Patent Application
20200339675
The present invention provides, inter alia, antibodies that modulate binding between Lrp5 and WISE or Lrp6 and WISE, but do not modulate binding between Lrp4 and WISE.
This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file “0339588pct.txt”, file size of 389 KB, created on Dec. 20, 2013. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
Wnt signaling plays an important role in a variety of processes, including development and maintenance of various organs and tissues, such as the bones. Mutations in Wnt genes or Wnt pathway components lead to specific developmental defects, including defects in the formation of mammary placodes, while various human diseases, including cancer and alterations in bone mass, are caused by abnormal Wnt signaling (For a review, see, e.g., Krishnan et al., 2006).
In the Wnt/β-catenin signaling pathway, interaction of Wnt ligands with Frizzled (Fz) receptors and Wnt co-receptors, Lrp5 and Lrp6, initiates a series of intracellular events leading to stabilization and nuclear accumulation of β-catenin. Subsequently, β-catenin forms complexes with TCF/LEF transcription factors and activates expression of target genes (MacDonald et al., 2009). Ectopic expression of the Wnt inhibitor Dickkopf 1 (Dkk1) blocks placode formation (Chu et al., 2004) and lack of Lef1, Lrp5 or Lrp6 disrupts normal placode development (van Genderen et al., 1994; Boras-Granic et al., 2006; Lindvall et al., 2006; Lindvall et al., 2009). It has been shown that Wnt/β-catenin signaling is initially activated in a broad domain along the mammary line, coincident with the expression pattern of a number of Wnt genes, but rapidly becomes restricted to mammary placodes (Chu et al., 2004; Veltmaat et al., 2004). This suggests that spatiotemporal control of the signaling activity is tightly coupled to placode formation. However, little is known about how precise control of Wnt signaling is achieved during embryonic mammary development.
Modulation of Wnt/β-catenin signaling in the extracellular space is often mediated by secreted Wnt antagonists, which interact with Wnt proteins, Fz receptors or Lrp5/6 co-receptors (MacDonald et al., 2009). For example, Dkk1, Sost and Wise (Sostdc1—Mouse Genome Informatics) can bind to the extracellular domain of Lrp5/6 and inhibit Wnt signaling presumably by disrupting the formation or activity of Wnt-induced Fz-Lrp5/6 complexes (Semenov et al., 2001; Itasaki et al., 2003; Li et al., 2005; Semenov et al., 2005). Another layer of complexity was added by recent findings on a low-density lipoprotein (LDL) receptor-related protein, Lrp4. The extracellular domain of Lrp4 resembles that of Lrp5/6, but its intracellular domain is distinct from that of Lrp5/6 suggesting that it may have different inputs on Wnt signaling (Herz and Bock, 2002; Weatherbee et al., 2006). In humans, LRP4 mutations cause limb, kidney and tooth malformations in Cenani-Lenz syndrome and are associated with bone overgrowth in two isolated cases of sclerosteosis (Li et al., 2010; Leupin et al., 2011). The role for Lrp4 appears to be conserved in mammals, because mice deficient for Lrp4 also display defects in limbs, kidney and teeth (Johnson et al., 2005; Weatherbee et al., 2006; Ohazama et al., 2008).
In Lrp4 mutant mice, limb and tooth defects were associated with abnormal Wnt signaling activity. Furthermore, Lrp4 can antagonize activation of Wnt signaling when over-expressed in cultured cells and this inhibitory activity is lost in mutant proteins (Johnson et al., 2005; Li et al., 2010). However, studies in bone and kidney development revealed no apparent elevation of Wnt signaling in Lrp4 mutants (Choi et al., 2009; Karner et al., 2010). In addition, Lrp4 is implicated in regulation of Bmp signaling in some contexts and functions as a co-receptor for Agrin in the neuromuscular junction (Kim et al., 2008; Ohazama et al., 2008; Zhang et al., 2008). Therefore, whether Lrp4 directly inhibits the Wnt pathway or it controls another pathway to indirectly affect Wnt signaling in vivo has been unclear.
