Compositions and methods useful for promoting milk production

Abstract

Methods, agents, and compositions for promoting milk production in mammals are provided. Agents useful for promoting milk production can include agents that inhibit NOTCH4 activity. The agent can inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent can inhibit NOTCH4 by competing with ROBO1 for binding to ROBO2, thereby making ROBO1 available to inhibit NOTCH4 activity. The agent can be a soluble ROBO1 extracellular domain or an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent can be an RNAi construct that inhibits NOTCH4 expression or an RNAi construct that inhibits ROBO2 expression. Also provided herein are transgenic mammals that have been genetically modified to express a soluble ROBO1 extracellular domain, inhibit ROBO2 expression, and / or inhibit NOTCH4 expression. Also provided are methods for promoting milk production in such transgenic mammals by administering one or more agents disclosed herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 837,590, filed Apr. 23, 2019, which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference of a Sequence Listing Provided as a Text File The sequence listing is provided herein as a text file named UCSC - 383PRV2 seq list_ST25.txt, created on Feb. 15, 2019, and having a size of 130 KB. This text file is hereby incorporated by reference in its entirety.

Background Art

[0003] The mammary gland, or breast, is a dynamic epithelial organ involved in milk production in mammals. 1 Beginning as a primordium located at the nipple, the mammary gland develops postnatally in response to hormonal cues generated during puberty and forms a ductal structure that branches into the underlying stromal fat pad. Each duct is bilayered, containing an outer layer of basal / myoepithelial cells (referred to herein as BC) and an inner layer of luminal cells (referred to herein as LC). Luminal cells can be further subdivided into two subpopulations, a ductal subpopulation that lines the lumen, and an alveolar subpopulation in which milk - producing alveoli are generated during pregnancy (FIG. 1A). When the offspring is weaned from breast milk, the mammary gland is remodeled back to its pre - pregnancy state in a process called regression. Within the alveolar cell subpopulation, there are alveolar progenitor cells (AVP). Currently, it is thought that alveologenesis during pregnancy results from the differentiation of alveolar progenitor cells into milk - producing alveolar cells (AV).

[0004] Notch is a major signaling pathway that regulates the maintenance and developmental fate determination of stem cells / progenitor cells. There are four NOTCH receptors: NOTCH1, NOTCH2, NOTCH3, and NOTCH4 - all of which are expressed in the mammary gland. 2 During mammary gland development, Notch signaling promotes the developmental fate of luminal cells at the expense of the basal cell fate. 3~6 Furthermore, results showing that overexpression of the constitutively active NOTCH4 intracellular domain (ICD) significantly reduces alveolar development suggest that inhibition of NOTCH4 activity is required for alveolar expansion and differentiation. 7~9 This indicates that Notch signaling via NOTCH4 must be inhibited in alveolar progenitor cells for them to differentiate into alveolar cells.

[0005] Roundabout (ROBO) receptors are members of the conserved immunoglobulin (Ig) superfamily involved in many developmental processes. They bind to a family of conserved secreted glycoprotein extracellular matrix ligands called SLIT (e.g., SLIT2 and SLIT3 in the mammary gland), which are expressed throughout the mammary epithelium (Figure 1B). 10、11 This signaling system has been shown to regulate cell fate determination in the mouse nervous system and the Drosophila intestine. 12、13 . SUMMARY OF THE INVENTION

[0006] To build a milk supply with each pregnancy, cell growth and differentiation need to be significantly accelerated. Disclosed herein is a method of promoting such accelerated cell growth and differentiation by treating with an agent that affects a derepressive signaling circuit, whereby ROBO2 inhibits ROBO1, which in turn inhibits NOTCH4 activation. ROBO1 is expressed in both BC and LC of virgin mammary glands, but is upregulated in LC during pregnancy. Expression of ROBO2 is restricted to a subset of luminal cells. Disclosed herein for the first time is the following finding: Loss (or deletion) of the Robo1 gene results in inhibition of mammary alveolar differentiation. This has been demonstrated in both the HC11 cell lactation model and in vivo in the mammary gland. Loss (or deletion) of Robo2 results in the opposite phenotype in both models, i.e., greater mammary alveolar differentiation. ROBO1 has been shown to specifically bind to NOTCH4 and inhibit its signaling. ROBO2 has been shown to specifically bind to ROBO1 and prevent ROBO1 from inhibiting NOTCH4. The interaction between ROBO1 and ROBO2 is enhanced by SLIT2. Disclosed herein is a ROBO1 receptor fragment comprising a portion of the ROBO1 extracellular domain that inhibits NOTCH4 signaling. The experiments disclosed herein show that SLIT / ROBO signaling regulates mammary alveologenesis by governing NOTCH4 activation and controlling the number of alveolar progenitor cells that differentiate into milk-producing alveolar cells.

[0007] Methods, agents, and compositions are provided for promoting milk production in mammals. Agents useful for promoting milk production can include agents that inhibit NOTCH4 activity. The agent can be a soluble ROBO1 extracellular domain, or the agent can inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent can inhibit NOTCH4 by competing with ROBO1 to release it from binding to ROBO2, thereby making ROBO1 available to inhibit NOTCH4 activity. The agent can be an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent can be an RNAi construct that inhibits the expression of NOTCH4. The agent can be an RNAi construct that inhibits the expression of ROBO2. Also provided herein are transgenic mammals genetically modified for the expression of a soluble ROBO1 extracellular domain, inhibition of the expression of ROBO2, and / or inhibition of the expression of NOTCH4. Also provided is a method for promoting milk production in such transgenic mammals by administering one or more of the agents disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0008]

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[0009] Sequence Listing SEQ ID NO: 1 - Bovine ROBO1 - Ecto SEQ ID NO: 2 - Bovine ROBO1 - Ig5 SEQ ID NO: 3 - Bovine ROBO1 - Ig2 SEQ ID NO: 4 - Homo sapiens ROBO1 - Ecto SEQ ID NO: 5 - Homo sapiens ROBO1 - Ig5 SEQ ID NO: 6 - Homo sapiens ROBO1 - Ig2 SEQ ID NO: 7 - American bison ROBO1 - Ecto SEQ ID NO: 8 - American bison ROBO1 - Ig5 SEQ ID NO: 9 - American bison ROBO1 - Ig2 SEQ ID NO: 10 - Yak ROBO1 - Ecto SEQ ID NO: 11 - Yak ROBO1 - Ig5 SEQ ID NO: 12 - Yak ROBO1 - Ig2 SEQ ID NO: 13 - Goat ROBO1 - Ecto SEQ ID NO: 14 - Goat ROBO1 - Ig5 SEQ ID NO: 15 - Goat ROBO1 - Ig2 SEQ ID NO: 16 - Sheep ROBO1 - Ecto SEQ ID NO: 17 - Sheep ROBO1 - Ig5 SEQ ID NO: 18 - Sheep ROBO1 - Ig2 SEQ ID NO: 19 - Yak ROBO1 - Ecto SEQ ID NO: 20 - Yak ROBO1 - Ig5 SEQ ID NO: 21 - Yak ROBO1 - Ig2 SEQ ID NO: 22 - Harvest Mouse ROBO1 - Ecto SEQ ID NO: 23 - Harvest Mouse ROBO1 - Ig5 SEQ ID NO: 24 - Harvest Mouse ROBO1 - Ig2 SEQ ID NO: 25 - Brown Rat ROBO1 - Ecto SEQ ID NO: 26 - Brown Rat ROBO1 - Ig5 SEQ ID NO: 27 - Brown Rat ROBO1 - Ig2 SEQ ID NO: 28 - Brown Rat DCC Ig2 SEQ ID NO: 29 - Brown Rat DCC Ig4 SEQ ID NO: 30 - Robo1 shRNA Forward Strand SEQ ID NO: 31 - Robo1 shRNA Reverse Strand SEQ ID NO: 32 - Notch4 shRNA Forward Strand SEQ ID NO: 33 - Notch4 shRNA Reverse Strand SEQ ID NO: 34 - Robo2 shRNA Forward Strand SEQ ID NO: 35 - Robo2 shRNA Reverse Strand

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] Methods, agents, and compositions for promoting milk production in mammals are provided. Agents useful for promoting milk production can include agents that inhibit NOTCH4 activity. The agent can be a soluble ROBO1 extracellular domain, and the agent can inhibit NOTCH4 activity by binding to ROBO2 and / or by binding to NOTCH4. The agent can inhibit NOTCH4 activity by competing with ROBO1 to release it from binding to ROBO2, thereby making ROBO1 available for inhibiting NOTCH4 activity. The agent can be an anti-NOTCH4 antibody that inhibits NOTCH4 activity. The agent can be an RNAi construct that inhibits the expression of NOTCH4. The agent can be an RNAi construct that inhibits the expression of ROBO2. Also provided herein are transgenic mammals genetically modified for the expression of a soluble ROBO1 extracellular domain, inhibition of the expression of ROBO2, and / or inhibition of the expression of NOTCH4. Also provided is a method for promoting milk production in such transgenic mammals by administering one or more of the agents disclosed herein.

[0011] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relevant to the places where the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and the provided publication date may be different from the actual publication date that may need to be independently confirmed. Therefore, the present methods, compositions, and transgenic animals should not be construed as admitting a right to precedence over such publications.

[0012] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude optional elements. Thus, this description is intended to serve as a basis for the use of exclusive terms such as "solely", "only", etc., or the use of "negative" limitations in connection with the recitation of claim elements.

[0013] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from, or combined with, the features of any of several other aspects without departing from the scope or spirit of the method. The methods described can be performed in the order of the recited events or in any other logically possible order.

[0014] Definitions As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and binds a target through at least one antigen-binding site. "Antibody" is used herein in its broadest sense and encompasses a variety of antibody structures including, but not limited to, polyclonal antibodies, recombinant antibodies, monoclonal antibodies, chimeric antibodies (chimeras of antibody sequences from two or more different species such as human, bovine, ovine, caprine, equine), humanized antibodies, human antibodies, bovineized antibodies, ovineized antibodies, caprine antibodies, equineized antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single-chain Fv (scFv) antibodies, single-domain antibodies (e.g., camelid / llama antibodies), and antibody fragments.

[0015] The term "intact antibody" or "full-length antibody" refers to an antibody having a structure substantially similar to the native antibody structure. This includes antibodies that include two light chains, each including a variable region and a light chain constant region (CL), and two heavy chains, each including a variable region and at least heavy chain constant regions CH1, CH2, and CH3.

[0016] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally includes an antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, disulfide-bonded Fv (sdFv), Fd, linear antibodies, single-chain antibody molecules (e.g., scFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies, and multispecific antibodies formed from antibody fragments.

[0017] As used herein, the term "variable region" refers to the region of an antibody light chain or the region of an antibody heavy chain that is involved in binding of the antibody to an antigen. The variable regions of the antibody heavy chain and the antibody light chain have similar structures and generally include four framework regions and three complementarity-determining regions (CDRs) (also known as hypervariable regions).

[0018] The term "framework region" refers to amino acid residues other than the CDR residues within the variable region. The variable region typically includes four framework regions, FR1, FR2, FR3, and FR4.

[0019] As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of antibodies that are involved in the highly specific recognition and binding of a single antigenic determinant or epitope. The individual antibodies that make up the population are identical, except for naturally occurring mutations that may be present in small amounts. The term "monoclonal antibody" includes intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv), single-chain (scFv) antibodies, antibody fragments, and fusion proteins that include other modified immunoglobulin molecules that contain an antigen-binding site. Further, "monoclonal antibody" refers to such antibodies made by any number of techniques including, but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0020] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0021] As used herein, the term "humanized antibody" generally refers to a chimeric antibody that includes a human immunoglobulin (e.g., a recipient antibody), wherein the native CDR residues are replaced by residues from the corresponding CDRs from a non-human species such as a mouse, rat, rabbit, or non-human primate, where the donor antibody has the desired specificity, affinity, and / or activity. Optionally, one or more residues within one or more framework regions of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications can be made to further refine and / or optimize the characteristics of the antibody. A humanized antibody may contain a variable region that includes all or substantially all of the CDRs corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions corresponding to those of a human immunoglobulin. In some embodiments, a humanized antibody will include at least a portion of the immunoglobulin Fc region (e.g., the hinge region, CH1, CH2, and / or CH3), typically that of a human immunoglobulin. Similar definitions apply to bovineized, ovineized, caprineized, and equinized antibodies.

[0022] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or an antibody made using any of the techniques known to those of skill in the art for making human antibodies. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, and B cell hybridoma technology. A human antibody as defined herein excludes humanized antibodies that contain residues from non-human sources.

[0023] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen or target that can be recognized and bound by a particular binding agent or binding agents (e.g., an antibody). When the antigen or target is a polypeptide, an epitope can be formed from both adjacent and non - adjacent amino acids juxtaposed by the three - dimensional folding of the protein. Epitopes formed from adjacent amino acids (also called linear epitopes) are usually retained upon protein denaturation, whereas epitopes formed by three - dimensional folding (also called conformational epitopes) are usually lost upon protein denaturation. Epitopes typically contain at least 3, more commonly at least 5, 6, 7, or 8 - 10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a number of software bioinformatics tools available on the Internet. X - ray crystallography can be used to characterize epitopes on a target protein by analyzing the amino acid residue interactions of the antigen / antibody complex.

[0024] As used herein, the term "specifically binds" refers to a binding agent (e.g., an antibody) that interacts more frequently, more rapidly, for a longer duration, with higher affinity, or in some combination of the above, with a particular antigen, epitope, protein, or target molecule than with alternative substances. Antibodies that specifically bind an antigen can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR) technology (e.g., Biacore), FACS, or other techniques known to those of skill in the art.

