のある抗体クローン
^ｂＬ−ＳＩＧＮおよびＤＣ−ＳＩＧＮの両方と反応性のある抗体クローン
^ｃ２．１〜２．３は、類似した軽鎖だが独特の重鎖を示す
Ｌ−ＳＩＧＮおよびＤＣ−ＳＩＧＮでの配列決定のために選択したクローンの反応性（ＯＤ値）を、下記で表２に示す。

^a Antibody clone reactive only with L-SIGN
^b Antibody clones reactive with both L-SIGN and DC-SIGN
^c 2.1-2.3 show similar light chains but unique heavy chains The reactivity (OD values) of clones selected for sequencing with L-SIGN and DC-SIGN are shown below. It is shown in 2.

  (Table 2)

^＊＝Ｌ−ＳＩＧＮおよびＤＣ−ＳＩＧＮでの配列決定のために選択したクローン
図８に示すように、ヒトＤＣ−ＳＩＧＮＲに結合する抗体の重鎖ＣＤＲ３領域は、以下のアミノ酸配列

^* = Clone selected for sequencing with L-SIGN and DC-SIGN As shown in FIG. 8, the heavy chain CDR3 region of the antibody that binds to human DC-SIGNR has the following amino acid sequence:

の１つを有することがわかった。図９に示されるように、抗体のＤＣ−ＳＩＧＮＲに結合する抗体の軽鎖ＣＤＲ３領域は、以下のアミノ酸配列

It was found to have one of As shown in FIG. 9, the light chain CDR3 region of the antibody that binds to DC-SIGNR of the antibody has the following amino acid sequence:

の１つを有することがわかった。

It was found to have one of

(Example 10)
Using the procedure described above in Example 7, further clones of Example 7 above were examined for their ability to bind to L-SIGN. Sequencing was determined by techniques known to those skilled in the art. The sequences of these additional clones are shown in Figure 10 (SEQ ID NOs 63-82). As shown in FIG. 10, the five additional heavy chain CDR3 regions of an antibody that binds human DC-SIGNR have the following amino acid sequence:

の１つを有することがわかった。ヒトＤＣ−ＳＩＧＮＲに結合するこれらのクローンのＣＤＲ２領域は、以下のアミノ酸配列

It was found to have one of The CDR2 region of these clones that bind to human DC-SIGNR has the following amino acid sequence:

の１つを有することがわかった。

It was found to have one of

  Table 3 shows additional IgG1k antibody clones selected based on reactivity with cells expressing L-SIGN.

  (Table 3)

以下の表４は、さらなるＩｇＧ１ｋ抗体クローンの、ＬＳＩＧＮを発現する細胞（幾何平均蛍光）ならびに組み換えＬ−ＳＩＧＮおよびＤＣ−ＳＩＧＮタンパク質（ＯＤ値）との反応性を示す。

Table 4 below shows the reactivity of additional IgG1k antibody clones with cells expressing LSIGN (geometric mean fluorescence) and with recombinant L-SIGN and DC-SIGN proteins (OD values).

  (Table 4)

図１１に示されるように、ヒトＤＣ−ＳＩＧＮＲに結合するさらなるクローンを同定した（配列番号９６〜１１５）。これらのクローンは、以下のアミノ酸配列

As shown in FIG. 11, additional clones that bind to human DC-SIGNR were identified (SEQ ID NOs: 96-115). These clones have the following amino acid sequences:

の１つを有するＩｇＧ１ｋ軽鎖ＣＤＲ３領域を有することがわかった。ヒトＤＣ−ＳＩＧＮＲに結合するこれらのクローンのＣＤＲ２領域は、以下のアミノ酸配列

It was found to have an IgG1k light chain CDR3 region with one of the following: The CDR2 region of these clones that bind to human DC-SIGNR has the following amino acid sequence:

の１つを有することがわかった。

It was found to have one of

  Table 5 shows additional IgG2ak antibody clones selected based on reactivity with cells expressing L-SIGN.

  (Table 5)

^＊これらの配列は停止コドンを含む
表６は、さらなるＩｇＧ２ａｋ抗体クローンの、ＬＳＩＧＮ（幾何平均蛍光）ならびに組み換えＬ−ＳＩＧＮおよびＤＣ−ＳＩＧＮタンパク質（ＯＤ値）を発現する細胞との反応性を示す。

^* These sequences contain stop codons Table 6 shows the reactivity of additional IgG2ak antibody clones with cells expressing LSIGN (geometric mean fluorescence) and recombinant L-SIGN and DC-SIGN proteins (OD values).

  (Table 6)

図１２に示されるように、ヒトＤＣ−ＳＩＧＮＲに結合するさらなる重鎖クローンを同定した（配列番号１３３〜１５４）。これらのクローンは、以下のアミノ酸配列

As shown in FIG. 12, additional heavy chain clones that bind to human DC-SIGNR were identified (SEQ ID NOs: 133-154). These clones have the following amino acid sequences:

の１つを有するＩｇＧ２ａｋ重鎖ＣＤＲ３領域を有することがわかった。ヒトＤＣ−ＳＩＧＮＲに結合したこれらのクローンのＣＤＲ２領域は、以下のアミノ酸配列

It was found to have an IgG2ak heavy chain CDR3 region with one of The CDR2 region of these clones bound to human DC-SIGNR has the following amino acid sequence:

の１つを有することがわかった。

It was found to have one of

  As shown in FIG. 13, additional light chain clones that bind to human DC-SIGNR were identified (SEQ ID NOs: 169-189). These clones have the following amino acid sequences:

の１つを有するＩｇＧ２ａｋ軽鎖ＣＤＲ３領域を有することがわかった。ヒトＤＣ−ＳＩＧＮＲに結合したこれらのクローンのＣＤＲ２領域は、以下のアミノ酸配列

It was found to have an IgG2ak light chain CDR3 region with one of the following: The CDR2 region of these clones bound to human DC-SIGNR has the following amino acid sequence:

