WO2022056329A1

WO2022056329A1 - Snx9 subfamily-targeting antibodies

Info

Publication number: WO2022056329A1
Application number: PCT/US2021/049967
Authority: WO
Inventors: Lauren A. PITT; Karen Lundgren; Jamie STEELE; Casey KILPATRICK; Ben HARMAN; Fang Shen
Original assignee: Immunome, Inc.
Priority date: 2020-09-10
Filing date: 2021-09-10
Publication date: 2022-03-17

Abstract

This invention relates to antibodies specific for members of the human Sorting nexin-9 (SNX9), SNX9 subfamily of Sorting nexin proteins, including SNX18 and SNX33. Antibodies of the invention may be characterized as having complementary determining region (CDR) loops H1, H2, H3, L1, L2, and L3, wherein: the H1 loop amino acid sequence comprises SEQ ID NO: 15; the H2 loop amino acid sequence comprises SEQ ID NO: 16; the H3 loop amino acid sequence comprises SEQ ID NO: 17; the L1 loop amino acid sequence comprises SEQ ID NO: 18; the L2 loop amino acid sequence comprises SEQ ID NO: 19; and the L3 loop amino acid sequence comprises SEQ ID NO: 20. The antibodies are characterized by relatively enhanced internalization by SNX9 subfamily-expressing tumor cells, as well as by enhanced binding at the surface of tumor cells in the presence of SNX9, SNX18, or SNX33 in the tumor microenvironment.

Description

SNX9 Subfamily-Targeting Antibodies

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Application No. 63/076,462, filed on September 10, 2020 which is incorporated by reference herein in its entirety.

Sequence Listing

[0002] The Sequence Listing submitted herewith is an ASCII text file (2021-09-10_Sequence listing_ST25, created on September 10, 2021, 33,392 bytes) via EFS-Web is hereby incorporated by reference.

Field of the Invention

[0003] The field of this invention relates to Sorting nexin-9 (SNX9) subfamily-targeting antibodies.

Background

[0004] The human adaptive immune system responds through both cellular (T cell) and humoral (B cell) processes. The humoral response results in selection and clonal amplification of B cells that express surface bound immunoglobulin (Ig) molecules capable of binding to antigens. The processes of somatic hypermutation and class switching take place concordant with the clonal amplification. Together these processes lead to secreted antibodies that have been affinity matured against a target antigen and contain a constant domain belonging to one of the four general classes (M, D, A, G, or E). Each class of antibody (IgM, IgD, IgA, IgG, and IgE) interact in distinct ways with the cellular immune system.

Hallmarks of antibodies that have been affinity matured against a target antigen can include: 1) nucleotide, and subsequent amino acid, changes relative to the germline gene, 2) high binding affinity for the target antigen, 3) binding selectivity for the target antigen as compared to other proteins.

[0005] It is well understood that oncology patients can mount an immune response against tumor antigens. Those antigens can result either from genetic changes within the tumor that lead to mutated proteins or aberrant presentation, of otherwise normal, proteins to the immune system. Aberrant presentation may occur through processes that include, but are not limited to, ectopic expression of neonatal proteins, over-expression of proteins to a high level, mis-localization of intracellular proteins to the cell surface, or lysis of cells. Aberrant glycosylation of proteins, which may occur because of changes in the expression of enzymes, such as, but not limited to, glycosyltransferases, can also result in generation of non-self antigens that are recognized by the humoral immune system.

[0006] Antibodies, which bind selectively to disease-related proteins, including proteins related to cancer, and more specifically to disease-related proteins that regulate critical cellular processes, have proven successful at modulating the functions of their target proteins in ways that lead to therapeutic efficacy. The ability of the human immune system to mount antibody responses against mutated, or otherwise aberrant proteins, including proteins that are overexpressed or mislocalized, suggests that patients' immune responses may include antibodies that are capable of recognizing, and modulating the function of, critical tumor-drivers.

[0007] The cell membrane is the physical barrier between a cell and its environment, and is comprised of a lipid bilayer that is interspersed with proteins in a mosaic manner (Singer and Nicolson, 1972). "Membrane dynamics" is used to describe the fluid and multifunctional aspect of the membrane that is involved in mediating many cell functions including communication within the cell, with neighboring cells, with the extracellular matrix and maintenance of the cell structure (through biochemical signaling) (Lundmark and Carlsson, 2009. Journal of Cell Science 122, 5-11).

[0008] Sorting nexin-9 (SNX9), a cell membrane-associated protein, was originally discovered as an accessory protein involved in clathrin-mediated endocytosis. SNX9 was subsequently shown to be a multifunctional scaffold protein that spatially and temporally coordinates multiple membrane trafficking events and supports remodeling with changes in actin dynamics. SNX9 has more than 30 binding partners through which it regulates the actin cytoskeleton, membrane signaling, endocytosis, invasion, migration and completion of the cell division cycle (Bendris and Schmid, Trends Cell Biol. 2017 Mar;27(3):189-200). Among more than 30 members of the SNX family, SNX9, SNX18 and SNX33 belong to a subfamily characterized by the presence of an SH3 domain at the N-terminus, a low complexity (LC) domain at the center, and a Bin-Amphiphysin-Rvs (BAR) domain at the C-terminus in addition to a phox- homology (PX) domain (see Figure 1; Sievers et al. Mol. Syst. Biol. 2011, 7: 539; Goujon, et al. Nucleic Acids Res. 2010, 38: W695-W699). The overall homology of SNX9 to SNX18 and SNX33 is 43% and 35%, respectively, with highest homology observed in the PX domain. Given the high sequence similarity, it is possible that functional redundancy exists among the three subfamily members.

[0009] Overexpressed, mislocalized and mutated cell surface proteins are hallmarks of cancer cells. Indeed, SNX9 and related subfamily members have been shown, in some instances, to be mutated, and, in other instances, to be overexpressed or misexpressed in multiple forms of cancer (Trends Cell Biol. 2017 Mar;27(3):189-200). For example, SNX9, SNX18, and SNX33 expression is elevated in multiple types of tumors compared to their adjacent normal tissues in TCGA dataset (Fig. 2A-C; statistical significance computed by Wilcoxon test indicated with stars where *, **, and *** represent p-values of < 0.05, <0.01, and <0.001, respectively; Taiwen et al. Nucleic Acids Res. 2020; Taiwen et al. Cancer Res. 2017 7(21): elO8-ellO; Li et al. Genome Biol., 2016, 17(1): 174). In addition, the TCGA dataset indicates multiple patient samples that show mutations in SNX9, most of which are missense mutations. Such characteristics are consistent with cancer cells acquiring mutations that enable uncontrolled cell division and metastasis to distant sites. Overexpression of SNX9 specifically in lung, cervical, and colorectal cancer correlates with poor prognosis, suggesting SNX9 may drive tumor growth or survival in vivo.

