111111
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111
`US 20040146948Al
`
`(19) United States
`(12) Patent Application Publication
`Britton et al.
`
`(10) Pub. No.: US 2004/0146948 Al
`Jul. 29, 2004
`(43) Pub. Date:
`
`(54) COMPOSITIONS AND METHODS FOR
`TARGETING ANTIGEN-PRESENTING
`CELLS WITH ANTIBODY SINGLE-CHAIN
`VARIABLE REGION FRAGMENTS
`
`(75)
`
`Inventors: Warwick Britton, Bardwell Park (AU);
`Caroline Demangel, Paris (FR)
`
`Correspondence Address:
`SPECKMAN LAW GROUP PLLC
`1501 WESTERN AVE
`SEATTLE, WA 98101 (US)
`
`(73) Assignee: CENTENARY INSTITUTE OF CAN(cid:173)
`CER MEDICINE AND CELL BIOL(cid:173)
`OGY, Camperdown (AU)
`
`(21) Appl. No.:
`
`10/689,921
`
`(22) Filed:
`
`Oct. 17, 2003
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/420,232, filed on Oct.
`18, 2002.
`
`Publication Classification
`
`(51)
`
`Int. Cl? ....................... G01N 33/53; GOlN 33/567;
`C07K 16/28
`(52) U.S. Cl. ................... 435/7.2; 530/391.1; 530/388.22
`
`(57)
`
`ABSTRACT
`
`Provided are single-chain Fv (scFv) fragment-based com(cid:173)
`positions and methods for targeting antigens to antigen(cid:173)
`presenting cells (APCs) such as, for example, dendritic cells
`(DC). Compositions and methods disclosed herein are useful
`in the treatment of diseases including infectious diseases and
`cancer.
`
`1
`
`AMG1037
`
`

`

`Patent Application Publication
`
`Jul. 29, 2004 Sheet 1 of 9
`
`US 2004/0146948 A1
`
`polyhedrin promoter
`
`ISS
`
`linker
`
`VL
`
`BCV/NLDC145
`
`Figure lA
`
`2
`
`

`

`Patent Application Publication Jul. 29, 2004 Sheet 2 of 9
`
`US 2004/0146948 Al
`
`ISS
`
`pCMV
`
`Amp
`
`pc0NA3/NLDC145-Ag85b
`
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`
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`
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`
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`Figure lB
`
`3
`
`

`

`Patent Application Publication Jul. 29, 2004 Sheet 3 of 9
`
`US 2004/0146948 Al
`
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`Figure 2
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`4
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`

`

`Patent Application Publication Jul. 29, 2004 Sheet 4 of 9
`
`US 2004/0146948 Al
`
`Figure 3A
`
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`Patent Application Publication Jul. 29, 2004 Sheet 5 of 9
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`US 2004/0146948 Al
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`Patent Application Publication Jul. 29, 2004 Sheet 6 of 9
`
`US 2004/0146948 Al
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`Patent Application Publication Jul. 29, 2004 Sheet 7 of 9
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`US 2004/0146948 Al
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`
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`
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`

`Patent Application Publication Jul. 29, 2004 Sheet 8 of 9
`
`US 2004/0146948 Al
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`

`Patent Application Publication Jul. 29, 2004 Sheet 9 of 9
`
`US 2004/0146948 Al
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`10
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`