の１つを有することがわかった。

It was found to have one of

(Example 11)
Selective reactivity of candidate soluble L-SIGN antibodies with L-SIGN receptor but not DC-SIGN receptor Based on phage-Fab screening, DNA sequencing (FIGS. 10 and 11), L-SIGN receptor The six antibodies (clone names, C7, D12, E4, E10, G3, G10) (Table 4) with the best reactivity with the cells expressing the protein were subcloned to remove the coat III protein. Each of these six candidate antibodies was expressed and purified as a soluble Fab portion and tested again for their ability to recognize L-SIGN receptors on cells but not DC-SIGN. As shown in FIG. 14, all six antibodies showed very good binding to the L-SIGN receptor, but the three antibodies (D12, G3 and E10) were both DC-SIGN, Reacted at a substantially lower level. All antibodies (20 ug / ml) were transferred to 0.5 × 10 ⁶ cells (K562, K562 / DC-SIGN, K562 / in FACS buffer (1% BSA, DPBS containing 0.1% azide)). L-SIGN) for 1 hour at 4 ° C., washed with FACS buffer, and 1:50 diluted phycoerythrin (PE) conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, Pa.) With 4 Incubated for 30 minutes at 0 C, washed, resuspended in 1% formaldehyde and analyzed with BD FACSCalibur (Becton Dickinson, Mountain View, CA).

(Example 12)
Relative affinity and epitope characteristics of L-SIGN antibodies The six candidate antibodies were further characterized with respect to their affinity for the L-SIGN protein and the nature of the recognized epitope. As shown in FIG. 15, all antibodies react with the L-SIGN receptor with nanomolar affinity, while the E10 clone showed binding even in the picomolar range. In ELISA, the epitope specificity of various antibodies was characterized by competing with an L-SIGN specific monoclonal antibody (mab162) that binds to the L-SIGNFc fusion protein. As shown in FIG. 16, four antibody clones, C7, D12, G10 and E4, competed with mab162 in a concentration-dependent manner, while clones G3 and E10 did not. These data indicate that clones C7, D12, G10 and E4 either share the same epitope or bind to overlapping epitopes, as shown by slight differences in competition studies. Implications. To characterize the nature of the epitope bound by the six antibodies (stereo versus linear, monomer versus multimer), the entire cell lysate of K562 / L-SIGN cells prepared under denaturing and reducing conditions was used. , Separated by SDS-PAGE, and membranes were probed with the respective antibodies. Two antibodies (clone D12 and E10) recognized a protein band corresponding to the monomer, while clone G3 recognized a protein band corresponding to the receptor in the trimeric form. These data also indicate that the epitope recognized by clones D12, E10 and G3 is linear, but the epitope recognized by antibody clones C7, E4 and G10 is steric. The nature of the epitope recognized by the L-SIGN fab was delineated by Western blotting studies. Briefly, 1 million K562 / L-SIGN cells were treated with 50 ul lysis buffer (150 mM NaCl, 25 mM Tris, pH 7.4, 2 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1% Triton X −100, 0.5 mM PMSF, 10 μg / ml aprotinin and 10 μg / ml leupeptin). Lysis was achieved by gentle rotation for 20 minutes at 4 ° C. Cell lysates were centrifuged (14,000 × g, 10 minutes) to remove cell debris and boiled in SDS sample buffer containing 1 mM DTT for 5 minutes. Protein lysates were separated on a 4-15% SDS-PAGE gradient gel (Bio-Rad # 116-1158), transferred to a nitrocellulose membrane, and then probed with an L-SIGN specific fab (1 μg / ml), respectively. . Protein transfer was monitored with a prestained molecular weight standard (Bio-Rad # 161-0324). Immunoreactive bands were detected by enhanced chemiluminescence (Super signal West Pico kit, Pierce, Rockford, Ill.) Using HRP-conjugated goat anti-mouse IgG (Bio-Rad # 170-6516).

(Example 13)
When L-SIGN antibodies bind to receptors on human liver non-parenchymal cells, they undergo internalization of antigen in L-SIGN-expressing sinusoidal endothelial cells (LSEC), which are known to cause immune tolerance. For delivery, antibody internalization ability was assessed by flow cytometry and confocal microscopy. Freshly isolated human liver non-parenchymal cells were incubated with 6 candidate antibodies. As shown in FIG. 17, the three antibodies (C7, E10 and G10) showed over 40 percent internalization within 2 hours. This level of internalization was found to be statistically significant (p <0.05). Furthermore, antibody internalization was also confirmed directly by confocal microscopy studies. All six antibodies were found inside K562 / L-SIGN cells after 90 minutes incubation, but no uptake was observed in K562 / DC-SIGN cells used as controls in this study. These studies show that all of the L-SIGN antibodies can be internalized, but the three antibodies (clone C7, E10 and G10) are most efficiently internalized.

Antibody Internalization Assay-The assay was performed as described by Takahara K et al. (International Immunology, 2004). Briefly, 0.5 × 10 ⁶ cells (fresh human LSEC or K562 / L-SIGN) were incubated with 20 μg / ml L-SIGN fab in DPBS / 1% BSA at 4 ° C. for 30 minutes. Unbound antibody was washed away and then incubated at 37 ° C. for an additional 2 hours to allow internalization. Two sets of samples maintained at 4 ° C. in DPBS / 1% BSA / 0.1% azide were used as controls. At the end of the incubation period, the cells are incubated with phycoerythrin (PE) conjugated goat anti-mouse antibody in DPBS / 1% BSA / 0.1% azide for 30 minutes at 4 ° C., washed and in 1% formaldehyde. Fixed and analyzed with FACSCalibur (Becton Dickinson).