[0010] SNX9 expression knockdown can induce a delay in chromosome alignment and segregation, as well as cytokinesis defects and multinucleation suggesting that it is a regulator of cell division (Ma et al 2012. Journal of Cell Science 125, 4372-4382). Conversely, SNX9 expression can facilitate cell invasion in breast cancer cells through modulation of RhoGTPases involved in metastasis (Brendis et al. Mol Biol Cell. 2016 May;l(9):1409-1419).

[0011] Recent data also point to SNX9 as being a novel angiogenic factor, as SNX9 is essential for human umbilical vein endothelial cells (HUVECs) cell spreading and tube formation, which mimics in vivo angiogenesis. Additionally, overexpression of SNX9 in the tumor endothelial cells of colorectal patients correlates with a poor prognosis (J Cellular Physiology 234: 17280-94; 2019).

[0012] Differential expression of SNX9 in different stages of cancer, and its involvement in cell division, metastasis and apoptotic cellular clearance, make SNX9 a relevant therapeutic target (Lu et al. Mol Biol Cell. 2011 Feb;l(3):354-374, Tanigawa et al. Cellular Physiology. 2019 Jan;(9):17280-17294). Therefore, methods to inhibit, or otherwise modify SNX9 function, or more broadly the SNX9 sub-family including of SNX9, SNX18 and SNX33, including with antibodies specific for SNX9, SNX18 or SNX33, could induce therapeutic effects.

Summary of the Invention

[0013] This invention relates to antibodies specific for human Sorting nexin-9 (SNX9), but may include antibodies that bind to the closely related family members SNX18 and/or SNX33 in addition to SNX9. In that regard, antibodies of the invention include isolated antibodies, or antigen-binding fragments thereof that possess a variable heavy chain (VHC) and a variable light chain (VLC), wherein the VHC contains an amino acid that shares at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 7; and the VLC contains an amino acid that shares at least 90% homology with the amino acid sequence corresponding to SEQ. ID NO: 10.

[0014] More particularly, some antibodies of the invention have complementary determining region (CDR) loops H1, H2, H3, L1, L2, and L3, wherein: the H1 loop amino acid sequence comprises SEQ ID NO: 15; the H2 loop amino acid sequence comprises SEQ ID NO: 16; the H3 loop amino acid sequence comprises SEQ ID NO: 17; the L1 loop amino acid sequence comprises SEQ ID NO: 18; the L2 loop amino acid sequence comprises SEQ ID NO: 19; and the L3 loop amino acid sequence comprises SEQ ID NO: 20.

[0015] Specific binding of some antibodies of the invention to SNX9, SNX18, and/or SNX33, can also occur when the SNX9, SNX18, and SNX33 are components of a multi-protein complex.

[0016] Antibodies of the invention also partially or fully block, inhibit, or neutralize a biological activity of one or more of SNX9, SNX18, and SNX33. Moreover, binding of an antibody of the invention to SNX9, SNX18, or SNX33 at the surface of a cell can be enhanced in the presence of complexed or uncomplexed SNX9, SNX18, or SNX33 in the microenvironment, (e.g., a tumor microenvironment) of the cell, which was not produced or released from the antibody-bound target cell. Indeed, such additional SNX9, SNX18, or SNX33 may be released, or otherwise derived from other cells, including dead or dying tumor cells. For example, the dead or dying tumor SNX9 subfamily-releasing cells may have undergone apoptosis and/or been successfully targeted by a chemotherapeutic agent. Therefore, the invention also includes methods of treating cancers in which an antibody of the invention is administered in combination with one or more chemotherapeutic agents.

[0017] Furthermore, binding of antibodies of the invention to SNX9, SNX18, or SNX33 at the surface of a cell may be followed by internalization of the antibody into a cell, wherein the internalization is enhanced relative to what is observed on normal cells. Thus, antibodies of the invention are well-suited for delivering conjugated drugs into tumor cell targets, including, but not limited to hepatocellular carcinoma (HCC), glioblastoma (GBM), lung adenocarcinoma (LUAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIRP). Brief Description of the Figures

[0018] Fig. 1 shows the sequence alignment of human SNX9, SNX18, and SNX33.

[0019] Fig. 2 A-C displays gene expression in tumor and adjacent normal tissues for all tumors within the TCGA database (compiled using the TIMER2.0 platform) for:

[0020] Fig. 2A SNX9;

[0021] Fig. 2B SNX18; and

[0022] Fig. 2C SNX33.

[0023] Fig. 3 is a graph depicting the signal strength of PR045-25G1 hybridoma-produced antibodies in a primary screen to isolate antibodies that bind to the surface of cancer cells.

[0024] Fig. 4 shows two histograms demonstrating that IMM20065 binds to the surface of HepG2 and Huh7 cancer cells, respectively, as measured by flow cytometry.

[0025] Fig. 5 shows enhanced binding of IMM20065 and a control anti-SNX9 antibody (15721-1-AP) to the surface of multiple cancer cell lines compared to normal cell lines, as measured by flow cytometry.

[0026] Fig. 6 depics binding of IMM20065 to various fragments of recombinant SNX9 by sandwich ELISA.

[0027] Fig. 7 is a graph depicting the ability of IMM20065 to inhibit the growth of HepG2 hepatocellular carcinoma cells, relative to control, in standard cell proliferation assay.

[0028] Fig. 8 is a graph depicting the ability of IMM20065 to inhibit the growth of U87 glioblastoma cancer cells, relative to control, in standard cell proliferation assays.

[0029] Fig. 9 is a graph depicting the ability of IMM20065 to inhibit the growth of A549 lung cancer cells, relative to control, in standard cell-proliferation assays.

[0030] Figs. 10 A-C are photomicrographs of A549 lung cancer cells at 0 and 24 hours after treatment with:

[0031] Fig. 10A buffer, as a negative control;

[0032] Fig. 10B nocodazole, as a positive control; and

[0033] Fig. 10C IMM20065, to evaluate the ability of IMM20065 to inhibit proliferation of cells.

[0034] Fig. 11 is a graph depicting the ability of IMM20065 to induce ADCC against Huh7 and HepG2 hepatocellular carcinoma cells. [0035] Fig. 12 shows enhanced binding of IMM20065 to the surface of Huh7 hepatocellular carcinoma cells with increasing concentrations of full-length and truncated SNX9 (aa 1-362) and freeze-thaw lysate from Huh7 cells.

[0036] Fig. 13 A-C are immunofluorescent photomicrographs of Huh7 hepatocellular carcinoma cells stained with anti-EGFR, IMM20065, and Hoescht stain as well as an overlay at:

[0037] Fig. 13A one hour post-antibody addition;

[0038] Fig. 13B two hours post-antibody addition; and

[0039] Fig. 13C four hours post-antibody addition.

[0040] Fig. 14 A-B depicts binding of IMM20065 to tumor cell leads to internalization relative to an isotype control antibody into:

[0041] Fig. 14A Huh7 hepatocellular carcinoma cells ; and [0042] Fig. 14B OE19 oesophageal carcinoma cells.