`

`US 2004/0146948 Al
`
`Jul. 29, 2004
`
`1
`
`COMPOSITIONS AND METHODS FOR
`TARGETING ANTIGEN-PRESENTING CELLS
`WITH ANTIBODY SINGLE-CHAIN VARIABLE
`REGION FRAGMENTS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims priority to U.S. Provisional
`Patent Application No. 60/420,232, filed Oct. 18, 2002.
`
`BACKGROUND OF THE INVENTION
`
`[0002] 1. Technical Field of the Invention
`
`[0003] The present invention relates generally to the fields
`of immunology and molecular biology. More specifically,
`the present invention is directed to antibody single-chain
`variable region fragment (scFv)-based compositions and
`methods for targeting antigens to antigen-presenting cells
`(APCs) such as, for example, dendritic cells (DCs). Com(cid:173)
`positions and methods disclosed herein are useful in the
`treatment of diseases including infectious diseases and can(cid:173)
`cers.
`
`[0004] 2. Description of the Related Art
`
`Immunization has proved one of the most cost
`[0005]
`effective strategies for the improvement of human health.
`Most of the effective vaccines against bacterial, parasitic,
`and viral pathogens depend on the production of antibodies.
`Protective immunity against a number of important human
`and veterinary pathogens depends, however, upon the devel(cid:173)
`opment of cellular immune responses. In addition, applica(cid:173)
`tion of therapeutic and prophylactic immunization method(cid:173)
`ology to vaccines directed against cancers also depends
`upon the stimulation of cellular immune responses to vac(cid:173)
`cine components. Accordingly, effective strategies for elic(cid:173)
`iting cellular immunity will prove widely applicable to the
`development of vaccines against infectious diseases and
`cancers.
`
`[0006] The in vivo processes involved in the development
`of cellular immunity continue to be more clearly delineated.
`One class of antigen-presenting cell, the dendritic cell (DC),
`is critical in sensing the presence of foreign organisms that
`play a central role in the induction of antimicrobial immu(cid:173)
`nity. Scattered throughout the body, they constitute the first
`line of defence against invading pathogens. Innate immune
`recognition by DCs is based on the recognition of microbial
`motifs by specialised receptors, the identification of which is
`a field of growing interest. Moll, Cellular Microbiology
`5:493-500 (2003); Figdor et al., Nature Reviews Immunol(cid:173)
`ogy 2:77-84 (2002); and Demangel et al., Immunology &
`Cell Biology 78:318-324 (2000). Following interaction with
`antigen, DC undergo a maturation process resulting in the
`up-regulation of expression of co-stimulatory, adhesion and
`MHC molecules enhancing their capacity to present pep tides
`to na1ve T cells.
`
`[0007] DCs migrate to specialized lymphoid organs, the
`lymph nodes, to stimulate immunity and undergo maturation
`to become effective antigen-presenting cells capable of
`stimulating T lymphocytes (T-cells). This process has been
`studied in mycobacterial infections such as TB. Infection of
`DC by M. tuberculosis or BCG induces the co-ordinate
`processes of DC maturation and secretion of the cytokine
`
`interleukin 12 (IL-12). These events are critical in the
`development of mycobacteria-specific T-cells.
`
`[0008] DCs represent a minor cell subset of the peripheral
`tissues. In steady state conditions, lung DCs constitute less
`than 1% of the total cell population, a low incidence rate
`considering their sentinel role against incoming pathogens.
`Moll et al., Cellular Microbiology 5:493-500 (2003). This
`sparse distribution is compensated for by a high sensitivity
`to environmental signals, delivered by damaged endogenous
`tissues or by pathogens. Austyn, Nature Medicine 5:1232-3
`(1999). Microbial products (cell wall components, non(cid:173)
`methylated CpG motifs, double stranded RNA) are potent
`inducers of DC activation. Sousa et al., Current Opinion in
`Immunology 11:392-399 (1999). Moreover, model antigens
`expressed in recombinant bacteria are presented by MHC
`Class I and Class II molecules on DC much more efficiently
`than the same antigens in soluble form. Svensson et al., J.
`Immunol. 158:4229-36 (1997); Rescigno et al., Proc. Natl.
`Acad. Sci. USA 95:5229-34 (1998). This strongly suggests
`that enhanced antigen presentation could be achieved by
`selective targeting of subunit vaccines to the DC receptors,
`which are specialized in the recognition of bacterial prod(cid:173)
`ucts.
`
`Immature DCs display receptors on their surface
`[0009]
`membranes that permit them to bind to and, in some cases,
`internalize a diverse array of antigens. Internalized protein(cid:173)
`antigens are processed into short peptides that are presented
`in the context of MHC Class I and Class II molecules.
`Following the interaction of DC receptors with antigens, DC
`undergo a maturation that results in the increased expression
`of co-stimulatory and MHC molecules that enhance their
`capacity to present peptides to na1ve T-cells.
`
`[0010] A number of receptor molecules have been identi(cid:173)
`fied on the surface of DCs. DEC-205, a homologue of the
`macrophage mannose receptor, and the integrin CDllc are
`surface receptors that are restricted to DCs. Immunological
`evidence suggests that targeting antigens to DEC-205 or to
`CDllc may improve antigen presentation by DCs. Thus, it
`is likely that DEC-205 and CDllc play an important role in
`antigen capture. Rat antibodies directed to mouse DEC-205
`are more efficiently internalized than non-specific rat anti(cid:173)
`bodies and are 100-fold more effective at generating T-cell
`responses to the anti-DEC-205 antibody than to the non(cid:173)
`specific rat antibodies. Jiang et al., Nature 375:151-5 (1995).
`Similar results have been obtained by immunizing mice with
`anti-CDllc antibodies.
`
`[0011] The ~2 integrin CDllc is an attractive candidate
`for investigating the impact of antigen targeting to DCs
`because it is a DC-restricted surface molecule expressed by
`all subsets of mouse DCs and all human DCs of myeloid
`origin. Wilson et al., Immunology and Cell Biology 81:239-
`246 (2003); and Pulendran et al., Trends in Immunology
`22:41-7 (2001). Although its function is still unclear, there
`is immunological evidence that CDllc is involved in anti(cid:173)
`gen capture and delivery to antigen processing compart(cid:173)
`ments. Finkelman et al.,J. Immunol. 157:1406-1414 (1996).
`So is DEC-205, a lectin receptor expressed by mouse DC
`subpopulations of the spleen, Peyer's patches, lymph nodes
`and skin, and by some human DC subsets. Anjuere et al.,
`Blood 93:590-8 (1999) and Guo et al., Human Immunology
`61:729-738 (2000). Despite significant sequence homology
`with the macrophage mannose receptor (MMR) and the
`
`11
`
`

