US20220251157A1

US20220251157A1 - IgM-MEDIATED RECEPTOR CLUSTERING AND CELL MODULATION

Info

Publication number: US20220251157A1
Application number: US17/674,438
Authority: US
Inventors: Larry R. Pease; Moses Rodriguez
Original assignee: Mayo Foundation for Medical Education and Research
Current assignee: Mayo Foundation for Medical Education and Research
Priority date: 2007-10-18
Filing date: 2022-02-17
Publication date: 2022-08-11
Also published as: WO2009052374A3; US20100291675A1; US20190256568A1; CA2734173C; US10202430B2; CA2734173A1; WO2009052374A2; US11274131B2

Abstract

Materials and methods for using multivalent molecules (e.g., antibodies) to modulate cellular function. A molecule can be targeted to a particular type of cell, either through direct binding to an epitope on the surface of the cell, or through a linker that recognizes both the multivalent molecule and a marker on the cell surface.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of co-pending application Ser. No. 16/233,773 filed Dec. 27, 2018, now U.S. Pat. No. 11,274,131, which is a Continuation of co-pending application Ser. No. 12/738,774 filed Jun. 28, 2010, now U.S. Pat. No. 10,202,430, which is a National Stage application under 35 U.S.C. § 371 claiming priority from PCT Application No. PCT/US2008/080304 having an International Filing Date of Oct. 17, 2008, which claims benefit of priority from U.S. Provisional Application Ser. No. 60/999,403 filed Oct. 18, 2007. Applicants claim the benefits of 35 U.S.C. § 120 as to the U.S. applications, and priority under 35 U.S.C. § 119 as to the said PCT Application and the U.S. Provisional application, and the entire disclosures of all applications are incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. CA096859 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This document relates to materials and methods for using multivalent molecules (e.g., antibodies) to modulate cellular function.

BACKGROUND

Dendritic cells (DC) are efficient antigen-presenting cells (APC). These cells express class I and class II major histocompatibility complex (MHC) peptide-presenting molecules on their cell surfaces, along with a series of costimulatory molecules (Banchereau and Steinman (1998) Nature 392:245-252). Naive T cells express receptors for these DC ligands. Following recognition of peptide-antigen presented in the context of class I or class II molecules, the structure of the T cell membrane is reorganized, bringing together the elements of the T cell receptor with other cell-surface molecules, including the co-receptors CD4 or CD8 and the costimulatory receptors CD28 and CTLA-4 (Monks et al. (1998) Nature 395:82-86; and Wulfing and Davis (1998) Science 282:2266-2269). Interactions within the newly formed macromolecular complexes determine the outcome of inductive events transduced into T cells by DC.
DC reside in a variety of tissues and display distinct tissue-associated phenotypes (Strunk etal. (1997) J. Exp. Med. 185:1131-1136; Caux et al. (1996) J. Exp. Med. 184:695-706; Wu et al. (1996) J. Exp. Med. 184:903-911; and Vremec et al. (1992) J. Exp. Med. 176:47-58). The relationships among the cell lineages of these different subsets of cells are not firmly established. A large body of work has emerged focusing on DC generated in vitro from bone marrow or blood precursors (Mayordomo et al. (1995) Nat. Med. 1:1297-1302; Nonacs et al. (1992) J. Exp. Med. 176:519-529; Steinman and Witmer (1978) Proc. Natl. Acad. Sci. USA 75:5132-5136; and Young and Steinman (1990) J. Exp. Med. 171:1315-1332). The cells generated in vitro express high levels of class I antigens and the series of costimulatory ligands associated with endogenous DC (Fagnoni et al. (1995) Immunology 85:467-474; and Banchereau et al. (2000) Annu. Rev. Immunol. 18:767-811). Importantly, they are able to efficiently activate naive T cells, a function that is the signature of the DC.
Decavalent IgM antibodies display measurable binding avidity to antigens, even though binding affinity may be low. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble antibodies with biological potential not normally associated with immune function. One such IgM antibody has been shown to bind and cross-link B7-DC on the surface of DC. This monoclonal antibody, which is referred to herein as B7-DC XAb but also has been called sHIgM12, rHIgM12, and Lym12, was originally isolated from a Waldenstrom's macroglobulinemia patient. As described in U.S. Pat. No. 7,052,694, U.S. Ser. No. 10/881,661, and U.S. Ser. No. 10/983,104 (all of which are incorporated herein by reference in their entirety), B7-DC XAb can, for example, activate DC, potentiate immune responses, modulate existing states of immune responsiveness, and treat or inhibit development of allergic asthma.

SUMMARY

As described herein, IgM antibodies and other multivalent molecules can be used to cluster and cap cell surface molecules on a variety of cell types, resulting in intracellular signaling and modulation of the targeted cells' functions. These methods takes advantage of certain IgM molecules' ability to bind with very low affinity to sets of endogenous ligands. Cell function can be modulated by targeting an IgM to a particular cell type via (1) a typical antibody interaction with an antigen normally expressed on the targeted cell, (2) a transgenic molecule expressed on the cell surface containing an epitope recognized by the IgM, or (3) a linker construct (e.g., a peptide or an antibody) with the ability to bind to the IgM and to a cell surface protein specific to the given cell type. Once the IgM has been recruited to the cell surface, its low affinity interaction with other endogenous ligands can result in receptor and cell surface molecule clustering, initiating intracellular signaling and modulating cell functions.
In one aspect, this document features a method for targeting a multivalent molecule to a cell, comprising: (a) contacting the cell with a linker molecule, wherein the linker molecule includes (i) an amino acid sequence comprising an epitope to which the multivalent molecule specifically binds and (ii) an amino acid sequence that binds specifically to a marker on the outer surface of the cell; and (b) contacting the cell with the multivalent molecule. The multivalent molecule can be an antibody (e.g., an IgM antibody). The linker molecule can consist of a polypeptide. The linker molecule can be a chimeric antibody.
In another aspect, this document features a method for targeting a multivalent molecule to a cell, comprising: (a) contacting the cell with a nucleic acid encoding a polypeptide, wherein the polypeptide includes (i) an amino acid sequence that directs the polypeptide to the cell's plasma membrane and (ii) an amino acid sequence comprising an epitope to which the multivalent molecule specifically binds; (b) culturing the cell under conditions in which the polypeptide is expressed and localized to the plasma membrane such that the epitope is located on the exterior of the cell; and (c) contacting the cell with the multivalent molecule. The multivalent molecule can be an antibody (e.g., an IgM antibody).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing IgM binding to a cell surface receptor. IgM antibodies can interact with cells via transgenic molecules expressed on the cell surface that contain an epitope recognized by the IgM (A), or a linker peptide (B) or antibody (C) having the ability to bind to the IgM and to a cell surface protein specific to the given cell type. Once recruited to the cell surface, the interaction of the IgM with other endogenous ligands can result in receptor and cell surface molecule clustering (D).

FIGS. 2A to 2G are a series of pictures showing that a kinase activated pathway regulates antibody-induced antigen uptake by matured DC. Matured human monocyte-derived DC were treated with the MTAb B7-DC XAb for varying time periods and evaluated for the phosphorylation status of select signaling pathway intermediates by immunoprecipitation with DAP12 (MC457), Syk (4D10), and PLCy1 (MC490) reactive antibodies followed by western blot analysis using the phosphotyrosine reactive antibody 4G10 as a probe (FIGS. 2A-2C). Samples of the cells blocked with indicated concentration of piceatannol (FIG. 2D) or with 10 mM of piceatannol (FIG. 2E) were analyzed for phosphorylation of Syk (FIG. 2D) and PLCγ1 (FIG. 2E). To investigate the functional importance of these signaling intermediates in the regulation of antigen uptake by matured DC activated with B7-DC XAb, varying concentrations of pharmacologic inhibitors were used to block the activity of p72Syk (piceatannol) and PLCγ1 (U73122) (FIGS. 2F and 2G, respectively). Antigen uptake was assessed by pulsing cultures with FITC-conjugated ovalbumin and measuring intracellular fluorescence 16 hours later in CD11c positive cells by flow cytometry.

FIG. 3 is a plot showing that matured human monocyte-derived DC take up ovalbumin when activated by the MTAb B7-DC XAb. Human DC were generated from peripheral blood CD14⁺ monocytes by incubation in GM-CSF and IL-4 for five days. Cultures were allowed to proceed untreated (immature DC) or were treated overnight with the TLR-3 agonist poly I:C (matured DC). On day six, the DC cultures were activated the isotype control antibody sHIgM39 or with the MTAb B7-DC XAb, as indicated. All cultures were pulsed with OVA-FITC at the time of treatment with IgM antibodies, and of OVA-FITC was assessed 24 hours later by flow cytometry.

FIGS. 4A and 4B are a series of plots showing the effects of kinase inhibitors on OVA-FITC uptake by matured human DC, as determined by flow cytometry. For FIG. 4A, day 6 matured human monocyte-derived DC were incubated in the presence or absence of the Src kinase inhibitor PP2 for 30 minutes prior to activation with isotype control antibody sHIgM39 or the MTAb B7-DC XAb, as indicated. All cells were pulsed with OVA-FITC at the time of treatment with IgM antibody, and CD11c+ cells were analyzed for OVA-FITC uptake 24 hours later by flow cytometry. For FIG. 4B, human DC were pretreated with the indicated inhibitors, activated on day 6 with the MTAb B7-DC XAb, pulsed with FITC-OVA, and analyzed the next day for uptake of FITC-OVA by flow cytometry Inhibitors used were 50 nM Bim 1 (PKC inhibitor); 25 μM Y-27632 (Rho A inhibitor); 10 μM LY294002 (PI3 kinase inhibitor); 10 μM U73122 (PLCy inhibitor); 10 μM PD98509 (MEK inhibitor); and 1 μM SB203580 (p38 MAP kinase inhibitor).

FIG. 5A to 5F is a series of histograms and blots indicating that tightly organized macromolecular caps assemble following B7-DC XAb treatment. In FIG. 5A, the distribution of class II (labeled with APC-conjugated antibody) and CD80/CD86 molecules (labeled with PE-conjugated antibodies) was visualized by FRET following treatment of human monocyte derived DC with B7-DC XAb (open histograms) or with isotype control antibody (filled histograms). For FIG. 5B, mouse bone marrow derived DC were labeled, treated, and analyzed as in FIG. 5A. FIG. 5C indicates the inability of the IA^bspecific IgM antibody 25-9-3 (open dark histograms), B7-DC XAb (open light histograms), and isotype control antibody treated samples (filled histograms) to induce FRET (top left and middle panels). Binding of the class II specific IgM antibody to the mouse DC analyzed is shown (top right panel). FIG. 5D indicates the ability of B7-DC-specific IgG antibody (open histograms on left of each panel) or no IgG antibody (open histograms on right of each panel) to inhibit FRET induced by the MTAb B7-DC XAb. Absence of FRET induced by Isotype control antibody is shown in reference (filled histograms). For FIGS. 5E and 5F, immunoprecipitates of membrane proteins from B7-DC XAb or control antibody treated DC were isolated using biotin tagged IA^b-specific KH74 antibody. The precipitates were analyzed by Western blot using mouse-specific TREM-2 and CD40 antibodies as probes.