Similar to Lrp5/6, Lrp4 can bind in vitro to Dkk1, Sost and Wise suggesting that roles for Lrp4 in Wnt signaling may be modulated by binding of these antagonists (Ohazama et al., 2008; Choi et al., 2009; Karner et al., 2010). This is consistent with the observation that Lrp4 facilitates the Wnt inhibitory function of Sost in in vitro bone mineralization (Leupin et al., 2011). In addition to this potential cell-autonomous role as a membrane receptor, Lrp4 is also postulated to modulate Wnt signaling by releasing its extracellular domain, and hence sequestering Wnt antagonists (Choi et al., 2009; Dietrich et al., 2010). It remains to be determined whether interaction between Lrp4 and the Wnt antagonists plays a significant role in vivo.
Similar to other LDL receptor-related proteins, Lrp4 is implicated in regulating different signaling pathways (May et al., 2007; Willnow et al., 2007). With its multiple ligand binding motifs in the extracellular domain, Lrp4 has the ability to bind in vitro to secreted Wnt and Bmp antagonists (Ohazama et al., 2008; Choi et al., 2009). Interestingly, in both humans and mice Lrp4 mutations phenocopy defects caused by deficiency of individual Wnt antagonists in a tissue-specific manner. For example, limb defects of Lrp4 mutants are similar to those of Dkk1 mutant mice (MacDonald et al., 2004), and bone overgrowth of human patients with LRP4 mutations is reminiscent of bone defects caused by SOST and Dkk1 mutations (Balemans et al., 2001; Morvan et al., 2006). Lastly, Lrp4 and Wise mutant mice share defects in tooth, mammary glands and other skin appendages (Ohazama et al., 2008). These observations imply that interplay between Lrp4 and the Wnt antagonists may play an important role in modulating Wnt/β-catenin signaling in many developmental and physiological contexts.
Wise is known as a context-dependent modulator of Wnt signaling and an inhibitor of Bmp signaling (Itasaki et al., 2003; Laurikkala et al., 2003; Lintern et al., 2009). The strong genetic interaction of Wise with Lrp5 and Lrp6 suggested that Wise controls tooth number and patterning by inhibiting Wnt signaling (Ahn et al., 2010).
Genetic studies in mouse have provided insights on signaling pathways required for embryonic mammary development (Robinson, 2007). Skin appendages such as teeth, hair and mammary glands develop from the surface ectoderm and underlying mesenchyme during embryogenesis. Despite the differences in the final structures, these skin appendages arise through similar morphological processes and tissue interactions in the early stages of their development (Mikkola and Millar, 2006). The future site of appendage development is initially marked by a thickening of the epithelium, which gives rise to a more localized placode. Subsequently, invagination of the placodal epithelium and condensation of the underlying mesenchymal cells leads to bud formation. Interactions within and between epithelial and mesenchymal tissues are essential for the proper growth and patterning of placode development. Genetic disruptions of genes encoding components of signaling pathways (Wnt, FGF, BMP, Eda, etc.) often cause developmental defects in multiple skin appendages suggesting that patterning processes are shared among these appendages at the molecular level (Pispa and Thesleff, 2003; Mikkola and Millar, 2006).
While many aspects of early patterning are similar, the spatial and temporal dynamics of placode development appear to be unique among the appendages. For example, hair placode formation begins with broad, regularly spaced epithelial thickenings, which are gradually refined to smaller circular placodes (Schmidt-Ullrich and Paus, 2005). In contrast, mammary placodes develop along the mammary lines, two lines of transient epithelial thickening, which appear between the fore and hind limb buds. Within a day, five pairs of mammary placodes form in a defined order as the mammary lines resolve (Robinson, 2007; Cowin and Wysolmerski, 2010) (
Accordingly, there is a need to clarify the relationship between Lrp4 and Wise, provide insights into interplay between Lrp4 and Wnt antagonists in Wnt inhibition, and to provide improved regulation of the interaction between Lrps and Wnt antagonists. The present invention is directed to meeting these and other needs.
One embodiment of the present invention is an antibody that modulates binding between Lrp5 and WISE or Lrp6 and WISE, but does not modulate binding between Lrp4 and WISE.
Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises one or more antibodies of the present invention and at least one pharmaceutically acceptable excipient or diluent.
A further embodiment of the present invention is a method for preventing WISE binding to Lrp5 or Lrp6, but not WISE binding to Lrp4, comprising contacting Lrp5 or Lrp6 with an agent that binds to Lrp5 or Lrp6 but not Lrp4.
An additional embodiment of the present invention is a method for preventing human WISE from binding to human Lrp5 or human Lrp6 comprising contacting human WISE with a monoclonal antibody, which antibody specifically binds to human Lrp5 or human Lrp6 but not to human Lrp4.