[0025] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The term "peptide" can be used to refer to polymers of less than 50 amino acids, for example, 5 - 50 amino acids. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non - amino acids. These terms also can encompass amino acid polymers that are naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations or modifications. The definition includes polypeptides containing, but not limited to, one or more analogs of amino acids, including, for example, non - natural amino acids, as well as other modifications known in the art. Since some of the polypeptides of the present disclosure can be antibody - based, it is understood that the term "polypeptide" encompasses polypeptides as single chains and polypeptides of two or more associated chains.

[0026] The terms "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase.

[0027] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that have nucleotides or amino acid residues that are identical or identical to a specified percentage when compared and aligned (introducing gaps if necessary) to obtain maximal correspondence without considering conservative amino acid substitutions as part of their sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, the GCG Wisconsin Package, and variations thereof. In some embodiments, two polynucleotides or polypeptides of the present disclosure are substantially identical when compared and aligned to obtain maximal correspondence as measured using a sequence comparison algorithm or by visual inspection, which means that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, the identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 40-60 nucleotides or amino acid residues, at least about 60-80 nucleotides or amino acid residues in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 nucleotides or amino acid residues, for example at least about 80-100 nucleotides or amino acid residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.

[0028] As used herein, the phrase "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are generally defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, a substitution from tyrosine to phenylalanine is considered a conservative substitution. In general, conservative substitutions in the sequences of polypeptides and / or antibodies do not impair the binding of the polypeptide or antibody to the target binding site. Methods for identifying conservative substitutions of nucleotides and amino acids that do not impair binding are well known in the art.

[0029] As used herein, the term "vector" means a construct that can deliver one or more genes or sequences of interest in a host cell and can usually be expressed. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0030] As used herein, the term "isolated" refers to a polypeptide, peptide, soluble protein, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. An "isolated" antibody substantially lacks substances from its cell source. In some embodiments, an isolated polypeptide, peptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is purified to the extent that it is no longer in the form found in nature. In some embodiments, the polypeptide, peptide, soluble protein, antibody, polynucleotide, vector, cell, or composition to be isolated is substantially pure. A polypeptide, peptide, soluble protein, antibody, polynucleotide, vector, cell, or composition can be isolated from a natural source or from a source such as an engineered cell line.

[0031] As used herein, the term "substantially pure" refers to a substance that is at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0032] As used herein, in the context of a polypeptide, the term "derived from" refers to a polypeptide having a sequence based on the sequence of a protein from a particular source. A polypeptide derived from a protein from a particular source can be a variant of the protein from that particular source. For example, a polypeptide derived from a protein from a particular source can have a sequence modified with respect to the sequence of the protein from which it is derived. A polypeptide derived from a protein from a particular source shares at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity with the protein from which it is derived.

[0033] As used herein, the term "effective amount" refers to an amount of an agent (e.g., an antibody, polypeptide, nucleic acid, etc.) sufficient to produce the intended effect in a subject, such as a mammal.

[0034] As used herein, reference to a value or parameter of "about" or "approximately" includes (and describes) the aspect directed to that value or parameter. For example, a description that refers to "about X" includes the description of "X".

[0035] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise.

[0036] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B, A or B, A alone, as well as B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0037] As used herein, the term RNAi construct includes RNA molecules and vectors that, upon their presence within a cell, result in RNA interference (RNAi) leading to a reduction in the expression of the transcript targeted by the RNAi construct. This term includes siRNA, shRNA, and RNAi-inducing vectors.

[0038] As used herein, an RNAi-inducing vector is a vector that, upon its presence within a cell, results in the transcription of one or more RNAs that self-hybridize or hybridize to each other to form shRNA or siRNA. The term encompasses plasmids, e.g., DNA vectors or viral vectors. The vector can contain a nucleic acid operably linked to an expression signal such that, when the vector is present within a cell, one or more RNA molecules that hybridize or self-hybridize to form siRNA or shRNA are transcribed. Thus, the vector provides a template for intracellular synthesis of one or more RNAs or precursors thereof.

[0039] Short interfering RNA (siRNA) comprises an RNA duplex of about 19 base pairs in length and optionally further comprises one or two single-stranded overhangs. siRNA can be formed from two RNA molecules that hybridize together or can be generated from a single RNA molecule that contains a self-hybridizing portion. The duplex portion of siRNA can contain one or more unpaired nucleotides. One strand of the siRNA contains a portion that hybridizes to a target transcript with perfect complementarity or with one or two mismatches. In embodiments where perfect complementarity is not achieved, the mismatch can be located at or near the siRNA terminus.

[0040] The term short hairpin RNA refers to an RNA molecule that comprises at least two complementary portions that hybridize or are capable of hybridizing to form a double-stranded (duplex) structure of sufficient length (usually at least 19 base pairs in length) to mediate RNAi and at least one single-stranded portion, usually about 1 to 10 nucleotides in length, that forms a loop. The duplex portion can contain one or more bulges consisting of one or more unpaired nucleotides, but usually does not.

[0041] Disclosed herein is an investigation of the role of ROBO receptors during mammary gland alveologenesis. In particular, loss of Robo1 inhibits alveologenesis, while loss of Robo2 promotes alveologenesis. Also disclosed are biochemical studies in cell lines that demonstrate that ROBO1 specifically binds to NOTCH4 and inhibits NOTCH4 activation. ROBO1 has been shown to be widely expressed throughout the mammary epithelial compartment, while ROBO2 expression is restricted to alveolar progenitor cells and basal / myoepithelial cells (BC). Also disclosed is a ROBO1 receptor fragment that includes a portion of the ROBO1 extracellular domain (ECD) that inhibits NOTCH4 signaling and promotes alveologenesis. It is also disclosed that alveologenesis is enhanced by treating cells and mammals with an antibody that inhibits the binding of ROBO2 to ROBO1. Without being bound by theory, the discoveries disclosed herein indicate an inhibitory circuit mechanism (ROBO2―|ROBO1―|NOTCH4) that regulates NOTCH4 signaling and, as a result, regulates the number of alveolar progenitor cells that differentiate into milk-producing alveoli with each pregnancy.

[0042] Method for enhancing milk production in a mammal The present disclosure provides a method for promoting milk production in a mammal. In certain embodiments, the method can include administering to the mammal a first agent that inhibits NOTCH4 activity, wherein the first agent is administered in an amount sufficient to inhibit NOTCH4 activity, thereby promoting milk production. The first agent can inhibit NOTCH4 activity by directly binding to the NOTCH4 protein, by inhibiting the binding of ROBO2 to ROBO1, by promoting the binding of ROBO1 to NOTCH4, by inhibiting the expression of NOTCH4, or by inhibiting the expression of ROBO2.

[0043] In certain embodiments, the first agent may comprise a soluble ROBO1 extracellular domain (ECD). In certain embodiments, the soluble ROBO1 ECD may comprise the entire extracellular domain of ROBO1 or a ROBO2-binding fragment thereof. In certain embodiments, the soluble ROBO1 ECD may comprise at least two immunoglobulin (Ig) domains of ROBO1, such as the first two Ig domains of ROBO1. In certain embodiments, the soluble ROBO1 ECD may comprise at least five immunoglobulin domains of ROBO1. In certain embodiments, the soluble ROBO1 ECD may be derived from the extracellular domain of mouse, bovine, ovine, caprine, or human ROBO1. In certain embodiments, the soluble ROBO1 ECD may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-27. In certain embodiments, the soluble ROBO1 ECD may comprise a sequence of any one of SEQ ID NOs: 1-27 having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 20) conservative amino acid substitutions thereto. In certain embodiments, the soluble ROBO1 ECD administered to a mammal may be derived from the sequence of the ROBO1 protein expressed by that mammal in order to reduce the immune response to the soluble ROBO1 ECD.

[0044] A soluble ROBO1 ECD that may contain the entire extracellular region of ROBO1 or its ROBO2-binding fragment can be identified by any means. For example, a soluble ROBO1 ECD effective to inhibit NOTCH4 activity can be identified by performing an assay to measure the binding of the soluble ROBO1 ECD to ROBO2. The assay can include determining whether the soluble ROBO1 ECD binds to ROBO2 in the presence of competition, such as a soluble ROBO1 ECD having the amino acid sequence set forth in either full-length ROBO1 or any one of SEQ ID NOs: 1-27. In certain embodiments, a soluble ROBO1 ECD effective to inhibit NOTCH4 activity can be identified by performing an assay to measure the binding of the soluble ROBO1 ECD to NOTCH4. The binding of the soluble ROBO1 ECD to ROBO2 and / or NOTCH4 can be measured by detecting the formation of ROBO1 ECD::ROBO2 and / or ROBO1 ECD::NOTCH4 complexes. Other methods for identifying the binding of the soluble ROBO1 ECD to ROBO2 and / or NOTCH4 can also be used.

[0045] In some embodiments, soluble ROBO1 is fused or linked to a heterologous polypeptide. In some embodiments, the heterologous polypeptide is linked to the amino terminus, carboxyl terminus, or both termini of the soluble ROBO1 ECD. As used herein, the term soluble, when used in the context of ROBO1 ECD, means that the ROBO1 ECD is not and cannot be localized to the cell surface because it lacks the transmembrane region required for cell surface localization. The soluble ROBO1 ECD also lacks the sequence of the intracellular region of ROBO1. In certain embodiments, the soluble ROBO1 ECD polypeptide can be fused to an immunoglobulin Fc polypeptide (e.g., a human IgG1 Fc such as IgG1 Fc), serum albumin (e.g., human serum albumin, cynomolgus serum albumin or bovine serum albumin), or maltose binding protein. In certain embodiments, the soluble ROBO1 ECD can be fused to a protein tag that facilitates polypeptide purification or tracking. Such protein tags include His tag, hemagglutinin tag, Fc region (derived from Ig from human, bovine, ovine, or caprine antibody, e.g., IgG, IgM, IgA, IgE, or IgD), or Myc tag.

[0046] In some embodiments, the first agent can be an anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity. As used herein, the term antibody includes its antigen-binding fragments unless the context clearly indicates otherwise. In some embodiments, the antibody comprises a plurality of polyclonal antibodies that bind to different epitopes on the antigen. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In certain embodiments, the antibody is modified to provide a reduction in immunogenicity in the mammal that receives the antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a bovineized antibody. In some embodiments, the antibody is a bovine antibody. In some embodiments, the antibody is a ovineized antibody. In some embodiments, the antibody is an ovine antibody. In some embodiments, the antibody is a camelized antibody. In some embodiments, the antibody is a camel antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is an antibody fragment that comprises at least one antigen-binding site. In some embodiments, the antibody is a scFv. In some embodiments, the antibody is a disulfide-bonded scFv. In some embodiments, the antibody is a Fab. In some embodiments, the antibody is a bispecific or multispecific antibody.

[0047] In some embodiments, the first agent is a polyclonal antibody that binds to NOTCH4. The polyclonal antibody can be prepared by any method known to those skilled in the art. In some embodiments, the polyclonal antibody is produced by immunizing an animal (e.g., cow, sheep, camel, rabbit, rat, mouse, goat, donkey) with the antigen of interest (e.g., a purified peptide fragment, a recombinant protein, or a fusion protein) using multiple subcutaneous or intraperitoneal injections. In some embodiments, the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. The antigen (with or without the carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., complete or incomplete Freund's adjuvant) to form a stable emulsion. After a certain period, the polyclonal antibody is recovered from the immunized animal (e.g., from blood or ascites). In some embodiments, the polyclonal antibody is purified from serum or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and / or dialysis.

[0048] In some embodiments, the first agent is a monoclonal antibody that binds to NOTCH4. The monoclonal antibody can be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using the hybridoma method known to those skilled in the art. A mouse, rat, rabbit, hamster, or other suitable host animal is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. Following immunization, the lymphocytes are isolated and fused, for example, using polyethylene glycol, with a suitable myeloma cell line. Hybridoma cells are selected using special media known in the art, and unfused lymphocytes and myeloma cells do not survive this selection process. Hybridomas that produce monoclonal antibodies specific for the selected antigen can be identified by various methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution or other techniques. Hybridomas can be grown either in in vitro culture using standard methods or in vivo as ascites tumors in animals. Monoclonal antibodies can be purified from the culture medium or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0049] In some embodiments, monoclonal antibodies are produced using recombinant DNA techniques known to those of skill in the art. For example, polynucleotides encoding the antibodies are isolated from mature B cells or hybridoma cells by, e.g., RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using standard techniques. Next, the isolated polynucleotides encoding the heavy and light chains are cloned into appropriate expression vectors that produce monoclonal antibodies when transfected into host cells that do not normally produce immunoglobulin proteins (e.g., E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, etc.).

[0050] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries that express the variable domains or CDRs of a desired species (e.g., bovine or human). Screening of phage libraries can be accomplished by a variety of techniques known in the art.

[0051] In some embodiments, monoclonal antibodies are modified by generating alternative antibodies using recombinant DNA techniques. In some embodiments, the constant domains of the light and heavy chains of a murine monoclonal antibody are replaced with the constant regions of a human antibody, ovine antibody, bovine antibody, caprine antibody, or camel antibody to generate a chimeric antibody. In some embodiments, the constant regions are cleaved or removed to generate a desired antibody fragment of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable regions is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0052] In some embodiments, the anti-NOTCH4 antibody is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, the humanized antibody comprises one or more amino acid residues introduced into its sequence from a non-human source. In some embodiments, humanization is performed by substituting one or more amino acids of the CDR sequences of a human antibody with the corresponding amino acids from a non-human antibody (e.g., a mouse antibody). In some embodiments, the humanized antibody is constructed by substituting all six CDRs of a human antibody with the corresponding amino acids from the CDRs of a non-human antibody (e.g., a mouse antibody).

