2. FIELD

Provided herein are compositions, methods, and uses involving antibodies that specifically bind to human Nectin-4 and detect the expression level of Nectin-4.

3. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application incorporates by reference a Computer Readable Form (CRF) of a Sequence Listing in ASCII text format submitted with this application, entitled 14369-208-228_SEQ_LISTING.txt, was created on May 31, 2018, and is 37,280 bytes in size.

4. BACKGROUND

Nectin-4, also known as poliovirus receptor-related protein 4 (PVRL4), is a single pass type I transmembrane protein of about 52 kDa in size that belongs to the Nectin family of cellular adhesion molecules. Nectins mediate Ca2+-independent cell-cell adhesion at adherens junctions via both homophilic interaction where one Nectin-4 interacts with another Nectin-4, and heterophilic interactions where Nectin-4 interacts with another Nectin family protein such as Nectin-1, Nectin-2, or Nectin-3. (Miyoshi J et al., 2007, Am J Nephrol27:590-604; Takai et al., 2003, Cancer Sci94:655-667; Reymond N. et al., 2001, Journal of Biological Chemistry,276:43205-15). The extracellular domain of Nectin-4 has three Ig-like subdomains designated as V, C1 and C2. It has been shown that the C1 domain in Nectin-2 is responsible for homophilic interactions, while V domains of most Nectin molecules contribute to heterophilic interactions and cell-cell adhesion. (Miyoshi J et al., 2007, Am J Nephrol27:590-604; Takai et al., 2003, Cancer Sci94:655-667; Reymond N. et al., 2001, Journal of Biological Chemistry,276:43205-15)

Nectins are expressed in various tissues, including, for example, hematopoietic, neuronal, endothelial, and epithelial cells. (Mendelsohn C et al., 1989, Cell56:855-865; Lopez M et al., 1995, Gene(Amst.) 155:261-265; Lopez M et al., 1998, Blood92:4602-4611; Reymond N et al., 2000, Gene(Amst.) 255:347-355; Cocchi F. et al., 1998, Journal of Virology.72:9992-10002; Takahashi K. et al., 1999, Journal of Cell Biology145:539-549; Miyoshi J et al., 2007, Am J Nephrol27:590-604; Takai et al., 2003, Cancer Sci94:655-667).

Besides mediating cell-cell adhesions by homophilic interactions or heterophilic interactions with other Nectin family proteins, Nectins can recruit other cell adhesion molecules such as cadherins, or other cell surface receptors such as the prolactin receptor. By recruiting other cell surface receptors, Nectins can also serve as a stimulatory co-receptor and thus have a signaling function. For example, in the mouse mammary gland, Nectin-4 interacts with the prolactin receptor through its extracellular and transmembrane domain and binds to and sequesters suppressor-of-cytokinesignaling-1 (SOCS-1), which normally inhibits the kinase activity of the Janus Kinase-2 (JAK2), thereby enhancing prolactin induced JAK2 activation and signaling. (Maruoka et al., 2017, Journal of Biological Chemistry, doi: 10.1074/jbc.M116.769091, jbc.M116.769091, Epub Ahead of Print; Kitayama et al., 2016, Journal of Biological Chemistry291:5817-5831). Nectins can also cooperate with cadherins to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion. (Khameeka et al., 2011, PLoS ONE6: e17841).

There has been a lack of success in anti-Nectin-4 antibodies developed so far for specifically detecting and assessing Nectin-4 expression in various biological samples. Thus there is need to identify an anti-Nectin-4 antibody that can specifically detect and assess Nectin-4 expression in biological samples.

5. SUMMARY

The present disclosure provides proteins that bind to Nectin-4 (e.g., human Nectin-4, SEQ ID NO:43), including binding proteins such as antibodies that bind to Nectin-4. Such binding proteins, including antibodies, may bind to a Nectin-4 polypeptide, a Nectin-4 fragment, and/or a Nectin-4 epitope.

The present disclosure also provides, in certain embodiments, binding proteins, including antibodies or fragments thereof, that binds specifically to a cell expressing Nectin-4.

Also provided herein are polynucleotides and vectors comprising sequences encoding such antibodies, cells (e.g., host cells) comprising such polynucleotides or vectors, and compositions, reagents, and kits comprising such antibodies. In another aspect, provided herein are methods for modulating Nectin-4 activity (e.g., activating Nectin-4 signaling) or Nectin-4 expression levels, diagnostic methods and uses of such anti-Nectin-4 antibodies.

In some embodiments, a binding protein (e.g., an anti-Nectin-4 antibody) comprises six complementarity determining regions (CDRs) or fewer than six CDRs. In other embodiments, a binding protein (e.g., an anti-Nectin-4 antibody) comprises one, two, three, four, five, or six CDRs selected from heavy chain variable region (VH) CDR1, VH CDR2, VH CDR3, light chain variable region (VL) CDR1, VL CDR2, and/or VL CDR3. In certain embodiments, a binding protein (e.g., an anti-Nectin-4 antibody) comprises one, two, three, four, five, or six CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibody designated as M22-321b41.1 as described herein, or a humanized variant thereof. In some embodiments, a binding protein (e.g., an anti-Nectin-4 antibody) further comprises a scaffold region or framework region (FR), including a VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or a variant thereof.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising VL CDR1, VL CDR2, and VL CDR3 of the antibody M22-321b41.1 as set forth in Table 1.

In other embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising VH CDR1, VH CDR2, and VH CDR3 of the antibody M22-321b41.1 as set forth in Table 1.

In other embodiments, the antibody or antigen-binding fragment thereof comprises:(a) a VL comprising VL FR1, VL FR2, VL FR3, and VL FR4 of the antibody M22-321b41.1 as set forth in Table 2; and(b) a VH comprising VH FR1, VH FR2, VH FR3, and VH FR4 of the antibody M22-321b41.1 as set forth in Table 2.