`

`US 2004/0146948 Al
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`Jul. 29, 2004
`
`2
`
`presence of eight C-type carbohydrate recognition domains,
`DEC-205 does not bind mannose and its specific ligands
`have yet to be defined. Jiang et al., Nature 375:151-5 (1995).
`Both MMR and DEC-205 receptors mediate adsorptive
`uptake of antigen in coated vesicles, direct antigen loaded
`vesicles to the endosomal compartment end recycle to the
`cell surface. However, whereas MMR recycles through early
`endosomes, DEC-205 targets antigens to the MHC Class II
`rich late endosomal compartment, leading to enhanced anti(cid:173)
`gen presentation to CD4+ T cells. Guo et al., Human
`Immunol. 61:729-738 (2000). Improving the delivery of
`antigens to DEC205 or CDllc receptors may thus result in
`enhanced T cell priming by DC.
`
`[0012] Antigen targeting to sites of immune induction is
`an efficient means of enhancing immune responses to DNA
`vaccines. Directing antigens to B7-expressing cells using
`cytotoxic T-lymphocyte antigen-4 (CTLA4) promotes the
`development of immune responses to fusion antigen in mice.
`Boyle et al., Nature 392:408-11 (1998). B7 molecules are
`expressed by a broad spectrum of leukocytes, including
`professional antigen presenting cells such as DCs, but also
`B and T lymphocytes. Products fused to L-selectin, a lym(cid:173)
`phocyte surface molecule mediating cell entry in the lymph
`nodes, are less efficient than the CTLA-4 fused ones in
`promoting T-cell proliferative responses suggesting that
`selective antigen targeting to cell subsets specialised in
`antigen presentation is more effective for immune stimula(cid:173)
`tion. The stimulatory effect of scNLDC may also relate to
`the fact that DEC-205-endocytosis pathway is highly effi(cid:173)
`cient for antigen presentation to CD4+ T cells. Mahnke et al.,
`J. Cell Bioi. 151:673-683 (2000).
`
`[0013] Protein antigen targeting to DEC-205 using chemi(cid:173)
`cally-coupled antibody molecules has been shown to induce
`T cell unresponsiveness in vivo under steady state condi(cid:173)
`tions. Hawiger et al., J. Exp. Med. 194:769-779 (2001) and
`Bonifaz et al., J. Exp. Med. 196:1627-1638 (2002). Toler(cid:173)
`ance was, however, converted into prolonged T cell stimu(cid:173)
`lation if the antigen was co-administered with an additional
`stimulus (such as an anti-CD40 antagonist).
`
`[0014] Tuberculosis (TB) is an intracellular bacterial
`infection, the control of which is dependent upon cellular
`immunity. TB remains the single most prevalent bacterial
`infection world-wide, with one third of the world's popula(cid:173)
`tion currently being infected with Mycobacterium tubercu(cid:173)
`losis. From this pool of 2 billion infected individuals, 8-9
`million new cases of clinical tuberculosis develop a year
`resulting in the death of at least 2 million people. Because
`of the interaction of M. tuberculosis and HIV, about half the
`deaths associated with HIV/AIDS in developing countries
`occur because of active tuberculosis. The meta-analysis of
`clinical trials with the only currently available vaccine, M.
`bovis Bacille Calmette Guerin (BCG), has led to the con(cid:173)
`clusion that BCG confers about 50% protective efficacy
`against the common pulmonary form of tuberculosis. This
`level of efficacy has proven insufficient to control the spread
`of tuberculosis and underscores the need for new immuni(cid:173)
`zation strategies.
`
`[0015] Despite the progress that has been made in identi(cid:173)
`fying receptors and other molecules on the surface of APCs