Detailed Description

[0043] The invention described herein is directed to compositions and methods related to antibodies and antigen-binding fragments thereof that bind members of the Sorting nexin-9 ("SNX9") subfamily of the SNX family of proteins. In general, antibodies and antigen-binding fragments of the invention are characterized by having an amino acid sequence corresponding to SEQ ID NO: 7 and SEQ ID NO: 10, or a portion thereof. Indeed, an antibody or antigen-binding fragment according to the invention may have an amino acid sequence that shares at least 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or sequence identity with an amino acid sequence corresponding to SEQ ID NO: 7, and/or that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence corresponding to SEQ ID NO: 10, or a portion thereof, or both. As used herein, the term "sequence identity" refers to the similarity between two, or more, amino acid or nucleic acid sequences. Sequence identity is typically measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

[0044] In addition to containing an amino acid sequence corresponding with SEQ ID NO: 7 or 10, an antibody according to the invention also specifically binds to SNX9, but may cross-react to the closely related family members SNX18 and/or SNX33. For example, an antibody according to the invention binds SNX9 expressed on the surface of various cell types, including carcinoma cells of epithelial origin or normal endothelial cells. An antibody according to the invention can also bind SNX9, which has been secreted into an extracellular environment by, for example, carcinoma cells, normal epithelial cells, or by cells of the immune system. SNX9 associated with exosomes, or membrane-bound vesicles is also bound by an antibody according to the invention. Therefore, antibodies described herein can be included in compositions, which are useful for methods of diagnosing or treating various diseases where SNX9 acts to modulate disease progression. Those include, but are not limited to, cancer and diseases resulting from aberrant angiogenesis.

[0045] With respect to structure, an "antibody" according to the invention refers to a polypeptide ligand composed of at least a light chain or heavy chain immunoglobulin variable region that specifically binds an epitope of an antigen. For example, an antibody may be an immunoglobulin molecule composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy ("VH") region and the variable light ("V_L") region. Together, a V_H region and a V_L region form a fragment variable "Fv" that is responsible for the specific binding of the antibody to its antigen.

[0046] Amino acid substitutions, such as, at least one, two, three, four, five, six, or more amino acid substitutions) can be made in the V_H and the V_L regions. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groupings of amino acids are examples of amino acids that are considered to be conservative substitutions for one another: i) Alanine (A), Serine (S), and Threonine (T); ii) Aspartic acid (D) and Glutamic acid (E); iii) Asparagine (N) and Glutamine (Q); iv) Arginine (R) and Lysine (K); v) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); and vi) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

[0047] An antibody according to the invention may be an intact immunoglobulin, or a variant of an immunoglobulin, or a portion of an immunoglobuilin. A naturally occurring immunoglobulin has two heavy (H) chains and two light (L) chains, each of which, contains a constant region and a variable region, and are interconnected by disulfide bonds. There are two types of light chains, which are termed lambda ("A") and kappa ("K"). There are five main heavy chain classes, also known as isotypes, which determine functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. In addition to its variable domain, an IgA, IgD, or IgG heavy chain has three constant domains (CHI, CH2, CH3). IgM and IgE heavy chains have four constant domains (CHI, CH2, CH3, CH4).

[0048] Light and heavy chain variable regions contain "framework" regions interrupted by three hypervariable regions, called complementarity-determining regions ("CDRs"). The CDRs are primarily responsible for binding to an epitope of an antigen. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, and serve to position and align the CDRs in three-dimensional space. The three CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are often identified by the chain in which the particular CDR is located. Accordingly, heavy chain CDRs are designated H-CDR1, H-CDR2, and H-CDR3; likewise, light chain CDRs are designated L-CDR1, L-CDR2, and L-CDR3. An antigen-binding fragment, one constant and one variable domain of each of the heavy and the light chain is refered to as an Fab fragment. An F(ab)'₂ fragment contains two Fab fragments, and can be generated by cleaving an immunoglobulin molecule below its hinge region.

[0049] Amino acid sequences of V_H and V_L framework and complementary regions of an antibody according to the invention correlate with SEQ ID NOS: 7 and 10, respectively. More particularly, based on the definition described by North, B. et al. (A new clustering of antibody CDR loop conformations, J Mol Biol (2011)), H-CDR1, H-CDR2, and H-CDR3 correspond to residues 23-35 (SEQ. ID NO: 15), 50-58 (SEQ. ID NO: 16), and 96 - 116 (SEQ ID NO: 17) of SEQ ID NO: 7. The analagous L-CDR1, L-CDR2, and L- CDR3 amino acid sequences of an antibody according to the invention correspond to residues 24-34 (SEQ ID NO: 18), 49-56 (SEQ ID NO: 19), and 89-97 (SEQ ID NO: 20) of SEQ ID NO: 10. An antibody according to the invention contains at least one of the foregoing CDR sequences; therefore, the combination of CDRs of an antibody may be, for example: (H-CDR1 and L-CDR1); (H-CDR2 and L-CDR2); (H-CDR3 and L-CDR3); (H-CDR1, L-CDR1, H-CDR2 and L-CDR2); (H-CDR1, L-CDR1, H-CDR3 and L-CDR3); (H-CDR2, L-CDR2, H-CDR3 and L-CDR3); or (H-CDR1, L-CDR1, H-CDR2, L-CDR2, H-CDR3 and L-CDR3). [0050] Antibodies according to the invention are monoclonal antibodies, meaning an antibody is produced by a single clonal B-lymphocyte population, a clonal hybridoma cell population, or a clonal population of cells into which the genes of a single antibody, or portions thereof, have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune lymphocyte cells.

[0051] Monoclonal antibodies according to the invention are also typically humanized monoclonal antibodies. More specifically, a "human" antibody, also called a "fully human" antibody, according to the invention, is an antibody that includes human framework regions and CDRs from a human immunoglobulin. For example, the framework and the CDRs of an antibody are from the same originating human heavy chain, or human light chain amino acid sequence, or both. Alternatively, the framework regions may originate from one human antibody, and be engineered to include CDRs from a different human antibody. [0052] An antibody according to the invention may also be an immunoglobulin fragment. Examples of immunoglobulin variants that are considered antibodies according to the invention include single- domain antibodies (such as VH domain antibodies), Fab fragments, Fab' fragments, F(ab)'₂ fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A VH single-domain antibody is an immunoglobulin fragment consisting of a heavy chain variable domain. An Fab fragment contains a monovalent antigen-binding immunoglobulin fragment, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. Similarly, an Fab' fragment also contains a monovalent antigen-binding immunoglobulin fragment, which can be produced by digestion of whole antibody with the enzyme pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per immunoglobulin molecule. An (Fab')₂ fragment is a dimer of two Fab' fragments, that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, so Fab' monomers remain held together by two disulfide bonds. An Fv fragment is a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains. A single chain ("sc") antibody, such as scFv fragment, is a genetically engineered molecule containing the V_L region of a light chain, the V_H region of a heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. A dimer of a single chain antibody, such as a scFV₂ antibody, is a dimer of a scFV, and may also be known as a "miniantibody". A dsFvs variant also contains a V_L region of an immunoglobulin and a V_H region, but the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0053] One of skill in the art will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V_H and the V_L regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules.