A further embodiment of the present invention is a kit. This kit comprises one or more antibodies of the present invention.
The patent or application file contains at least one drawing executed in color. Copies of this patentorpatent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
One embodiment of the present invention is an antibody that modulates binding between Lrp5 and WISE or Lrp6 and WISE, but does not modulate binding between Lrp4 and WISE.
As used herein, an “antibody” encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), as well as antigen-binding fragments thereof, (e.g., Fab′, F(ab′)2, Fab, Fv, and rgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301. Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies, are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference).
The term “antibody” includes both polyclonal and monoclonal antibodies. The term “monoclonal antibody”, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example.
Typically, an antibody has a heavy and a light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). Light and heavy chain variable regions contain four “framework” regions interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody. As used herein, “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
The phrase “single chain Fv” or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
For application in man, it is often desirable to reduce immunogenicity of antibodies originally derived from other species, like mouse. This can be done by construction of chimeric antibodies, or by a process called “humanization”. In this context, a “chimeric antibody” is understood to be an antibody comprising a domain (e.g. a variable domain) derived from one species (e.g. mouse) fused to a domain (e.g. the constant domains) derived from a different species (e.g. human).
As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-3′27 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
Furthermore, technologies have been developed for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (WO 90/05144; D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths and G. Winter (1991) “By-passing immunisation. Human antibodies from V-gene libraries displayed on phage.” J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, “Concepts in antibody phage display”. Briefings in Functional Genomics and Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. “Human antibodies from transgenic mice”. Int Rev Immunol. 1995; 13(1):65-93; Bruggemann M, Taussig M J. “Production of human antibody repertoires in transgenic mice”. Curr Opin Biotechnol. 1997 August; 8(4):455-8.). Such antibodies are “human antibodies” in the context of the present invention.
“Epitope” or “antigenic determinant” refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in an unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). A preferred method for epitope mapping is surface plasmon resonance.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
The phrase “binds specifically” or “specific binding” refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein sequence, thereby identifying its presence.
Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods of determining binding affinity and specificity are well known in the art (see, for example, Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co. 1976)).
Furthermore, a binding agent can interfere with the specific binding of a receptor, for example, an Lrp such as Lrp5 or Lrp6, and its ligand, for example, WISE, by various mechanism, including, for example, by binding to the ligand binding site, thereby interfering with ligand binding; by binding to a site other than the ligand binding site of the receptor, but sterically interfering with ligand binding to the receptor; by binding the receptor and causing a conformational or other change in the receptor, which interferes with binding of the ligand; or by other mechanisms. Similarly, the agent can bind to or otherwise interact with the ligand to interfere with its specifically interacting with the receptor. For purposes of the methods disclosed herein, an understanding of the mechanism by which the interference occurs is not required and no mechanism of action is proposed. An Lrp5 or Lrp6 antibody is characterized by having specific binding activity (Ka) for an Lrp5 or Lrp6 of at least about 105 mol−1, 106 mol−1 or greater, preferably 107 mol−1 or greater, more preferably 108 mol−1 or greater, and most preferably 109 mol−1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).
As used herein, “modulate”, “modulates”, “modulating”, or other grammatical variations thereof means to change.
Non-limiting representative protein sequences for Lrp5 are listed in SEQ ID NOs.53-56, and the corresponding cDNAs are listed in SEQ ID NOs:383-386. Preferably, the Lrp5 is human (such as SEQ ID NO:54, a mature form or an isoform thereof). As used herein, the “mature form” of a protein means the form after post-translational processing, including the removal of signal sequences, which, in the case of the Lrps, are the approximately 20 N-terminal amino acids. As used herein “isoform” means an alternative form of a protein resulting from differential transcription of the relevant gene either from an alternative promoter or an alternate splicing site.
Non-limiting representative protein sequences for Lrp6 are listed in SEQ ID NOs:57-60, and the corresponding cDNAs are listed in SEQ ID NOs. 387-390. Preferably, the Lrp6 is human such as SEQ ID NO:58, a mature form or an isoform thereof).
Non-limiting representative protein sequences for WISE are listed in SEQ ID NOs:61-64, and the corresponding cDNAs are listed in SEQ ID NOs:391-394. Preferably, the WISE is human (such as SEQ ID NO:62, a mature form or an isoform thereof).