`and DCs, there remains a need in the art for improved
`compositions and methods for the delivery of antigens to
`APCs and DCs in order to achieve improved therapeutic and
`
`prophylactic efficacy against diseases including infectious
`diseases, autoimmune diseases, and cancers.
`
`SUMMARY OF THE INVENTION
`
`[0016] The present invention addresses these and other
`related needs by providing, inter alia, compositions and
`methods for targeting antigen-presenting and dendritic cells
`with antigens, including protein-antigens. As disclosed
`herein, compositions and methods will find utility in the
`treatment of disease by enhancing the cellular immune
`response to antigens.
`[0017] Disclosed herein are single chain antibody frag(cid:173)
`ments (scFvs) from the monoclonal antibodies NLDC-145
`and N418, which are directed to DEC-205 and CDllc
`mouse DC receptors. Exemplary scFv presented herein have
`the typical structure of scFvs, with the variable domain of
`the immunoglobulin heavy chain (V a) linked to the light
`chain one (V L) via a flexible peptide linker in a V H-V L
`orientation. Nissim et al., EMBO J. 13:692-698 (1994).
`These scFvs bind to their target receptor comparably to the
`parental antibodies in vitro. Thus, scFv targeting, as pro(cid:173)
`vided herein, is a powerful means for eliciting strong
`immune responses in vivo.
`
`[0018] Within certain embodiments, the present invention
`provides antibody single-chain variable region fragments
`(scFv) for targeting antigen-presenting cells (APCs) such as,
`for example, dendritic cells (DC). scFv presented herein
`comprise an antibody heavy chain variable region (V a)
`operably linked to an antibody light chain variable region
`(V L) wherein the heavy chain variable region and the light
`chain variable region together or individually form a binding
`site for specifically binding to a molecule on the surface of
`anAPC and/or a DC. ScFv may comprise a VH region at the
`amino-terminal end and a V L region at the carboxy-terminal
`end. Equally suitable are scFv that comprise a V L region at
`the amino-terminal end and a VH region at the carboxy(cid:173)
`terminal end.
`
`[0019] An exemplary scFv is derived from monoclonal
`antibody NLDC-145 which antibody specifically binds to
`DEC-405 on the surface of DC. According to this embodi(cid:173)
`ment, the scFv comprises variants of the NLDC-145 heavy
`chain (V a) and light chain (V L) variable regions wherein
`each variant NLDC-145 heavy chain (Va) and light chain
`(VL) region is at least 70%, 80%, 90%, 95% or 98%
`identical to the sequences disclosed herein in SEQ ID NOs:
`5 and 6, respectively. A most preferred exemplary scFv,
`disclosed herein in SEQ ID NO: 7, comprises the NLDC-
`145 heavy chain (V a) and light chain (V L) variable regions
`disclosed herein in SEQ ID NOs: 5 and 6, respectively.
`
`[0020] An alternative preferred exemplary scFv is derived
`from monoclonal antibody N418 which antibody specifi(cid:173)
`cally binds to CDllc on the surface of DC. According to this
`embodiment, the scFv comprises variants of the N418 heavy
`chain (V a) and light chain (V L) variable regions wherein the
`variant N418 derived scFv is at least 70%, 80%, 90%, 95%
`or 98% identical to the sequences disclosed herein in SEQ
`ID NO: 2. A most preferred exemplary scFv comprises the
`N418 heavy chain (V H) and light chain (V L) variable regions
`which scFv is disclosed herein in SEQ ID NOs: 2.
`
`[0021] ScFv disclosed herein may, optionally, further
`comprise a polypeptide linker operably linked between the
`
`12
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`