[0054] An antibody according to the invention may also comprise a "tagged" immunoglobulin CH3 domain to faciliate detection of the biologic against a background of endogenous antibodies. More particularly, a tagged CH3 domain is a heterogenous antibody epitope that has been incorporated into one or more of the AB, EF, or CD structural loops of a human IgG-derived CH3 domain. CH3 tags are preferably incorporated into the structural context of an IgGl subclass antibody, other human IgG subclasses, including lgG2, lgG3, and lgG4, are also available according to the invention. Epitope-tagged CH3 domains, also referred to as "CH3 scaffolds" can be incorporated into any antibody of the invention having a heavy chain constant region, generally in the form of an immunoglobulin Fc portion. Examples of CH3 scaffold tags, and methods for incorporating them into antibodies are disclosed in PCT/US2019/032780. Antibodies used to detect epitope tagged CH3 scaffolds, and antibodies of the invention, that comprise epitope tagged CH3 scaffolds, are generally referred to herein as "detector antibodies".

[0055] Therapeutic and diagnostic effectiveness of an antibody according to the invention correlates with its binding affinity for its target antigen. Binding affinity may be calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Alternatively, binding affinity may be measured by the dissociation rate of an antibody from its antigen. Various methods can be used to measure binding affinity, including, for example, surface plasmon resonance (SPR), competition radioimmunoassay, ELISA, and flow cytometry. An antibody that "specifically binds" an antigen is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens. In general, an antibody exhibits "high affinity binding" if its dissociation constant value ("KD") is 10 nM, or less. Therefore, an antibody according to the invention exhibits high affintiy binding if the KD between the antibody and SNX9, SNX18, or SNX33 is 10 nM or less. For example, an antibody according to the invention exhibits high affinity binding to SNX9, SNX18, or SNX33 if the K_D value is 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less.

[0056] High affinity binding of an antibody to its antigen is mediated by the binding interaction of one or more of the antibody's CDRs to an epitope, also known as an antigenic determinant, of the antigen target. Epitopes are particular chemical groups or peptide sequences on a molecule that are antigenic, meaning they are capable of eliciting a specific immune response. An epitope that is specifically bound by an antibody according to the invention may be formed by a linear sequence of amino acids contained within SNX9, SNX18, or SNX33. Such an epitope is called a "linear epitope", and it may remain functional with respect to the specific binding of an antibody according to the invention to a denatured form of SNX9, SNX18, or SNX33. Alternatively, the specific binding of an antibody according to the invention may depend on a particular three-dimensional structure of the SNX9, SNX18, or SNX33 target, such that the contributing residues of an epitope are not necessarily in a linear sequence. In other words, an epitope of an antibody according to the invention may be a "conformational epitope". [0057] Therapeutic and diagnostic uses of antibodies according to the invention may include uses of immunoconjugates. As described herein, an immunoconjugate is a chimeric molecule, which comprises an effector molecule linked to an antibody according to the invention. As referred to herein, an effector molecule is the portion of an immunoconjugate that is intended to have a desired effect on a cell to which the immunoconjugate is targeted, or an effector molecule may serve to increase the half-life or bioavailability of an antibody according to the invention. General examples of effector molecules include therapeutic agents, (such as toxins, radionuclides, and chemotherapeutic drugs), diagnostic agents, (such as radionuclides and fluorescent markers), and half-life and bioavailability-enhancing molecules, (such as lipids or polyethylene glycol).

[0058] Effector molecules can be conjugated to antibodies according to the invention using any number of means known to those of skill in the art, including covalent and noncovalent attachment means. The procedure for attaching an effector molecule to an antibody may vary according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as a carboxylic acid (COOH) group, a free amine (--NH₂), and a sulfhydryl (SH) group, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, an antibody according to the invention can be derivatized to expose, or attach, additional reactive functional groups. Derivatization may involve attachment of any of a number of known linker molecules, which serve to join an antibody to an effector molecule.

[0059] A linker molecule is capable of forming covalent bonds to the antibody and effector molecule. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. If an effector molecule is a polypeptide, a linker may be joined to the constituent amino acids of the polypeptide through their side groups, such as through a disulfide linkage to cysteine, or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

Recombinant technology may be used to make two or more polypeptides, including linker peptides, into one contiguous polypeptide molecule.

[0060] An effector molecule may also be contained within a directly attached, or linked encapsulation system, that shields the effector molecule from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Pat. No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985).

[0061] The effector molecules of immunoconjugates according to the invention are generally useful for the treatment of cancer, and diseases characterized by abnormal cell growth, generally. Accordingly, the effector molecules of immunoconjugates according to the invention can be chemotherapeutic agents, including: small molecule drugs; nucleic acids, such as antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides; proteins; peptides; amino acids, and amino acid derivatives; glycoproteins; radioisotopes; lipids; carbohydrates; recombinant viruses; and toxins, such as, but not limited to, abrin, ricin, Pseudomonas exotoxin ("PE", such as PE35, PE37, PE38, and PE40), diphtheria toxin ("DT"), botulinum toxin, saporin, restrictocin, gelonin, bouganin, and modified toxins thereof.

[0062] In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of a linker to release the effector molecule from an antibody according to the invention may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. Alternatively, after specifically binding its target antigen, an antibody according to the invention can be internalized by the cell expressing the target antigen.

[0063] Therapeutic antibodies according to the invention, including therapeutic immunoconjugates, can be used in methods for preventing, treating, or ameliorating a disease in a subject. In certain embodiments of the invention, antibodies according to the invention can be used for preventing, treating, or ameliorating cancer in a subject. For example, antibodies according to the invention can be used to prevent, treat or ameliorate hepatocellular carcinoma (HCC), glioblastoma (GBM), lung adenocarcinoma (LUAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIRP). In other embodiments, antibodies according to the invention can be used for preventing, treating, or ameliorating diseases related to aberrant angiogenesis. Examples, of diseases related to aberrant angiogenesis include, but are not limited to, tumor growth and metastisis, diabetic retinopathy, macular degeneration, such as non-exudative or exudative age-related macular degeneration, or psoriasis.

[0064] "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease. With respect to the use of antibodies according to the invention to prevent, treat or ameliorate cancer, signs or symptoms of the disease may correlate to tumor burden or the number or size of metastases. Whereas, signs and symptoms of a disease associated with aberrant angiogenesis may correlate to aberrant vessel formation, or an abnormal vessel structure.