Non-limiting representative protein sequences for Lrp4 are listed in SEQ ID NOs:49-52, and the corresponding cDNAs are listed in SEQ ID NOs:379-382. Preferably, the Lrp4 is human such as SEQ ID NO:50, a mature form or an isoform thereof).
In one aspect of this embodiment, the antibody prevents binding between Lrp5 and WISE or Lrp6 and WISE. For example, the antibody may specifically bind to Lrp5 or Lrp6 at one or more sequences selected from the group consisting of SEQ ID NOs: 1-39, such as one or more of SEQ ID NOs: 1-15, one or more of SEQ ID NOs: 1-3 and 16-27, or one or more of SEQ ID NOs: 1-3 and 28-39. Preferably, the antibody binds specifically to Lrp5. In another preferred embodiment, the antibody binds specifically to Lrp6.
The details of SEQ ID NOs:1-39 are listed below.
In another aspect of this embodiment, the antibody specifically binds to Lrp5 or Lrp6 within a sequence selected from the group consisting of SEQ ID NOs:40-48. For example, such an antibody may specifically bind to Lrp5 or Lrp6 at SEQ ID NOs:65-378. It is noted that SEQ ID NOs: 46 and 47 contain the amino acid sequences just before the transmembrane domain of Lrp5 and Lrp6, respectively. The identified portions of the Lrp5 and Lrp6 are very similar to each other, but are very different from the sequence of Lrp4 (see
The details of SEQ ID NOs:40-48 are listed below.
In another aspect of this embodiment, Lrp4, Lrp5, and Lrp6 are human.
In an additional aspect of this embodiment, the antibody is monoclonal.
In a further aspect of this embodiment, the antibody is human, humanized, or chimeric.
Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises an antibody of the present invention such as, e.g., an antibody that modulates binding between Lrp5 and WISE or Lrp6 and WISE, but does not modulate binding between Lrp4 and WISE, and at least one pharmaceutically acceptable excipient or diluent.
A further embodiment of the present invention is a method for preventing WISE binding to Lrp5 or Lrp6, but not WISE binding to Lrp4, comprising contacting Lrp5 or Lrp6 with an agent that binds to Lrp5 or Lrp6 but not Lrp4.
A binding agent according to the present invention may be an antibody, or non-immunoglobulin “antibody mimics”, sometimes called “scaffold proteins”, may be based on the genes of protein A, the lipocalins, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin (Skerra, Current Opinion in Biotechnology 2007, 18(4): 295-304). A preferred embodiment in the context of the present invention are designed ankyrin repeat proteins (DARPin's; Steiner et al., J Mol Biol. 2008 Oct. 24; 382(5): 1211-27; Stumpp M T, Amstutz P. Curr Opin Drug Discov Devel. 2007 March; 10(2):153-9). Preferably, the agent is an antibody. More preferably, the antibody is monoclonal.
In one aspect of this embodiment, the agent is an antibody that specifically binds to Lrp5 or Lrp6. Preferred binding sites are as disclosed herein.
An additional embodiment of the present invention is a method for preventing human WISE from binding to human Lrp5 or human Lrp6 comprising contacting human WISE with a monoclonal antibody, which antibody specifically binds to human Lrp5 or human Lrp6 but not to human Lrp4. Preferred binding sites are as disclosed herein.
A further embodiment of the present invention is a kit. This kit comprises one or more of the antibodies of the present invention.
In the present invention, an “effective amount” or a “therapeutically effective amount” of a compound or composition disclosed herein is an amount of such compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a composition according to the invention will be that amount of the composition, which is the lowest dose effective to produce the desired effect. The effective dose of a compound or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
A suitable, non-limiting example of a dosage of antibody in the compositions disclosed herein is from about 0.1 mg/kg to about 150 mg/kg per day, such as from about 0.5 mg/kg to about 50 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 110 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, and 150 mg/kg per day. The effective dose of antibody in the compositions disclosed herein maybe administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
A composition of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a composition of the present invention may be administered in conjunction with other treatments. A composition of the present invention maybe encapsulated or otherwise protected against gastric or other secretions, if desired.
The compositions of the invention comprise one or more active ingredients i.e., antibodies of the present invention, in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.).
Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
The compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
Compositions of the present invention suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
Pharmaceutical compositions may be prepared by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-Methylglucosamine (so-called “Meglumine”), galactosamine and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X or polyethylene glycol (PEG).