[0053] The selection of the human heavy chain variable region and / or light chain variable region used to generate a humanized antibody can be made based on various factors and by various methods known in the art. In some embodiments, the "best fit" method is used, and the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to the sequence of the non-human (e.g., rodent) sequence is selected as the human variable region framework of the humanized antibody. In some embodiments, a particular variable region framework derived from the consensus sequence of all human antibodies of a particular subgroup of the light or heavy chain is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most abundant human subclass. In some embodiments, human germline genes are used as the source of the variable region framework sequence.

[0054] In some embodiments, the anti-NOTCH4 antibody is a human antibody. Human antibodies can be prepared using a variety of techniques known in the art. In some embodiments, the human antibody is generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, the human antibody is generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody against the target antigen can be generated and isolated. In some embodiments, the human antibody is selected from a phage library that expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from an immunoglobulin variable region gene repertoire from non-immunized donors. Techniques for the generation and use of antibody phage libraries are well known in the art. Once an antibody is identified, more affinity matured human antibodies can be generated using affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis. In some embodiments, the human antibody is produced in transgenic mice containing the human immunoglobulin locus. Upon immunization, these mice can produce a complete repertoire of human antibodies even in the absence of endogenous immunoglobulin production.

[0055] In certain embodiments, the antibody can be a bovineized antibody or a fully bovine antibody. Methods for producing a bovineized antibody from a non-bovine antibody can include forming a chimeric antibody that retains the CDRs from the non-bovine antibody, while other regions of the antibody can have one or more amino acid residues introduced from a bovine antibody and be replaced with the corresponding sequence from a bovine antibody. In certain embodiments, the non-bovine antibody can be bovineized by replacing the constant region with a constant region from a bovine antibody. In certain embodiments, the non-bovine antibody can be bovineized by replacing the constant region with a constant region from a bovine antibody and replacing the framework region with a framework region from a bovine antibody. In certain embodiments, a bovineized antibody can be generated by replacing the CDRs of a bovine antibody with CDRs from a non-bovine antibody. In some cases, the antibody can be a fully bovine antibody produced using a gene sequence encoding a bovine antibody. A fully bovine antibody can be produced in a bovine, a bovine cell line, a non-bovine cell line genetically modified to express a bovine antibody, or a transgenic non-bovine animal genetically modified to express a bovine antibody. Similar methods can be used to generate species-specific antibodies that reduce the immune response to the antibody when administered to a species. For example, ovine, caprine, and equine antibodies can be produced for the purpose of administering an antibody to sheep, goats, and horses, respectively.

[0056] The CDRs of an antibody are defined by those skilled in the art using various methods / systems. These systems and / or definitions have been developed and refined over the years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is commonly used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is the combination of Kabat and Chothia. Software programs (e.g., abYsis) are available for the analysis of antibody sequences and determination of CDRs and are known to those skilled in the art.

[0057] The specific CDR sequences defined herein are generally based on a combination of the Kabat and Chothia definitions (Exemplary system). However, it will be understood that references to one or more heavy chain CDRs and / or one or more light chain CDRs of a particular antibody encompass all CDR definitions known to those skilled in the art.

[0058] In some embodiments, the anti-NOTCH4 antibody comprises an antibody in which at least one or more constant regions are modified or deleted. In some embodiments, the antibody may comprise a modification of one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and / or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody comprises a plurality of human constant regions. In some embodiments, the modification of the constant region comprises an addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain is removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility normally provided by the absent constant region. In some embodiments, the modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, the modified antibody comprises a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0059] It is known in the art that the constant region of an antibody mediates several effector functions, and these effector functions can vary depending on the antibody isotype. For example, when the C1 component of complement binds to the Fc region of an IgG or IgM antibody (bound to an antigen), the complement system is activated. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates an inflammatory response and may be involved in autoimmune hypersensitivity. Further, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). There are many Fc receptors specific for various classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). When an antibody binds to an Fc receptor on the cell surface, many important and diverse biological responses are elicited, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell cytotoxicity or ADCC), release of inflammatory mediators, passage across the placenta, and regulation of immunoglobulin production.

[0060] In some embodiments, the anti-NOTCH4 antibody comprises a variant Fc region. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art. In some embodiments, the variant Fc region provides an altered effector function, which in turn affects the biological profile of the antibody. For example, in some embodiments, deletion or inactivation (by point mutation or other means) of the constant region reduces or eliminates Fc receptor binding of the modified antibody when the antibody circulates. In some embodiments, the constant region modification increases the serum half-life of the antibody. In some embodiments, the constant region modification decreases the serum half-life of the antibody. In some embodiments, the constant region modification decreases, reduces, or eliminates antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region having corresponding IgG2 or IgG4 residues can reduce effector functions (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions. In some embodiments, the antibody does not have ADCC activity and / or CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector functions (e.g., an "effectorless" antibody). In some embodiments, the constant region modification increases or enhances the effector function of the antibody. In some embodiments, the constant region modification increases or enhances the ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified by adding / substituting one or more amino acids to provide one or more cytotoxic agents, oligosaccharides, or carbohydrate attachment sites.

[0061] Modifications to the constant region of the antibodies described herein can be made using well-known biochemical or molecular engineering techniques. In some embodiments, antibody variants are prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesis of the desired antibody or polypeptide. Using this technology, it may be possible to disrupt the activity or effector function provided by a particular sequence or region while substantially maintaining the structure, binding activity, and other desired properties of the modified antibody.

[0062] The present disclosure further encompasses additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. One of ordinary skill in the art will understand that amino acid changes can result in changes to the post-translational processes of the antibody, such as changes in the number or location of glycosylation sites or changes in membrane anchoring properties. The mutation can be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared to the native antibody or polypeptide sequence. In some embodiments, the amino acid substitution is the result of substituting one amino acid with another amino acid having similar structural and / or chemical properties, such as substituting leucine with serine, for example, a conservative amino acid substitution. The variant antibodies or polypeptides described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0063] In some embodiments, the agents that inhibit NOTCH4 activity as described herein are chemically modified. In some embodiments, the soluble ROBO1 ECD and / or anti-NOTCH4 antibodies chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or conjugation to a cell ligand or other protein. Any of a number of chemical modifications can be carried out by known techniques.

[0064] In some embodiments, the method can include increasing milk production in a mammalian species, where the mammalian species includes humans, cows, sheep, goats, or camels, and the method can include administering to the mammalian species a soluble ROBO1 ECD derived from human ROBO1, bovine ROBO1, ovine ROBO1, caprine ROBO1, or camel ROBO1, respectively. In certain embodiments, the mammal is a female at a developmental stage suitable for milk production. For example, the mammal can be a human female with developed mammary glands. In certain embodiments, the mammal is a human female, dairy cow, heifer, ewe, or female camel. In certain embodiments, the mammal can be pregnant when an agent that inhibits NOTCH4 activity is administered to the mammal. In certain embodiments, the mammal can be an animal that has given birth prior to administration of the agent that inhibits NOTCH4 activity. For example, the mammal can be an animal that gave birth within 1 to 2 years of administration, such as within 3 months, 6 months, 1 year, or 18 months. In other embodiments, the mammal is not pregnant. In some embodiments, the mammal has not given birth prior to administration of the agent that inhibits NOTCH4 activity. For example, the mammal has not given birth within 1 to 2 years after administration, such as within 3 months, 6 months, 1 year, or 18 months.

[0065] In some embodiments, an agent that inhibits NOTCH4 activity as described herein can be an RNAi construct that binds to NOTCH4 mRNA and reduces the expression of NOTCH4. In some embodiments, an agent that inhibits ROBO2 activity as described herein can be an RNAi construct that binds to ROBO2 mRNA and reduces the expression of ROBO2. The RNAi construct can be a short interfering RNA (siRNA). The siRNA can be a short hairpin RNA (shRNA). The RNAi construct can be a microRNA (miRNA). Methods for making RNAi constructs for inhibiting the expression of known gene sequences are known to those of skill in the art. In certain embodiments, the siRNA for reducing the expression of NOTCH4 can comprise the nucleic acid sequence set forth in SEQ ID NO: 32 or 33. In certain embodiments, the siRNA for reducing the expression of ROBO2 can comprise the nucleic acid sequence set forth in SEQ ID NO: 34 or 35. In certain embodiments, the RNAi construct can be administered to a mammal. In other embodiments, the nucleic acid

[0066] In certain embodiments, a method for promoting milk production in a mammal can include administering one or more agents that inhibit NOTCH4 activity. In certain embodiments, the method can include administering at least one of a first agent and a second agent, where the first agent and the second agent are independently selected from a soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits expression of NOTCH4, or an RNAi construct that inhibits expression of ROBO2. In certain embodiments, the method can include administering at least one of a first agent, a second agent, and a third agent, where the first agent, the second agent, and the third agent are independently selected from a soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits expression of NOTCH4, or an RNAi construct that inhibits expression of ROBO2. In certain embodiments, the method can include administering a first agent, a second agent, a third agent, and a fourth agent, where the first agent, the second agent, the third agent, and the fourth agent are independently selected from a soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits expression of NOTCH4, or an RNAi construct that inhibits expression of ROBO2.

[0067] One or more agents that inhibit NOTCH4 activity can be administered to a mammal to promote milk production via any suitable route, including parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intra-articular, intraperitoneal, intracerebral (e.g., parenchymal) and intraventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., subcutaneous), sublingual and inhalation. In certain embodiments, one or more agents can be administered by direct injection, e.g., injection into mammary tissue, e.g., intraductal injection.

[0068] Agents and Compositions for Inhibiting NOTCH4 Activity Also provided herein are agents and compositions thereof that can be used to practice the methods disclosed herein.

[0069] In certain aspects, polypeptides are provided that include a soluble ROBO1 ECD polypeptide as disclosed herein. The soluble ROBO1 ECD polypeptide can be fused to a heterologous polypeptide as disclosed herein. In certain aspects, nucleic acids are provided that encode a soluble ROBO1 ECD polypeptide as disclosed herein. Descriptions of the soluble ROBO1 ECD polypeptide are provided in the previous section and elsewhere in this specification and are not repeated here for the sake of brevity. The soluble ROBO1 ECD can be produced using methods known in the art. The polypeptide can be produced in whole or in part using standard recombinant DNA techniques or chemical methods. Chemical methods for synthesizing polypeptides can include using various solid-phase techniques that can be performed using an automated synthesizer (e.g., Biotage equipment). Chemical methods for synthesizing polypeptides can include using combinatorial methodologies. Further, the polypeptide can be modified by a variety of chemical methods known to those of skill in the art. Mutations, substitutions, and / or modifications of the polypeptide sequence can be made using methods such as site-directed mutagenesis, alanine scanning, and / or PCR-based mutagenesis. Site-directed mutagenesis, cassette mutagenesis, restriction-selection mutagenesis, and other techniques can be performed on cloned DNA to produce soluble ROBO1 ECD, variants, fusions, chimeras, and other derivatives thereof. A "produced" or "synthesized" polypeptide sequence is a polypeptide made by any method involving human manipulation. Such methods include, but are not limited to, chemical synthesis, recombinant DNA techniques, biochemical or enzymatic fragmentation of larger molecules, and combinations of the foregoing.

[0070] When a polypeptide, such as a soluble ROBO1 ECD polypeptide, is produced using recombinant techniques, the polypeptide can be produced as an intracellular protein or a secreted protein using any suitable construct and any suitable host cell, which can be a prokaryotic cell or a eukaryotic cell, such as a bacterial (e.g., E. coli) or yeast host cell, respectively. In certain embodiments, eukaryotic cells used as host cells for the production of the polypeptide include insect cells, mammalian cells, and / or plant cells. In certain embodiments, mammalian host cells are used, including human cells (e.g., HeLa, 293, H9, and Jurkat cells), mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos1, Cos7, and CV1) as well as hamster cells (e.g., Chinese hamster ovary (CHO) cells). In certain embodiments, the polypeptides disclosed herein are produced in CHO cells or HEK cells. In certain embodiments, the polypeptides of the present disclosure, such as soluble ROBO1 ECD, are produced in cells cultured in the presence of heparin. For example, about 300 ng / ml of heparin can be included in the culture medium. In other embodiments, the polypeptides of the present disclosure, such as soluble ROBO1 ECD, are produced in cells cultured in a culture medium that does not contain a significant amount of heparin, for example, the culture medium can contain 300 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 10 ng / ml, or less than 1 ng / ml of heparin, or can be heparin-free.

[0071] A variety of host-vector systems suitable for the expression of polypeptides can be used according to standard procedures known in the art. See, for example, Sambrook et al., 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York, and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introducing genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate method, and the like. A method for transfer can be selected so as to provide stable expression of the nucleic acid encoding the introduced polypeptide. The nucleic acid encoding the polypeptide can be provided as an episomal element (e.g., plasmid) or integrated into the genome. A variety of suitable vectors for use in the production of polypeptides of interest are commercially available.

[0072] The vector can provide for extrachromosomal maintenance in the host cell or integration into the host cell genome. Expression vectors provide transcriptional and translational regulatory sequences and can provide inducible or constitutive expression, with the coding region being operably linked under the transcriptional control of a transcription initiation region and under transcriptional and translational termination regions. Generally, the transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. The promoter can be either constitutive or inducible and can be a strong constitutive promoter (e.g., T7).