[0065] A method for preventing, treating, or ameliorating cancer may require the administration of a composition, comprising an effective amount of an antibody according to the invention, to a subject to inhibit tumor growth or metastasis, comprising selecting a subject with a cancer characterized by tumor cells that expresses the antigen target of the antibody, or otherwise present cell membrane-associated target antigens of an antibody according to the invention. For example, an antibody according to the invention can contact a tumor cell via binding to its target antigen, to modulate, inhibit, or neutralize the target antigen's function. An antibody according to the invention may also deliver cytotoxic therapy upon binding to its target antigen on the surface of a tumor cell. An antibody according to the invention can also contact tumor-associated stromal cells via binding to its target antigen, to impair target- mediated functions including endothelial cell migration and proliferation, which are required for tumor angiogenesis.

[0066] An antibody according to the invention may also bind a target antigen that has been secreted, or otherwise released, from a cell, either as a soluble target antigen or in the context of a membrane bound vesicle, such as an exosome. Thus, an antibody according to the invention can bind to an extracellular target, either a soluble target antigen or a target in the context of a membrane-bound vesicle, in a fluid, such as, but not limited to, blood or a blood derivative, like plasma and serum, or a fluid within a tumor microenviroment. As is the case with a cell membrane-attached target antigen, binding of an antibody according to the invention to an extracellular target antigen can result in the modulation, inhibition, or neutralization of the target antigen's biological function. Thus, the binding of an antibody according to the invention to its extracellular target antigen, can, for example, modulate, inhibit, or neutralize its target antigen's cancer promoting, or angiogenic, activity in vivo. In some methods of the invention, a target antigen may be released from one cell, and then bind to surface of another cell. As such, in certain methods of the invention for treating a cancer, SNX9, SNX18, or SNX33 may be released from one cell, and then bind to the surface of a tumor cell, where, in turn, it is targeted by an antibody of the invention. Similarly, an antibody of the invention may bind SNX9, SNX18, or SNX33 that was mislocalized by the target tumor cell.

[0067] There are also uses of antibodies according to the invention, in which, an antibody binds a target antigen that is associated with an extracellular matrix ("ECM") or ECM protein. For example, an antibody according to the invention may bind to a target antigen that is, itself associated with an ECM that migrating or differentiating cancer or endothelial cells are attached, or would be expected to encounter, to modulate, inhibit, or neutralize its target antigen's role in angiogensis or metastasis. The presence of ECM-associated target antigen may correlate with various disease states, including diseases associated with aberrant angiogenesis, or the presence of ECM-associated target antigen in the tumor micro environment.

[0068] As stated above, antibodies disclosed herein can be administered to slow or inhibit the growth of primary tumors or inhibit the metastasis of various types of tumors. For example, antibodies according to the invention can be administered to slow or inhibit the growth or metastasis of cancers, including but not limited to, hepatocellular carcinoma (HCC), glioblastoma (GBM), lung adenocarcinoma (LUAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIRP). In these applications, a therapeutically effective amount of an antibody is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer, in which the tumor cells express a target antigen of an antibody according to the invention. A therapeutically effective amount of an antibody according to the invention will depend upon the severity of the cancer, and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified professional.

[0069] Antibodies according to the invention may be administered to a subject to attenuate the growth of blood vessels, or decrease the permeability of blood vessels, caused by expression of the target antigen. For example, antibodies according to the invention can be administered to attenuate the growth of blood vessels, thereby leading to a reduction of symptoms associated with aberrant angiogenesis, such as non-exudative or exudateive age-related macular degeneration. In another, non- limiting example, antibodies according to the invention can be administered to slow or inhibit the growth of blood vessels associated with diabetic retinopathy. In these applications, a therapeutically effective amount of an antibody is administered to a subject in an amount sufficient to normalize disease-associated blood vessels, or to inhibit a sign or a symptom of the disease. Suitable subjects may include those diagnosed with diabetic retinopathy, macular degeneration, such as non-exudative or exudative age-related macular degeneration, psoriasis, or a cancer, in which involved tissues express a target antigen of an antibody according to the invention. A therapeutically effective amount of an antibody according to the invention will depend upon the severity of the disease, the location of the disease, and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by a clinician or other qualified professional.

[0070] Antibodies according to the invention, that are administered to subjects in need thereof, are formulated into compositions. More particularly, the antibodies can be formulated for systemic administration, or local administration, such as intra-tumor administration. For example, an antibody according to the invention may be formulated for parenteral administration, such as intravenous administration. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.

[0071] Administration of antibodies according to the invention can also be accompanied by administration of other anti-cancer agents or therapeutic treatments, such as surgical resection of a tumor. Any suitable anti-cancer agent can be administered in combination with the antibodies disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti- survival agents, biological response modifiers, immune modulators, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0072] Administration of antibodies according to the invention can also be accompanied by administration of other modulators of blood vessel formation. Any suitable angiogenesis modulating agent can be administered in combination with the antibodies disclosed herein. Exemplary anti- angiogeneis agents include, but are not limited to, biological response modifiers, such as the anti-VEGF antibody bevacizumab.

[0073] The compositions for administration can include a solution of the antibody dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, or glycerol as a vehicle. For solid compositions, such as powder, pill, tablet, or capsule forms, conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. The foregoing carrier solutions are sterile and generally free of undesirable matter, and may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and toxicity adjusting agents such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.

[0074] Options for administering an antibody according to the invention include, but are not limited to, administeration by slow infusion, or administration via an intravenous push or bolus. Other options for antibody administration may be optimized for intraocular administration. Prior to being administered, an antibody composition according to the invention may be provided in lyophilized form, and rehydrated in a sterile solution to a desired concentration before administration. The antibody solution may then be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight. In one example of administration of an antibody composition accroding to the invention, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated. [0075] Antibody compositions according to the invention may also be controlled release formulations. Controlled release parenteral formulations, for example, can be made as implants, or oily injections.

Particulate systems, including microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles, may also be used to deliver antibody compositions according to the invention. Microcapsules, as referred to herein, contain an antibody according to the invention as a central core component. In microspheres, an antibody according to the invention is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. [0076] As described above, antibodies according to the invention may also be useful for diagnosing or monitoring the presence of a pathologic condition, such as, but not limited to, a cancer, diabetic retinopathy, macular degeneration, such as non-exudative or exudative age-related macular degeneration, or psoriasis. More particularly, methods of the invention are useful for detecting expression of the antigen target of an antibody according to the invention. Detection may be in vitro or in vivo. Any tissue sample may be used for in vitro diagnostic detection, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine.

[0077] A method determines if a subject has a disease by contacting a sample from the subject with an antibody according to the invention; and detecting binding of the antibody to its target antigen present in the sample. An increase in binding of the antibody to its target antigen in the sample, as compared to binding of the antibody in a control sample identifies the subject as having a disease associated with SNX9, SNX18, or SNX33 expression, such as, for example: cancer; diabetic retinopathy; macular degeneration, including non-exudative and exudative age-related macular degeneration; or psoriasis., or any other type of disease that expresses SNX9, SNX18, or SNX33. In general, a control sample is a sample from a subject without disease.