`

`US 2004/0146948 Al
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`Jul. 29, 2004
`
`3
`
`heavy chain variable region and the light chain variable
`region. Polypeptide linkers of the present invention gener(cid:173)
`ally comprise between 1 and 50 amino acids. More common
`are polypeptide linkers of at least 2 amino acids. Even more
`commonly, polypeptide linkers are between 3 and 12 amino
`acids. An exemplary linker peptide for
`incorporating
`between scFv heavy and light chains comprises the 5 amino
`acid sequence Gly-Gly-Gly-Gly-Ser. Alternative exemplary
`linker pep tides comprise one or more tandem repeats of the
`sequence Gly-Gly-Gly-Gly-Ser to create linkers comprising,
`for example, the sequences Gly-Gly-Gly-Gly-Ser-Gly-Gly(cid:173)
`Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(cid:173)
`Gly-Gly-Gly-Gly-Ser, and Gly-Gly-Gly-Gly-Ser-Gly-Gly(cid:173)
`Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.
`
`[0022] Surface molecules on APC and/or DC that may be
`targeted by scFv of the present invention include proteins
`and carbohydrates. Within certain embodiments, surface
`protein molecules include receptor proteins. Surface recep(cid:173)
`tor proteins may facilitate internalization of the specifically
`bound scFv into the APC and/or the DC. Within certain
`aspects, specifically bound scFv may be internalized by
`receptor-mediated endocytosis and/or by pinocytosis. Pre(cid:173)
`ferred surface protein molecules include, but are not limited
`to, the mannose receptor (MR), chemokine receptor 1
`(CCR1), B7-1 (CD80), B7-2 (CD86), CD40, CD11c, DEC-
`205, a Toll-like receptor (TLR), and the Fey receptor (FcyR).
`Most preferred are those surface protein molecules that are
`restricted to DCs such as CDllc and DEC-205.
`
`[0023] Other aspects of the present invention provide
`complexes between scFv and one or more antigens, includ(cid:173)
`ing protein-antigens. Antigens encompass protein-antigens
`that undergo
`in vivo post-translational modifications
`wherein the protein-antigen may be glycosylated, lipidated,
`phosphorylated or the like.
`
`[0024] Further aspects of the present invention provide
`complexes comprising scFv and a lipid. Thus, exemplified
`herein are scFv-lipid complexes wherein the scFv comprises
`a tag such as an affinity tag. Suitable affinity tags include, but
`are not limited to, the FLAG-tag and the hexahistidine tag.
`Thus, for example, a hexahistidine tagged scFv may form a
`complex directly with a lipid, such as a metal chelating lipid.
`An exemplary metal chelating lipid presented herein is
`nitrilotriacetic acid ditetradecylamine (NTA-DTDA).
`
`[0025] Within still further aspects of the present invention,
`scFv may be complexed directly with a lipid and/or with one
`or more antigen that is encapsulated by, incorporated within,
`and/or associated with a lipid membrane, a lipid bi-layer,
`and/or a lipid complex such as, for example, a liposome, a
`vesicle, a micelle and/or a microsphere. Thus, within these
`aspects of the invention, the term "antigen" encompasses
`such liposomes, vesicles, micelles and/or microspheres that
`comprise an antigen, such as a protein-antigen, including
`glycoprotein-antigens and/or lipoprotein-antigens.
`
`[0026] Complexes between scFv, a lipid, and/or an antigen
`may be achieved by chemical crosslinking or, alternatively,
`may be a fusion protein comprising scFv heavy and light
`chain variable regions and an antigen. Suitable scFv that
`may be employed in the complexes comprising an scFv,
`such as scFv/antigen, scFv!lipid, and scFv!lipid/antigen
`complexes, include those indicated above and as described