[0073] Also provided herein are nucleic acids encoding the polypeptides disclosed herein. In certain embodiments, the nucleic acids encoding the polypeptides disclosed herein are operably linked to a promoter sequence that confers expression of the polypeptide. In certain embodiments, the sequence of the nucleic acid is codon-optimized for expression of the polypeptide in mammalian cells. In certain embodiments, the nucleic acid is deoxyribonucleic acid (DNA). In certain embodiments, the nucleic acid is ribonucleic acid (RNA). Also provided herein are vectors comprising nucleic acids encoding polypeptides for promoting milk production as described herein. In certain embodiments, the vector is a viral vector.

[0074] In certain embodiments, anti-NOTCH4 antibodies as disclosed herein are provided. Descriptions of anti-NOTCH4 antibodies are provided in the previous section and elsewhere in this specification and are not repeated here for the sake of brevity. Anti-NOTCH4 antibodies for inhibiting NOTCH4 activity can be identified by using any suitable means such as the assays and / or cell and animal models disclosed herein.

[0075] In certain embodiments, RNAi constructs for inhibiting the expression of NOTCH4 or RNAi constructs for inhibiting the expression of ROBO2 as disclosed herein are provided. Descriptions of such RNAi constructs are provided in the previous section and elsewhere in this specification and are not repeated here for the sake of brevity. RNAi constructs for inhibiting NOTCH4 activity can be identified by using any suitable means such as the assays and / or cell and animal models disclosed herein.

[0076] Also disclosed herein is a pharmaceutical composition comprising one or more inhibitors of NOTCH4 activity as disclosed herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to substances that are approved or approvable by a regulatory agency or are listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeias for use in animals including humans.

[0077] As used herein, the term "pharmaceutically acceptable excipient, carrier, or adjuvant" or "acceptable pharmaceutical carrier" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one agent and that has no effect on the pharmacological activity of the agent. In general, those skilled in the art and the U.S. FDA consider pharmaceutically acceptable excipients, carriers, or adjuvants to be inert components of the formulation.

[0078] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form that enables the biological activity of an agent (e.g., an antibody) to be effective. Pharmaceutical formulations or compositions generally include additional components such as pharmaceutically acceptable excipients, carriers, adjuvants, buffers, etc.

[0079] In certain embodiments, the polypeptide and nucleic acid (e.g., encoding a polypeptide or RNAi) are present in a therapeutically effective amount in the pharmaceutical composition. The therapeutically effective amount can be determined based on the observed efficacy of the composition. The therapeutically effective amount can be determined using an assay that measures the desired effect in cells, e.g., in a reporter cell line in which reporter expression is regulated in response to a polypeptide of the present disclosure. The pharmaceutical composition can be administered to a mammal ex vivo or in vivo for practicing the methods and uses described herein.

[0080] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration, and exemplary routes of administration are described herein. Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, appropriate vehicles such as nuclease inhibitors, protease inhibitors, saline or citrate buffered saline.

[0081] Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base such as hydrochloric acid and sodium hydroxide. Parenteral formulations may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0082] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In the case of intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).

[0083] Sterile injectable solutions can be prepared by incorporating the active compound in the required amounts into a suitable solvent containing one or a combination of the ingredients enumerated above, followed by filter sterilization, as required. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any desired additional ingredients from its previously sterile filtered solution.

[0084] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be used in association with an excipient in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow promoters such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor. Formulations for oral delivery can advantageously incorporate agents for improving stability within the gastrointestinal tract and / or enhancing absorption.

[0085] For administration by inhalation, the composition is formulated with a suitable propellant (e.g., a gas such as carbon dioxide) in a pressurized container or dispenser, or with a delivery agent for delivery in the form of an aerosol spray from a nebulizer.

[0086] Systemic administration can also be by transmucosal or transdermal means. In the case of transmucosal or transdermal administration, a penetration enhancer suitable for the permeation barrier is used in the formulation. Such penetration enhancers are generally known in the art and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be effected by using a spray nasal preparation or a suppository. In the case of transdermal administration, the active compound and the delivery agent are formulated into an ointment, salve, gel, or cream as is generally known in the art. The composition can also be prepared in the form of a suppository (using conventional suppository bases such as cocoa butter and other glycerides) or a retention enema for rectal administration.

[0087] In one aspect, the composition is prepared using a carrier that protects the compound against rapid elimination from the body, such as a controlled release formulation including, for example, implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells together with monoclonal antibodies against viral antigens) can also be used as a pharmaceutically acceptable carrier.

[0088] Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to physically discrete units suitable as a single dosage for the intended subject, each unit containing a predetermined quantity of the active compound calculated to produce the desired effect in association with the required pharmaceutical carrier.

[0089] As described above, a nucleic acid molecule that functions as a template for the transcription of siRNA or shRNA can be inserted into a vector that can be used as a gene therapy vector. A nucleic acid molecule encoding a soluble ROBO1 ECD can also be inserted into a vector that can be used as a gene therapy vector. Generally, a gene therapy vector can be delivered to a subject by, for example, intravenous injection, local administration, or stereotactic injection. In certain embodiments, a composition comprising a gene therapy vector and a delivery agent can be delivered orally or by inhalation, can be encapsulated to protect them from degradation, etc., or can be otherwise manipulated. A pharmaceutical composition comprising a gene therapy vector can comprise an acceptable diluent or can comprise a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral or lentiviral vectors), the pharmaceutical preparation can comprise one or more cells that produce that gene delivery system.

[0090] A pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.

[0091] Transgenic mammal In certain embodiments, transgenic mammals are provided that comprise a genetic modification that results in one or more of the following phenotypes: expression of a soluble ROBO1 extracellular domain; inhibition of ROBO2 expression; and inhibition of NOTCH4 expression. In certain embodiments, the transgenic mammal can be a mouse, cow, sheep, goat, or camel.

[0092] In certain embodiments, the phenotype is limited to mammary tissue. In certain embodiments, the phenotype is limited to mammary tissue by using a mammary tissue-specific promoter to induce expression of the phenotype.

[0093] In certain embodiments, a transgenic mammal can comprise two genetic modifications that result in two of the described phenotypes. In certain embodiments, a transgenic mammal can comprise three genetic modifications that result in all three of the described phenotypes.

[0094] In certain embodiments, a method for promoting milk production, as disclosed herein, can comprise administering to a transgenic mammal at least one pharmaceutical composition that inhibits NOTCH4 activity, as disclosed herein.

[0095] In certain embodiments, the transgenic animal can comprise a genetic modification that results in the expression of the soluble ROBO1 extracellular domain, and the method can further comprise administering to the transgenic animal a pharmaceutical composition comprising an anti-ROBO1 antibody, and an anti-NOTCH4 antibody, or an RNAi construct that inhibits the expression of ROBO2 and / or NOTCH4.

[0096] In certain embodiments, the transgenic animal can comprise a genetic modification that results in the inhibition of the expression of ROBO2 and / or NOTCH4, and the method can further comprise administering to the transgenic animal a pharmaceutical composition comprising a soluble ROBO1 ECD as disclosed herein.

[0097] Transgenic mammals can be produced using methods known in the art. Exemplary methods for producing transgenic mammals can include the following steps: 1) Producing a gene construct comprising a nucleic acid encoding soluble ROBO1 ECD, or a nucleic acid sequence transcribed into an siRNA or shRNA targeting NOTCH4 or ROBO2, under the control of a promoter. The promoter can be a mammary gland-specific promoter or a ubiquitously active promoter. 2) Transfecting the gene construct into cells from a mammal, such as bovine cells, and selecting transgenic cells incorporating the gene construct. 3) Fusing the transgenic cells with enucleated oocytes from the same species as the transgenic cells (e.g., bovine) (e.g., by applying an electric pulse), and developing the oocytes into embryos. 4) Transferring the embryos into a recipient mammal of the same species as the embryos (e.g., bovine). 5) Confirming that the embryos have developed into transgenic mammals.

Example

[0098] Experiment ROBO1 is expressed in both the luminal compartment and the basal compartment and is upregulated during pregnancy. Previously published studies have focused on the role of SLIT / ROBO1 signaling during the period of branching morphogenesis in virgin animals 11、15、16 . To investigate the role of ROBO1 during pregnancy, Robo1 mRNA levels in cells isolated from the mammary gland were measured using RT qPCR (Figure 1C). Cells were harvested from adult virgin and wild-type (WT) mice on day 18 of pregnancy (PD18) (as shown in Figure 1C) and purified by fluorescence-activated cell sorting (FACS) into three subpopulations: luminal progenitor cells (LP, Lin-CD24 lo CD29 + CD61 + ), mature luminal (ML, Lin-CD24 lo CD29 + CD61 - ), and basal (BC, Lin-CD24 +CD29 hi ) 17、18 . The results show upregulation of Robo1 in both luminal progenitors and mature lumens, but not in the basal subpopulation (Figure 1C).

[0099] To evaluate the expression of ROBO1 and ROBO2 proteins in tissues, immunohistochemistry (Figure 1D) and β-galactosidase (lacZ) staining (Figure 1E) were performed on WT and Robo1 lacZ / + tissue sections of virgin mammary glands. Immunohistochemistry and β-gal staining were also performed on mammary gland sections at gestational day 16 (PD16) (Figure 1F) and lactation day 3 (LD3) (Figure 1G). ROBO1 protein is expressed in a subpopulation of luminal cells in the mammary glands of mature virgin and pregnant females (arrows in Figures 1D - F). Basal myoepithelial ROBO1 expression is also observed in pregnant and lactating mammary glands during pregnancy (arrows in Figures 1F, G).

[0100] ROBO1 promotes alveologenesis: To investigate ROBO1 function during alveologenesis, Robo1 gene expression was inhibited in HC11 cells (Robo1 KD). HC11 cells are a well - established prolactin - responsive model of lactation 19、20Cells in which Robo1 gene expression was not inhibited are herein called WT or Robo1+ / +. To measure milk production, cells were grown to confluence and then primed by treatment with epidermal growth factor (EGF, 10 ng / ml). EGF was administered for 3 days in combination with charcoal-stripped fetal bovine serum, followed by administration of charcoal-stripped fetal bovine serum for 1 day in the absence of EGF. Next, these primed cells were differentiated by treatment with dexamethasone (1 μg / ml), insulin (5 μg / ml), and prolactin (Prl, 5 μg / ml) medium (DIP medium) for 3 - 5 days (Figure 2A). Differentiation (Dif) resulted in the development of milk-filled domes (Figure 2B). In response to treatment with DIP medium, statistically significantly fewer milk domes were formed and statistically significantly less whey acidic protein (WAP) gene expression was observed (Figure 2B). When the cells were left undifferentiated (Undif), there was little dome formation in either WT cells or Robo1- / - cells (Figure 2B). Next, tissues from Robo1 knockout mice (Robo1- / -) and wild-type mice (WT or Robo1+ / +) were analyzed. Mammary glands were collected from WT and Robo1- / - animals on day 18 of pregnancy and alveologenesis was analyzed by quantifying the area occupied by alveoli in serial sections, carmine staining, and sections located in the upper, middle, and lower parts of the tissue. This analysis revealed that the alveolar area in Robo1- / - was significantly reduced compared to WT mammary glands (Figure 2C).

[0101] To confirm that this defect is due to inhibition of Robo1 in the mammary epithelium and not due to its systemic deletion that may affect hormone production 21 , tissues from Robo1- / - and littermate Robo1+ / + mice were processed using standard protocols 22It was transplanted contralaterally into a host from which endogenous mammary epithelium had been previously removed. Ten weeks later, the animals were mated and tissues were examined on the 18th day of pregnancy. In the transplanted Robo1- / -KO mammary glands, the alveolar area was significantly reduced (Figure 2D), and the same results as those observed in the mammary glands of intact Robo1- / - animals were obtained (Figure 2C). To evaluate the expression of specific markers regulated by pregnancy, Robo1- / - and Robo1+ / + tissues were collected on the first day of lactation, RNA was extracted, and RT-qPCR was performed for genes known to be involved in milk production. Significantly lower expression of WAP, lactalbumin alpha (Lalba), xanthine dehydrogenase (XDH), and butyrophilin (Btn1) was observed in Robo1- / - tissues (Figure 2E).

[0102] The selected markers were further evaluated using immunohistochemistry. WAP expression in the mammary glands of Robo1- / - and WT on the 18th day of pregnancy showed less WAP immunostaining in Robo1- / - tissues (Figure 2F). The transplanted tissues on the 16th day of pregnancy were immunostained with an antibody specific for the lipid-binding protein perilipin 2 (PLIN2). Little PLIN2 immunostaining was observed in Robo1- / - mammary tissues (Figure 2G).

[0103] Furthermore, clearing of whole-organ tissues was used to optimize tissue optical transparency and morphological preservation. Subsequently, double immunohistochemistry was performed using an antibody specific for the transcription factor ELF5 required for alveologenesis 6 and an antibody specific for the cell adhesion protein E-cadherin (CDH1) (Figure 2H). Significantly less ELF5 staining was observed in Robo1- / - compared to Robo1+ / + tissues (Figure 2H).

[0104] Loss of Robo1 impairs milk production in vivo: To evaluate the effect of Robo1 expression on milk production, matings were performed to generate heterozygous offspring that were nursed by either Robo1− / − or WT dams. Heterozygous offspring were generated by mating WT males with Robo1− / − females and Robo1− / − males with WT females. The litter size was limited to five, and the weights of these offspring were measured daily (Figure 2I). Heterozygous offspring nursed by WT dams gained weight linearly, whereas those nursed by Robo1− / − dams had less weight gain (Figure 2J).