[0078] Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Prognostic" is the probability of development (e.g., severity) of a pathologic condition, such as the development or metastasis of hepatocellular carcinoma (HCC), glioblastoma (GBM), lung adenocarcinoma (LUAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIRP).

[0079] Antibodies of the invention can be linked to a detectable label to form immunoconjugates that are useful as diagnostic agents. A detectable label, as referred to herein, is a compound or composition that is conjugated directly or indirectly to an antibody according to the invention, for the purpose of facilitating detection of a molecule that correlates to presence of a disease, such as, for example, a tumor cell antigen that is the antigen target of an antibody according to the invention. Detectable labels useful for such purposes are well known in the art, and include: radioactive isotopes, such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, technetium-99m ("^99mTc), ¹²⁴l, ¹³¹l, ⁸⁹Zr, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ¹¹¹ln and ¹²⁵l; fluorophores; chemiluminescent agents; enzymatic labels, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter, such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags; and magnetic agents, such as gadolinium chelates. A labeled antibody according to the invention may also be referred to as a "labeled antibody", or more specifcally a "radiolabeled antibody". For some antibodies according to the invention, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0080] A diagnostic method comprising a step of using an antibody according to the invention may, in certain applications, be an immunoassay. While the details of immunoassays may vary with the particular format employed, a method of detecting the antigen target of an antibody according to the invention in a biological sample generally includes the steps of contacting the biological sample with the antibody which specifically reacts with the antigen, under immunologically reactive conditions to form an immune complex. The presence of the resulting immune complex can be detected directly or indirectly. In other words, an antibody according to the invention can function as a primary antibody (1° Ab) in a diagnostic method, and a labeled antibody, specific for the antibody according to the invention, functions as the 2° Ab. In the case of indirect detection of an immune complex, the use of an antibody according to the invention for a diagnostic method will also include the use of a labelled secondary antibody (2° Ab) to detect binding of the primary antibody - the antibody according to the invention - to its target antigen. Suitable detectable labels for a secondary antibody include the labels, described above, for directly labled antibodies according to the invention. A 2° Ab, used in a diagnostic method according to the invention, may also be a "detector antibody", as defined, above, for use in conjunction with an antibody according to the invention that contains a CH3 epitope tag, as described in U.S. Provisional Patent Application No. 62/672,738.

[0081] Antibodies according to the invention can also be used for fluorescence activated cell sorting (FACS). A FACS analysis of a cell population employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Pat. No. 5,061,620). [0082] Reagents used in a diagnostic application of an antibody according to the invention, as described above, may be provided in a kit for detecting the antigen target of an antibody according to the invention a biological sample, such as a blood sample or tissue sample. Such a kit can be used to confirm a cancer diagnosis in a subject. For example, a diagnostic kit comprising an antibody according to the invention can be used to perform a histological examination for tumor cells in a tissue sample obtained from a biopsy. In a more particular example, a kit may include antibodies according to the invention that can be used to detect lung cancer cells in tissue or cells obtained by performing a lung biopsy. In an alternative, particular example, a kit may include antibodies according to the invention that can be used to detect pancreatic cancer cells in a tissue biopsy. Kits for detecting an antigen target of an antibody according to the invention will typically comprise an antibody according to the invention in the form of a monoclonal antibody, or a fragment thereof, such as an scFv fragment, a VH domain, or a Fab. The antibody may be unlabled of labeled by a detectable marker, such as a fluorescent, radioactive, or an enzymatic label, as described above. A kit also generally includes instructional materials disclosing means of use of an antibody according to the invention. The instructional materials may be written, in an electronic form, such as a portable hard drive, and the materials also be visual, such as video files. Instructional materials may also refer to a website or link to an application software program, such as a mobile device or computer "app", that provides instructions. A kit may also include additional components to facilitate the particular application for which the kit is designed. For example, a kit may also contain a means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). Buffers and other reagents, which are routinely in methods of using an antibody according to the invention for diagnositc purposes.

[0083] Antibodies according to the invention can be produced by various recombinant expression systems. In other words, the antibodies can be produced by the expression of nucleic acid sequences encoding their amino acid sequences in living cells in culture. An "isolated" antibody according to the invention is one which has been substantially separated or purified away from other biological components environment, such as a cell, proteins and organelles. For example, an antibody may be isolated if it is purified to: i) greater than 95%, 96%, 97%, 98%, or 99% by weight of protein as determined by the Lowry method, and alternatively, more than 99% by weight; ii) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; iii) homogeneity by SDS-PAGE, under reducing or nonreducing conditions, using Coomassie blue or silver stain. Isolated antibody may also be an antibody according to the invention that is in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. [0084] A variety of host-expression vector systems may be utilized to express an antibody according to the invention, by transforming or transfecting the cells with an appropriate nucleotide coding sequences for an antibody according to the invention. Examples of host-expression cells include, but are not limited to: Bacteria, such as E.coli and 8. Subtilis, which may be transfected with antibody coding sequences contained within recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors; Yeast, such as Saccharomyces and Pichia, transformed with recombinant yeast expression vectors containing antibody coding sequences; Insect cell systems, infected with recombinant virns expression vectors, such as baculovinls, containing antibody coding sequences; Plant cell systems infected with recombinant vims expression vectors, such as cauliflower mosaic virus ("CaMV"), or tobacco mosaic vims ("TMV"), containing antibody coding sequences; and Mammalian cell systems, such as, but not limited to COS, Chinese hamster ovary ("CHO") cells, ExpiCHO, baby hamster kidney ("BHK") cells, HEK293, Expi293, 3T3, NSO cells, harboring recombinant expression constructs containing promoters derived from the genome of mammalian cell, such as the metallothionein promoter or elongation factor I alpha promoter, or from mammalian viruses, such as the adenovirus late promoter, and the vaccinia virus 7.5K promoter. For example, mammalian cells such as Human Embryonic Kidney 293 (HEK293) or a derivative thereof, such as Expi293, in conjunction with a dual promoter vector that incorporates mouse and rat elongation factor 1 alpha promoters to express the heavy and light chain fragments, respectively, is an effective expression system for antibodies according to the invention, which can be advantageously selected, depending upon the use intended for the antibody molecule being expressed.

[0085] When a large quantity of an antibody according to the invention is to be produced for the generation of a pharmaceutical composition of the antibody, vectors which direct the expression of high levels of readily purified fusion protein products may be desirable. Such vectors include, but are not limited to: a pUR278 vector (Ruther et al. EMBOJ. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with a lac Z coding region so that a fusion protein is produced; a pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985) [0086] A host expression cell system may also be chosen which modulates the expression of inserted sequence(s) coding for an antibody according to the invention, or modifies and processes the gene product as desired. For example, modifications, including the glycosylation and processing, such as cleavage of protein products, may be important for the function of the protein. Indeed, different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of proteins and gene products. To this end, eukaryotic host cells, which possess appropriate cellular machinery for proper processing of a primary transcript, as well as the glycosylation and phosphorylation of a gene product according to the invention may be used.