Confocal microscopy 10 ⁵ K562 transductants at 90 ° C. in RPMI 1640 (Gibco, Life Technology, Breda, The Netherlands) supplemented with 10% fetal bovine serum with 10 μg / ml of various antibodies. Incubated for minutes. Cells were then washed with PBS, fixed in PBS / 4% paraformaldehyde, washed again and allowed to adhere to poly-L-lysine coated coverslips (20 minutes at room temperature). Cells with blocking buffer, PBS / 3% BSA (Sigma Chemical Company, St. Louis, MO) / 10 mM glycine (Merck, Darmstadt, Germany) /0.1% saponin (Riedeldehan, Germany, Seeles) for 1 hour at room temperature Incubated. Subsequently, the cells were incubated with 10 μg / ml rabbit anti-human CD55 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature, washed with blocking buffer, and 10 μg / ml goat anti-mouse IgG Alexa 647 ( (Molecular probe, Leiden, The Netherlands) and 10 μg / ml goat anti-rabbit IgG Alexa 488 (molecular probe) were incubated for 1 hour at room temperature in blocking buffer. The cells were then washed with blocking buffer, PBS, and finally 50 mM Tris-HCl. Finally, the cover glass was mounted on the slide glass with Mowiol (Calbiochem, Omnilab International, Breda, The Netherlands).

(Example 14)
L-SIGN antibodies block ligand binding to receptors Several viruses such as Ebola, SARS, HIV and HCV have been shown to utilize the L-SIGN receptor to increase cell entry Has been. Both envelope glycoproteins on the surface of viruses and ICAM-3 on the surface of host T cells are known to interact with the lectin binding domain of L-SIGN. To determine if the antibody can block L-SIGN receptor and ligand interactions, a ligand-coated fluorescent bead-blocking assay was performed. As shown in FIG. 18, the three most efficiently internalized antibodies (C7, G10 and E10) were also used without affecting the binding of ICAM-1 coated beads used as controls in the assay. , ICAM-3 binding to the L-SIGN receptor was best blocked (greater than 40% inhibition was observed). (See FIG. 18.)
Fluorescent bead adhesion assay for ligand blocking. As previously described for ICAM-1 beads ( ⁵ Geijtenbeek et al., 1999), carboxylic acid modified TransFluorSphere (488/645 nm, 1.0 μm, molecular probe) was prepared from ICAM-3 Fc protein (R & D Systems, Coated with Minneapolis, Minnesota). Briefly, streptavidin coated beads were incubated with biotinylated goat anti-human anti-Fc Fab2 fragment in PBS, 0.5% BSA for 2 hours at 37 ° C. The beads were washed and incubated overnight at 4 ° C. with human IgG1 Fc fusion ICAM-3 Fc (250 ng / mL). For adhesion to ICAM-3 beads, K562 / L-SIGN cells were treated with Tris-sodium-BSA (20 mmol / L Tris-HCl, pH 8.0, 150 mmol / L NaCl, 1 mmol / L CaCl2, 2 mmol / L MgCl2). , 0.5% BSA (weight / volume), 5 to about 106 cells / mL). Fifty thousand cells were pre-incubated in 96-well V-type bottom plates for 10 minutes at room temperature with or without L-SIGN fab (20 μg / mL). Ligand coated beads (20 beads / cell) were added and the suspension was incubated at 37 ° C. for 30 minutes. After washing, the cells were resuspended in Tris-sodium-BSA buffer. ICAM-3-mediated adhesion of K562 / LSIGN cells was measured by flow cytometry using a FACSCalibur (Becton Dickinson). The percentage of cells binding to ICAM-3 without antibody was set to 100 and the decrease in binding in the presence of L-SIGN antibody was expressed as ligand blocking.

(Example 15)
Summary of biological activity mediated by L-SIGN specific antibodies Based on the above studies (see Table 7 below), at least three antibody clones, C7, G10 and E10, are found in autoimmune and infectious diseases. It is an excellent candidate for therapeutic use.

(Table 7)
Biological activity mediated by L-SIGN Fab

^ａ（％）パーセント内在化は、（４℃での平均蛍光強度−３７℃での平均蛍光強度）／（４℃での平均蛍光強度）×１００として決定した。
^ｂ（％）パーセントブロッキングは、（Ｆａｂありでリガンドに結合している細胞の数−Ｆａｂなしでリガンドに結合している細胞の数）／（Ｆａｂなしでリガンドに結合している細胞の数）×１００として決定した。

^a (%) percent internalization was determined as (average fluorescence intensity at 4 ° C.−average fluorescence intensity at 37 ° C.) / (average fluorescence intensity at 4 ° C.) × 100.
^b (%) Percent blocking is (number of cells bound to ligand with Fab-number of cells bound to ligand without Fab) / (number of cells bound to ligand without Fab) X100 was determined.