`in further detail herein below. scFv/antigen complexes are
`
`capable of specifically binding to APC and/or DC thereby
`facilitating the targeting of the antigen to the APC and/or
`DC.
`
`[0027] An exemplary scFv/antigen complex presented
`herein is the scFv NLDC-145-85B encoded by the nucle(cid:173)
`otide sequence presented herein as SEQ ID NO: 8. Equally
`preferred are functional fragments, derivatives and variants
`of the scFv NLDC-145-85B encoded by the nucleotide
`sequence presented herein as SEQ ID NO: 8. Functional
`variants of scFv NLDC-145-85B preferably exhibit at least
`about 70%, more preferably at least about 80% or 90% and
`most preferably at least about 95% or 98% sequence identity
`to the polypeptide encoded by SEQ ID NO: 8.
`
`[0028] Another exemplary scFv/antigen complex pre(cid:173)
`sented herein is the scFv N418-85B encoded by the nucle(cid:173)
`otide sequence presented herein as SEQ ID NO: 3. Equally
`preferred are functional fragments, derivatives and variants
`of the scFv N418-85B encoded by the nucleotide sequence
`presented herein as SEQ ID NO: 3. Functional variants of
`scFv N418-85B typically exhibit at least about 70%, more
`typically at least about 80% or 90% and most typically at
`least about 95% or 98% sequence identity to the polypeptide
`encoded by SEQ ID NO: 3.
`
`[0029] Within certain aspects, antigens that may be com(cid:173)
`plexed with the inventive scFv include protein-antigens
`from an organism, including a virus, parasite or a bacterium,
`which is capable of causing an infectious disease in a
`human. Exemplary viral organisms include, but are not
`limited to, human immunodeficiency virus (HIV), a herpes
`virus, and an influenza virus. Exemplary parasitic organisms
`include, but are not limited to, Leishmania (e.g., L. major
`and L. donovani). Exemplary bacterial organisms include,
`but are not limited to, Mycobacteria (e.g., M. tuberculosis
`and M. bovis), Chlamydia (e.g., C. trachomatis and C.
`pneumoniae), and Ehrlichia (e.g., E. sennetsu, E. chaffeen(cid:173)
`sis, E. ewingii, and E. phagocytophila). Within certain
`aspects, the protein-antigen is an M. tuberculosis antigen
`selected from the group consisting of 85B, MPT64, and
`ESAT-6 disclosed herein in SEQ ID NO: 14, SEQ ID NO:
`16, and SEQ ID NO: 18, respectively. Other aspects provide
`that the protein-antigen is a fragment, derivative or variant
`of 85B, MPT64, or ESAT-6. Typical protein-antigens exhibit
`at least about 70%, more typically at least about 80% or 90%
`and most typically at least about 95% or 98% sequence
`identity to the polypeptide disclosed herein in SEQ ID NO:
`14, SEQ ID NO: 16, and/or SEQ ID NO: 18.
`
`[0030] The present invention also provides fusion pro(cid:173)
`teins, comprising an antigen-presenting cell binding protein
`and a protein-antigen wherein the fusion protein is capable
`of specifically binding to an antigen-presenting cell (APC)
`and/or a dendritic cell (DC) and in inducing a protein(cid:173)
`antigen specific T-cell response. According to certain
`aspects, the APC and/or DC binding protein specifically
`binds to a receptor on the APC and/or DC. Exemplary
`receptors include, but are not limited to, the mannose
`receptor (MR), chemokine receptor 1 (CCR1), B7-1, B7-2,
`CD40, CDllc, DEC-205, a Toll-like receptor (TLR), and the
`Fey receptor (FcyR). Preferred antigens are infectious dis(cid:173)
`ease antigens, autoimmune disease antigens, or cancer cell
`antigens, including tissue-specific and/or tumor-specific
`antigens, as indicated above and as described in further
`detail herein.
`
`13
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`