FIG. 6A to 6C is a series of histograms and pictures showing that TREM-2 is recruited into the MTAb-induced cap on mouse DC and expression of TREM-2 is important for MTAb-induced phosphorylation of the adapter DAP-12 and the protein kinase Syk. FIG. 6A: six day cultures of bone marrow-derived mouse DC were differentiated in GM-CSF and IL-4 were tagged with antibodies specific for the class II molecule IA^b(25-9-17-APC) and for TREM-2 (237916-PE) for 15 minutes prior to stimulation with the MTAb B7-DC XAb (open histograms) or with isotype control antibody sHIgM39 (filled histograms). Cells were fixed in 0.1% paraformaldehyde prior to analysis by flow cytometry. FRET between PE and APC was assessed collecting light emitted by APC following excitation of PE with a 488 nm laser. Expression of TREM-2 on the cells was assessed by staining with 237916-PE antibody alone (open histogram) or isotype control antibody (closed histogram, bottom-right panel). For FIG. 6B, mouse bone marrow-derived DC were transduced on day 2 of culture with a retrovirus expressing an hnRNA specific for TREM-2 or a control virus containing a scrambled sequence. On day 5 of culture, the transduced cells were not treated (0′ time), activated with the isotype control antibody sHIgM39, or activated with B7-DC XAb for 5 minutes. DAP12 was immunoprecipitated from cell lysates with antibody MC457, resolved by electrophoresis, and the blotted filters were probed with the phosphotyrosine-specific antibody 4G10. FIG. 6C: the lysates used in the experiments from FIG. 6B were immunoprecipitated with the Syk-specific antibody (4D10) and analyzed by western blot for the presence of phosphotyrosine.

FIGS. 7A and 7B is a series of pictures and a pair of graphs indicating that distinct pathways are activated by B7-DC XAb. FIG. 7A is a pair of pictures of western blot analysis for phosphorylation of DAP12 and Syk in B7-DC XAb or control antibody-treated mouse DC, as indicated. FIG. 7B is a graph plotting percent CD11c+inguinal lymph node cells in mice after OVA-FITC was introduced into the footpads of mice treated systemically with isotype control antibody or the MTAb B7-DC XAb (n=3/group). Cells were analyzed by flow cytometry 24 hours after OVA-FITC introduction.

FIG. 8 is a graph plotting IL-6 levels in supernatants of cultured DC, showing that CD40 KO DC do not secrete IL-6 following activation with B7-DC XAb. Six day mouse bone marrow-derived DC from wild type or IL-6 KO mice were stimulated with isotype control antibody sHIgM39 or B7-DC XAb. IL-6 levels in culture supernatants were assessed 48 hours later by ELISA.

FIGS. 9A-9B, 9C and 9D are a series of graphs and pictures demonstrating that MTAbs share activation mechanisms. In FIGS. 9A and 9B, free intracellular calcium levels were assessed by flow cytometry in Indo-1 labeled mouse DC (FIG. 9A) or human DC (FIG. 9B) following stimulation with control antibody (dark) or B7-DC XAb (light histograms). The bottom panels in FIGS. 9A and 9B show calcium levels in DC preincubated with B7-DC specific IgG antibody and treated with B7-DC XAb (dark) or treated with ionomycin (light histogram). FIG. 9C shows a series of histograms obtained using transfected Jurkat cells expressing the mouse class I gene K^b/L^d(top panels) or a chimeric class I gene containing the 5A peptide mimetope recognized by the MTAb rHIgM22 (bottom panels) were tagged with antibodies specific for human CD4 (PE) and CD28 (APC) prior to treatment for 15 minutes with the control antibody sHIgM39, the MTAb rHIgM22, or the IgM antibody 28-13-3, specific for the K^b/L^dmolecule. Cells were analyzed for FRET by flow cytometry. FIG. 9D is a picture of a western blot using whole cell lysates of Jurkat cells that were analyzed for global tyrosine phosphorylation by western blot.

FIG. 10 is a series of histograms showing recruitment of C28 and human MHC class I molecules into a molecular cap on Jurkat cells expressing a rHIgM22-reactive peptide mimetope. Human Jurkat cells were transiently transfected with a mouse class I carrier gene (K^b/L^d; top panels) or with a chimeric class I gene expressing an rHIgM22-reactive peptide mimetope (K^b/L^d-5A; middle panels). The transfected cells were tagged with a pan human class I-specific antibody (PE) and CD28-specific antibody (APC) 15 minutes prior to treatment with either the human isotype control antibody sHIgM39, the MTAb rHIgM22, or the mouse IgM antibody 28-13-3, specific for the K^b/L^dcarrier protein. The cells were fixed 15 minutes after treatment with the IgM antibodies and analyzed by flow cytometry for evidence of FRET between the PE and APC fluorochromes. Bottom panels: stable Jurkat transfectants expressing either the K^b/L^dcarrier protein (filled histograms) or the mimetope tagged K^b/L^d-5A protein (open histograms), were analyzed for FRET following treatment of cells pre-stained with pan anti-HLA-A.B,C-PE and anti-CD4-APC with MTAb rHIgM22. The treated cells were analyzed for FRET at the indicated time points.

FIGS. 11A and 11B depicts the amino acid sequences of B7-DC XAb. FIG. 11A shows the variable (Vk) and constant (Ck5454) domains (SEQ ID NOS:4 and 5, respectively) of the B7-DC XAb light chain. FIG. 11B shows the variable (Vh) and constant (CH1, CH2, CH3, CH4) domains (SEQ ID NOS:6, 7, 8, 9, and 10, respectively) of the sHIgM12 heavy chain.

FIG. 12 depicts the nucleic acid sequences of B7-DC XAb. Nucleic acids sequences encoding the Vk and Vh domains (SEQ ID NOS:11 and 12, respectively) B7-DC XAb.

FIG. 13 is a series of plots showing surface expression of Kb-peptide constructs in Balb/c bone-marrow derived DCs, as detected by staining with antibody B8-24-3.

FIG. 14 is a series of histograms showing induction of FRET between CD80 PE and Class II APC in B7DC−/− DCs expressing the Kb-peptide constructs. Wild type DCs or retrovirally transduced B7DC−/− were stained with CD80 PE and Class II APC. Cells were treated with hIgM12 (red), hIgM22 (blue), or 28-13-3 (green). FRET of CD80 PE with Class II APC was measured by fluorescence in channel FL3.

FIG. 15 is a series of histograms showing restoration of antigen persistence in CpG-treated DCs expressing Kb-peptide constructs upon treatment with the appropriate MTab. DCs were untreated (blue) or treated with 10 ug/mL CpG (red) 48 hours prior to harvesting. They were then pulsed with Ovalbumin-Alexa647 and either hIgM12, hIgM22, or 28-13-3, and Ovalbumin-647 levels were measured in CD11c positive cells by flow cytometry.

FIG. 16 is a series of plots showing conversion of OT-II CD4 Treg cells to a Th17 phenotype by co-incubation with DCs expressing the Kb-peptide constructs treated with MTabs. OT-II CD4+CD25+ splenic Tregs were isolated by MACS and added 1:1 to DCs pulsed with Ovalbumin and treated with hIgM12, hIgM22, or 28-13-3. After 48 hours, the T cells were harvested, stained with CD4, and internally stained with FoxP3 PE and IL-17 APC. Expression was assayed by flow cytometry, and cells were gated on CD4.

FIG. 17 is a graph depicting protection of mice from B16-Ova tumor challenge by injection of Ova-pulsed DCs expressing the Kb-peptide constructs treated with MTabs. Tumor area was measured daily, with tumor area on day 11 represented in the graph.