In certain embodiments, the VL CDR1, VL CDR2, and VL CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:7, 10, and 12, respectively, and the VH CDR1, VH CDR2, and VH CDR3 of the antibody or antigen-binding fragment thereof comprise amino acid sequences of SEQ ID NOS:15, 19, and 23, respectively.

In another embodiment, the antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence of SEQ ID NO:3. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence of SEQ ID NO:4. In some embodiments, the amino acid sequence comprises one or more conservative modifications thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a VL comprising an amino acid sequence of SEQ ID NO:3; and (b) a VH comprising an amino acid sequence of SEQ ID NO:4.

In some embodiments, the antibody comprises a human IgG1 Fc region. In other embodiments, the antibody comprises a mutant human IgG1 Fc region.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:33.

In other embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain Fc region comprising an amino acid sequence of SEQ ID NO:35.

In yet another embodiment, the antibody or antigen-binding fragment thereof further comprises a light chain constant region comprising an amino acid sequence of SEQ ID NO:33; and a heavy chain Fc region comprising an amino acid sequence of SEQ ID NO:35.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence of SEQ ID NO:5.

In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to Nectin-4, binds to at least one of residues 31-346 within an amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment thereof, when bound to Nectin-4, binds to at least one of residues 31-150 within an amino acid sequence of SEQ ID NO:1. In certain embodiments, the antibody or antigen-binding fragment thereof, when bound to Nectin-4, binds to at least one of residues 1-150 within an amino acid sequence of SEQ ID NO:1.

In one embodiment, the epitope of human Nectin-4 is distinct from the Nectin-4 ligand binding site.

In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to an epitope of human Nectin-4, wherein the antibody or antigen-binding fragment thereof binds specifically to a cell expressing Nectin-4.

In one embodiment, the antibody or antigen-binding fragment thereof that binds to an epitope of human Nectin-4 is used to assess Nectin-4 expression in a tissue sample from a subject suspected of having cancer, comprising: (a) contacting said tissue sample with the antibody or antigen binding fragment thereof; (b) detecting the binding of said antibody or antigen binding fragment thereof to said tissue sample; and (c) determining the expression of Nectin-4 in the tissue sample, wherein the expression level of Nectin-4 in the tissue sample is compared with a reference expression level of Nectin-4.

In certain embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized, human, or chimeric antibody. In another embodiment, the humanized antibody is a deimmunized antibody or a composite human antibody. In certain embodiments, the antibody is a humanized antibody. In specific embodiments, the antibody is a humanized antibody that specifically binds human Nectin-4.

In certain embodiments, the antibody or antigen-binding fragment thereof is a Fab, a Fab′, a F(ab′)2, a Fv, a scFv, a dsFv, a diabody, a triabody, or a tetrabody. In some embodiments, the antibody or antigen-binding fragment thereof is a multispecific antibody formed from antibody fragments.

In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an agent. In one embodiment, the agent is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.

Also provided herein is a composition comprising an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Also provided herein is a polynucleotide comprising nucleotide sequences encoding a VH, a VL, or both a VH and a VL of an antibody provided herein.

The present disclosure also provides, in certain embodiments, a polynucleotide comprising nucleotide sequences encoding a heavy chain, a light chain, or both a heavy chain and a light chain of an antibody provided herein.

In certain embodiments, the polynucleotide is operably linked to a promoter.

Also provided herein is a population of polynucleotides comprising: (a) a first polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an antibody provided herein, and (b) a second polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an antibody provided herein. In certain embodiments, the first polynucleotide is operably linked to a first promoter, and the second polynucleotide is operably linked to a second promoter.

Also provided herein is a vector comprising a polynucleotide provided herein.

The present disclosure also provides, in certain embodiments, a population of vectors comprising: (a) a first vector comprising nucleotide sequences encoding a VH or a heavy chain of an antibody provided herein, and (b) a second vector comprising nucleotide sequences encoding a VL or a light chain of an antibody provided herein.

The present disclosure further provides, in certain embodiments, a population of vectors comprising: (a) a first vector comprising nucleotide sequences encoding a VH or a heavy chain of an antibody provided herein operably linked to a first promoter, and (b) a second vector comprising nucleotide sequences encoding a VL or a light chain of an antibody provided herein operably linked to a second promoter.

The present disclosure provides, in certain embodiments, a cell comprising a polynucleotide or a population of polynucleotides provided herein.

The present disclosure also provides, in certain embodiments, a cell comprising a vector or a population of vectors provided herein.

The present disclosure further provides, in certain embodiments, an isolated cell producing an antibody or antigen-binding fragment thereof provided herein.

Also provided herein is a population of cells comprising: (a) a first host cell comprising a polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an antibody provided herein, and (b) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an antibody provided herein.

Further provided herein is a population of cells comprising: (a) a first host cell comprising a polynucleotide comprising nucleotide sequences encoding a VH or a heavy chain of an antibody provided herein operably linked to a first promoter, and (b) a second host cell comprising a polynucleotide comprising nucleotide sequences encoding a VL or a light chain of an antibody provided herein operably linked to a second promoter.

Also provided herein is a kit comprising an antibody or antigen-binding fragment thereof provided herein.

Also provided herein is a method of making an antibody or antigen-binding fragment thereof that specifically binds to an epitope of human Nectin-4. In certain embodiments, the method comprises culturing a cell provided herein to express the antibody or antigen-binding fragment thereof. In other embodiments, the method comprises expressing a polynucleotide provided herein. In one embodiment, the epitope of human Nectin-4 is distinct from the Nectin-4 ligand binding site.