[0105] ROBO1 interacts with and inhibits NOTCH4 signaling: Notch signaling is highly sensitive to dosage, and its outcome depends on the level of receptor activity 23 . After ligand binding, the Notch receptor is activated by cleavage. First, there is extracellular cleavage, followed by intracellular cleavage via γ-secretase, releasing the Notch intracellular domain (ICD), which enters the nucleus and regulates transcription. RNA sequencing analysis of FACS-purified subpopulations isolated from virgin mammary glands revealed higher expression of the Notch signaling effector Hey1 in Robo1− / − luminal progenitor (LP) subpopulations compared with Robo1+ / + (Figure 3A).

[0106] Luminal progenitor cells purified by FACS using the Sca / CD54 marker were enriched for alveolar progenitor cells (AVPs). Similar to the data from bulk luminal progenitor cells (Figure 3A), RT-qPCR analysis of alveolar progenitor cells (AVPs) revealed significantly higher expression of three downstream Notch effectors (Hey1, Hes1, and Hey2) in Robo1− / − compared to Robo1+ / + (Figure 3B). Similarly, in HC11 cells, significantly higher expression of Hey1, Hes1, and Hey2 was observed after inhibition of Robo1 expression compared to WT cells (Figure 3C). Inhibition of Robo1 expression in HC11 cells also resulted in significantly lower expression of the differentiation-promoting marker Elf5 compared to WT (Figure 3C). These data indicate that inhibition of Robo1 expression in both primary cells and tissue culture cells results in upregulation of Notch effector genes and downregulation of the differentiation-promoting Elf5 gene, further suggesting activation of Notch signaling in the absence of ROBO1.

[0107] Previous studies have shown that alveologenesis requires downregulation of Notch signaling 6 , specifically, NOTCH4 7~9 . Alveolar progenitor cells (AVPs) were FACS purified from virgin (Virg) and day 18 of pregnancy (PD18) animals, and the expression of the Notch4 target genes Hes1 and Hey1 was examined by RT-qPCR (Figure 3D). It was observed that both Notch target genes were significantly downregulated in alveolar progenitor cells isolated from the glands of pregnant animals compared to those isolated from the glands of virgin animals.

[0108] The effect of Notch in the HC11 cell differentiation assay was also evaluated. Inhibition of Robo1 expression (KD) resulted in a significant decrease in the formation of HC11 milk domes compared to the control (Scr) (Figure 3E). HC11 cells with inhibited Robo1 expression (siR1) were shown to have less expression of WAP and Lalba compared to the control (Scr) (Figure 3F). Both of these effects were rescued by treating the cells with a γ-secretase inhibitor (GSI, RO4929097) (siR1+GSI) (Figure 3E, F). This γ-secretase inhibitor acts to prevent Notch signaling, further supporting the concept that loss of Robo1 enhances Notch4 signaling and results in an effect that can be rescued by γ-secretase treatment.

[0109] In additional experiments, Notch4 expression was inhibited in HC11 cells (siN4). These cells showed greater WAP and Lalba expression compared to control cells (Scr). In further experiments, the expression of both Robo1 and Notch4 was inhibited in HC11 cells (dKD), and the expression of WAP and Lalba increased compared to control cells (Scr), similar to the expression levels of WAP and Lalba observed with Robo1 inhibition plus GSI treatment (siR1+GSI) (Figure 3F). Notch4 knockdown resulted in a significantly higher number of domes compared to control cells (Scr) (Figure 3G), which was consistent with greater expression of milk genes (Figure 3F). These data support a model in which NOTCH4 inhibits alveologenesis. Inhibition of Robo1 expression (siR1) led to a substantial decrease in the number of milk domes formed compared to control cells (Scr) (Figure 3G), but simultaneous inhibition of the expression of both Robo1 and Notch4 (dKD) led to the formation of more milk domes, which was the same effect observed with inhibition of Notch4 expression alone (siN4) (Figure 3G). Collectively, these data suggest that ROBO1 and NOTCH4 function in the same pathway to regulate alveologenesis by ROBO1 inhibiting NOTCH4, which in turn inhibits alveologenesis.

[0110] Activation of Notch receptors can be regulated through direct interaction with binding partners 24As a result, ROBO1 may bind to NOTCH4 and directly inhibit the cleavage and activation of NOTCH4. To investigate this possibility, co-immunoprecipitation experiments were performed using MBA-MD-231 cell lysates that express all detectable levels of the four Notch receptors (NOTCH1-4). Endogenous ROBO1 co-immunoprecipitated with NOTCH4, but not with NOTCH1, NOTCH2, or NOTCH3 (Figure 3H, data not shown). Next, the expression and intracellular localization of the NOTCH4 intracellular domain (N4-ICD) and HES1 were examined in control (Scr) and Robo1 (siR1) knockdown HC11 cells. The expression of Robo1 was inhibited in HC11 cells, and then the HC11 cells were primed for differentiation as described above. Robo1 knockdown cells showed significantly higher expression of the nuclear NOTCH4 intracellular domain (N4-ICD) and HES1 compared to control cells (Scr). This effect was not observed in control Robo1 knockdown cells engineered to overexpress Robo1 (siR1+o / e) or cells treated with the γ-secretase inhibitor GSI (siR1+GSI) (Figures 3I-3K).

[0111] Additional studies addressed how the formation of the ROBO1 / NOTCH4 complex is regulated over time during HC11 differentiation. Expression analysis was performed during the stages of HC11 differentiation (confluence, priming, milk dome formation). 19、20 Analysis of the intracellular domains of ROBO1, pSTAT5, and NOTCH4 by Western blot over this time course revealed that the levels of ROBO1 (R1) and pSTAT5 were high during the milk dome formation stage compared to other stages. In contrast, the NOTCH4 intracellular domain (N4-ICD) was expressed at low levels during the milk dome formation stage (Figure 3L). This finding is consistent with previous studies showing that NOTCH4 signaling attenuates during alveologenesis. 7~9Co-immunoprecipitation with anti-ROBO1 was used to pull down NOTCH4 in primed cells and differentiated (Dif) HC11 cells at the early (+EGF) and late (-EGF) stages, both in the absence and presence of SLIT2 and SLIT3 (Figure 3M). ROBO1 / NOTCH4 complex formation does not seem to be affected by SLIT2 / SLIT3 treatment in differentiated (Dif) HC11 cells. However, less ROBO1 / NOTCH4 complex formation was observed in the presence of SLIT2 and SLIT3 in late-primed cells (-EGF) compared to untreated cells. The ROBO1 / NOTCH4 complex was not detected in early-primed cells (+EGF) nor in control IgG immunoprecipitates. Taken together, these data suggest that ROBO1 directly binds and inhibits the cleavage and signaling of NOTCH4 during alveologenesis, interfering with the differentiation of mammary epithelial cells into milk-producing cells.

[0112] ROBO1 inhibits Notch signaling in primary cells and mammals: Deletion of Robo1 enhanced NOTCH4 signaling and inhibited the differentiation of HC11 cells, so this process was further evaluated in primary cells and animals. Alveolar progenitor cells were FACS-purified and seeded at single-cell density in Matrigel, and then grown for 5 days in medium supplemented with neuregulin (100 ng / ml) and R-spondin (42.5 ng / ml). Next, the cells were switched to DIP medium and differentiated for an additional 5 days. 25。Colonies grown from Robo1− / − alveolar progenitor cells were observed to be smaller than colonies grown from WT alveolar progenitor cells, and Robo1− / − colonies did not produce WAP (Figure 4A). Immunostaining was performed on cultured WT and Robo1− / − primary luminal cells. Significantly higher levels of the NOTCH4 intracellular domain (N4-ICD) were detected in the nuclei of Robo1− / − primary cells compared with WT cells (Figure 4B). These studies indicate that Robo1− / − alveolar progenitor cells (AVPs) contain high levels of the NOTCH4 intracellular domain in the nucleus and do not generate milk-producing organoids like their WT counterparts. This finding is consistent with the alveologenesis defect observed in Robo1− / − mammary glands (Figure 2).

[0113] In further studies, Notch signaling was inhibited in an attempt to reverse the Robo1− / − phenotype. The mammary 29 gland, along with several other different organs 26~28 in which γ-secretase inhibitors had been successfully used previously in vivo to inhibit Notch were selected. Virgin animals at maturity were treated with 10 mg / kg of GSI or vehicle control 29 for 7 days (Figure 4C). After treatment, mammary glands were harvested and analyzed by FACS and qPCR. Robo1− / − mammary glands were observed to contain more alveolar progenitor cells compared with WT controls (Figure 4D). When Robo1− / − animals were treated with a gamma-secretase inhibitor, these animals had the same number of alveolar progenitor cells as WT animals (Figure 4D). GSI treatment did not affect the number of alveolar progenitor cells in WT animals (Figure 4D).

[0114] Investigation of the expression of Notch effector genes (Hey1 and Hes1) revealed that the expression of Notch effectors was lower in animals treated with GSI compared to animals treated with vehicle (Figure 4E). Although the GSI inhibitor does not specifically target the Notch receptor, this result indicates that the drug acts in the mammary gland to reduce Notch signaling. Robo1− / − alveolar progenitor cells (AVPs) expressed higher levels of Hey1 and Hes1 when treated with vehicle compared to AVPs of WT animals treated with vehicle (Figure 4E). This result is similar to that seen in primary alveolar progenitor cells and HC11 cells (Figure 3B, C). Robo1− / − alveolar progenitor cells (AVPs) treated with vehicle expressed lower levels of Elf5 compared to AVPs of WT animals treated with vehicle (Figure 4E). This is consistent with the observation of lower levels of ELF5 expression in Robo1− / − compared to Robo1+ / + mammary glands (Figure 2H) and lower levels of Elf5 observed upon Robo1 knockdown compared to control (Scr) HC11 cells (Figure 3C). Further observations showed that treating Robo1− / − animals with GSI reversed the altered AVP gene expression compared to Robo1− / − animals treated with vehicle, with the expression of Hey1 and Hes1 being lower in AVPs from Robo1− / − animals treated with GSI compared to Robo1− / − animals treated with vehicle, while Elf5 expression was higher in AVPs from Robo1− / − animals treated with GSI compared to Robo1− / − animals treated with vehicle (Figure 4E). Collectively, this study indicates that ROBO1 restricts NOTCH4 signaling. In the absence of Robo1, NOTCH4 is activated, and this effect is reversed by either pharmacological inhibition of Notch signaling (Figure 4E, 3E, 3F, 3I - K) or knockdown of Notch4 gene expression (Figure 3F, G).

[0115] ROBO2 inhibits alveologenesis: Inhibition of Robo2 in animals and cells resulted in phenotypes opposite to those resulting from inhibition of Robo1 expression. In HC11 cells, inhibition of Robo2 expression (Robo2 KD) led to faster differentiation, higher WAP expression, and more milk domes compared to control cells (Scr). Inhibition of Robo1 and Robo2 in the same cells made the number of milk domes indistinguishable from the negative control (Figure 5A).

[0116] Alveologenesis was evaluated in both intact Robo2− / − mammary glands and contralaterally transplanted Robo2− / − derivatives. When measured by alveolar area, significantly faster alveologenesis was observed in both intact Robo2− / − mammary glands and Robo2− / − grafts compared to Robo2+ / + control mammary glands (Figure 5B). Expression of milk genes in intact virgin Robo2− / − mammary glands (MG) was higher than in Robo2+ / + controls, while expression of Notch effector genes (Hey1, Hes1, and Hey2) in FACS-purified alveolar progenitor cells (AVP) from Robo2− / − intact mammary glands was lower than in Robo2+ / + controls (Figure 5C).

[0117] Expression of Robo2 was evaluated by RT-qPCR in FACS-purified subpopulations from virgin mammary epithelial cells. Unlike Robo1, which is expressed in all subpopulations, expression of Robo2 is more restricted, being expressed at high levels in alveolar progenitor cells (AVP) and at lower levels in basal cells (BC) (Figure 5D). Expression in ductal progenitor cells (DP) was indistinguishable from that in mature luminal cells (ML); expression in ML was used for normalization. Expression of Robo2 in tissue was examined by β-galactosidase (lacZ) staining of mammary sections of the gland. Expression of Robo2 was observed in a subpopulation of luminal cells of alveoli on day 18 of pregnancy (Figure 5E, top), and in a subpopulation of cells located basally along ducts of reproductively senescent breeder animals (Figure 5E, bottom). lacZ / + Expression of Robo2 was examined by β-galactosidase (lacZ) staining of mammary sections of the gland. Expression of Robo2 was observed in a subpopulation of luminal cells of alveoli on day 18 of pregnancy (Figure 5E, top), and in a subpopulation of cells located basally along ducts of reproductively senescent breeder animals (Figure 5E, bottom).

[0118] One interpretation of these phenotypic and expression data is that ROBO2 inhibits ROBO1 in alveolar progenitor cells. During differentiation, ROBO2 is downregulated, releasing ROBO1, which inhibits NOTCH4, creating a derepression circuit (ROBO2―|ROBO1―|NOTCH4) (Figure 5F, left). In other words, by inhibiting the expression of Robo2, ROBO1 can promote alveolar differentiation. Inhibiting the expression of Robo1 allows NOTCH4 to inhibit alveolar differentiation.