DETAILED DESCRIPTION

Described herein are multivalent molecules that can activate the function of particular cell types. This work has implications for treatment of a wide variety of human diseases. For example, monoclonal IgM therapeutic antibodies (MTAbs) have been identified that can activate cells in the dendritic cell (DC) and oligodendrocyte lineages, inducing, e.g., immune modulation and remyelination of denuded axons (Nguyen et al. (2002) J. Exp. Med. 196:1393-1398; Warrington et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:6820-6825; and Miller and Rodriguez (1995) J. Immunol. 154:2460-2469). A remarkable feature of MTAbs is their tendency to bind to and activate homologous cells in rodents and humans (Warrington et al., supra; Radhakrishnan et al. (2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2007) J. Immunol. 178:1426-1432), facilitating the use of animal models in preclinical studies. These antibodies are present in the normal human repertoire, and can be identified in patients with monoclonal gammopathies.
MTAbs can function at very low concentrations, similar to what is observed with conventional growth factors (Warrington et al. (2007) J. Neurosci. Res. 85:967-976). While the clinical application of growth factors has been hampered by their short half life and difficulty of delivery, these obstacles can be overcome by MTAbs. IgM antibodies are natural blood products and have half lives in humans of days. In addition, these molecules activate cells to perform inherent “luxury” functions. For example, MTAbs can activate DC to stimulate killing of tumors by CD8+ T cells (Radhakrishnan et al. (2004) Cancer Res. 64:4965-4972; and Heckman et al. (2007) Eur. J. Immunol. 37:1827-1835), reprogram cellular immunity to block allergic airway inflammation (Radhakrishnan et al. (2004) J. Immunol. 173:1360-1365; and Radhakrishnan et al. (2005) J. Allergy Clin. Immunol. 116:668-674), or activate oligodendrocytes to make new myelin wraps around axons (Warrington et al. (2000), supra; and Miller et al. (1994) J. Neurosci. 14:6230-6238). MTAbs thus represent a new class of clinically useful reagent which may have wide applicability.
The mechanism by which MTAbs activate targeted cells has remained obscure, as cell surface molecules bound by these antibodies are not well defined. As disclosed herein, however, mechanisms underlying DC activation by the human MTAb B7-DC XAb have been characterized. Using these principles, it has been demonstrated that a second human MTAb, rHIgM22, an antibody that induces myelin repair in models of multiple sclerosis, can activate cells in the same fashion. Both therapeutic antibodies can rapidly induce formation of multimolecular caps on the surface of targeted cells, recruit signaling molecules that are known to control important cellular functions, and activate a series of intracellular signals in response.
MTAbs can modulate immune responsiveness and oligodendrocyte maturation by targeting cells in situ and inducing preprogrammed cellular functions. MTAbs capable of inducing anti-tumor immunity, blocking allergic airway inflammation, and inducing remyelination of denuded axons can employ a common cellular activation mechanism. For example, these antibodies can cross-link cell surface molecules and assemble macromolecular signaling complexes by recruiting receptor and adapter molecules into clusters, thereby activating key signaling pathways. Additional MTAbs that target cells specifically throughout the body can be identified within the normal human antibody repertoire, providing the basis for the development of novel therapeutic approaches to treat disease.
As described herein, IgM antibodies or other multivalent molecules can be used to cluster and cap cell surface molecules on a variety of cell types, resulting in intracellular signaling and modulation of the targeted cells' functions. These methods take advantage of certain IgM molecules' ability to bind with very low affinity to sets of endogenous ligands. In some embodiments, cell function can be modulated by targeting the IgM to a particular cell type via a “typical” antibody interaction with an antigen normally expressed on the targeted cell, or a transgenic molecule expressed on the cell surface containing an epitope recognized by the IgM (e.g., as depicted in FIG. 1A). In some cases, an IgM antibody can be targeted to a particular cell type via a linker molecule. A linker can interact with both an IgM via a mimetope recognized the IgM, and with a cell surface molecule specific to the given cell type. A linker can be, for example, a specially designed peptide or nucleic acid (FIG. 1B), or an antibody linked to a mimetope recognized by IgM (FIG. 1C). Once the IgM has been recruited to the cell surface, its low affinity interaction with other endogenous ligands can result in clustering/capping of the receptors, cell surface molecules, and adapter proteins (FIG. 1D), initiating intracellular signaling and modulating cell functions.
This ability to target a “generic” MTAb to a variety of different cell types using a linker can have a number of advantages. For example, because every IgM does not have sufficient affinity to bind effectively to endogenous ligands, a single MTAb can be used against a variety of different cell types. This can be especially useful if an IgM is identified that has no affinity for a specific receptor or a particular cell type. In addition, other types of multivalent molecules (e.g., tetramers, specially designed beads, and other constructs) can be designed to bind a specific linker and to have low affinity for endogenous ligands found on all cell types.
Linker polypeptides can be designed using any suitable means, including phage display. Such methods can allow the design of linkers specific to cell types where a cell-type specific receptor/surface molecule has not been identified. Moreover, linker polypeptides can be designed using very small peptides, such that the full construct is about 15 to about 40 amino acids in length (e.g., about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, or about 40 amino acids in length). Such linkers can be synthesized using standard techniques.
Further, the ability to target MTAbs to different cell types using a linker system can have enormous therapeutic potential. For example, a linker can be used to target an antibody specifically to a T_regcell or to a stem cell, enhancing or inhibiting its activation for therapeutic purposes. For example, a stem cell could be activated using an MTAb and a linker to promote differentiation either in vivo or ex vivo, providing a means for creating more differentiated stem cells for a variety of treatments (e.g., cardiac stem cells for treating myocardial infarction, or pancreatic islet cells for treating diabetes).
In addition, the ability to use a linker can be combined with other uses of therapeutic antibodies, such that the antibodies have multiple effects. For example, a linker peptide could be used to make the “B7-DC XAb” antibody described herein simultaneously therapeutic against dendritic cells through its ability to bind B7-DC and T_regcells (or any other cell type) through the linker.

Polypeptides and Antibodies

The molecules provided herein typically are polypeptides, and antibodies can be particularly useful (see below), but other multivalent molecules that can bind and cross-link molecules on the surface of cells also can function in this capacity. Examples of such molecules include, without limitation, multivalent RNA or DNA aptamers.
As used herein, a polypeptide is an amino acid chain, regardless of length or post-translational modification (e.g., phosphorylation or glycosylation). A polypeptide can contain an amino acid sequence that is similar to the amino sequence of B7-DC AXb, for example. A polypeptide can contain, e.g., an amino acid sequence that is at least 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to the amino acid sequence set forth in SEQ ID NO 4 or SEQ ID NO:6. In some embodiments, a polypeptide can further contain an amino acid sequence that is at least 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence.
To determine percent sequence identity, a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ.
B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: −i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); −j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set to blastn; −o is set to any desired file name (e.g., C:\output.txt); −q is set to −1; −r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq c:\seq1.txt −j c:\seq2.txt −p blastn −o c:\output.txt −q −1−r 2. To compare two amino acid sequences, the options of B12seq are set as follows: −i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); −j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); −p is set to blastp; −o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq c:\seq1.txt −j c:\seq2.txt −p blastp −o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:4), or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 98 matches when aligned with the sequence set forth in SEQ ID NO:4 is 92.5 percent identical to the sequence set forth in SEQ ID NO:4 (i.e., 98/106*100=92.5). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It is also noted that the length value will always be an integer.
The amino acid sequences of the polypeptides provided herein can have substitutions, deletions, or additions with respect to the amino acid sequences set forth in SEQ ID NOS:4 and 6. A polypeptide having an amino acid sequence that is modified (e.g., by substitution) with respect to SEQ ID NO:4 and/or SEQ ID NO:6 can have substantially the same or improved qualities as compared to a polypeptide containing the amino acid sequence identical to that set forth in SEQ ID NO:4 and SEQ ID NO:6. A substitution can be a conserved substitution. As used herein, a “conserved substitution” is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution typically can be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall peptide essentially retains its spatial conformation but has altered biological activity. Examples of conserved changes include, without limitation, Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu, and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains (see, e.g., Stryer, Biochemistry (2^ndedition) W. H. Freeman and Co. San Francisco (1981), pp. 14-15; and Lehninger, Biochemistry (2^ndedition, 1975), pp. 73-75). Conservative substitutions can include substitutions made within these groups.
Molecules provided herein can be antibodies. The terms “antibody” and “antibodies” encompass intact molecules as well as fragments thereof that can bind to a particular antigen. Antibodies can be polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)₂fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, while monoclonal antibodies are homogeneous populations of antibodies to a particular epitope contained within an antigen.
An antibody can be of any immunoglobulin (Ig) class, including IgM, IgA, IgD, IgE, and IgG, and any subclass thereof. Antibodies of the IgM class (e.g., B7-DC XAb) typically are pentavalent and can be particularly useful. Immune complexes containing Ig molecules that are cross-linked (e.g., cross-linked IgG) and are thus multivalent also can be particularly useful.
As used herein, an “epitope” is a portion of an antigenic molecule to which an antibody binds. Antigens can present more than one epitope at the same time. For polypeptide antigens, an epitope typically is about four to six amino acids in length, and can include modified (e.g., phosphorylated or glycosylated) amino acids. Two different immunoglobulins can have the same epitope specificity if they bind to the same epitope or set of epitopes.
Polyclonal antibodies are contained in the sera of immunized animals. Monoclonal antibodies can be prepared using, for example, standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture as described, for example, by Kohler et al. (1975) Nature 256:495-497, the human B-cell hybridoma technique of Kosbor et al. (1983) Immunology Today 4:72, and Cote et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the EBV-hybridoma technique of Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96 (1983). A hybridoma producing monoclonal antibodies can be cultivated in vitro or in vivo.
Antibodies also can be isolated from, for example, the serum of an individual. The B7-DC XAb antibody, for example, was isolated from human serum as described in U.S. Pat. No. 7,052,694. Suitable methods for isolation include purification from mammalian serum using techniques that include, for example, chromatography.
Antibodies also can be produced by, for example, immunizing host animals (e.g., rabbits, chickens, mice, guinea pigs, or rats) with an immunogen (e.g., an antigen or epitope). An immunogen can be produced recombinantly, by chemical synthesis, or by purification of the native protein, and then used to immunize animals by injection of the polypeptide. Adjuvants can be used to increase the immunological response, depending on the host species. Suitable adjuvants include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin (KLH), and dinitrophenol. Standard techniques can be used to isolate antibodies generated in response to the immunogen from the sera of the host animals.
Antibodies such as B7-DC XAb also can be produced recombinantly. The amino acid sequence (e.g., the partial amino acid sequence) of an antibody can be determined by standard techniques, and a cDNA encoding the antibody or a portion of the antibody can be isolated from the serum of the subject (e.g., the human patient or the immunized host animal) from which the antibody was originally isolated. The cDNA can be cloned into an expression vector using standard techniques. The expression vector then can be transfected into an appropriate host cell (e.g., a Chinese hamster ovary cell, a COS cell, or a hybridoma cell), and the antibody can be expressed and purified. See, for example, U.S. Ser. No. 10/983,104. Antibody fragments that have specific binding affinity for an antigen also can be generated by techniques such as those disclosed above. Such antibody fragments include, but are not limited to, F(ab′)2 fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al. (1989) Science 246:1275-1281. Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. Single chain Fv antibody fragments can be produced through standard techniques, such as those disclosed in U.S. Pat. No. 4,946,778. Such fragments can be rendered multivalent by, for example, biotinylation and cross-linking, thus generating antibody fragments that can cross-link a plurality of B7-DC polypeptides.

Nucleic Acids, Vectors, and Host Cells

Nucleic acids encoding polypeptides and antibodies also are provided herein.
The term “nucleic acid” refers herein to both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acids include, for example, cDNAs encoding antibody light and heavy chains.
An “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that normally flank one or both sides of the nucleic acid in the genome in which it is normally found. The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in its naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.
The isolated nucleic acids disclosed herein can encode polypeptides as described herein. For example, an isolated nucleic acid can encode a polypeptide containing an amino acid sequence that is similar or identical to an amino acid sequence found in the variable or constant regions of B7-DC XAb (e.g., SEQ ID NOS:4, 5, 6, 7, 8, 9, 10, 11, and 12, shown in FIGS. 11A, 11B, and 12). In some embodiments, a nucleic acid can encode a polypeptide containing an amino acid sequence that is at least 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:6. The encoded polypeptide can further contain an amino acid sequence that is at least 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In some cases, an isolated nucleic acid can contain a nucleotide sequence that is at least 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to the nucleotide sequence set forth in SEQ ID NO:11 or SEQ ID NO:12. The method for determining percent sequence identity is provided above.
Isolated nucleic acid molecules can be produced using standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid molecule encoding an antibody. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to 5′ direction using phosphoramidite technology) or as a series of polynucleotides. For example, one or more pairs of long polynucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the polynucleotide pair is annealed. DNA polymerase is used to extend the polynucleotides, resulting in a single, double-stranded nucleic acid molecule per polynucleotide pair.
Vectors containing nucleic acids such as those described herein also are provided. A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
In the expression vectors provided herein, a nucleic acid (e.g., a nucleic acid encoding the light and/or heavy chains of an antibody such as B7-DC XAb) can be operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 to 500 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence. Expression vectors thus can be useful to produce antibodies as well as other multivalent molecules.
Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).
An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
Host cells containing vectors also are provided. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells are found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2^ndedition), Cold Spring Harbor Laboratory, New York (1989), and reagents for transformation and/or transfection are commercially available (e.g., LIPOFECTIN® (Invitrogen); FUGENE® (Roche, Indianapolis, Ind.); and SUPERFECT® (Qiagen, Valencia, Calif.)).
Also provided herein are cells (e.g., DC) contacted in vitro with a multivalent polypeptide (e.g., an IgM antibody) as described herein.