[0087] The vector used to produce an antibody according to the invention comprises a nucleic acid molecule encoding at least a portion of that particular antibody. For example, such a nucleic acid sequence can comprise a DNA sequence corresponding to SEQ. ID NO: 5, or a portion thereof. Thus, a first nucleic acid encoding at least a portion of an antibody according to the invention, that is operably linked with a second nucleic acid sequence that is placed in a functional relationship with the first nucleic acid sequence, such as a promoter, is a nucleic acid according to the invention. An operable linkage exists if a linked promoter sequence affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, may also join two, or more, protein-coding regions, in the same reading frame.

[0088] When a nucleic acid comprising a DNA sequence according to the invention is substantially separated or purified away from other biological components in the environment, such as a cell, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles, it may be considered to be an "isolated nucleic acid" according to the invention. For example, a nucleic acid, which has been purified by standard purification methods, is an isolated nucleic acid.

[0089] Nucleic acids according to the invention also include degenerate variants of nucleotides encoding an antibody according to the invention. More particularly, a "degenerate variant" refers to a polynucleotide, which encodes an antibody according to the invention, but is degenerate as a result of the genetic code. All degenerate nucleotide sequences are included, according to the invention, as long as the amino acid sequence of the encoded antibody specifically binds the antigen target of an antibody according to the invention.

Examples

[0090] The following Examples describe the isolation and characterization of an antibody, IMM20065, which binds to an epitope on SNX9. SNX9, can, in certain contexts, be associated with a cell membrane, including at a cell surface. Specific binding of IMM20065 is also described. [0091] Example 1. Isolation of a human hybridoma producing an antibody that binds the surface of intact human cancer cells. PR045-25G1 hybridoma cells were generated from the fusion of human B cells isolated from the lymph node of a head and neck cancer patient, with the B56T fusion partner. Fusion of human B cells with B56T was carried out by electrofusion essentially as described in U.S. Pat. No. 8,999,707. Post-fusion, hyridomas were plated and allowed to grow for approximately two weeks. Conditioned medias from IgG/A-positive hybridomas were then collected and screened for the ability of the antibodies to bind to the surface of cancer cell lines. Binding of PR045-25Gl-produced Abs to pools of live, intact cancer cell lines was detected using fluorophore-labelled anti-human IgG secondary Abs and a LI-COR Odyssey™ Sa imaging system configured for 96-well plates. Prior to screening, the cancer cells were mixed in equal proportions and the pools were aliquoted into 96-well plates and allowed to attach for 24 hours. Hybridoma supernatants were incubated with cells and binding to the cancer cell lines was assessed relative to a positive control comprising a mixture of anti-basigin, anti-EGFR, and anti- ERBB2 (BCH) antibodies in equal ratios. The BCH positive control was incubated with cells at 33 and 3 ng/mL of each antibody. An anti-CD29 antibody was also used as a positive control. The anti-CD29 antibody was incubated at 100 and 10 ng/mL of each antibody. Conditioned media from an Ig-negative hybridoma, plated in four different wells was used as a negative control to determine assay background. The combination of controls provides a range of absolute signal intensities across both the cell line pools and the detection range of the LI-COR instrument. Signals for PR045-25G1 exhibited a signal above background against all cell pools tested. The background subtracted signal for PR045-25G1, against cell pool A, was approximately 4.2-fold and 1.75-fold higher than the signals for the lowest two positive controls, BCH (3ng/mL) and anti-CD29 (lOng/mL), respectively (Fig. 3).

[0092] Example 2. The PR045-25G1 hybridoma produces an IgG comprising IGHV/IGV variable domains. Nucleotide sequences, encoding the variable heavy chain (VH) and variable light chain (V_L) domains of PR045-25G1, were obtained by RT-PCR amplification of RNA isolated from cells of the hybridoma line that produced PR045-25G1, and subjecting the resulting antibody cDNA to sequencing reactions. SEQ ID NO: 1 corresponds to the nucelotide sequence of the V_H and SEQ ID NO: 3 corresponds to the nucleotide sequence of the V_L of PR045-25G1 isolated from the hybridoma. SEQ ID NO: 2 and SEQ ID NO: 4 correspond to the corresponding amino acid sequences of the V_H and V_L of PR045-25G1 isolated from the hybridoma. IGHV4-34 and IGKV1-5 gene assignments were predicted based upon homology to known germline gene sequences, and were used to generate the 5' ends of the V_H and V_L to create full-length coding sequences, represented by SEQ ID NO: 5 and SEQ ID NO: 8, which encode the amino acid sequences SEQ ID NO: 7 and SEQ ID NO: 10, respectively. A two plasmid system was used to facilitate recombinant expression of antibodies that contain the variable domains of PR045- 25G1 with IgGl heavy chain and kappa light chain constant domains. Codon optimization was carried out on SEQ. ID NO: 5 and a nucleotide fragment corresponding to SEQ ID NO: 6, which encodes the amino acid sequence correpsonding to SEQ ID NO: 7 was synthesized to faciliate expression of antibodies comprising the VH domain of PR045-25G1. Codon optimization was also carried out on SEQ ID NO: 8 and a nucleotide fragment corresponding to SEQ ID NO: 9, which encodes SEQ ID NO: 10, was synthesized to facilitate expression of antibodies comprising the VL domain of PR045-25G1. Vectors expressing either the heavy chain or light chain of PR045-25G1 were generated by synthesis and cloning of V_H and V_L domains into a two vector system that encode for a full-length IgGl antibody comprised of amino acid sequences corresponding to SEQ ID NO: 12 and SEQ ID NO: 14. Antibodies containing the PR045-25G1 V_H and V_L domains, were expressed recombinantly by transient transfection into mammalian cell lines, such as Chinese Hampster Ovary (CHO) and human embryonic kidney (HEK), using standard conditions. Recombinant antibodies, referred to as I M M20065, were purified from conditioned media by affinity chromatography using techniques well known to one of ordinary skill in the art.

[0093] IMM20065 antibodies displayed binding activity consistent with the original PR045-25G1 hybridoma-produced antibody. As depicted in Fig. 4 IMM20065-IMM binds to the surface of live HepG2 and Huh7 cells when analyzed by flow cytometry. More broadly, IMM20065 demonstrates enhanced binding to the surface of a range of cancer cell lines compared to a panel of normal cell lines, as shown in Fig. 5 and Table 1. Enhanced binding to cancer over normal cell lines is also observed for a control anti-SNX9 antibody (Fig. 5; catalog number 15721-1-AP from Proteintech). The values for relative change mean fluorescent intensity (MFI) in Fig. 5 are calculated by subtracting the MFI of the isotype control from the antibody of interest and then normalizing to the MFI of the isotype control.