[0119] The interaction between ROBO1 and ROBO2 is enhanced by SLIT. The interaction between SLIT proteins and ROBO proteins is evolutionarily conserved, as shown by studies indicating that human SLIT2 binds to Drosophila Robo1 with an affinity similar to that of mammalian receptors, and conversely, Drosophila Slit binds to rat ROBO1 and ROBO2. 30 Biochemical studies have shown that the interaction between this receptor / ligand pair involves the highly conserved second LRR domain of Slit and the conserved Ig1 domain of Robo, while the Ig2 - Ig5 domains and all FN3 domains of ROBO1 are not seen to be essential for binding. 31~34 Furthermore, studies have shown that ROBO1 and ROBO2 can bind to each other both in cis 32、35、36 and in trans. 37 This interaction also depends on the Ig domain. Recent crystallography experiments have shown that ligand - unbound ROBO forms a compact homodimer that opens in response to SLIT, enabling the formation of a "dimer of dimers" between ROBOs. 38

[0120] The disclosed model shows that ROBO2 inhibits ROBO1. To examine whether this inhibition is due to direct interaction, co-immunoprecipitation experiments were performed on endogenous proteins in HEK cells using a ROBO1 antibody. A band (seen as the glycosylated form) bound by the ROBO2 antibody co-immunoprecipitated with ROBO1. When immunoprecipitation was performed using cells in which Robo1 expression was inhibited, the intensity of this band decreased (Figure 5G). When cells were treated with SLIT2 and SLIT3 (1 μg each) for 4 hours before lysate preparation and co-immunoprecipitation using an anti-ROBO1 antibody, two strongly stained bands for ROBO2 were observed. This suggests that SLIT2 and SLIT3 promote a more efficient interaction between ROBO1 and ROBO2 (Figure 5G).

[0121] The ROBO1 receptor extracellular domain fragment binds to ROBO2: The experiments disclosed herein suggest that ROBO1 and NOTCH4 form a complex that inhibits the activation of NOTCH4, suggesting a direct interaction between the two proteins. Previous studies have shown that soluble extracellular domain fragments of many transmembrane receptors act to block both homophilic and heterophilic interactions between transmembrane receptors, as well as interactions between transmembrane receptors and their ligands 39 . It can be hypothesized that the soluble ROBO1 extracellular domain (ECD) may similarly interfere with the interaction between ROBO1 and ROBO2. A construct containing ROBO1 ECD competes with endogenous ROBO1 for binding to ROBO2, thereby enabling endogenous ROBO1 to bind to NOTCH4 and inhibit the activation of NOTCH4, thereby promoting alveolar differentiation and promoting milk production (right panel of Figure 5F). Soluble ROBO1 ECD can also directly bind to NOTCH4 and inhibit NOTCH4 activation in a non-mutually exclusive manner, which also results in promoting alveolar differentiation (right panel of Figure 5F). Thus, ROBO1 ECD can directly and indirectly inhibit the activation of NOTCH4.

[0122] Three recombinant ROBO1 ECD constructs were generated: one containing two immunoglobulin (Ig) domains (ROBO1-Ig2), another containing all five Ig domains (ROBO1-Ig5), and another containing the entire extracellular domain (ROBO1-Ecto) (Figure 6A). In other constructs, HA (hemagglutinin), Myc, and human and mouse immunoglobulin Fc were fused to the Robo1 ECD (Figure 6A, right panel of 5F). The extracellular domain of Deleted in Colorectal Cancer (DCC), an Ig superfamily member structurally similar to ROBO1, containing either two Ig (DCC-Ig2) or four Ig (DCC-Ig4) domains and tagged with HA, was generated for use as a negative control (Figure 6A, right panel of 5F). Expression and secretion of the constructs were confirmed by overexpressing the constructs in HEK293 cells and performing Western blots on cell lysates and media (Figure 6B). Previous studies have shown that incubating cells with heparin, a highly sulfated variant of heparan sulfate, promotes the secretion of several extracellular proteins. 40 Robo1-Ig5, as used herein, was expressed in HEK-293 cells in the absence and presence of heparin (300 ng / ml). Media were harvested from these ROBO1-Ig5 overexpressing cells at 2, 4, and 6 days after plasmid transfection. To assess the relative secretion of this soluble ROBO1 ECD, a dot blot dilution assay of the collected media was performed (Figure 6C). Soluble ROBO1-Ig5 secretion increased over this time course, and heparin treatment resulted in a trend towards more secretion (Figure 6C). Media samples from these ROBO1-Ig5 transfected cells were TCA-precipitated and also analyzed by Western blot, which showed intact ROBO1-Ig5 protein in the media at 6 days in both the absence and presence of heparin (Figure 6D).

[0123] The soluble ROBO1 ECD fragments ROBO1-Ig2 and ROBO1-Ig5 generated in the presence of heparin were used in the dome assay. The results showed that the soluble ROBO1 ECD fragments generated in the presence of heparin formed fewer domes than the same fragments generated in the absence of heparin (Figure 6E). Treatment with heparin had only a modest positive effect on ROBO1 ECD production and had a detrimental effect on their function (Figures 6C, E), so the use of heparin to generate soluble ROBO1 ECD fragments was not pursued. The ability of ROBO1 ECD fragments to bind to the ROBO2 receptor was tested by overexpressing Robo2 in Cos7 cells, treating the cells with sodium azide to prevent protein internalization, and incubating the cells with ROBO1-Ecto-HA 1H before fixation and immunostaining. The results showed that ROBO1-Ecto-HA binds to ROBO2, while DCC-Ig2-HA does not bind to ROBO2 (Figure 6G).

[0124] ROBO1 extracellular domain fragments promote the differentiation of HC11 cells: To determine whether ROBO1 ECD fragments affect NOTCH4 signaling, the HC11 assay was performed and dome formation was monitored using both phase contrast microscopy (Figures 7A, B, top) and fluorescence microscopy using the hydrophobic Bodipy493 / 503 that binds to neutral lipids (Figures 7A, B, bottom). Undifferentiated (Undif) cells are distinguished by interconnected processes visible by phase contrast and little / no Bodipy staining (Figure 7A). During differentiation and prolactin treatment, small lipid droplets accumulate, appearing as dark-edged circles by phase contrast (Figures 7B, top) and as punctate green circles by Bodipy staining (Figures 7B, bottom). Treatment with ROBO1-Ecto was revealed by Bodipy493 / 503 staining to increase the number of cells completely surrounded by lipid droplets (Figure 7B).

[0125] The formation of domes in response to titration of ROBO1 ECD fragments was quantified. The higher the concentration of ROBO1-Ig2, ROBO1-Ig5, and ROBO-1-Ecto, the higher the dome formation rate. This result was not observed in response to treatment with either DCC-Ig2 or DCC-Ig4 control ECD fragments (Figures 7C - G). The bovine ROBO1-Ig5 construct was also tested in this assay and, similar to the rat construct, more domes were formed in response to treatment with high concentrations of ROBO1-Ecto ECD (Figure 7H). Collectively, these studies have shown that ROBO1-ECD promotes dome formation in HC11 cells.

[0126] To determine whether the promotion of dome formation also results in higher milk production, HC11 cells were differentiated in the presence or absence of ROBO1-ECD fragments added to the cells simultaneously with DIP medium. Cells were harvested and the expression of WAP and Lalba was evaluated by RT-qPCR. Treatment with different ROBO1-ECDs resulted in 6 - 9-fold higher expression compared to untreated controls (Figures 7I, J). In cells treated with DCC-Ig4, the WAP expression level did not increase (Figure 7K). Next, Western blot analysis of WAP and PLIN2 was performed on cells treated with ROBO1-Ig5 and ROBO1-Ecto. In cells treated with ROBO-1 ECD, an approximately 2-fold increase in the expression of WAP and PLIN2 proteins was observed (Figures 7L - O).

[0127] The ROBO1 extracellular domain fragment inhibits Notch signaling: To examine the effect of the ROBO1-ECD fragment on Notch signaling, HC11 cells were treated with the ROBO1-ECD fragment and the expression of Notch effectors was evaluated. Treatment with ROBO1-Ig5 and ROBO1-Ecto resulted in a decrease in Hey1 and Hes1 expression (Figure 8A), whereas this effect was not observed in cells treated with ROBO1-Ig2 (Figure 8A). Furthermore, HC11 cells were treated with ROBO-Ig5 during differentiation. These cells were then fractionated and western blotting was performed to detect HES1 and NOTCH4-ICD (Figure 8B). Treatment with ROBO1-Ig5 led to lower levels of both HES1 and NOTCH4-ICD (N4-ICD) proteins in the nuclear fraction compared to control treatment, and NOTCH4-ICD was also low in the cytoplasmic fraction by ROBO1-Ig5 treatment. Collectively, these results suggest that ROBO1-ECD inhibits Notch signaling.

[0128] Disclosed herein is a model in which the soluble ROBO1 ECD fragment binds to ROBO2, preventing ROBO2 from binding to endogenous transmembrane ROBO1, thereby promoting the formation of a ROBO1 / NOTCH complex that interferes with Notch signaling (right panel of Figure 5F). However, the ROBO1-ECD fragment may also directly bind to NOTCH4 and inhibit it. Therefore, to test whether the ROBO1-ECD fragment inhibits Notch in the absence of ROBO1, the expression of Robo1 was inhibited in HC11 cells. Next, cells lacking Robo1 expression were treated with the ROBO1-ECD fragment, and their ability to form domes was evaluated. As observed above (Figures 7C-E, H), treatment with the ROBO1-ECD fragment increased dome formation in control cells (Scr) (Figure 8C). Inhibition of Robo1 expression (shRobo1) also inhibited dome formation in control cells, as observed above (Figures 1B, 3E) (Figure 8C). However, treatment of cells with inhibited Robo1 expression (shRobo1) with ROBO1-Ig5 resulted in dome formation at the same level as control cells (Scr) treated with the ROBO1-ECD fragment (Figure 8C). Inhibition of Notch4 expression (shNotch4) in the absence of the ROBO1-Ig5 fragment resulted in greater dome formation compared to control cells (Scr), as observed above (Figure 3G) (Figure 8C). Treatment of these cells (shNotch4) with ROBO1-Ig5 led to dome formation at the same level as untreated Notch4 knockdown cells (shNotch4) (Figure 8C). This suggests that ROBO1-Ig5 treatment does not further increase HC11 dome formation in the absence of NOTCH4. Collectively, these results suggest that NOTCH4 is a direct target of ROBO1-Ig5.

[0129] The ROBO1 extracellular domain fragment enhances organoid formation and mammary branching: The effects of ROBO1 ECD fragments on in vitro primary alveolar progenitor cell proliferation and in vivo ductal morphogenesis were tested. FACS-purified mouse and bovine alveolar progenitor cells (AVPs) were plated as single cells in Matrigel and grown for 10 days in the absence and presence of ROBO1-ECD fragments (Figures 9A, B). Treatment with ROBO1-ECD resulted in more mouse organoids compared to untreated controls (Figure 9A). Treatment with ROBO1-Ig5 resulted in larger bovine organoids compared to untreated controls (Figure 9B). The ROBO1-Ig5 fragment was also tested in vivo by subcutaneous injection (7.5 μg mammary gland / kg / day) into ovariectomized animals orally administered the following hormones in Nutella: estrogen (E, 1 μg / day), progesterone (P, 1 mg / day mammary gland / day), and prolactin (Prl, 0.2 mg / day mammary gland / day) (Figure 9C). Mammary glands were harvested 14 days after ROBO1-Ig5 fragment treatment and stained with carmine for evaluation. ROBO1-Ig5 treatment resulted in significantly larger areas and a greater number of primary (1°) branches compared to untreated controls (Figure 1D). More secondary (2°) and tertiary (3°) branches of the gland were also observed. However, the overall branching density of the treated glands did not differ from that of the controls because the size of the glandular area was larger for glands with larger branches (Figure 1E). In summary, this study shows that ROBO1-Ig5 treatment in vivo resulted in mammary glands with significantly more branches. In other embodiments, ROBO1-ECD constructs tagged with mouse-Fc sequences may be included. This tag is recognized by endogenous receptors that facilitate transport into tissues 41 。

[0130] The ROBO1 extracellular domain fragment increases lobuloalveolar mammary gland development and milk production. The in vivo effect of ROBO1 ECD-Fc fragment on the development of lobular alveoli during pregnancy was investigated. ROBO1 ECD fragment (7.5 mg / kg) and sham-injected control were subcutaneously injected three times during pregnancy (at gestational day (PD) 8.5, PD11.5, and PD14.5) (Figure 10A). Mammary glands were harvested at PD17.5, and alveologenesis was analyzed by serial sectioning and hematoxylin and eosin (H&E) staining, and then the area occupied by alveoli was quantified in sections from the upper, middle, and lower parts of the tissue. As observed above, the alveolar area in Robo1− / − was significantly reduced compared to that in WT and sham-injected mammary glands, and the Robo1− / − alveolar size was reduced (Figure 10B, C, F, arrows, asterisks). Injection of ROBO1 ECD-Fc fragment into both WT and Robo1− / − animals showed a significant increase in alveolar area and the number of alveoli filled with milk droplets compared to sham-injected controls.

[0131] To further evaluate milk production, RT-qPCR of whey acidic protein (WAP), xanthine dehydrogenase (XDH), and beta-casein (CSN2) of milk protein genes was performed. Milk protein gene expression was significantly increased in ROBO1 ECD-Fc treatment compared to control treatment (Figure 11A–C). For milk expression, protein levels were also evaluated by immunohistochemistry of sectioned tissues using an antibody against milk (#YNRMTM, Accurate Chemical and Scientific Corp). Again, a significant increase in milk was observed when ROBO1 ECD was injected into either WT or Robo1− / − animals (Figure 11D–H). Collectively, these data indicate that subcutaneous injection of ROBO1 ECD fragment into pregnant animals increases lobular alveolar development, milk protein genes, and milk production.