Compositions

The molecules described herein (e.g., antibodies such as B7-DC XAb and nucleic acids encoding linkers or transgenic receptors) can be incorporated into compositions, as can isolated cells that have been contacted with one or more molecules as described herein. Compositions provided herein also can contain a molecule (e.g., a polypeptide) that is immobilized on a solid substrate, such as a bead. The compositions can be administered to a subject in order to modulate cellular function (e.g., to enhance DC function and potentiate an immune response).
Methods for formulating and subsequently administering therapeutic compositions are well known to those skilled in the art. Dosages typically are dependent on the responsiveness of the subject to the molecule, with the course of treatment lasting from several days to several months, or until a suitable immune response is achieved. Persons of ordinary skill in the art routinely determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of an antibody, and generally can be estimated based on the EC₅₀found to be effective in in vitro and/or in vivo animal models. Dosage typically is from 0.01 μg to 100 g per kg of body weight (e.g., from 1 μg to 100 mg, from 10 μg to 10 mg, or from 50 μg to 500 μg per kg of body weight). Compositions containing the molecules provided herein may be given once or more daily, weekly, monthly, or even less often.
In addition to the molecules provided herein, the compositions described herein further can contain antigens that will elicit a specific immune response. Suitable antigens include, for example, polypeptides or fragments of polypeptides expressed by tumors and pathogenic organisms. Killed viruses and bacteria, in addition to components of killed viruses and bacteria, also are useful antigens. Such antigens can stimulate immune responses against tumors or pathogens.
The molecules (e.g., antibodies, other polypeptides, or nucleic acids) can be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures, or mixtures of compounds such as, for example, liposomes, receptor targeted molecules, or oral, topical or other formulations for assisting in uptake, distribution and/or absorption.
In some embodiments, a composition can contain a molecule provided herein in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering antibodies to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, without limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
Pharmaceutical compositions containing molecules provided herein can be administered by a number of methods, depending upon whether local or systemic treatment is desired. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous (i.v.) drip); oral; topical (e.g., transdermal, sublingual, ophthalmic, or intranasal); or pulmonary (e.g., by inhalation or insufflation of powders or aerosols). Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). For administration to the central nervous system, antibodies can be injected or infused into the cerebrospinal fluid, typically with one or more agents capable of promoting penetration across the blood-brain barrier.
Compositions and formulations for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions (e.g., sterile physiological saline), which also can contain buffers, diluents and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers).
Compositions and formulations for oral administration include, for example, powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Such compositions also can incorporate thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders.
Formulations for topical administration include, for example, sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents and other suitable additives. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be useful.
Pharmaceutical compositions include, but are not limited to, solutions, emulsions, aqueous suspensions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, for example, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Emulsion formulations are particularly useful for oral delivery of therapeutic compositions due to their ease of formulation and efficacy of solubilization, absorption, and bioavailability. Liposomes can be particularly useful due to their specificity and the duration of action they offer from the standpoint of drug delivery.
Molecules featured herein can encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a subject, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, this document provides pharmaceutically acceptable salts of molecules such as antibodies, prodrugs and pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. A prodrug is a therapeutic agent that is prepared in an inactive form and is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the antibodies useful in methods provided herein (i.e., salts that retain the desired biological activity of the parent antibodies without imparting undesired toxicological effects). Examples of pharmaceutically acceptable salts include, but are not limited to, salts formed with cations (e.g., sodium, potassium, calcium, or polyamines such as spermine); acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid); salts formed with organic acids (e.g., acetic acid, citric acid, oxalic acid, palmitic acid, or fumaric acid); and salts formed with elemental anions (e.g., bromine, iodine, or chlorine).
Compositions additionally can contain other adjunct components conventionally found in pharmaceutical compositions. Thus, the compositions also can include compatible, pharmaceutically active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or additional materials useful in physically formulating various dosage forms of the compositions described herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. Furthermore, the composition can be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, penetration enhancers, and aromatic substances. When added, however, such materials should not unduly interfere with the biological activities of the other components within the compositions.
Pharmaceutical formulations as disclosed herein, which can be presented conveniently in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients (i.e., the antibodies) with the desired pharmaceutical carrier(s). Typically, the formulations can be prepared by uniformly and intimately bringing the active ingredients into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Formulations can be sterilized if desired, provided that the method of sterilization does not interfere with the effectiveness of the molecules(s) contained in the formulation.

Methods

This document provides methods for targeting multivalent molecules to a particular cell type. Such methods can include, for example, contacting a cell with a multivalent polypeptide (e.g., an IgM antibody) that can bind specifically to a particular epitope. In some embodiments, the epitope can be present on the surface of the cell. In such cases, the epitope can be contained within a native polypeptide that is expressed on the cell surface, or can be a mimetope contained within a transgenic polypeptide that is expressed on the cell surface. In the latter case, the method can include contacting the cell with a nucleic acid encoding the transgenic polypeptide, such that the transgenic polypeptide is expressed on the cell surface. In some embodiments, the epitope can be included in a linker molecule (e.g., a polypeptide or antibody) that interacts with both the multivalent molecule and with a cell surface molecule. In such cases, the method can include contacting the cell with the linker or with a nucleic acid encoding the linker.
The methods provided herein can be used to modulate the function(s) of the cells to which the multivalent molecules and other components are administered. In some embodiments, for example, a multivalent molecule (e.g., an antibody such as B7-DC XAb) or a composition containing the multivalent molecule or a nucleic acid encoding the molecule can be administered to a mammal (e.g., a dog, a cat, a horse, a cow, a rabbit, a rat, a mouse, or a human). As described above, the molecule(s) or composition can be administered using any suitable systemic or local method. Systemic methods of administration include, without limitation, oral, topical, or parenteral administration, as well as administration by injection. Local methods of administration include, for example, direct injection into a tumor.
Methods also are provided that include contacting an isolated cell (e.g., a dendritic cell, or any other type of cell) in vitro with a multivalent polypeptide (e.g., an IgM antibody) as described above, and administering the cell to a subject.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1—Materials and Methods