The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization), however, solutions comprising antibacterial agents also may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.
Exemplary antibody concentrations in the pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL. For clarity reasons, it is emphasized that the concentrations as indicated herein relate to the concentration in a liquid or in a liquid that is accurately reconstituted from a solid form.
An aqueous formulation of the antibody may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
A tonicity agent may be included in the antibody formulation to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. Preferably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as a physiological salt solution and the blood serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, in particular in an amount of 105 mM to 305 nM.
A surfactant may also be added to the antibody formulation to reduce aggregation of the formulated antibody and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulphate (SDS). Preferred polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Preferred polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Preferred Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v.
A lyoprotectant may also be added in order to protect the labile active ingredient (e.g. a protein) against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants are generally used in an amount of about 10 mM to 500 nM.
In one embodiment, the formulation contains the above-identified agents (i.e. antibody, surfactant, buffer, stabilizer and/or tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In another embodiment, a preservative may be included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
Compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
Compositions of the present invention for rectal or vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
Lrp4mdig, Lrp4mitt, Lrp4mte, Wise, TopGal, Lrp5, Lrp6, Ctnnb1fx, K14cre, Ptch1LacZ and R26-floxstop-LacZ mice were described previously (DasGupta and Fuchs, 1999; Milenkovic et al., 1999; Soriano, 1999; Dassule et al., 2000; Pinson et al., 2000; Brault et al., 2001; Kato et al., 2002; Simon-Chazottes et al., 2006; Weatherbee et al., 2006; Ahn et al., 2010; Ferrer-Vaquer et al., 2010). All experiments involving mice were approved by the Institutional Animal Care and Use Committee of the Stowers Institute for Medical Research (Protocol 2010-0062).
Generation of Lrp4-LacZ, K14-tTA, TCF-tTA and tetO-Wise Transgenic Mice
For Lrp4-LacZ BAC reporter, a mouse BAC clone, RP23-276H15, was modified to contain an 134 kb genomic region which covers the whole Lrp4 coding region and neighboring upstream (36 kb) and downstream (44 kb) sequences using the bacterial recombination technology (Lee et al., 2001). LacZ was then inserted in-frame into the first coding exon of Lrp4. The K14-tTA was generated by inserting the K14 promoter (Ahn et al., 2010) and a synthetic intron (IVS) (Clontech) upstream of VP22-tTA-SV40 pA (Gossen and Bujard, 1992). For TCF-tTA, the K14 promoter of K14-tTA was removed except the basal promoter region (−120 to +13) and replaced with the multiple TCF binding sites from TOPFLASH vector (Millipore). To make tetO-Wise, G-CaMP2 of tetO-G-CaMP2 (He et al., 2008) was replaced with a Wise ORF, and then IRES-eGFP was subcloned between Wise and SV40 pA. Transgenic founders were generated by pro-nuclear injection of linearized constructs into C57Bl/10J xCBA-F1 embryos.
The Wise-LacZ construct was generated by inserting a LacZ-SV40 pA in-frame into the first coding exon of Wise in the 24 kb EcoR I-Sal I genomic fragment from a mouse BAC clone, RP23-98E22. Three of five Wise-LacZ lines mimicked the known expression pattern of Wise in the skin appendages and used for this study (
The K14-Wise construct has been previously described (Ahn et al., 2010). Two of the 9 K14-Wise transgenic founders showed severely deformed limbs and hair loss (data not shown). By in vitro fertilization using sperm from one of the founders, transgenic progeny which phenocopied their parents were generated (
To detect β-Galactosidase activity, embryos were fixed in either 0.1% paraformaldehyde/0.2% glutaraldehyde (E11.5-E13.5) or 4% paraformaldehyde (PFA) (E14.0 or older) for 30-60 minutes on ice. After several washes in phosphate buffered saline, samples were stained in X-Gal for 4-20 hours at 4° C. or at room temperature. Whole-mount in situ hybridization was performed with embryos fixed in 4% PFA overnight according to standard protocols using DIG-labeled anti-sense riboprobes. Histological samples were paraffin-embedded after post-fixation in 4% PFA, sectioned at 8 μm and counterstained with nuclear fast red. For analysis of cell proliferation and cell death, embryos were harvested 2 hours after intraperitoneal injection of BrdU (50 μg/g body weight) into pregnant females, sectioned and stained with a mouse anti-BrdU antibody (Amersham), a mouse E-cadherin antibody (BD Biosciences), or a rabbit caspase 3 antibody (Cell Signaling).