[0132] ROBO1 is required for alveolar differentiation and milk production in mammary basal cells: The mammary gland is a two - layer tissue composed of outer basal cells (basal compartment) and inner luminal cells (luminal compartment) (Figure 1A). ROBO1 expression was detected in both luminal and basal cells of the mammary gland (Figures 1D - G). To determine in which cell type ROBO1 functions to enable the differentiation of mammary progenitor cells into milk - producing alveolar cells, mosaic organoids were generated in which either the basal or luminal compartment - containing cells were composed of Robo1− / − cells (Figures 12C, D). As controls, WT and KO cells formed WT organoids and Robo1− / − organoids that consisted of both basal and luminal compartments (WT / WT and KO / KO) (Figures 12A, B). To distinguish WT cells from KO cells, ACTb - EGFP mice were used for WT tissue (GFP+ / +). Organoids were generated by differential trypsin treatment to separate the two populations, followed by mixing the separated basal and luminal sub - populations (WT / WT, KO / KO, WT / KO, KO / WT), and then differentiating them for 5 days after culturing in Matrigel. WT / WT organoids containing both GFP+ / + basal cells and GFP+ / + luminal cells formed large two - layer organoids that, upon differentiation, produced milk filling the lumen (Figure 12A). In contrast, KO / KO organoids containing both Robo1− / − basal cells and Robo1− / − luminal cells produced smaller two - layer structures that produced little or no milk upon differentiation (Figure 12B). When Robo1− / − basal cells were mixed with WT luminal cells (KO / WT), the resulting mosaic organoids produced little / no milk upon differentiation (Figure 12C). However, when WT basal cells were mixed with Robo1− / − luminal cells (WT / KO), the resulting organoids produced milk similar to milk production in WT / WT organoids (Figure 12A) (Figure 12D). These data indicate that for luminal cells to produce milk upon hormonal stimulation, ROBO1 expression is required in the basal rather than the luminal compartment of the mammary gland.

[0133] ROBO1 inhibits the expression of Jagged1 in basal cells: One way to regulate Notch expression is to control the expression levels of the Notch ligands Jagged1, Jagged2, or Delta. To examine whether ROBO1 regulates Notch ligand expression, cells were transfected while increasing the amount of plasmid expressing Robo1. After 48 hours, the cells were harvested and immunoblotted with antibodies against ROBO1, JAGGED1 (JAG1), and GAPDH (loading control) (Figure 13A). The data show that an increase in ROBO1 expression led to a decrease in JAGGED1 expression. Next, siRNA was used to knockdown the expression of Robo1. In cells after 48 hours, JAGGED1 and JAGGED expression were evaluated by immunoblot. An increase in JAGGED1 expression and no change in JAGGED2 expression were observed (Figures 13B, C). To examine whether this regulation of JAGGED1 occurs in vivo, subpopulations of primary WT and Robo1− / − mammary epithelial cells were purified into basal, luminal, and stromal subpopulations using fluorescence-activated cell sorting (FACS) and then analyzed by immunoblot against JAGGED1 and Cytokeratin14 (CK14) (loading control) (Figure 13D). More JAGGED1 was observed in Robo1− / − compared to Robo1+ / + basal cells. There was no detectable expression in luminal cells and only moderate expression in stromal cells, and these data are similar to the results obtained by knocking down Robo1 expression in cell lines (Figure 13B). Also, JAGGED1 expression was evaluated by immunostaining Robo1+ / + and Robo1− / − organoids (Figures 13E, F). More JAGGED1 expression was observed in Robo1− / − basal cells compared to Robo1+ / + organoids. Collectively, these data indicate that ROBO1 inhibits JAGGED1 expression in mammary basal cells and that JAGGED1 increases when Robo1 is lost. When JAGGED1 expression increases, NOTCH signaling in adjacent alveolar progenitor cells is enhanced and their differentiation into milk-producing alveolar cells is inhibited.Thus, one mechanism by which ROBO1 regulates milk production is by governing the levels of the Notch ligand JAGGED1 within the basal compartment of the mammary gland.

[0134] Materials and Methods Animals: All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz (UCSC). All Robo1 mice were generated and genotyped as described above 11 。

[0135] Clearing and Transplantation of Mammary Fat Pads: Small mammary tissue fragments from 8-week-old WT and ROBO1 KO mice were transplanted contralaterally into pre-cleared Foxn1 nu fat pads. The contralateral derivatives were harvested on gestational day 18.5 and subjected to carmine staining.

[0136] Mammary Whole Mount Carmine-Aluminum Assay: Mouse mammary glands were surgically dissected, spread on glass slides, and fixed in Carnoy's solution (25% glacial acetic acid and 75% ethanol). After brief dehydration, the glands were stained overnight with 0.2% carmine and 0.5% aluminum potassium sulfate, dehydrated in a graded series of ethanol solutions (70%, 95%, 100%), cleared in toluene, and mounted in Permount.

[0137] Fat Pad Filling Analysis: Paraffin-embedded Robo1 KO or WT littermate tissues or contralateral derivatives were sectioned and subjected to hematoxylin and eosin (H&E) staining. Images were analyzed using ImageJ, and the filling rate of the fat pad was calculated by measuring the area occupied by alveoli.

[0138] Immunohistochemistry and Beta-Galactosidase Staining: The tissues were fixed with 4% paraformaldehyde. Paraffin-embedded tissues were sectioned at 6 μm and mounted continuously. For immunohistochemistry, standard protocols were followed. For beta-galactosidase staining, 40 mg / ml of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside was prepared in 1 M phosphate buffer containing 1 M MgCl2 and 10 mM potassium ferricyanide. Frozen sections of the tissues were treated with the staining solution at 37°C for 1.5 - 24 hours, washed with PBS, dehydrated with ethanol, cleared with xylene, and covered with a coverslip. 42 。

[0139] Microscopic observation: Bright-field imaging was performed with a Biorevo BZ-9000 digital microscope (Keyence), and confocal microscopy was performed with a Nikon C2 Confocal and a Leica SP5 confocal. The collected data were analyzed using ImageJ.

[0140] Co-immunoprecipitation: Adherent cells were lysed in 1 mL of 1X lysis buffer (137 nM NaCl, 10 mM Tris-HCl (pH 8), 2 nM EDTA, 1 mM sodium orthovanadate) supplemented with 1% Igepal NP40 (Sigma), 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM leupeptin, 1 mM aprotinin, and a phosphatase inhibitor (Roche Complete). The cell lysates were incubated at 4°C for 15 minutes with gentle rocking and then centrifuged at 12,000 rpm for 10 minutes. The soluble phase was incubated with Dynabeads Protein A (Thermo-fisher) conjugated with 1 μg of antibody at room temperature for 1 hour or at 4°C for 4 hours according to the company protocol. The samples were washed and eluted according to the protocol. The eluted protein complexes were mixed with 2X Lysis buffer and incubated at 70°C for 10 minutes and at 100°C for 5 minutes.

[0141] Western blotting: Adherent cells were directly lysed with 1X sample buffer supplemented with 5% beta-mercaptoethanol and boiled for 5 minutes to prepare a protein lysate. The protein lysate was separated by SDS-PAGE and transferred to PVDF at 400 mA for 90 minutes or at 30 mA overnight. Primary antibodies were used at the concentrations shown in Table 1 and incubated overnight at 4°C. HRP-conjugated secondary antibody (Jackson Labs) was used at 1:3000 and incubated for 45 minutes at room temperature. All proteins were detected using Clarity ECL (BioRad) with a BioRad Chemi-Doc MP Imager and quantified using ImageLab software as described above 43 。

[0142] 2D cell culture: All cell lines were obtained from the American Type Culture Collection. MDA-MB-231 cells were cultured in DMEM growth medium (Gibco) supplemented with 10% heat-inactivated FBS (Seradigm) and 1X antibiotic-antimycotic (Gibco). Undifferentiated HC11 cells were cultured in RPMI-1640 growth medium (Gibco) supplemented with 10 μg / mL bovine insulin (Sigma-Aldrich) and 10 ng / mL human EGF (Peprotech). Primary LECs were harvested from 8-week-old Robo1 KO or WT littermates as previously described 15 。

[0143] 3D cell culture: FACS-purified AVP was cultured in Matrigel (BD Bioscience) at a density of 5000 cells / 100 μL and cultured for 5 days in basal medium DMEM: F12 phenol-free, 10 mM HEPES, N2 (Gibco), B27 (Gibco) supplemented with 100 ng / mL neuregulin (R&D) and 42.5 ng / mL R-Spondin1 (Peprotech). To differentiate the cultured AVP, 10 -6They were further grown for 5 days in a basal medium supplemented with M dexamethasone (Sigma), 10 μg / mL bovine insulin (Sigma), and 5 μg / mL prolactin (National Hormone and Peptide program). The acini were fixed and processed as previously described 44 .

[0144] HC11 dome assay: HC11 cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (BioFluid Technologies), 5 μg / mL insulin (Sigma), and 10 ng / ml epidermal growth factor (EGF; Sigma). To induce differentiation in HC11 cells, confluent plates were given fresh medium (RPMI 1640 medium supplemented with 5% charcoal-stripped fetal bovine serum (BioFluid Technologies), 5 μg / mL insulin, and 10 ng / ml epidermal growth factor (EGF; Sigma)) for 3 days, followed by priming with priming medium (RPMI 1640 medium supplemented with 5% charcoal-stripped fetal bovine serum (BioFluid Technologies) and 5 μg / mL insulin) for 24 hours. After priming, DIP medium (RPMI 1640 medium supplemented with 5% charcoal-stripped fetal bovine serum (BioFluid Technologies), 10 -6 M dexamethasone (Sigma), 5 μg / mL insulin, and 5 μg / mL prolactin (National Hormone and Peptide program) was added to the RPMI 1640 medium) was added with fresh medium every 24 hours.

[0145] Lentivirus production: For the generation of lentiviral particles for the knockdown experiments of Scramble, Robo1, Robo2, and Notch4, a combined transfection of psPAX2, pMD2.G, and pLVTHM-Scramble-GFP (SCR) or pLVTHM-sh-target GFP into HEK293T cells was included. Next, the filtered (0.45um) viral particles were diluted in the medium and used to infect the target breast cell lines (MDA-MB-231 and HC11 cells).

[0146] Isolation and flow cytometry of mammary epithelial cells: Mechanically dissociated inguinal and thoracic mammary fat pads were prepared in cell suspension for FACS as described. 17 . AVP was isolated using FITC-CD14 (clone Sa14-2; BioLegend) and ACP-Cy7-CD117 (clone 2B8; BioLegend) as described. 14 .

[0147] In vivo gamma secretase inhibitor (GSI): The GSI inhibitor (RO4929097; MedchemExpress) was orally administered at 10 mg / kg for 5 days as described. 29 . The mammary glands were harvested 5 days after GSI or vehicle treatment and prepared for single cell analysis. The purified populations were collected and processed for RNA. FlowJo was used to analyze the number of purified populations.

[0148] RNA extraction and RT-qPCR: Total RNA was recovered from cells lysed with TRIzol reagent (Invitrogen), and RNA precipitation was added overnight in ethanol to separate the phases according to the manufacturer's protocol (Macias et al., 2011). RNA was further purified by TURBO DNase (Ambion) treatment. The quality of total RNA was analyzed by agarose gel electrophoresis and quantified with an ND-1000 spectrophotometer (NanoDrop). A cDNA library was prepared from 1 μg of total RNA using the iScript cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed in triplicate using LightCycler 480 SYBR Green I Master (Roche) and quantified using the BioRad CFX’Connect Real-Time System and CFX Manager software (BioRad). Results were normalized to GAPDH.

[0149] ROBO1 extracellular domain generation: To generate protein fragments, HEK cells were transfected with plasmids corresponding to the fragments of interest. PEI transfection was performed according to the Cytographica protocol. Twenty-four hours after transfection, the medium was replaced with OptiMEM. Eight days after transfection, the medium was collected and centrifuged at 3000 x g for 10 minutes. Next, the supernatant was filtered through a 0.45 μm PVDF filter.

[0150] TCA precipitation: Add 1 volume of TCA stock to 4 volumes of protein sample. Incubate at 4 °C for 10 minutes. Spin the tube in a microcentrifuge at 14K rpm for 5 minutes. Remove the supernatant leaving the protein pellet intact and wash the pellet with 200 μl of cold acetone. Spin in a microfuge at 14K rpm for 5 minutes. Repeat steps 4 - 6 for a total of 2 acetone washes. Dry the pellet before suspending in sample buffer.

[0151] Bodipy 493 / 503 staining: Place the cells in buffer or medium at half the volume. Prepare a 2X solution (2 μg / ml = 7.6 μM) of Invitrogen™ BODIPY™ 493 / 503 dye in 0.5 mL of pre-warmed same buffer or medium (without cells, without BSA and without serum), mix vigorously and mechanically emulsify this solution. Immediately add it to the cell solution, mix, and incubate for up to 30 minutes.

[0152] CUBIC immunofluorescence method: The glands were harvested and fixed overnight at 4°C in 10% neutral buffered formalin (Sigma). The fixation was quenched with PBST (0.1% Triton X-100; Sigma) containing 0.2% glycine (Fisher Scientific) for 2 x 10 minutes. Next, the glands were incubated in CUBIC reagent 1A at 37°C for 48 hours, followed by washing with PBST for 3 x 10 minutes as described. 45 The glands were blocked with PBST / 10% donkey serum (Sigma) overnight at 4°C and then incubated with the primary antibody in PBST / 5% donkey serum for 48 hours at 4°C. Next, the glands were washed with PBST for 3 x 1 hour and incubated with the secondary antibody diluted in PBST / 5% donkey serum for 24 hours at 4°C. To counterstain DNA, the glands were incubated with Hoechst diluted in PBST for 1 hour and then washed with PBST for 2 x 1 hour. Finally, the glands were incubated with CUBIC reagent 2 at 4°C for up to 24 hours until they became transparent.