Mice: C57BL/6J, B6.129s4-CD80−/−CD86−/−, CNCr.129P2-Cd40tm1Kik/J, and B6.12952-IL6tmlKopf/J mice, 6-8 weeks old, were obtained from Jackson Laboratories (Bar Harbor, Me.) and used for generation of bone marrow derived DC. Class II knock out mice (L. Madsen et al. (1999) Proc. Natl. Acad. Sci. USA 96:10338-10343) were a gift from Dr. Chella David, Mayo Clinic. TREM-2 knock out mice (Turnbull et al. (2006) J. Immunol. 177:3520-3524), bred in the mouse colony at Washington University School of Medicine, St. Louis, Mo., were provided by Dr. Marco Colonna. Pregnant rats were purchased from Harlan Sprague (Indianapolis, Ind.). All animals were maintained at the Mayo Clinic animal facility for at least one week prior to use.
Reagents: Appropriate fluorophore labeled antibodies against murine I-A^b(25-9-17), murine class II specific IgM (25-9-3), APC labeled anti-mouse CD11c (HL3) FITC labeled anti-human class II (TU39), FITC labeled anti-human CD28 (CD28.2), APC labeled anti-human CD28 (CD28.2), PE labeled anti-human CD4 (RPA-T4) and PE labeled anti-human HLA A, B, C (G46-2.6) were purchased from BD PharMingen (San Jose, Calif.). Appropriate fluorophore labeled antibodies against mouse class II (M5/114.15.2), CD80 (16.10A1), CD86 (GL-1), CD11c (N418), CD40 (IC 10), APC labeled antibody against human DR (LN3), PE labeled anti-human CD80 (2D10.4), CD86 (IT2.2), murine B7-DC specific IgG antibody (TY25), and human B7-DC IgG antibody (MIH18) were purchased from eBioscience (San Diego, Calif.). All secondary appropriately fluorophore labeled F(ab)²fragment antibodies were obtained from Jackson Immunoresearch (Westgrove, Pa.). An IgM antibody (28-13-3) specific for mouse class I H-2 K^bwas obtained from a hybridoma cell line from ATCC(HB-41) (Manassas, Va.). Antibodies against the C terminal portion of NF-κB (sc372) and the protein kinase Syk (4D10) were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). DAPI and DNAse were obtained from Sigma Aldrich (St. Louis, Mo.). Anti phosphotyrosine, 4G10 and goat anti-mouse antibody were obtained from Upstate Cell Signaling Solutions (Lake Placid, N.Y.). Anti-mouse TREM-2 antibodies (237920) for flow cytometry and (237916) for western blot analysis were purchased from R&D systems (Minneapolis, Minn.). Rabbit antibodies against PLC yl (MC490), and DAP12 (MC457) were developed by Dr. Paul Leibson, Mayo Clinic. Ovalbumin labeled with FITC or APC was purchased from Molecular Probes (Eugene, Oreg.). Protein A Sepharose was purchased from Pierce Biotechnology (Rockford, Ill.). All inhibitors were obtained from Calbiochem (San Diego, Calif.) unless otherwise indicated. Piceatenol was obtained from Sigma Aldrich. Rac-1 inhibitor, NSC23766 was a gift from Dr. Daniel Billadeau, Mayo Clinic. LPS was obtained from Sigma Aldrich. CpG oligonucleotides (Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443) were synthesized at the Mayo core facility. The polynucleotide pI:C was purchased from Calbiochem (San Diego, Calif.). All MTABs were purified as described (Warrington et al. (2000), supra; and Radhakrishnan et al. (2003) J. Immunol. 170:1830-1838) and used at 10 μg/ml.
Generation of DC: DC from mouse bone marrow were generated as previously described (Inaba et al. (1992) J. Exp. Med. 176:1693-1702). Briefly, bone marrow was isolated from the long bones of the hind legs. Erythrocytes were lysed by treatment with ammonium chloride/potassium bicarbonate/EDTA at 37° C. The remaining cells were plated 1×10⁶cells/ml in six-well plates (BD Biosciences, San Jose, Calif.) in RPMI containing 10 ng/ml of murine GM-CSF and 1 ng/ml of murine IL-4 (PeproTech, Rocky Hill, N.J.). The cells were incubated at 37° C. with 5% CO2. After 48 hours, the cells were washed and replated with RPMI containing the same concentration of GM-CSF and IL-4 for another 5 days. Human DC were derived from CD14+ mononuclear cells isolated from peripheral blood using magnetic bead sorting (Miltenyi Biotec, Auburn, Calif.). Briefly, a buffy coat was obtained from a unit of blood donated by a normal human donor. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, N.J.) and the CD14+ cells were separated by positive magnetic cell sorting. The isolated cells were incubated in RPMI 1640 supplemented with 5% human AB serum (HP 10220, Valley Biomedical, Winchester, Va.), 1% sodium pyruvate (Mediatech, Herndon, Va.), 1% non-essential amino acids (Mediatech), 1 ng/ml IL-4 (R&D Systems, Minneapolis, Minn.) and 50 ng/ml GM-CSF (Berlex, Richmond, Calif.) at 1×10⁶cells/ml, 3 ml/well in six well plates for 6-8 days at 37° C. with 5% CO₂. Maturation of DC was achieved by addition of TLR ligands for a period of 24 hours before being used for antigen uptake assays.
Generation of mixed glial cultures: Oligodendrocytes from rat pups were obtained as per the protocol previously described (Warrington et al. (2000), supra). Briefly, tissue culture plates coated with poly-D-lysine (25 ug/mL) prepared in water for 1-2 hours at 37° C. were used for culturing the cells. Rat pup brains were removed under sterile conditions. Cerebral hemispheres, hindbrain, cerebella, thalamus, hippocampus, and meninges were removed and minced into 1 mm chunks with a sterile single-edge razor blade. Tissue chunks were trypsinized, subjected to shaking for 30-40 minutes at 37° C., and heat inactivated fetal calf serum was added to a final concentration of 10% to inactivate the trypsin. DNAse at 1:50 was added to the solution and incubated for 5-10 minutes. This process was repeated until a single cell suspension was achieved. The cell suspension was layered over a cushion of 4% BSA and then centrifuged. Supernatant was collected and the cells were resuspended in DMEM containing 10% FCS at a density of 20×10⁷cells/plate. Media was changed 4 days post-seeding and every 3 days thereafter. The plates were shaken at day 9 to obtain a heterogenous population of oligodendrocytes.
Production of shTREM2 and shControl virus: Oligos containing the shTREM2 sequence (5′-TGATGCTGGAGATCTCTGGGTTCAAGAGACCCAGAGATCTCCAGCATCTTTTTTC-3; SEQ ID NO:1) and shControl sequence (5′-TGACTGCTGAAGGTCGCTTGTTTCAAGAGACCAAGCGACCTCCAGCATCTTTTTTC-3′; SEQ ID NO:2) (Warrington et al. (2000), supra) were synthesized and cloned into the pSUPER RNAi System (provided by Dr. Daniel Billadeau, Mayo Clinic) using the key restriction sites Bgl II and Hind III. The sequence was confirmed by automated sequencing of the vectors. The resulting vectors were co-transfected with VSV-G and gagpol plasmids (both provided by Dr. Richard Vile, Mayo Clinic) into 293T cells. Supernatant was collected at 48 and 72 hours, pooled, filtered through a 0.45 micron filter and frozen until used for transduction.
Transduction of DC: For transducing DC with the virus, 1 ml of supernatant containing the scrambled virus or shDNA containing dominant negative TREM-2 virus was mixed with 2 ml of RPMI. Cytokines were added to a final concentration of 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4 at day 2 of DC culture. Cells were maintained for another 3 days before using the DC for antigen uptake assay as mentioned above or for analysis of phosphorylation status of DAP12 and Syk proteins.
Immunoblots: In experiments involving assessment of the phosphorylation status of various protein kinases using whole cell lysate, dendritic cells of mouse or human origin or Jurkat cells were stimulated at the indicated times with control antibody or B7-DC XAb, and were lysed on ice for 10 minutes in 1 ml buffer containing 20 mM Tris-HCl, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na₄P₂O₇, 0.1% BSA, 1 mM Na₃VO₄, 1 mM PMSF, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 1% Triton X-100. Cellular debris was removed by centrifugation at 20,800×g for 5 minutes at 4° C. and the supernatant was used in SDS-PAGE analysis for phosphorylated tyrosine proteins. For immunoprecipitation, antibody against mouse Syk (4D10) or PLC γ1 (MC490) or DAP12 (MC457) at 10 μg per sample was bound to protein A-Sepharose beads at 4° C. for 2 hours under constant rotation. Supernatants from cell lysate after stimulation were incubated with antibody coupled beads for 2 hours at 4° C. with constant rotation. In experiments involving inhibition of Syk kinase, cells were incubated with 10 μM of Piceatenol for 30 minutes before being stimulated with control antibody or B7-DC XAb. For suppression of TREM-2, DC were transduced as described above and were stimulated with control antibody or B7-DC XAb on day six, lysed, and subjected to immunoprecipitation. Protein complexes were eluted in 40 μl of SDS sample buffer, resolved by SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). Tyrosine-phosphorylated proteins were detected using the anti-phosphotyrosine specific antibody, 4G10, followed by goat anti-mouse IgG coupled to Horse Radish Peroxidase (Santa Cruz Biotechnology) and the SuperSignal detection system (Pierce Biotechnology, Rockford, Ill.). Thereafter, total protein was visualized by staining the membrane with Ponceau staining solution (Pierce Biotechnology) for 30 seconds for analysis of whole cell lysate. For immunoprecipitation assays, the membrane was stripped with 7M guanidine, blocked with BSA, and probed with the antibody against the whole protein followed by protein A coupled to HRP (Amersham Biosciences) and the SuperSignal detection system. For pull down assays from the macro molecular complex, affinity purified antibody against mouse Class II (I-A^b) (KH74) was used for immunoprecipitation. The supernatants were resolved by SDS-PAGE, probed with anti-mouse TREM-2 antibody (237920) and detected by Goat-anti mouse antibody coupled to HRP, or probed with affinity purified anti-mouse CD40 (1C10) and detected by Goat-anti mouse antibody coupled to HRP.