Fluorescent images were obtained by the LSM 710 confocal microscope (Carl Zeiss). Nuclei with fluorescence above basal level were counted using the Imaris software (Bitplane).
Lrp4 is known to be expressed in placodes of skin appendages such as mammary glands, hair follicles and vibrissae (Weatherbee et al., 2006; Fliniaux et al., 2008). Thus, potential roles for Lrp4 in development of these tissues were examined. Mice homozygous for null alleles of Lrp4 (Lrp4mitt and Lrp4mte) die after birth, but mice homozygous for a hypomorphic allele (Lrp4mdig) survive to reach adulthood (Simon-Chazottes et al., 2006; Weatherbee et al., 2006). The analyses of both Lrp4mitt/dig and Lrp4mdig/mdig females revealed a variety of abnormalities in the number, position and morphology of nipples (
The mammary defects in Lrp4 mutants suggest that Lrp4 plays a role in embryonic mammary development. The number and position of the nipples and associated mammary glands is primarily determined around embryonic day 12 (E12) when the mammary placodes develop (Cowin and Wysolmerski, 2010). The TopGal reporter mouse line (DasGupta and Fuchs, 1999) was used to follow the progress of mammary development and to monitor changes in the activity of Wnt/β-catenin signaling (
In Lrp4mdig/mdig embryos, TopGal expressing cells were more loosely organized around the developing placodes at E12.0 suggesting that placode assembly was delayed (compare
Whether the abnormal mammary patterning in Lrp4 mutants is associated with changes in the number of mammary epithelial cells was investigated using the TCF/LEF:H2B-GFP reporter, which marks mammary placodes similar to TopGal (Ferrer-Vaquer et al., 2010). Confocal imaging of the placode 2/3 region revealed a 40% increase in the total number of GFP-expressing cells (
Although mammary placodes #2 and 3 were developmentally delayed and morphologically abnormal in Lrp4 mutants, they were centered at fairly normal positions at E12.0 (
With respect to cell death, in control mice, a small number of apoptotic cells were observed mostly around the neck of the buds, but not in the interplacodal epithelium (
To examine which signaling pathways were misregulated in the mammary placodes of Lrp4 mutants, placodes 2 and 3 were dissected from E12.5 embryos and expression analysis was performed using qPCR assays designed for components of Wnt, FGF, TGFβ/BMP and Eda pathways (FIG. 16). Differential expression of genes in Wnt (Dkk1, Dkk4 and Lef1) and TGFβ/BMP (Bmp3, Msx1 and Msx2) pathways suggests that signaling activity of the two pathways is changed in Lrp4 mutants.
The increased number and abnormal distribution of cells expressing the Wnt reporters (
It has been shown that embryonic mammary development is delayed or severely impaired in Lrp5−/− and Lrp6−/− mice, respectively, in association with reduced Wnt signaling activity. (Lindvall et al., 2006; Lindvall et al., 2009). Therefore, whether reduced doses of Lrp5 and Lrp6 can rescue the mammary defects of Lrp4 mutants was investigated (
Whether the normal timing of placode initiation is restored in Lrp4mdig/mdig;Lrp5+/−;Lrp6+/− mice was investigated. Indeed, the compound mutants displayed placodes with almost normal morphology and size with few TopGal-expressing cells in the interplacodal region at E12.5 (
To further explore a role for Wnt/β-catenin signaling in controlling the number of mammary epithelial cells, the β-catenin gene (Ctnnb1) was inactivated in the epithelium after placode initiation using a conditional allele of β-catenin combined with a Cre line driven by a Keratin 14 promoter (K14cre). K14cre can induce recombination in a subset of epithelial cells along the mammary line at E11.5-E12.0 (
Whether inactivation of β-catenin has effects on the Lrp4 mutant phenotypes was tested (
Whether the findings in mammary gland development reflect related roles for Lrp4 in other skin appendages was investigated. Primary hair follicles were marked by Wnt10b transcripts at E14.5 in control mice, but in the Lrp4 mutant skin, Wnt10b was undetectable (
Groups of vibrissae develop in different regions of the mouse head (Yamakado and Yohro, 1979), and supernumerary vibrissal follicles were observed for each group in Lrp4 mutants (
Because Wise is a potential ligand for Lrp4 and mice deficient for Wise or Lrp4 displayed similar tooth defects, roles for Wise in the mammary glands and other skin appendages were investigated. Earlier studies have shown that in developing skin appendages Wise is excluded from the epithelial signaling centers where Lrp4 is expressed (Laurikkala et al., 2003; Weatherbee et al., 2006). During mammary placode formation, Lrp4 was expressed in the placodal epithelial cells similar to Lef-1 while Wise expression was strong in the surrounding epithelial and mesenchymal cells (