(Example 16)
Selecting antibodies that can block viral binding by blocking ligand binding to the receptor Some viruses, such as Ebola, SARS, HIV and HCV, to increase cell entry It has been shown to utilize DC-SIGN and L-SIGN receptors. Both ICAM-3 on T cells and envelope glycoproteins on viruses were found to interact with the carbohydrate recognition domain (CRD) of the SIGN receptor in an overlapping but distinct manner. To determine whether a CRD-reactive Fab capable of blocking ligand binding has been isolated, ligand-coated fluorescent beads—as essentially described by ⁵ Geijtenbeek et al. (1999) A blocking assay was performed. Briefly, 50,000 K562 / L-SIGN transfected cells are preincubated in 96 well V-bottom plates for 10 minutes at room temperature with or without L-SIGN Fab (20 μg / mL). Fluorescent beads (20 beads / cell) coated with viral envelope proteins such as HCV E1 / E2 or HIV gp120 are added and the suspension is incubated at 37 ° C. for an additional 30 minutes. After washing, the cells are resuspended in Tris-sodium-BSA buffer. The degree of blocking of the virus-coated beads on the K562 / L-SIGN cells by the antibody is measured using FACSCalibur (Becton Dickinson). The percentage of cells bound to virus beads without antibody (negative control) is taken as 100, and the decrease in binding in the presence of L-SIGN antibody is expressed as% blocking. Fluorescent beads coated with ligand not only mimic ligand multimeric binding to cell surface receptors, but also allow easy quantification of ligand binding by flow cytometry. First, the adhesion of fluorescent beads coated with Ebola and HIV envelope glycoproteins to K562 / DC-SIGN and K562 / L-SIGN was evaluated without antibodies. As shown in FIG. 19A, Ebola envelope glycoprotein binds equally well to both DC-SIGN and L-SIGN expressing cells, but binding of HIV envelope glycoprotein to L-SIGN is dependent on DC-SIGN expression. It was much weaker than binding to cells. These differences in viral protein binding to the SIGN molecule had a profound effect on the ability of the Fab to block viral protein adhesion. All six Fabs were able to block HIV gp120 binding to L-SIGN (33% -66%), but only two clones, C7 and E10, showed significant blocking of Ebola gp binding (75% each). And 64%) (see FIG. 19B). Of the three DC-SIGN cross-reactive clones, D12, E10 and G3, only E10 blocked the binding of both viral proteins to DC-SIGN. In contrast, the other three Fabs that were uniquely reactive with L-SIGN had no blocking effect on ligand binding to DC-SIGN (see FIG. 19C). In addition, the three Fabs that best blocked viral protein binding, clones C7, E10 and G10, also best prevented ICAM-3 binding to K562 / L-SIGN cells (see Table 7).

Converting Fab to IgG Improves Blocking of Receptor Binding and Viral Protein Adhesion Based on receptor binding, internalization and ICAM-3 blocking results, the murine variable doin of Fab can be humanized using conventional methods. Two Fab clones, E10 and G10, were converted to complete IgG by fusing to the constant region of IgG1. These chimeric IgGs were evaluated for blocking receptor binding and viral protein adhesion. As shown in FIG. 20A, Fab to IgG conversion greatly improved the receptor binding of both clones, E10 and G10. Also, clone G10 did not show any binding to DC-SIGN as Fab, which showed some binding to DC-SIGN as IgG, which may be a result of increased affinity for the receptor. Is expensive. Complete IgG also showed increased blocking of viral protein binding compared to their Fab counterparts (see FIGS. 19B and 19C). The most profound effect of Fab to IgG conversion was observed in clone G10, where complete IgG produced over 90% blocking of the Ebola envelope gp to L-SIGN, whereas Fab counterparts. Little to no blocking was observed in (see FIG. 19C). Fab to IgG conversion greatly improved the blocking ability of clone G10, but had a milder effect on clone E10, which is a strong blocking agent as Fab molecule as clone C7.

  The three most efficiently internalized Fab clones, C7, E10 and G10, also best blocked ligand binding. Binding of antibodies to the carbohydrate recognition domain of the receptor makes them excellent candidates for their use in modulating immune responses and preventing viral transmission. Clones, C7 and E10, consistently and effectively blocked the binding of ICAM-3 and viral proteins to the L-SIGN receptor (p <0.05). Clone G10 was able to block ICAM-3 and HIV gp120 binding, but not Ebola envelope glycoprotein binding (summarized in Table 7 above). Based on the foregoing, clone G10 blocks ligand binding by some mechanism of steric interference, but the blocking effect produced by clone C7 and clone E10 is due to direct competition to the ligand binding site.

(Example 17)
Blocking virus entry After selecting antibodies that can block virus binding to the receptor in Example 16 above, the ability of these antibodies to block virus entry is tested. L-SIGN transfected K-562 cells are pre-incubated with antibody for 30 minutes before adding reporter virus expressing the desired envelope protein or, if practicable, serum from virus + or virus-donor . After 1 hour incubation at 37 ° C., the cells are washed 5 times with phosphate buffered saline and viral RNA is extracted from the cells using Qiagen's viral RNA minispin kit. The viral RNA thus obtained is amplified by RT-PCR according to the procedure of Gardner et al. (2003) and Southern blotting is performed.

(Example 18)
Preventing virus transmission To test whether the antibodies identified in Example 16 can prevent viral transfer from receptor-positive endothelial cells to either human T cells or hepatocytes, K562 / L -SIGN cells or freshly isolated human hepatic sinusoidal endothelial cells (L-SIGN +) or dendritic cells (DC-SIGN +) are incubated with the antibody of Example 16 for 30 minutes before the desired envelope protein, eg HCV- Add luciferase or green fluorescent protein reporter virus expressing E2, HIV gp120, Ebola (Alvarez et al., 2002) or Sindbis (Klimstra et al., 2003). After washing with medium, the cells are co-cultured with T cells (C8166) or human hepatocytes (Huh-7). Measurement of luciferase activity in target cell lysates (relative luminescence units) or flow cytometric analysis of GFP positive target cells in combination with suitable surface marker double staining on target cells (eg CD3 on T cells) Reporter virus transmission is assessed by either.

(Example 19)
Assessing the Role of Antibodies in Blocking Infection from Mycobacterium tuberculosis Mannosylated lipoarabinomannan (ManLAM), a carbohydrate-rich structure present on the surface of Mycobacterium tuberculosis, is known as DC-SIGN ( ⁴ Geijtenbeek et al. , 2003) and L-SIGN (Koppel et al., 2004). High antibody titers against ManLAM have been observed in people with active tuberculosis and have been shown to reduce bacterial burden in passive protection experiments (Hamasur et al., 2004). Mycobacterial binding and infection is inhibited using L-SIGN antibodies or L-SIGN peptidomimetics that can bind with high affinity to ManLAM. Bacterial strains such as M. bovis and M. tuberculosis are labeled with fluorescein isothiocyanate (FITC) as detailed in ⁴ Geijtenbeek et al. (2003). K562 / L-SIGN cells are incubated at a ratio of 1-20 with FITC-conjugated bacteria with or without L-SIGN peptide mimics (50 μg / ml). The degree of blocking by antidotes or mimetics (decrease in fluorescence) is determined by flow cytometric analysis.