NFκB activation assay: DC of murine or human origin were stimulated with 10 μg/ml of control antibody or the MTAb B7-DC XAb for 30 minutes, while stimulation of oligodendrocytes was achieved by addition of 10 μg/ml of MTAb sHIgM39, 04, or sHIgM22 for same period of time. All groups were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD PharMingen, San Diego, Calif.) for 20 minutes on ice. Subsequently, rabbit antibody against a C terminal peptide of NF-κB (sc372) was added, incubated for 30 minutes at 4° C. and washed three times with Cytoperm Buffer. Anti-rabbit FITC was used to detect the bound sc372 antibody by incubating the cells for 30 minutes (green). Cells were washed three times before the nuclei were stained with DAPI (blue) (Sigma Aldrich). The cells were then harvested from the plate, cytospin slides were made, and cells were visualized using a LSM510 Laser scanning confocal microscope (Carl-Zeiss Inc, Oberkochen, Germany) at 40.times. magnification.
IL-6 ELISA: DC of wild type origin or from CD40 deficient mice were stimulated with 10 μg/ml control antibody or B7-DC XAb for 48 hours. Supernatants from the different treatment groups were harvested and quantified for the amount of IL-6 by sandwich ELISA as per the manufacturer's protocol (eBioscience, San Diego, Calif.).
Live cell imaging for visualization of macromolecular complex: DC of murine origin were stained with anti-Class II-FITC (MF/114.15.2), and either anti-CD80-PE (16.10A1)/CD86-PE (GL-1), or anti-CD11c-PE (N418). DC of human origin were stained with anti-Class II-FITC (LN3) and anti-CD80-PE (2D10.4)/CD86-PE (IT2.2). All incubations were carried out for 15 minutes at 37° C. The cells were subsequently stimulated with 10 μg/ml of control (sHIgM39) or B7-DC XAb, and were observed every 5 minutes using time lapse confocal imaging at 40.times. magnification with a LSM510 Laser scanning confocal microscope having a 37° C. stage (Carl-Zeiss Inc, Oberkochen, Germany). Jurkat cells were pre stained with anti-human CD28-FITC (CD28.2) for 15 minutes, followed by addition of 10 μg/ml of MTAb control antibody sHIgM39, MTAb sHIgM22, or anti-K^bIgM (28-13-3), and were and analyzed in the same manner as the DC.
Flow Cytometry and FRET: The flow cytometry approach for FRET was used as a way of quantifying molecular aggregation on the cell surface and was carried out as described previously (Block et al. (2001) J. Immunol. 167:821-826). Briefly, DC of murine origin were stained with anti-Class II APC (M5/114.15.2) and anti-CD80-PE (16.10A1)/CD86-PE(GL-1) for 15 minutes to visualize FRET between these molecules. Monitoring for FRET induced by interaction between TREM-2 and Class-II molecules was achieved by incubating the cells with the abovementioned anti-Class-II antibody and anti-TREM-2-PE (237920). DC of human origin were stained for 15 minutes with APC-anti class II (LN3) and anti-CD80-PE (2D10.4)/CD86-PE (IT2.2). In experiments involving blocking of B7-DC, both fluorophore labeled antibodies and purified anti-mouse B7-DC (TY-25) or purified anti-human B7-DC (MIH18) IgG monoclonal antibody was added at 10 μg/ml for 15 minutes. Jurkat cells were stained with anti-human Class I-PE (G46-2.6) or anti-human CD4 (RPA-T4) and anti-human CD28-APC (CD28.2) for 15 minutes. Cells were stimulated with control antibody or B7-DC XAb or purified anti-mouse class II IgM (25-9-3) (experiments involving DC) or sHIgM22 (experiments involving Jurkat cells) and aliquots from different groups were taken at different time points. After 15 minutes of incubation, the cells were washed and fixed in 2% paraformaldehyde prior to analysis by FACS using a FACSCALIBER™ (BD Biosciences, Franklin Lakes, N.J.). Data collected as log₁₀fluorescence were analyzed using CELLQUEST™ (BD Biosciences). The FRET signal was visualized in FL3 channel (650-670 nm LP).
Antigen uptake assay: Antigen uptake experiments were carried out as described previously (Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443). Day 5 DC were matured with TLR ligand CpG-ODN for mouse DC, or with pI:C for human DC. The matured cells were incubated with ovalbumin labeled with FITC or APC and control antibody or B7-DC XAb for two hours, washed and analyzed by FACSCALIBUR™ (Becton Dickinson). In studies involving inhibitors, cells were pretreated 30 minutes as indicated or at 10 μM concentration prior to addition of control antibody or B7-DC XAb. In vivo antigen uptake assays involving wild type or TREM-2−/− mice were carried out by intravenous injection of 10 μg of control antibody or B7-DC XAb on three consecutive days (days −1, 0 and +1). Ovalbumin coupled to FITC was injected in the right foot pad at 1 mg/ml in PBS at 100 μA volume. The draining lymph node cells were harvested 48 hours after injection of antigen, and stained with anti-mouse CD11c-PE. The data are expressed as percent green cells (ovalbumin-FITC) that are positive for CD11c.
Calcium flux assays: Changes in the levels of intracellular Ca²⁺ were assessed in Indol-loaded cells by flow cytometry as described previously (Takahashi et al. (2005) J. Exp. Med. 201:647-657). Briefly, 5×10⁶DC/ml were incubated with 5 μM Indo-1 (Calbiochem) at 37° C. for 30 minutes. The cells were washed twice in serum free media and resuspended at 10^6/ml in PBS containing 0.5% BSA. The samples were analyzed by flow cytometry using a UV laser. Violet (390 nm) and blue (500 nm) fluorescence emissions were recorded. Baseline was recorded for two minutes. Antibody was added to the cell suspension (10 μg/ml of control antibody or B7-DC XAb) and the measurements were recorded for 8 additional minutes. In blocking experiments, mouse or human DC were pre-incubated with 10 μg/ml of purified anti-mouse B7-DC antibody (TY-25) or purified anti-human B7-DC antibody (MIH18) for 15 minutes, followed by collection of data for calcium flux as described above. Ionomycin added at 1 μg/ml after 2 minutes served as a positive control. The blue to violet ratio was calculated using FlowJo software (Tristar, Ashland, Oreg.).
Phage Display Library: A disulfide constrained heptapeptide phage display library from New England Biolabs (Ipswich, Mass.) was used for phage display (Felici et al. (1991) J. Mol. Biol. 222:301-310). The randomized sequence in the library is flanked by cysteine residues, allowing disulfide cross-link that results in phage display of cyclized peptides. Host strain ER2738 and phage titering were followed per New England Biolab's instruction manual. Briefly, a tissue culture dish was coated with 30 μg/ml of sHIgM22 in 2 ml of PBS overnight at 4° C. The following day, an exponential culture of ER2738 was grown in Tetracycline LB media. After blocking and washing, the phage library was diluted to 2×10¹¹in 1 ml of 1×TBS containing Tween-20 buffer. Upon removal of unbound phage by repeated washing, bound phage was eluted with 1.5 ml of 0.2M Glycine pH 2.2. After neutralizing with 1M Tris pH 9.0, eluted phage was added to an exponentially growing culture of ER 2738 bacteria and was allowed to amplify for 4.5 hours at 37° C. PEG was used to precipitate the culture supernatant. After 4 rounds, the amplified phage was cloned by limiting dilution. Twenty single-phage containing colonies were picked for sequence analysis. A peptide sequence (5A) present in 7 of 20 colonies was identified.
Generation of K^b/L^d-5A class I-peptide chimera: The 5A consensus peptide (PPWQSWI; SEQ ID NO:3) coding sequence was introduced into the K^b/L^dgene (Pullen et al. (1989) J. Immunol. 143:1674-1679) by site directed mutagenesis (Stratagene, Cedar Creek, Tex.) as per manufacturer instructions. Briefly, two complementary oligonucleotides were generated. The 5′ strand included a sequence encoding the consensus 5A peptide (PPWQSWI; SEQ ID NO:3) flanked by a HSAC (SEQ ID NO:13) spacer on the 5′ end and 20 bases of the K^bintron sequence. The 3′ end included a CG spacer followed by 20 bases of the alpha one domain of the class I gene. Oligonucleotides were isolated by PAGE purification. A mixture consisting of 290 ng of each oligo with 500 ng of 5A7-K^btemplate DNA was amplified with Ultra HF polymerase for 18 cycles at 95° C. for 1 minute and 55° C. for 1 minute, followed by one cycle of elongation at 68° C. for 10 minutes. After treatment with the endonuclease DpnI, the samples were transformed into XL-10 competent cells and plated on Ampicillin plates, and 12 randomly selected colonies were sequenced with ABI's 3730XL capillary sequencer. Positive colonies were grown up using Qiagen's Endotoxin Free Mega plasmid purification kit.
Transfection of Jurkat T cells: The day prior to transfection, plasmid DNA of each construct was purified by ethanol precipitation. Jurkat cells (15×10⁶cells) were resuspended in 250 μl RPMI, and 50 μl of resuspended DNA was added. After 10 minutes of incubation at room temperature, the whole suspension was transferred to a #640 BTX 4 mm Gapped cuvette and electroporation was carried out at 315 volts, 1 pulse, 10 pulse length, and at low voltage using a BTX Model 820 electroporator. Cells (1×10⁶/ml) were resuspended in RPMI containing 10% FCS. After overnight incubation at 37° C., cells were harvested and used for assays.