In Wise-null females, changes in the position and number of nipples were observed (
In addition to the mammary defects, Wise-null mice displayed supernumerary vibrissal follicles with a frequency lower than that of Lrp4mitt/mitt mice (
Whether changes in Wnt/β-catenin signaling account for the Wise-null mammary defects were tested genetically. Wise-null mice were crossed with Lrp5 mutants to generate double homozygous mutants. It was found that removing both copies of Lrp5 significantly rescues the abnormal spacing and ectopic TopGal expression of Wise-null mammary buds (
To complement and validate predicted roles for Wise based on loss-of-function analyses, whether over-expression of Wise using the Keratin 14 promoter (K14-Wise) (Ahn et al., 2010) can reduce the number of placodal epithelial cells was investigated. K14-Wise embryos showed defects in development of hair/vibrissal follicles, mammary placodes and limbs with reduced TopGal expression (
Using a strong (#32) or a moderate (#87) K14-tTA driver, the mammary defects of the K14-Wise embryos were reproduced (
The overall similarities in skin defects and elevated Wnt signaling in Lrp4 and Wise mutants raise the question of whether Lrp4 and Wise act through a common or parallel pathways. To genetically test this idea, combinatorial mutants of the two genes were generated. No mammary defect was observed in transheterozygotes, and defects in double homozygous mutants were indistinguishable from those of Lrp4mdig/mdig mice during embryonic mammary development (
Considering the close genetic interaction of both genes with the components of Wnt/β-catenin pathway, this lack of synergy or additive effect between the two mutants suggests that Lrp4 and Wise may be acting on the same pathway to inhibit Wnt/β-catenin signaling and Lrp4 acts downstream of Wise. Alternatively, it is possible that Lrp4 and Wise function independent of each other, but Lrp4 has a larger role in modulating the level of signaling activity. To distinguish between these possibilities, Wise was over-expressed in Lrp4 mutants. Elevated Wise expression would rescue the Lrp4 mutant phenotypes if Wise and Lrp4 function primarily in an independent manner. However, over-expression of Wise resulted in no changes in the limb and mammary defects of Lrp4 mutants (
The genetic analyses have revealed that Lrp4 and Wise play stage-specific roles for proper patterning and morphogenesis of the murine mammary glands and other skin appendages through their ability to modulate Wnt/β-catenin signaling. Lrp4 has an early role in facilitating placode initiation and together Lrp4 and Wise have later roles in induction and/or maintenance of precursor cells. Through loss-, gain-of-function and epistasis analyses, it was found that Wise requires Lrp4 to exert its activity. Together the data suggest a model whereby Wise and Lrp4 work in concert to modulate the activity of Wnt signaling though a common mechanism. These findings have important implications for a mechanistic understanding of how Wnt antagonists participate in the precise control of Wnt signaling to regulate cellular processes involved in ectodermal placode formation.
Development of mammary glands provides an opportunity to study spatiotemporal patterning of ectodermal organs since multiple placodes form along the mammary lines in a fairly well-defined order. The analyses of Lrp4 and Wise mutant mice have provided insight on the cellular processes that control the transition from stretches of thickened epithelium into precisely spaced placodes.
First, initiation of the placodes requires assembly of the precursor cells. In Lrp4 mutants, even when comparable numbers of cells were present around the site of placode formation, they were loosely assembled with a smaller degree of invagination compared with those of control mice. This delay in placode assembly suggests that Lrp4 normally facilitates aggregation of the precursor cells.
Second, the number of the precursor cells needs to be tightly controlled for proper morphogenesis of individual placodes and maintenance of spacing between them. The significant increase in the number of Wnt reporter-positive cells in Lrp4 and Wise mutants suggests that both Lrp4 and Wise have a role in limiting the mammary fate to a defined number of epithelial cells. This may be achieved by suppressing maintenance of mammary fate in existing precursor cells or by blocking induction of new precursor cells as mammary epithelial cells tend to proliferate at a very low rate.