[0153] Intratracheal injection: Preparation for injection: The mice were anesthetized using an isoflurane chamber and eye lubricant was applied. The mice were continuously anesthetized with 2 - 4% isoflurane in oxygen via a nose cone. Hair was removed from the nipple area using Nair chemical hair remover. Injection: On the 7th day of pregnancy, PBS or ROBO2 mAb was injected bilaterally into the nipples of glands #3, #4, and #5 using a 33 - gauge bevel needle (Hamilton) attached to a 50 μl syringe. The injection was performed very slowly (about 40 μl / min) to minimize damage that could be caused by fluid rapidly moving within the lumen. After injection: The animals were removed from the nose cone and transferred to another cage for recovery. 46 。

[0154] Ovariectomy, hormonal treatment, and subcutaneous injection: C57BL mice (8 - 10 weeks old) were bilaterally ovariectomized and allowed to recover for 1 week. 47 。During recovery, the mice were trained with oral administration of Nutella. The mice were given Nutella mixed with 17 beta - estradiol (E, 1 μg, Sigma) + progesterone (P, 1 mg, Sigma) daily for 3 weeks. In the case of ROBO1 - Ig5 ECD, prolactin (Prl, 200 μg, NHPP) was administered starting 1 week after E + P and given daily for 1 week by oral administration of Nutella. In the case of ROBO2 mAb, prolactin (Prl, 50 μg) was injected starting 1 week after E + P and given daily by intraperitoneal injection for 2.5 weeks. ROBO1 - Ig5 ECD was administered by subcutaneous injection starting 1 week after E + P and given daily for 2 weeks (7.5 mg / kg, ROBO1 - Ig5 ECD or PBS). ROBO2 mAb or IgG isotype control mAb (250 μg / mouse) was administered by subcutaneous injection twice a week starting 1 week after E + P and given for 17 days. 48 。

Table 1

Table 2

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[0156] Preferred embodiments of the present invention have been illustrated and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various alternative embodiments of the embodiments of the present invention described herein may be used in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0157] For reasons of completeness, certain aspects of the polypeptides, compositions, and methods of the present disclosure are set forth in the clauses numbered below. 1. A method for promoting milk production in a mammal, comprising: administering to the mammal a first agent that inhibits NOTCH4 activity in an amount sufficient to inhibit NOTCH4 activity, thereby promoting milk production. 2. The method of clause 1, wherein the first agent inhibits NOTCH4 activity by directly binding to the NOTCH4 protein, by inhibiting the binding of ROBO2 to ROBO1, by promoting the binding of ROBO1 to NOTCH4, by inhibiting the expression of NOTCH4, or by inhibiting the expression of ROBO2. 3. The method of clause 1, wherein the first agent comprises a soluble ROBO1 extracellular domain (ECD). 4. The method of clause 3, wherein the soluble ROBO1 ECD is a mouse, bovine, ovine, caprine, equine, or human ROBO1 ECD. 5. The method of clause 3 or 4, wherein the ROBO1 ECD comprises a heterologous polypeptide. 6. The method of clause 5, wherein the heterologous polypeptide comprises a His tag, a hemagglutinin tag, an immunoglobulin (Ig) Fc region, or a Myc tag. 7. The method of clause 1, wherein the first agent comprises an RNAi construct that inhibits the expression of NOTCH4 or ROBO2. 8. The method of clause 7, wherein the RNAi construct is a short interfering RNA. 9. The method according to clause 1, wherein the first agent comprises an anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof. 10. The method according to clause 9, wherein the first agent comprises a plurality of polyclonal anti-NOTCH4 antibodies. 11. The method according to clause 9, wherein the anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof is a monoclonal antibody or a NOTCH4-binding fragment thereof. 12. The method according to clause 10, wherein the polyclonal anti-NOTCH4 antibody is a mouse, bovine, sheep, goat, camel, or human polyclonal antibody, and the species in which the polyclonal antibody is produced is the same as the species of the mammal to which the first agent is administered. 13. The method according to clause 11, wherein the monoclonal antibody or a NOTCH4-binding fragment thereof is a bovine, sheep, goat, or human monoclonal antibody or a NOTCH4-binding fragment thereof, and the species from which the monoclonal antibody is derived is the same as the species of the mammal to which the first agent is administered. 14. The method according to clause 13, wherein the anti-NOTCH4 monoclonal antibody or a NOTCH4-binding fragment thereof is bovineized, ovineized, caprineized, camelized, or humanized. 15. The method according to clause 1, wherein the first agent comprises a soluble ROBO1 extracellular domain, and the method further comprises administering to the mammal, in an amount sufficient to inhibit NOTCH4 activity, a second agent that inhibits NOTCH4 activity. 16. The method according to clause 15, wherein the second agent comprises an RNAi construct that inhibits the expression of NOTCH4 or ROBO2. 17. The method according to clause 16, further comprising a third agent comprising an RNAi construct that inhibits the expression of NOTCH4 or ROBO2. 18. The method according to claim 1, comprising administering at least one of a first agent, a second agent, a third agent, and a fourth agent that inhibits NOTCH4 activity, wherein each of the first agent, the second agent, the third agent, and the fourth agent is independently selected from a soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits the expression of NOTCH4, and an RNAi construct that inhibits the expression of ROBO2. 19. A polypeptide comprising a soluble ROBO1 extracellular domain fused to a heterologous polypeptide. 20. The polypeptide according to claim 19, wherein the soluble ROBO1 ECD is a mouse, bovine, ovine, caprine, or human ROBO1 ECD. 21. The polypeptide according to claim 20, wherein the heterologous polypeptide comprises a His tag, a hemagglutinin tag, a human or mouse Fc region, a Myc tag, or a fluorescent protein. 22. A pharmaceutical composition comprising the polypeptide according to any one of claims 19-21 and a pharmaceutically acceptable carrier. 23. The pharmaceutical composition according to claim 22, for use in promoting milk production in a mammal. 24. An anti-NOTCH4 antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity. 25. The antibody according to claim 24, wherein the antibody comprises a plurality of polyclonal antibodies. 26. The antibody according to claim 24, wherein the antibody is a monoclonal antibody or a NOTCH4-binding fragment thereof. 27. The antibody according to any one of claims 24-26, wherein the antibody comprises a bovine, ovine, caprine, camelid, or human polyclonal or monoclonal antibody, and at least a portion of the monoclonal antibody comprises an antibody sequence from a bovine, ovine, caprine, or human antibody. 28. The antibody according to claim 26, comprising a bovineized, ovineized, caprineized, camelidized, or humanized antibody or any antigen-binding fragment thereof. 29. A pharmaceutical composition comprising an antibody according to any one of clauses 24 to 28 and a pharmaceutically acceptable carrier. 30. The pharmaceutical composition according to clause 29 for use in promoting milk production in a mammal. 31. A polynucleotide comprising an RNAi construct that inhibits the expression of ROBO2 or NOTCH4. 32. The polynucleotide according to clause 31, comprising at least one non-naturally occurring nucleotide. 33. The polynucleotide according to clause 31 or 32, comprising one or more of SEQ ID NOs: 32 to 35. 34. A pharmaceutical composition comprising the polynucleotide according to any one of clauses 31 to 33. 35. The pharmaceutical composition according to clause 34 for use in promoting milk production in a mammal. 36. A transgenic mammal comprising a genetic modification that results in one or more of the following phenotypes: expression of a soluble ROBO1 extracellular domain; inhibition of ROBO2 expression; and inhibition of NOTCH4 expression. 37. The transgenic animal according to clause 36, wherein the phenotype is limited to mammary tissue. 38. The transgenic mammal according to clause 36 or 37, wherein the transgenic animal is a cow, sheep, goat, or camel. 39. The transgenic mammal according to any one of clauses 36 to 38, comprising two genetic modifications that result in two of the described phenotypes. 40. The transgenic mammal according to any one of clauses 36 to 38, comprising three genetic modifications that result in all three of the described phenotypes. 41. A method for promoting milk production, comprising administering to a transgenic mammal according to any one of clauses 36 to 40 a pharmaceutical composition that inhibits NOTCH4 activity. 42. The method according to clause 41, wherein the pharmaceutical composition is the composition according to any one of clauses 22, 23, 29, 30, 34, and 35. 43. The transgenic animal comprises a genetic modification that results in the expression of the soluble ROBO1 extracellular domain, and the method according to clause 41 further comprises administering to the transgenic animal a pharmaceutical composition according to any one of clauses 22, 23, 29, 30, 34 and 35. 44. The transgenic mammal comprises a genetic modification that results in the inhibition of the expression of ROBO2 and / or NOTCH4, and the method according to clause 41 further comprises administering to the transgenic animal a pharmaceutical composition according to clause 34 or 35.

Claims

1. A method for promoting milk production in non - human mammals, comprising: administering to the mammary gland tissue of said mammal a first agent that inhibits NOTCH4 activity by directly binding to the NOTCH4 protein, by inhibiting the binding of ROBO2 to ROBO1, by promoting the binding of ROBO1 to NOTCH4, by inhibiting the expression of NOTCH4, or by inhibiting the expression of ROBO2, (i) soluble ROBO1 extracellular domain (ECD); (ii) an RNAi construct that inhibits the expression of NOTCH4 or ROBO2; and (iii) an anti - NOTCH4 antibody or a NOTCH4 - binding fragment thereof selected from the group consisting of, in an amount sufficient to inhibit NOTCH4 activity, thereby promoting milk production.

2. The method according to claim 1, wherein the first agent is a soluble ROBO1 extracellular domain (ECD).

3. The method according to claim 1 or 2, wherein the soluble ROBO1 ECD is a mouse, bovine, ovine, caprine, equine, or human ROBO1 ECD.

4. The method according to any one of claims 1 to 3, wherein the ROBO1 ECD comprises a heterologous polypeptide.

5. The method according to claim 4, wherein the heterologous polypeptide comprises a His - tag, a hemagglutinin tag, an immunoglobulin (Ig) Fc region, or a Myc - tag.

6. The method according to claim 1, comprising administering to the mammary tissue at least one of a first agent, a second agent, a third agent, and a fourth agent that inhibit NOTCH4 activity, each of the first agent, the second agent, the third agent, and the fourth agent being independently selected from a soluble ROBO1 ECD, an anti-NOTCH4 antibody, an RNAi construct that inhibits the expression of NOTCH4, and an RNAi construct that inhibits the expression of ROBO2.

7. A composition for use in promoting milk production in a mammal, comprising a polypeptide comprising a soluble ROBO1 extracellular domain (ECD).

8. The composition according to claim 7, wherein the polypeptide is fused to a heterologous polypeptide, and optionally, the soluble ROBO1 ECD is a mouse, bovine, ovine, caprine, or human ROBO1 ECD.

9. The composition according to claim 7 or 8, wherein the soluble ROBO1 ECD is fused to a heterologous polypeptide comprising a His tag, a hemagglutinin tag, a human or mouse Fc region, a Myc tag, or a fluorescent protein.

10. A pharmaceutical composition for use in promoting milk production in a mammal, comprising the composition according to claim 7 or 8 and a pharmaceutically acceptable carrier.

11. A composition comprising the polynucleotide for use in promoting milk production in a mammal by administering the polynucleotide to the mammary tissue, wherein the polynucleotide comprises an RNAi construct that inhibits the expression of ROBO2 or NOTCH4, and the RNAi construct comprises one or more of SEQ ID NOs: 32 to 35.

12. A pharmaceutical composition comprising the polynucleotide for use in promoting milk production in a mammal by administering the polynucleotide to the mammary tissue, The pharmaceutical composition, wherein the polynucleotide comprises an RNAi construct that inhibits the expression of ROBO2 or NOTCH4, and the RNAi construct comprises one or more of SEQ ID NOs: 32 to 35. **Claim 13** A non-human transgenic mammal comprising a genetic modification that results in one or both of the following phenotypes in mammary tissue: expression of the soluble ROBO1 extracellular domain; and inhibition of the expression of ROBO2 **Claim 14** The non-human transgenic mammal according to claim 13, wherein the genetic modification results in the expression of the soluble ROBO1 extracellular domain. **Claim 15** The non-human transgenic mammal according to claim 13, wherein the genetic modification results in inhibition of the expression of ROBO2. **Claim 16** The non-human transgenic mammal according to any one of claims 13 to 15, wherein the phenotype is limited to mammary tissue. **Claim 17** The non-human transgenic mammal according to any one of claims 13 to 15, wherein the transgenic animal is a cow, sheep, goat, or camel. **Claim 18** The non-human transgenic mammal according to claim 17, wherein the phenotype is limited to mammary tissue. **Claim 19** A method for promoting milk production, comprising administering to the mammary tissue of a non-human transgenic mammal according to any one of claims 13 to 18 a pharmaceutical composition that inhibits NOTCH4 activity, wherein the pharmaceutical composition comprises the pharmaceutical composition according to claim 10 or 12, or comprises an anti-NOTCH antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity. **Claim 20** The transgenic animal comprises a genetic modification that results in the expression of the soluble ROBO1 extracellular domain in mammary tissue, and the pharmaceutical composition comprises the pharmaceutical composition according to claim 10 or 12, or comprises an anti-NOTCH antibody or a NOTCH4-binding fragment thereof that inhibits NOTCH4 activity, the method according to claim 19.

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