Example 2—Effects of B7-DC XAb Binding

Human MTAb B7-DC XAb binds to the costimulatory molecule B7-DC (PD-L2) on the surface of DC and in a B7-DC-dependent manner activates DC functions (Nguyen et al., supra; Radhakrishnan et al. (2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443). To determine the mechanism of cellular activation for MTAbs, intracellular events rapidly triggered by B7-DC XAb binding were examined. As shown in FIG. 2, phosphorylation of cellular proteins was induced as early as one minute after treatment of human DC with B7-DC XAb. As no signaling domains are evident in the structures of mouse or human B7-DC (Tseng et al. (2001) J. Exp. Med. 193:839-846), association of B7-DC with molecules containing signaling domains may be required for this activation. The adaptor molecule DAP12 can couple receptors lacking innate signaling capability to downstream signaling pathways (Tomasello et al. (2000) Immunity 13:355-364; Bouchon et al. (2001) J. Exp. Med. 194:1111-1122; and Snyder et al. (2004) Immunol. 173:3725-3731), and was rapidly phosphorylated upon cross-linking B7-DC on human DC (FIG. 2A). In other systems, Src family kinases are linked to Syk family kinases by DAP12 (Obergfell et al. (2002) J. Cell Biol. 157:265-275). Analysis of immunoprecipitated Syk, before and after treatment of human DC with B7-DC XAb, revealed phosphorylation of the protein tyrosine kinase (FIG. 2B) concomitant with the phosphorylation of PLCγ1 (FIG. 2C), a frequent downstream substrate for Syk (Obergfell et al., supra).
Similar to the previously observed response by matured mouse DC (Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443), matured human DC responded to B7-DC XAb treatment by regaining the ability to take up antigen (FIG. 3). To address the importance of the signaling intermediates generated by cross linking B7-DC in the regulation of antigen uptake in mature DC, pharmacologic inhibitors of Src, Syk, and PLCy were used to block steps in the activation pathway. As shown in FIG. 4A, blockade of Src kinases with PP2 resulted in inhibition of antigen uptake by matured DC in response to B7-DC cross-linking Pretreatment of DC with piceatannol inhibited phosphorylation of Syk and PLCγ 1 (FIGS. 2D and 2E), and also inhibited uptake of tagged proteins by matured DC in a dose responsive manner (FIG. 2F), as did inhibition of PLCγ activation using the inhibitor U73122, but not when the cells were treated with the inactive analogue U73343 (FIG. 2G). Taken together, these findings indicate that the MTAb B7-DC XAb activates a Src.fwdarw.PLCγ1 pathway that is required for activation of antigen uptake in matured DC induced by B7-DC XAb treatment. Using similar modes of pharmacological inhibition, it was observed that calcium dependent PKC activity, PI3 kinase, and the Rho family GTPase RAC1 are important factors regulating antigen uptake by matured DC activated with cross-linking B7-DC (FIG. 4B). In contrast, neither Rho-A nor MAP family kinases p38 appear to influence this function (FIG. 4B).
To address how B7-DC might be coupled to these downstream signaling pathways, the possibility that binding B7-DC with B7-DC XAb might recruit other membrane proteins into a complex on the membrane was evaluated. DC cell-surface membrane molecules, including MHC class II, CD80, and CD86, were tagged with fluoresceinated antibodies prior to activation with cross-linking antibody. The distribution of the antibody tags reorganized into a distinctly capped cluster on the cell membranes of both mouse and human DC soon after treatment with B7-DC XAb, but not after treatment with the irrelevant IgM isotype control antibody sHIgM39. To establish a quantitative and dynamic assessment of cap formation, recruitment of molecules into the cap was measured using resonance energy transfer (FRET), as described previously (Block et al. (2001) J. Immunol. 167:821-826). Within ten minutes of B7-DC cross-linking with the MTAb B7-DC XAb, class II, CD80, and CD86 molecules moved into close juxtaposition on the DC membrane, as visualized by a strong FRET signal among the fluorescently tagged molecules (FIGS. 5A and 5B). The molecules remained close enough for FRET during the next 20 minutes of observation, indicating that a stable macromolecular complex was formed.
An antibody of IgM isotype that binds to class II I-A^bmolecules expressed by C57BL/6 mice failed to induce any co-localization of CD80 and CD86 on mouse DC (FIG. 5C), underscoring the specialized capability of B7-DC XAb to induce these marked membrane rearrangements. The ability of B7-DC XAb to induce the cap was abolished by pretreating human or mouse DC with B7-DC-specific IgG antibodies (FIG. 5D). This observation was consistent with previous findings that the B7-DC XAb-induced functional changes in DC are dependent on B7-DC (Radhakrishnan et al. (2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443). The importance of CD80, CD86, and class II molecules expressed on DC to B7-DC XAb-induced cap formation was investigated using DC derived from CD80/CD86 double knock out and MHC class II knock out mice. Following treatment with B7-DC XAb, co-capping of CD80 and CD86 or CD11c with class II molecules still occurred, indicating that these particular molecules are not required for cap formation.
TREM-2, a pattern recognition receptor expressed on monocytes and cultured DC, is known to activate DAP12 (Obergfell et al., supra; and Daws et al. (2001) Eur. J. Immunol. 31:783-791). As visualized by FRET, an association of TREM-2 with class II molecules was observed within 5 minutes, and increased by 15 minutes after treatment of DC with B7-DC XAb (FIG. 6A). This association of TREM-2 with class II molecules was confirmed by co-immunoprecipitation of TREM-2 with the class II molecule IA^bin lysates isolated from DC 5 minutes after B7-DC XAb treatment (FIG. 5E). In contrast, TREM-2 was not associated with class II molecules on DC following treatment with isotype control antibody in mock activation.
To evaluate the functional importance of TREM-2 in DC activation with the MTAb B7-DC XAb, antigen uptake by matured, TREM-2 deficient DC was assessed using an RNA knockdown strategy. A retrovirus containing a dominant negative shDNA for TREM-2 was transduced into mouse bone marrow-derived DC, substantially reducing the expression of TREM-2 on the cell surface. This reduction was associated with the absence of phosphorylation of DAP12 and Syk (FIGS. 6B and 6C). Furthermore, using matured DC, the shDNA-transduced cells were not induced to take up ovalbumin when treated with the MTAb B7-DC XAb. When DC were transduced with virus containing a scrambled shDNA sequence, expression of TREM-2, phosphorylation of DAP12 and Syk (FIGS. 6B and 6C), and enhanced ovalbumin uptake proceeded in the same manner described previously with wild type DC not infected with retrovirus (Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443).
The important contribution of TREM-2 in the transduction of B7-DC XAb induced signals was confirmed using DC derived from TREM-2 KO bone marrow. While phosphorylation of DAP12 and Syk was readily induced in wild type DC activated with the MTAb B7-DC XAb, phosphorylation of these same signaling intermediates was not induced in TREM-2 KO mice (FIG. 7A), and the matured KO DC did not regain the ability to take up ovalbumin after antibody treatment. Together, these findings indicate that B7-DC cross-linking leads to recruitment of TREM-2 to the macromolecular cap, and activation of DAP12. DAP12 is to recruit Syk, a kinase functionally linked to PLCγ1, which is an upstream regulator of antigen uptake in matured DC activated by B7-DC XAb. These findings using mouse DC, coupled with those described in FIG. 2 and FIG. 5 using human cells, underscore the mechanistic parallels governing DC activation in these two species by the MTAb B7-DC XAb.
To determine whether the role of TREM-2 in mediating signals induced by B7-DC XAb in DC generated in vitro also applies to cells in vivo, experiments were conducted to compare the ability of CD11c+ draining lymph node cells in wild type and TREM-2 KO mice treated systemically with B7-DC XAb or control antibody to take up labeled proteins introduced subcutaneously after antibody treatment. Whereas B7-DC XAb treatment significantly activated uptake of labeled ovalbumin in the lymph nodes of wild type mice, the antibody had no effect on uptake of this antigen by CD11c+ cells in the lymph nodes of TREM-2 KO mice (FIG. 7B). These findings indicate that the importance of TREM-2 in the signaling pathway described for DC generated in vitro from bone marrow precursors also applies to the response of natural DC in vivo.
DC can be activated by B7-DC XAb in the absence of TREM-2, as shown by the mobilization of NF-κB in MTAb treated TREM-2 KO cells. To determine what other receptors regulating NF-κB activation might also be recruited to the signaling complex by B7-DC XAb, the role of CD40 was examined. These experiments revealed that this receptor also is recruited to the B7-DC XAb-induced cap as indicated by its co-precipitation with class II molecules following activation of DC with cross-linking antibody, but not following treatment with isotype control IgM antibody (FIG. 5F). The functional importance of CD40 in B7-DC XAb-mediated DC activation is indicated by the absence of NF-κB activation and IL-6 secretion (FIG. 8) by bone marrow derived DC generated from CD40 KO mice, while DC from wild type mice responded to the cross-linking antibody by activating NF-κB and producing IL-6. DC from CD40 KO mice displayed enhanced antigen uptake in response to treatment with B7-DC XAb, indicating that TREM-2 expression was required for up-regulation of antigen uptake in matured DC following B7-DC cross-linking, while the presence of CD40 was required for activation of NF-κB and secretion of IL-6.
The delineation of signaling pathways linking the binding of B7-DC XAb to the DC cell surface to downstream cellular functions provides a basis for evaluating the mechanisms of activation employed by other MTAbs. The monoclonal MTAbs 04 (Asakura et al. (1998) J. Neurosci. 18:7700-7708) and rHIgM22 (Warrington (2000), supra; and Mitsunaga et al. (2002) FASEB J. 16:1325-1327) bind oligodendrocytes and induce remyelination of denuded axons when administered systemically to mice. Primary oligodendrocytes and oligoglial cell lines mobilize a calcium response when exposed to the MTAbs 04 and rHIgM22 (Pas Soldan et al. (2003) Mol. Cell. Neurosci. 22:12-24; and Howe et al. (2004) Neurobiol. Dis. 15:120-131). Treatment of both mouse (FIG. 9A) and human DC (FIG. 9B) with the MTAb B7-DC XAb also resulted in prolonged calcium signals, which were blocked when the DC were first treated with B7-DC specific IgG antibodies. Furthermore, CG4 cells are protected from apoptotic signals enhancing their longevity under stressed conditions (Howe et al., supra), a phenotype also observed in culture when DC were treated with the MTAb B7-DC XAb (Nguyen et al., supra). Another parallel among these cells treated with MTAbs is the mobilization of activated NF-κB. These strong parallels observed among cells activated with the MTAbs B7-DC XAb, 04, and rHIgM22 suggest a common mechanism of membrane rearrangement that leads to recruitment of signaling molecules into macromolecular caps, mobilizing specific tissue responses in vitro and vivo. The precise nature of the molecules mediating MTAb induced signals in oligodendrocytes remains undefined. Several different IgM antibodies share the ability to activate oligodendrocytes (Warrington et al. (2000), supra; Miller and Rodriguez, supra; Asakura et al., supra; Pas Soldan et al., supra; and Howe et al., supra). These antibodies appear to target more than one cell surface molecule, as indicated by blocking a cellular activation of one IgM by its IgG switch variant, but the inability of that IgG antibody to block cellular activation by other IgM antibodies that target the same cells (Howe et al., supra).
One hypothesis is that the MTAbs are targeted to cells by moderate specific binding affinity for molecules distinctly expressed by specific cell types. Once bound to these cell restricted epitopes, the antibodies may interact by secondary, weak cross-reactivity with other cell surface molecules that are moving on the membrane, drawing them into a tight cluster. The structure of the induced clusters may lead to activation of cell surface molecules with intrinsic signaling capability, initiating preprogrammed changes in cellular function.
To test this hypothesis, a molecule displaying low but measurable binding for the oligodendrocyte-specific MTAb rHIgM22 was engineered and introduced into Jurkat cells to serve as bait and attract the MTAb to the surface of a cell not normally targeted by the IgM antibody. To construct this antibody target, a phage display peptide library was screened to identify a peptide mimetope that binds weakly to the remyelinating antibody sHIgM22. The peptide sequence was incorporated into the coding sequence for the N-terminus of a K^b/L^dchimeric mouse class I molecule (Pullen et al. (1989) J. Immunol. 143:1674-1679) and the gene was expressed in human Jurkat T cells by transient transfection. Previous studies had demonstrated that peptides introduced at the amino terminus of a mouse class I extracellular domain can be expressed without disrupting the expression and structural integrity of the class I molecule (Hedley et al. (1989) J. Immunol. 143:1026-1031). Treatment of the transfected Jurkat cells with the MTAb sHIgM22 induced a molecular cap on the cell surface, and promoted a FRET signal between the B7-family receptor CD28 and the class II co-receptor CD4 (FIG. 9D) and with a set of human class I molecules (FIG. 10). The ability of the sHIgM22 antibody to promote assembly of a macromolecular complex on Jurkat cells also was confirmed using stable transfectants (FIG. 10). Enhanced tyrosine phosphorylation of Jurkat cellular proteins also was detected after treating cells transfected with the peptide-class I chimeric genes with the MTAb rHIgM22, but not following treatment with an IgM antibody that binds to the carrier K^b/L^dmouse class I heavy chain that carries the peptide mimetope. In cells transfected with the parental class I carrier gene (not encoding the peptide mimetope), none of the IgM antibodies induced caps, FRET signals, or phosphorylation of cellular proteins (FIG. 9D). Thus, by introducing an artificial target capable of attracting rHIgM22 onto the surface of Jurkat cells, a signaling complex was assembled that resulted in activation of cellular tyrosine kinases. This experimental system validates the model, and demonstrates that two different MTAbs function by recruiting cell surface proteins to a macromolecular complex.
The concept that antibodies can modulate cell functions by their ability to bind cell surface receptors is well established (Sege and Peterson (1978) Proc. Natl. Acad. Sci. USA 75:2443-2447; and Taub and Greene (1992) Biochem. 31:7431-7435). In addition to specific engagement of receptors by antibodies raised by direct immunization, observed interactions between naturally-occurring autoreactive antibodies and cells have given rise to the notion that natural antibodies may function as ligands supporting normal development and homeostatic maintenance of tissue physiology (Ando et al. (1994) J. Biol. Chem. 269:19394-19398; Coutinho and Avrameas (1992) Scand. J. Immunol. 36:527-532; and Avrameas (1991) Immunol. Today 12:154-159). Until now, however, there has been little mechanistic insight into how antibodies might function in this way. IgM antibodies with their decavalent binding structure would seem to be natural cross-linkers, however, many studies using IgM antibodies have required secondary cross-linkers to assemble molecular caps. The molecules described herein represent a subset of naturally occurring IgM autoreactive antibodies that are highly somatically mutated, distinguishing them in some respect from traditional natural antibodies (Miller and Rodriguez (1995), supra; Asakura et al., supra; Mitsunaga et al., supra; and Van Keulen et al. (2006) Clin. Exp. Immunol. 143:314-321), that have the capacity to induce macromolecular caps and activate signaling pathways without secondary cross-linking.
The accumulating evidence leads to the prediction that IgM antibodies capable of specifically activating a wide variety of cells in the human body can be identified. Recently, an IgM antibody has been described that activates neurons in culture, promoting neurite outgrowth (Warrington et al. (2004) J. Neuropathol. Exp. Neurol. 63:461-473). The IgM antibodies already described from mice and human patients have tended to bind to equivalent cell types in rodents and humans (Warrington et al. (2000), supra; Radhakrishnan et al. (2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2007) J. Immunol. 178:1426-1432), indicating that the recognized epitopes are evolutionarily conserved structures. These shared products are likely the products of enzymatic modification of surface molecules. This cross-species reactivity demonstrates the potential clinical application of MTAbs using animal models of multiple sclerosis, cancer, and asthma (Warrington et al. (2000), supra; Radhakrishnan et al. (2004) Cancer Res. 64:4965-4972; Radhakrishnan et al. (2004) J. Immunol. 173:1360-1365; Miller et al. (1997) J. Neuroimmunol. 75:204-209; and Bieber et al. (2002) Glia 37:241-249). An important feature of the human MTAbs is that they were identified in patients with high titer monoclonal gammopathies. While each of these patients has high titers of IgM antibody in their serum, none of them have developed antibody-associated pathologies causing abnormal kidney, liver, cardiovascular, or neurologic functions. By identifying MTAbs from monoclonal gammopathy patients who are free of these antibody-associated pathologies, one can select IgM antibodies for further development that may behave well as therapeutic agents when administered to patients. Once MTAbs are identified in patient serum and tested for therapeutic activity using animal models of disease, recombinant sources of antibody can be generated using reverse genetics, as described previously (Mitsunaga et al., supra; and Van Keulen et al., supra), and prepared as GMP-grade reagents for clinical study.