(Example 20)
Treatment of transplant patients with HCV infected liver Once virus transmission is prevented, the donor can potentially use the antibody in a transplant environment with HCV infection. To test this, a mild HCV-infected human donor liver is transplanted into immune deficient mice such as NOD / SOID along with injection of primary blood lymphocytes from healthy HLA-compatible human donors. Mice are treated with antibodies over a period of 1-6 months. One to six months after transplantation, mice are sacrificed and the extent of HCV infection in the liver is assessed. T cells are also examined for viral infection by PCR.

(Example 21)
Identification of cancer types expressing L-SIGN Malignant tissue and paired normal tissue are collected and either fixed in para-formaldehyde or snap frozen in OCT. For the presence of L-SIGN by preparing sections using a microtome and using either L-SIGN antibody directly conjugated to a suitable fluorescent dye such as FITC, or secondary antibody fluorescent dye conjugated anti-mouse IgG Stain sections. The staining result of malignant tissue significantly stronger than normal tissue staining is an indicator of a cancer type expressing L-SIGN.

Commercial cell lines for these cancer types expressing L-SIGN are then obtained and the presence of L-SIGN is assessed by FACS analysis. Briefly, 1 million cells are incubated with 1 μg anti-L-SIGN antibody in PBS containing 1% BSA and 0.1% NaN ₃ . After a 30 minute incubation, the cells are washed and incubated with fluorescent dye-conjugated anti-mouse IgG for an additional 30 minutes before being analyzed on a FACSCalibur (Becton Dickinson).

(Example 22)
Therapeutic use of anti-L-SIGN antibodies Direct cell killing As identified in Example 21 above, identifying L-SIGN-expressing cell tumor types identified them as anti-L-SIGN antibodies, which induce ADCC or CDC in vitro. Test for ability to do. In order to evaluate ADCC, target cells (tumor cells) are first labeled with ⁵¹ Cr. The antibody against L-SIGN is then added at a concentration between 1-50 μg and target cell lysis by PBMC is measured after 4 hours at an effector to target ratio of 1:10 to 1: 100. Tumor cells are incubated with human complement and L-SIGN antibody for assessment of CDC. Cell death is assessed by FACS analysis after addition of propidium iodide, a reagent that can only enter dead cells and not live cells. For antibodies that do not induce ADCC or CDC, some radioisotope labeling or toxic reagent remains conjugated to the antibody.

  The ability of either bare or conjugated anti-L-SIGN antibodies to arrest tumor growth is evaluated in a xenograft model. Briefly, tumor cells expressing L-SIGN are injected subcutaneously, intraperitoneally or intravenously. Animals are treated with either control or anti-L-SIGN antibody. Tumor growth is measured by size for subcutaneous treatment or by survival time for intraperitoneal or intravenous treatment. If tumor growth in the anti-L-SIGN treated group is reduced by more than 30% compared to the control group, the antibody can be used as a cancer therapy.

Blocking negative regulatory interactions of cancer cells in the immune system As described in Example 14, antibodies that block the interaction of L-SIGN with immune cells are identified using a fluorescent bead assay. To do. The antibody is evaluated for its therapeutic utility with respect to the ability of the immune system to eradicate cancer cells by preventing negative regulation of the immune system by L-SIGN expressing cancer cells.

  L-SIGN expressing tumor cells are transplanted subcutaneously, intraperitoneally or intravenously into immune deficient mice such as NOD / SOID. Mice are also given 2 million PBMCs from healthy donors (or any number of PBMCs that are not sufficient to spontaneously cause tumor rejection). Tumor growth with or without anti-L-SIGN antibody is compared to control antibody. Tumor growth is measured by size for subcutaneous treatment or by survival time for systemic tumors. If tumor growth in the anti-L-SIGN treated group is reduced by more than 30% compared to the control group, the antibody can be used as a cancer therapy.

(Example 23)
Use of L-SIGN Antibodies to Isolate L-SIGN Expressing Cells Fresh human non-parenchymal hepatocytes are obtained from commercial sources (eg, Cells Direct, Tucson, Ariz.). Non-parenchymal hepatocytes include sinusoidal endothelial cells, red blood cells, Kupffer cells and a mixture of a few other cell populations. To isolate hepatic sinusoidal cells, erythrocytes are lysed using ammonium chloride. Dead cells are removed using a commercially available dead cell removal kit (Miltenyi Biotech, Germany). Cells are counted and labeled with 0.1 μg / million cells of anti-L-SIGN antibody. After washing the cells, they are resuspended in MACS buffer (Milteny) and the cells of interest are isolated using anti-mouse IgG conjugated beads according to the manufacturer's instructions (Milteny). Isolation quality is monitored by FACS analysis using a panel of antibodies against molecules such as CD54, mannose receptor, LYVE-1, CD40, asialoglycoprotein and others.