`US 2004/0146948 Al
`
`Jul. 29, 2004
`
`4
`
`[0031] Further aspects of the present invention provide
`polynucleotides that encode one or more of the scFv pre(cid:173)
`sented herein. Within certain embodiments, the polynucle(cid:173)
`otide is a component of a vector, such as a plasmid vector or
`a viral vector, wherein the vector comprises a transcriptional
`promoter operably linked to the scFv encoding polynucle(cid:173)
`otide.
`
`[0032] Related aspects provide polynucleotides
`that
`encode an s cFv/antigen fusion protein which polynucle(cid:173)
`otides comprise a first polynucleotide that encodes an scFv
`and a second polynucleotide that encodes one or more
`protein-antigen wherein the first polynucleotide and the
`second polynucleotide are operably linked such that together
`they encode a fusion protein comprising an scFv and a
`protein-antigen. More preferred embodiments provide that
`the first polynucleotide and the second polynucleotide are
`operably linked by a third polynucleotide that encodes a
`polypeptide linker between the scFv and the protein-antigen.
`Within certain embodiments, the polynucleotide encoding
`the scFv/protein antigen fusion protein is a component of a
`vector, such as a plasmid vector or a viral vector, wherein the
`vector comprises a transcriptional promoter operably linked
`to the s cFv encoding polynucleotide. Particularly preferred
`vectors comprising a polynucleotide encoding an scFv and
`an scFv/antigen are, respectively, the pcDNA3-NLDC-145
`and pcDNA3-NLDC-85 plasmid vectors presented herein in
`FIG. 1 as well as pcDNA3-N418-85. The nucleotide
`sequences encoding scFv NLDC-145-85B, scFv N418-85B,
`and the nucleotide sequence of pcDNA3 are presented
`herein in SEQ ID NO: 8, SEQ ID NO: 3, and SEQ ID NO:
`9, respective! y.
`
`[0033] The present invention also provides compositions
`comprising scFv, scFv/lipid, scFv/antigen, and/or scFv/
`lipid/antigen complexes as well as compositions comprising
`polynucleotides encoding scFv and/or scFv/antigen com(cid:173)
`plexes and compositions comprising vectors comprising one
`or more polynucleotides encoding an scFv and/or an scFv/
`antigen complex. Exemplary compositions may, optionally,
`further comprise a cytokine such as interleukin-12 (IL-12),
`IL-6, IL-4, IL-l, interferon-y (IFNy), GM-CSF, tumor necro(cid:173)
`sis factor (TNF), and/or the CD40 ligand CD154, and/or
`may comprise a lipopolysaccharide (LPS) or other inducer
`of the DC response to antigen, such as other cell wall
`components, non-methylated CpG motifs, and/or double(cid:173)
`stranded RNA
`
`[0034] Other aspects of the present invention provide
`methods for introducing an antigen into an antigen-present(cid:173)
`ing cell (APC) and/or a dendritic cell (DC), the methods
`comprising the steps of: (a) isolating from a patient sample,
`an APC and/or a DC; and (b) contacting the APC and/or DC
`with an scFv/antigen complex, wherein the scFv/antigen
`complex is in contact with the APC and/or DC under
`conditions and for such a time as required to permit the
`antigen to enter the APC and/or DC.
`
`[0035] Related aspects of the present invention provide
`methods for introducing an antigen into an APC and/or a DC
`of a patient, the methods comprising the step of adminis(cid:173)
`tering to a patient a composition comprising an scFv/antigen
`complex, thereby inducing an interaction with an APC
`and/or a DC of the patient.
`
`[0036] Still further related aspects provide methods for
`introducing a protein-antigen into an APC and/or a DC of a
`
`patient, the methods comprising the step of administering to
`the patient a composition comprising a polynucleotide
`encoding an scFv/antigen complex.
`
`[0037] Still further aspects of the present invention pro(cid:173)
`vide methods for treating a disease and/or modulating an
`immune response in a patient, the methods comprising the
`steps of: (a) obtaining from the patient a sample comprising
`an antigen-presenting cell (APC) and/or a dendritic cell
`(DC); (b) contacting the sample with an scFv/antigen com(cid:173)
`plex under conditions and for such a time as required to