In addition, migration of the mammary precursor cells may play an important role in placode initiation and morphogenesis. The sustained presence of the precursor cells in the interplacodal regions of Lrp4 and Wise mutants suggest that these cells fail to migrate to the normal sites of placode formation. These ectopic precursor cells then interfere with morphogenesis of normal placodes and give rise to supernumerary placodes. The extent of migration along the mammary line is not well characterized. It is possible that cell movement is limited to cells near the sites of placode formation and cells farther away from the placodes lose their potential to become mammary epithelial cells.
Disruption in any of the above processes would lead to defects in the number, morphogenesis and position of the mammary placodes. Mutant phenotypes suggest that initially Lrp4 is predominantly required for assembly of the placodes, and later both Lrp4 and Wise play a role in the number of the precursor cells (
Consistent with the idea that the molecular mechanisms for early morphogenesis are shared among the skin appendages, both Lrp4 and Wise mutants display similar abnormalities in patterning of hair and vibrissal follicles with stronger defects observed in Lrp4 mutants. Interestingly, the formation of supernumerary vibrissal follicles is preceded by delayed placode morphogenesis with a broader distribution of the Wnt-active precursor cells in Lrp4 mutants. A delay in placode formation was also observed in the primary hair follicles of Lrp4 mutants. These delays are reminiscent of the defects observed during the mammary placode formation. Focalization of the epithelial precursor cells and associated Wnt activity are commonly seen during the formation of the skin placodes as well as AER (Mikkola and Millar, 2006; Fernandez-Teran and Ros, 2008). It is possible that Lrp4 and Wise modulate Wnt signaling in those precursor cells to control cellular processes such as cell movement, cell shape change, cell-cell adhesion and cell proliferation, which are important for patterning and morphogenesis of the skin placodes (Jamora et al., 2003).
The data showed that the mammary defects of Lrp4 and Wise mutants can be rescued by reducing the dose of Lrp5/6 and β-catenin. This genetic interaction indicates that elevated Wnt/β-catenin signaling is responsible for the mammary defects and suggests that Lrp4 and Wise directly antagonize Wnt/β-catenin signaling instead of acting indirectly via another signaling pathway. This is consistent with the previous studies which provided genetic evidence that Wise functions as a Wnt inhibitor in tooth development (Munne et al., 2009; Ahn et al., 2010).
The genetic analyses also demonstrate that Wnt/β-catenin signaling is essential for induction and/or maintenance of mammary precursor cells, but its activity needs to be tightly controlled to achieve a proper number of these cells (
Similar to other LDL receptor-related proteins, Lrp4 is implicated in regulating different signaling pathways (May et al., 2007; Willnow et al., 2007). With its multiple ligand binding motifs in the extracellular domain, Lrp4 has the ability to bind in vitro to secreted Wnt and Bmp antagonists (Ohazama et al., 2008; Choi et al., 2009). Interestingly, in both humans and mice Lrp4 mutations phenocopy defects caused by deficiency of individual Wnt antagonists in a tissue-specific manner. For example, limb defects of Lrp4 mutants are similar to those of Dkk1 mutant mice (MacDonald et al., 2004), and bone overgrowth of human patients with LRP4 mutations is reminiscent of bone defects caused by SOST and Dkk1 mutations (Balemans et al., 2001; Morvan et al., 2006). Lastly, Lrp4 and Wise mutant mice share defects in tooth, mammary glands and other skin appendages (Ohazama et al., 2008; this study). These observations imply that interplay between Lrp4 and the Wnt antagonists may play an important role in modulating Wnt/β-catenin signaling in many developmental and physiological contexts.
While genetic evidence for such interplay has been lacking, the observation that Wise gain-of-function phenotypes depend on Lrp4 in various tissue contexts provides important insight on this issue. Based on loss- and gain-of-function analyses in this study, without being bound by a particular theory, it is believed that Lrp4 and Wise act through a common mechanism where Lrp4 lies downstream of Wise in the pathway leading to inhibition of Wnt/β-catenin signaling (
All documents cited in this application are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.
The present invention claims benefit to U.S. provisional application Ser. No. 61/745,007 filed Dec. 21, 2012, the entire contents of which are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61745007 | Dec 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14653759 | Jun 2015 | US |
| Child | 16793780 | US |