  The following references are hereby incorporated by reference in their entirety:

上述の記載は限定として解釈されるべきではなく、単に好ましい実施形態の例示として解釈されるべきである。本明細書中に開示される実施形態に種々の変更がなされ得ることが理解されよう。例えば、当業者が認めるように、本明細書中に記載される特定の配列は、抗体または抗体断片の機能性に必ずしも有害に影響を及ぼすことなく、わずかに改変することができる。例えば、抗体配列における単一または複数のアミノ酸の置換は、抗体または断片の機能性を破壊することなく、頻繁に行うことができる。したがって、本明細書中に記載される特定の抗体に対して７０％より高い程度の相同性を有する抗体が、本開示の範囲内であることが理解されるべきである。特に有用な実施形態において、本明細書中に記載される特定の抗体に対して約８０％より高い相同性を有する抗体が企図される。他の有用な実施形態においては、本明細書中に記載される特定の抗体に対して約９０％より高い相同性を有する抗体が企図される。したがって、上述の記載は限定として解釈されるべきではなく、単に好ましい実施形態の例示として解釈されるべきである。当業者は、本開示の範囲内および精神内の、他の変更を構想するであろう。

The above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. It will be understood that various modifications may be made to the embodiments disclosed herein. For example, as those skilled in the art will appreciate, certain sequences described herein can be altered slightly without necessarily adversely affecting the functionality of the antibody or antibody fragment. For example, single or multiple amino acid substitutions in an antibody sequence can frequently be made without destroying the functionality of the antibody or fragment. Accordingly, it is to be understood that antibodies having a degree of homology greater than 70% to the particular antibodies described herein are within the scope of this disclosure. In particularly useful embodiments, antibodies having greater than about 80% homology to the specific antibodies described herein are contemplated. In other useful embodiments, antibodies having greater than about 90% homology to the specific antibodies described herein are contemplated. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the present disclosure.

FIG. 1 schematically illustrates the interaction of an antibody / autoantigen construct according to the present disclosure with antigen presenting cells (APC) and T cells. FIG. 2A shows the light chain amino acid sequence (SEQ ID NO: 1-6) and heavy chain amino acid sequence of rabbit anti-mSIGNR1 scFV antibody. FIG. 2B shows the heavy chain amino acid sequences (SEQ ID NOs: 7-12) of rabbit anti-mSIGNR1 scFV antibodies. FIG. 3 is a schematic diagram of a portion of a vector for production of antibody peptide constructs. FIG. 4 is a graphical depiction of the results of an in vitro experiment according to the present disclosure showing the reactivity of IgG1 clones with human DC-SIGNR. FIG. 5 is a graphical depiction of the results of an in vitro experiment according to the present disclosure showing the reactivity of IgG2a clones with human DC-SIGNR. FIG. 6 is a graphical depiction of the results of an in vitro experiment according to the present disclosure showing the reactivity of IgG1 clones with human DC-SIGNR and DC-SIGN. FIG. 7 is a graphical depiction of the results of an in vitro experiment according to the present disclosure showing the reactivity of IgG2a clones with human DC-SIGNR and DC-SIGN. 8A-8C show the amino acid sequences of heavy chain clones reactive with human DC-SIGNR (SEQ ID NOs: 17-36). 8A-8C show the amino acid sequences of heavy chain clones reactive with human DC-SIGNR (SEQ ID NOs: 17-36). 8A-8C show the amino acid sequences of heavy chain clones reactive with human DC-SIGNR (SEQ ID NOs: 17-36). 9A-9B show the amino acid sequences of light chain clones that are reactive with human DC-SIGNR (SEQ ID NOs: 37-55). 9A-9B show the amino acid sequences of light chain clones that are reactive with human DC-SIGNR (SEQ ID NOs: 37-55). FIG. 10 shows additional amino acid sequences of IgG1 heavy chain clones reactive with human DC-SIGNR (SEQ ID NOs 63-82). FIG. 10 shows additional amino acid sequences of IgG1 heavy chain clones reactive with human DC-SIGNR (SEQ ID NOs 63-82). FIG. 11 shows additional amino acid sequences of IgG1 light chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 96-115). FIG. 11 shows additional amino acid sequences of IgG1 light chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 96-115). FIG. 12 shows additional amino acid sequences of IgG2a heavy chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 133-154). FIG. 12 shows additional amino acid sequences of IgG2a heavy chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 133-154). FIG. 13 shows additional amino acid sequences of IgG2a light chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 169-189). FIG. 13 shows additional amino acid sequences of IgG2a light chain clones that are reactive with DC-SIGNR (SEQ ID NOs: 169-189). FIG. 14 shows that six antibodies (clone names C7, D12, E4, E10, G3, G10) showed very good binding to the L-SIGN receptor, but three antibodies (D12, G3 and E10). Indicates that DC-SIGN also reacted at a substantially lower level. FIG. 15 shows the affinity of various antibodies for the L-SIGN protein. FIG. 16 shows the epitope specificity for various antibodies characterized by competing with an L-SIGN specific monoclonal antibody (mab162) that binds to an L-SIGNFc fusion protein in an ELISA. FIG. 17 shows the results of antibody internalization by hepatic sinusoidal endothelial cells. FIG. 18 shows the results of a fluorescent bead adhesion assay for ligand blocking used to measure ICAM-1 and ICAM-3-mediated adhesion of K562 / LSIGN cells as measured by flow cytometry. FIG. 19A shows the adhesion of fluorescent beads coated with Ebola K562 / DC-SIGN and K562 / L-SIGN envelope glycoproteins. Without antibody, 50,000 K562 / L-SIGN cells and K562 / DC-SIGN cells were incubated with fluorescent beads coated with HIV and Ebola envelope glycoproteins (20 beads / cell) for 30 minutes, virus The degree of binding by the protein-coated beads was measured by flow cytometry in FL-3. FIG. 19B shows the ability of Fab to block adhesion of HIV gp120 and Ebola gp binding to L-SIGN. Incubate 50,000 K562 / L-SIGN cells with fluorescent beads coated with HIV and Ebola envelope glycoproteins (20 beads / cell) for 30 min in the presence of L-SIGN Fab (20 ug / ml) The number of cells bound to the beads coated with the viral protein was determined by flow cytometry. FIG. 19C shows the ability of Fab to block the binding of both viral proteins to DC-SIGN. Incubate 50,000 K562 / DC-SIGN cells with fluorescent beads coated with HIV and Ebola envelope glycoproteins (20 beads / cell) in the presence of L-SIGN Fab (20 ug / ml) for 30 minutes The number of cells bound to the beads coated with the viral protein was determined by flow cytometry. The percent binding in FIGS. 19A-C is the number of cells bound to the protein-coated beads with Fab divided by the number of cells bound to the protein-coated beads without Fab. It was decided to multiply by 100. Bars represent the mean + SD of two independent experiments. FIGS. 20A-C show binding of full IgG versions of Fab, E10 and G10 to the receptor and blocking viral protein adhesion. To generate the data in FIG. 20A, 5 × 10 ⁵ K562, K562 / DC-SIGN and K562 / L-SIGN cells were incubated with purified Fab (20 μg / mL) for 1 hour to determine their binding. The extent was assessed by flow cytometry using PE-conjugated goat anti-mouse (Fab detection) or goat anti-human (IgG detection) secondary antibodies. mAb 162 (L-SIGN specific) and mAb 16211 (DC-SIGN / L-SIGN cross-reactivity) were used as positive controls. To generate the data in FIG. 20B, 50,000 K562 / L-SIGN and K562 / DC-SIGN cells were coated with HIVgp120 fluorescent beads (20 beads / ml) in the presence of Ab (20 ug / ml). Cells) for 30 minutes and the number of cells bound to the beads coated with the viral protein was determined by flow cytometry. To generate the data of FIG. 20C, 50,000 K562 / L-SIGN and K562 / DC-SIGN cells coated with Ebola envelope glycoprotein (20 cells) in the presence of Ab (20 ug / ml). The number of cells bound to the beads coated with the viral protein was determined by flow cytometry. mAb 162 (L-SIGN specific) and mAb AZND1 (DC-SIGN specific) were used as positive controls. Percent binding is determined as 100 times the number of cells bound to protein-coated beads with Ab divided by the number of cells bound to protein-coated beads without Ab. did. Bars represent the mean + SD of two independent experiments.