Example 3—A Surrogate Target System for Investigation of Human MTAbs

A phage display system (Ph.D.-C7C Peptide Library Kit, New England Biolabs) was used to select 7-mer peptides in a cysteine loop for either hIgM12 or hIgM22. Briefly, a random peptide bacteriophage library was panned against immobilized antibody. Unbound bacteriophage was washed away, and bound bacteriophage was eluted, regrown and re-panned against the antibody. Multiple (three to seven) rounds of panning led to development of a peptide consensus sequence for each antibody. Two similar consensus sequences were identified for hIgM22, while one consensus sequence was identified for hIgM12 (Table 1).

TABLE 1

Amino acid sequences of consensus peptide targets

Name	Sequence	No. of clones	SEQ ID NO:

hIgM22 peptides

22p3	CPSEHQWIC	4x	14

22p5a	CPPWQSWIC	7x		15

hIgM12 peptides

12p4	CARNSTPPC	1x		16

12p9a	CHQTEKLTC	11x	17

Peptides 22p3 and 12p9A, which represented 4 and 11 clones, respectively, were selected. Each peptide was joined to the N-terminal sequence of the mouse MHC I molecule Kb with an Asp-Ser-Ala linker, permitting surface expression of the peptide. Kb was chosen due to its high level surface expression, previous work showing that peptides could be attached to Kb's N terminus without disrupting its structure, and possession of an anti-Kb IgM (28-13-3). In addition, a Kb construct with both peptides was constructed, with 22p3 N-terminal to 12p9A.
Kb, Kb-12p9A, Kb-22p3, and Kb-22p12p were placed in a VSV-G pseudotyped pBabe Puro retroviral vector, a vector based on the Maloney Murine Retrovirus. Previous experiments had shown that a high rate of transduction can be achieved, as measured by GFP expression, and that infection with the retrovirus does not alter the gross biology of DCs. Retrovirus was produced by transient transduction of 293T cells, and supernatant was concentrated 100:1 by ultracentrifugation on a 10% sucrose cushion. The retrovirus was titered with a colony forming unit assay of HT-1080 cells transduced with limiting dilutions of the supernatant concentrate, followed by selection with 2 ug/mL puromycin. To make DCs, bone marrow was harvested from Balb/c, C57BL6 mice or B7DC−/− mice on a C57BL6 background.
Bone marrow was placed in RPMI+10% Cosmic Calf Serum with 1 ng/mL IL-4 and 10 ng/mL GM-CSF, and plated at 10⁶/mL in 24 well plates. On day 2 post-plating, media was aspirated; fresh media was added with 0.5 MOI retrovirus and 4 ug/mL polybrene. Using this system, the peptide constructs were expressed in cultured DCs (FIG. 13).
To directly compare the activity of the MTabs hIgM12 and hIgM22, a series of well-defined biological outputs of treatment of wild type DCs with hIgM12 was used. FRET was used as an output for formation of the multimolecular cap seen upon binding of hIgM12 to wild type DCs. The emission of phycoerythrin (PE) was used to excite APC, which normally emits in the FL4 channel but under FRET emits in the FL3 channel. Experiments were conducted to test FRET of CD40-PE to Class II APC, and CD80-PE to Class II APC (FIG. 14). Either wild type C57BL6 DCs or B7DC−/− DCs transduced with either B7DC or the Kb constructs were used. DCs were stained with the fluorochrome-labeled antibodies, and then treated for 15 minutes at 37° C. with 10 μg/mL of either hIgM12, hIgM22, or 28-13-3. Wild type DCs responded to hIgM12 but not hIgM22 or 28-13-3, and knockouts with restored B7DC expression responded as well. Knockouts transduced only with Kb did not respond to any antibody, while the Kb-peptide constructs restored activity of the target antibody, such that Kb-12p9A DCs responded to hIgM12, Kb-22p3 DCs responded to hIgM22, and Kb-22p12p DCs responded to both antibodies.
The ability of Kb-peptide expressing DCs to alter antigen processing was then tested. Untreated DCs take up fluorochrome-labeled protein, and the fluorochrome labeled protein persists over 24 hours, as measured by flow cytometry. DCs treated with a Toll receptor agonist (LPS, CpG, or PolyI:C) 24 hours prior to addition of protein lose their ability to take up and maintain this antigen. Addition of hIgM12, but not other MTabs, has been shown to restore the ability of TLR agonist-treated DCs to take up and maintain antigen. The ability of B7DC−/− DCs transduced with the Kb-peptide constructs was compared to wild type DCs or knockouts transduced with B7DC. As previously shown, antigen persistence in CpG-treated wild type DCs or B7DC−/− DCs could be restored to untreated levels by addition of hIgM12, but not hIgM22 or 28-13-3. Knockouts transduced with Kb did not respond to any antibody, while those transduced with the Kb-peptide constructs responded to the appropriate MTAb (FIG. 15).
Previous experiments demonstrated the ability of DCs treated with hIgM12 to convert CD4+CD25+Treg cells to a Th17 phenotype in an antigen-specific manner. This was measured by loss of FoxP3 expression and gain of IL-17 expression, corresponding to loss of a repressive phenotype and gain of an effector phenotype. Studies were conducted to compare the ability of wild type DCs or B7DC−/− B7DC reconstituted DCs to B7DC−/− transduced with the Kb-peptide constructs to convert Tregs to Th17 polarized T cells. DCs were pulsed with 10 ug/mL ovalbumin and treated with 10 ug/mL of either hIgM12, hIgM22, or 28-13-3, 24 hours prior to addition of T cells. Splenic OT-II Tregs were isolated by MACs and added to DC cultures at a 1:1 ratio. After 48 hours of co-incubation, T cells were harvested, stained with CD4 PerCP, and permeabilized and stained with FoxP3 PE and IL-17 APC. Samples were run by flow cytometry and cells were gated by high CD4 expression. Conversion was measured by loss of FoxP3 expression and gain of IL-17 expression. Wild type DCs and B7DC reconstituted knockouts treated with hIgM12 but not hIgM22 or 28-13-3 converted Tregs to Th17 polarized cells (FIG. 16). Knockouts transduced with Kb did not respond to any antibody, while those transduced with the Kb-peptide constructs responded only to the appropriate antibody—hIgM12 when expressing Kb-12p9A and hIgM22 when expressing Kb-22p3.
DCs treated with hIgM12 can induce potent anti-tumor immunity. It has been shown (e.g., as described in U.S. Pat. No. 7,052,694) that mice injected intravenously with either hIgM12 directly or DCs pre-treated with hIgM12 and tumor antigen are protected against challenge by B16 melanoma cells, while untreated or control IgM treated mice had to be sacrificed due to disease progression. A variation of this model was used to determine whether Kb-peptide expressing DCs could induce protective tumor immunity. C57BL6 mice were injected subcutaneously in the right flank with 5×10⁵B16-Ova cells, a B16 variant engineered to express ovalbumin. Wild type DCs or B7DC−/− DCs transduced with mB7DC or the Kb constructs were pulsed with 10 ug/mL ovalbumin and treated with 10 ug/mL of either hIgM12 or hIgM22, 24 hours prior to injection. Before injection, DCs were washed and resuspended in 200 μL HBSS. 10⁶DCs were injected intravenously by tail vein concurrent to B16 injection. Three mice were used in each treatment group. Tumor area was measured daily, and mice were sacrificed when tumor area reached 289 mm². Mice treated with wild type DCs stimulated with hIgM12 or B7DC reconstituted knockout mice were successfully protected from tumor outgrowth, while hIgM22 failed to protect (FIG. 17). Mice treated with knockout DCs transduced with Kb did not respond to either treatment. Mice injected with DCs expressing Kb-12p9A and treated with hIgM12, or DCs expressing Kb-22p3 and treated with hIgM22 were protected, while the reciprocal antibody treatment failed to offer protection. Thus, DC expressing Kb-peptide fusions were able to induce protective tumor immunity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A method for targeting a multivalent molecule to a cell, comprising:

(a) contacting the cell with a linker molecule, wherein the linker molecule includes (i) an amino acid sequence comprising an epitope to which the multivalent molecule specifically binds and (ii) an amino acid sequence that binds specifically to a marker on the outer surface of the cell; and

(b) contacting the cell with the multivalent molecule.

2. The method of claim 1, wherein the multivalent molecule is an antibody.

3. The method of claim 2, wherein the antibody is an IgM antibody.

4. The method of claim 1, wherein the linker molecule consists of a polypeptide.

5. The method of claim 1, wherein the linker molecule is a chimeric antibody.

6. A method for targeting a multivalent molecule to a cell, comprising:

(a) contacting the cell with a nucleic acid encoding a polypeptide, wherein the polypeptide includes (i) an amino acid sequence that directs the polypeptide to the cell's plasma membrane and (ii) an amino acid sequence comprising an epitope to which the multivalent molecule specifically binds;

(b) culturing the cell under conditions in which the polypeptide is expressed and localized to the plasma membrane such that the epitope is located on the exterior of the cell; and (c) contacting the cell with the multivalent molecule.

7. The method of claim 6, wherein the multivalent molecule is an antibody.

8. The method of claim 7, wherein the antibody is an IgM antibody.

9. A chimeric polypeptide comprising a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a Kb amino acid sequence, and where the second amino acid sequence comprises an epitope to which a multivalent molecule binds.

10. The chimeric polypeptide of claim 9, wherein the multivalent molecule is an antibody.

11. The chimeric polypeptide of claim 10, wherein the antibody is an IgM antibody.

12. The chimeric polypeptide of claim 11, wherein the IgM antibody is hIgM12 or hIgM22.

13. The chimeric polypeptide of claim 9, wherein the second amino acid sequence comprises SEQ ID NO:14 or SEQ ID NO:17.