`allow binding of the scFv/antigen complex to the APC
`and/or DC; and (c) administering the scFv/antigen APC
`and/or DC-bound complex to the patient. Modulation of the
`immune response may include enhancing, stimulating, sup(cid:173)
`pressing, and/or blocking the immune response in the
`patient.
`
`[0038] Within methods for the present invention, the dis(cid:173)
`ease may be selected from the group consisting of an
`infectious disease, an autoimmune disease and a cancer.
`More preferred methods provide that the infectious disease
`is caused by an organism selected from the group consisting
`of Leishmania, Mycobacteria, Chlamydia, and Ehrlichia.
`Equally preferred methods provide that the cancer is
`selected from the group consisting of soft tissue sarcomas,
`lymphomas, and cancers of the brain, esophagus, uterine
`cervix, bone, lung, endometrium, bladder, breast, larynx,
`colon/rectum, stomach, ovary, pancreas, adrenal gland and
`prostate.
`
`[0039] Other aspects provide methods for inhibiting,
`reducing, suppressing and/or blocking the activity of a target
`antigen on the surface of an antigen-presenting cell (APC)
`and/or a dendritic cell (DC), the methods comprising the
`steps of: (a) obtaining a sample comprising and APC and/or
`a DC; (b) contacting the APC and/or DC with an scFv
`capable of specifically binding to the target antigen on the
`surface of the APC and/or DC under conditions and for such
`a time as required to permit binding of the scFv to the APC
`and/or DC, wherein binding of the scFv to the APC and/or
`DC blocks or substantially reduces the activity of the target
`antigen, thereby inhibiting, reducing, suppressing and/or
`blocking an immune response.
`
`[0040] By any of the methods disclosed herein, the scFv
`may bind to a molecule, including a carbohydrate molecule
`or a protein molecule, on the surface of the APC and/or DC.
`Preferred surface protein molecules include, but are not
`limited to, the mannose receptor (MR), chemokine receptor
`1 (CCRl), B7-1, B7-2, CD40, CDllc, DEC-205, a Toll-like
`receptor (TLR), and the Fey receptor (FcyR).
`
`[0041] Within certain methods, the scFv may be com(cid:173)
`plexed to an antigen wherein scFv/antigen complexes are
`achieved by chemical crosslinking or wherein scFv/antigen
`complexes are scFv/antigen fusion proteins.
`
`[0042] Suitable antigens that may be employed in any of
`the methods disclosed herein include, but are not limited to,
`antigens from an organism, including a virus, a parasite, or
`a bacterium, which is capable of causing an infectious
`disease in a human. Exemplary viral organisms include, but
`are not limited to, human immunodeficiency virus (HIV), a
`herpes virus, and an influenza virus. Exemplary bacterial
`organisms include, but are not limited to, Mycobacteria,
`Chlamydia, and Ehrlichia. Exemplary parasitic organisms
`
`14
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`

`

`US 2004/0146948 Al
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`Jul. 29, 2004
`
`5
`
`include, but are not limited to, Leishmania. Within certain
`aspects, the antigen is an M. tuberculosis antigen selected
`from the group consisting of 85B, MPT64, and ESAT-6
`disclosed herein in SEQ ID NO: 14, SEQ ID NO: 16, and
`SEQ ID NO: 18, respectively.
`
`[0043] These and other aspects of the present invention
`will become apparent upon reference to the following
`detailed description and attached drawings. All references
`disclosed herein are hereby incorporated by reference m
`their entirety as if each was incorporated individually.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`AND SEQUENCE IDENTIFIERS
`
`[0044] FIG. 1 depicts plasmid maps for (A) pcDNA3-
`NLDC-145 expressing an scFv derived from anti-DEC-205
`hybridoma NLDC-145, and (B) pcDNA3-NLDC-85 in
`which the gene for theM. tuberculosis antigen 85B is fused
`to the anti-DEC-205 derived scFv.
`[0045] FIG. 2 depicts a plasmid map of DNA vectors used
`for transfections and immunizations exemplified within the
`examples disclosed herein. Two vectors were constructed in
`which the ScNLDC or ScN418 sequences were fused to the
`Ag85B gene vi