Claims (78)

  An antibody that recognizes the L-SIGN receptor and shows at least 40 percent internalization into isolated human liver non-parenchymal cells within 2 hours.   An antibody that internalizes in isolated human liver non-parenchymal cells and blocks ICAM-3 binding to the L-SIGN receptor without affecting ICAM-1 binding.   A composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.   A vaccine comprising the antibody of claim 1.   An antibody that recognizes an L-SIGN receptor on a cell, which can effectively block virus binding to the cell.   6. The antibody of claim 5, which can effectively block the binding of a virus selected from the group consisting of HIV, HCV, Ebola, SARS, CMV and Sindbis to cells.   An antibody that recognizes an L-SIGN receptor on a cell, which can effectively block infection of the cell by a virus.   8. The antibody of claim 7, which can effectively block infection of cells with a virus selected from the group consisting of HIV, HCV, Ebola, SARS, CMV and Sindbis.   An antibody that recognizes an L-SIGN receptor on a cell that can effectively block the spread of a virus from one cell to another.   10. The antibody of claim 9, which can effectively block the transmission of a virus selected from the group consisting of HIV, HCV, Ebola, SARS, CMV and Sindbis from one cell to another.   A vaccine comprising the antibody according to claim 5.   An antibody that recognizes an L-SIGN receptor on a cell, which can effectively block the binding of a bacterium of the genus Mycobacterium to the cell.   13. The antibody of claim 12, which can effectively block the binding of bacteria selected from the group consisting of M. tuberculosis and M. bovis to cells.   An antibody that recognizes an L-SIGN receptor on a cell, which can effectively block infection of the cell by a bacterium of the genus Mycobacterium.   The antibody according to claim 14, which can effectively block infection of cells by a bacterium selected from the group consisting of M. tuberculosis and M. tuberculosis.   An antibody that recognizes an L-SIGN receptor on a cell that can effectively block the transmission of Mycobacterium bacteria from one cell to another.   17. The antibody of claim 16, which can effectively block the transmission of bacteria selected from the group consisting of M. tuberculosis and M. tuberculosis from one cell to another.   An antibody that recognizes the L-SIGN receptor on a cell, which can effectively block the binding of Schistosoma mansoni to the cell.   An antibody that recognizes an L-SIGN receptor on a cell, which can effectively block infection of the cell by Schistosoma mansoni.   An antibody that recognizes an L-SIGN receptor on a cell that can effectively block the transmission of Schistosoma mansoni from one cell to another.   A diagnostic agent for a tumor characterized by increased L-SIGN expression, comprising an antibody that recognizes the L-SIGN receptor.   A diagnostic kit comprising the diagnostic agent according to claim 21. Obtaining a tissue sample from a subject suspected of having cancer, and measuring the extent to which the tissue sample binds to an antibody that recognizes the L-SIGN receptor;
An increased degree of binding compared to the corresponding normal tissue indicates the presence of cancer,
A method for diagnosing cancer.   24. The method of claim 23, wherein the measuring step comprises staining for the presence of L-SIGN.   A therapeutic agent for treating cancer characterized by increased L-SIGN expression, comprising an antibody recognizing L-SIGN receptor.   A method for treating cancer comprising administering to a subject a composition comprising an antibody that recognizes the L-SIGN receptor in an amount that kills cancer cells.   27. The method of claim 26, wherein the antibody recognizing the L-SIGN receptor induces antibody-dependent cytotoxicity of cancer cells.   27. The method of claim 26, wherein the antibody recognizing the L-SIGN receptor induces complement dependent cytotoxicity of cancer cells.   27. The method of claim 26, wherein the antibody recognizing the L-SIGN receptor prevents negative regulation of the immune system by L-SIGN expressing cancer cells.   27. The method of claim 26, wherein the antibody recognizing the L-SIGN receptor is fused to a toxin.   25. The method of claim 24, wherein the antibody recognizing the L-SIGN receptor is fused to a high energy radiation emitter.   A method for treating an inflammatory disease comprising administering to a subject a composition comprising an antibody that recognizes an L-SIGN receptor in an amount that kills L-SIGN-expressing cells.   The method according to claim 32, wherein the antibody recognizing the L-SIGN receptor induces antibody-dependent cytotoxicity of L-SIGN-expressing cells.   33. The method of claim 32, wherein the antibody recognizing the L-SIGN receptor induces complement dependent cytotoxicity of L-SIGN expressing cells.   33. The method of claim 32, wherein the antibody recognizing the L-SIGN receptor prevents negative regulation of the immune system by L-SIGN expressing cells.   35. The method of claim 32, wherein the antibody recognizing the L-SIGN receptor is fused to a toxin.   35. The method of claim 32, wherein the antibody recognizing the L-SIGN receptor is fused to a high energy radiation emitter.   An antibody having high affinity for L-SIGN but low affinity for DC-SIGN.   An antibody that binds to a linear epitope on L-SIGN and a steric epitope on L-SIGN.   An antibody that blocks L-SIGN-T cell interactions, thereby causing proliferation of regulatory T cells.   41. The antibody of claim 40, wherein proliferation of regulatory T cells occurs in the liver.   An antibody that blocks L-SIGN-T cell interactions and thereby suppresses the proliferation of autoreactive T cells.   43. The antibody of claim 42, wherein suppression of autoreactive T cell proliferation occurs in a lymph node.   Antibody-peptide constructs that deliver autoantigens to hepatic sinusoidal endothelial cells, thereby stimulating the growth of regulatory cells.   An antibody-peptide construct that delivers autoantigens to hepatic sinusoidal endothelial cells, thereby inhibiting the activity of autoreactive T cells.   An antibody-peptide construct that delivers vaccine antigens to sinusoidal endothelial cells in the lymph nodes, thereby stimulating the proliferation of antigen-specific T cells. Obtaining a mixture of L-SIGN expressing cells in combination with cells not expressing L-SIGN,
Exposing the mixture to an anti-L-SIGN antibody and allowing the L-SIGN expressing cells to bind to the anti-L-SIGN antibody, thereby causing the L-SIGN expression from cells not expressing the L-SIGN. A method for isolating L-SIGN expressing cells, comprising isolating cells.   A composition comprising the antibody of claim 2 and a pharmaceutically acceptable carrier.   A vaccine comprising the antibody according to claim 2.   A vaccine comprising the antibody according to claim 7.   A vaccine comprising the antibody of claim 9.   An antibody that recognizes the L-SIGN receptor and blocks the binding of HIV gp120 to L-SIGN.   An antibody that recognizes the L-SIGN receptor and blocks the binding of the Ebola envelope protein to L-SIGN.   An antibody that recognizes the L-SIGN receptor and blocks the binding of HIV gp120 to DC-SIGN.   An antibody that recognizes the L-SIGN receptor and blocks the binding of the Ebola envelope protein to DC-SIGN.   An antibody that recognizes both L-SIGN and DC-SIGN receptors and blocks the binding of HIV gp120 to DC-SIGN.   An antibody that recognizes both L-SIGN and DC-SIGN receptors and blocks Ebola envelope protein binding to DC-SIGN.   58. A composition comprising the antibody of any of claims 52 to 57 and a pharmaceutically acceptable carrier.   58. A vaccine comprising the antibody of any of claims 52 to 57.   54. The antibody of claim 53, wherein the antibody and the Ebola envelope protein bind to the same epitope.   An antibody that recognizes the L-SIGN receptor on a cell that can block the binding of the virus to the cell.   62. The antibody of claim 61, which can block the binding of a virus selected from the group consisting of HIV, HCV, Ebola, SARS, CMV and Sindbis to a cell.   62. The antibody of claim 61, which also recognizes a DC-SIGN receptor.   A chimeric antibody comprising a non-human variable domain and a human IgG1 constant region, wherein the non-human variable domain binds to a receptor on any antigen presenting cell.   65. The chimeric antibody of claim 64, wherein the non-human variable region recognizes the L-SIGN receptor.   66. The chimeric antibody of claim 65, wherein the non-human variable region further recognizes a DC-SIGN receptor.   65. The chimeric antibody of claim 64, wherein the non-human variable region is that of a mouse.   65. The chimeric antibody of claim 64, wherein the human IgG constant region is a human IgG1 constant region.   65. The chimeric antibody of claim 64, which blocks binding of HIV gp120 to L-SIGN.   65. The chimeric antibody of claim 64, which blocks Ebola envelope protein binding to L-SIGN.   65. The chimeric antibody of claim 64, which blocks at least about 90% of Ebola envelope protein binding to L-SIGN.   65. The chimeric antibody of claim 64, which blocks Ebola envelope protein binding to L-SIGN better than non-human variable domains alone.   65. The chimeric antibody of claim 64, which blocks Ebola envelope protein binding to DC-SIGN better than non-human variable domains alone.   65. The chimeric antibody of claim 64, which blocks binding of HIV gp120 to L-SIGN better than a non-human variable domain alone.   65. The chimeric antibody of claim 64, which blocks binding of HIV gp120 to DC-SIGN better than a non-human variable domain alone.   An antibody that recognizes both L-SIGN and DC-SIGN receptors, wherein L-SIGN receptor recognition is stronger than DC-SIGN receptor recognition.   77. The antibody of claim 76, wherein L-SIGN receptor recognition is about 1.1 to about 20 times stronger than DC-SIGN recognition.   77. The antibody of claim 76, wherein L-SIGN receptor recognition is about 1.2 to about 10 times stronger than DC-SIGN recognition.

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