[0094] Example 3. IMM20065 Ab binds an epitope on SNX9. To identify the target antigen bound by IMM20065, the antibody was screened against CDI HuProt arrays where target proteins were spotted in native format. More specifically, IMM20065 (1 microgram /mL) was incubated overnight at 4 degrees C against the native GDI HuProt array. Slides were washed and IMM20065 binding was detected with an Alexa-647 conjugated anti-H+L secondary antibody. Non-specific hits that were bound by the secondary were eliminated from any analysis. Selective binding to target proteins were analyzed by a combination of overall signal intensity, Z-score to determine reproducibility of binding to replicates on each slide, and S-score to determine difference in selectivity versus possible targets. An S-score >3 between the top and second-ranked hits is considered as highly specific for the top hit.

[0095] As shown in Table 2. IMM20065 bound in a highly reproducible and selective manner to SNX9. Binding to SNX9 exhibited an approximate 10-fold higher absolute intensity and an approximate 10-fold higher Z-score than observed for CABP4, the second-ranked target on the array. This was coupled with an S-score of 132, which is 44-fold greater than the baseline cut-off for a highly selective hit.

[0096] Binding of IMM20065 to recombinant SNX9 was further quantified by surface plasmon resonance using a BIAcore4000 and an anti-huFc coated CM5 sensor chip. Experiments were run at 25 degrees C in running buffer comprising 10nM HEPES, pH7.4, 150mM NaCI, 0.0005% Tween-20, 0.2% BSA. IMM20065 was diluted in running buffer to 100nM and 25nM and captured at four different surface densities from 200 - 800 response units (RU). Human SNX9 (Origene, catalog number TP302822) was then tested in a 3-fold dilution series up to 150nM across all four IMM20065 surface densities. The data from all four surfaces were fit to a 1:1 interaction model, demonstrating high affinity binding with kinetic constants listed in Table 3. Further SPR analysis was carried out to examine specificty for human SNX9 as compared to closely related family members SNX18 and SNX33. As with the initial SPR experiment, IMM20065 was captured at four different densities on a CM5 sensor chip. Recombinant human SNX9, 18 (Origene, catalog number TP319205), and 33 (Origene, catalog number TP305879) were run over the surfaces in 3-fold dilution series up to 100nM. At all four surface densities, SNX9 exhibited dose-dependent binding that could be fit to a 1:1 binding model. SNX18 and SNX33 failed to generate interpretable binding curves at the lowest, and two lowest, surface densities of IMM20065, respectively. At the higher surface densities binding could be observed (Table 4). Human SNX9 was re- run on all four surfaces under identical conditions and exhibited reproducible binding curves, supporting the fact that the lack of binding observed with SNX18 and SNX33 at lower surface densities was not due to inactive IMM20065 surfaces. The eight replicates of SNX9 binding, the 3 replicates of SNX18 binding, and 2 replicates of SNX33 binding yielded binding constants listed in Table 4. The data support at least a 10-fold selectivity for SNX9 as compared to the other two SNX proteins tested.

[0097] Binding of IMM20065 to various fragments of recombinant SNX9 was performed by ELISA in an attempt to determine the epitope of the antibody (Fig. 6). His- or FLAG-tagged recombinant SNX9 was captured on anti-His or anti-FLAG-coated high-binding plates, respectively (aa 6-75, Atlas Antibodies; catalog number APrEST91791; aa 154-243, Atlas Antibodies, catalog number APrEST77526; aa 1-362, Proteintech, catalog number Ag8446; aa 250-595, LSBio, catalog number LS-G25539; full-length SNX9, Origene, catalog number TP302822). IMM20065 and an isotype control were added followed by an appropriate HRP-labeled secondary antibody and OPD substrate and binding was quantified using an Enspire plate reader. As shown in Figure 6, IMM20065 bound to a fragment of SNX9 encompassing amino acids 1-362 and to another fragment containing amino acids 250-595 in addition to full-length protein. This data suggest that IMM20065 binds to a region within the PX domain of SNX9, which is made up of amino acids 250-361.

[0098] Example 4. IMM20065 inhibits growth of antigen-positive cancer cell lines. Various antigen- positive cancer cell lines were seeded at a sufficient density to ensure the cells have undergone three to four doublings throughout the experiment in black-walled clear bottom 384-well plates (Corning™ #3764). Cells were seeded on day 0 at the following densities in 50 μL of pre-warmed media: HepG2: 1000 cells/well, A549 250 cells/welI, U87MG 250 cells/well. Cells were attached for 24 h, 25 μL of media was removed and replaced with media containing a serial dilution of IMM20065 starting at a final concentration of 100 μg/mL down to 0.4 μg/mL or a parallel vehicle only control. Proliferation was monitored over five days using the cell confluency mask on an IncuCyte® Live Cell Analysis Systems (Satorius). At the end of the experiment cell viability was assessed using CelITiter-Glo® Luminescent Cell Viability Assay (Promega #G7570) and normalized to untreated cells. As depicted in Figures 7 - 9, IMM20065 was able to inhibit the growth of all three cell lines in a dose-dependent manner. Images of the A549 cells at 0 and 24 hours after addition of test agents (Figures 10A- 10C) are consistent with the dose-response curve of IMM20065. Within 24 hours of treating cells with IMM20065, inhibition of growth can be observed.

[0099] Example 5. IMM20065 can direct ADCC activity against antien-positive cancer cells.

Opsinization of cancer cells with an antibody specific for a cell surface protein can engage immune effector cells, for example Natural Killer (NK) cells, and direct the killing of the cancer cells through the process of antibody-dependent cellular cytocixity (ADCC). Using standard assays, such as those exemplified by Promega's ADCC Reporter Assay (catalog # G7010), IM20065 was assessed for its ability to direct ADCC against antigen-positive cancer cells. As shown in Fig. 11, IMM20065 induced a dose- dependent ADCC effect against Huh7 and HepG2 cells, relative to an isotype control.

[00100] Example 6. Recombinant SNX9 protein and SNX-9 containing cancer cell lysate enhance IMM20065 binding to the surface of antigen-positive cancer cells. Ectopic expression, mislocalization, or cell lysis within the tumor microenvironment could all plausibly result in realeasing of SNX9 in extracellular matrix. Given BAR domains in SNX9 tend to dimerize, the extracellular SNX9 may bind to SNX9 on the tumor cell surface. To investigate whether binding of IMM20065 to tumor cells can be enhanced in the presence of extracellular SNX9 protein, recombinant SNX9 or SNX9-containing cancer cell lysate were incubated with IMM20065 and applied to cancer cells. SNX9-containing lysate was prepared via a freeze-thaw of Huh7 hepatocellular carcinoma cells in the absence of detergent. Briefly, cells were grown in a monolayer to high confluency, washed once with ice-cold 1x PBS, scraped into 1x PBS, and transfered to a conical tube for pelleting at top speed for 45 seconds. After spinning cells and removing the PBS, the cells were resuspended in freeze-thaw lysis buffer (600 mM KCI, 20 mM Tris-HCI pH 7.8, 20% glycerol, 1x protease inhibitor) and resuspended by pipetting up and down. The sample was then successively frozen and thawed for a total of three times with vortexing upon each thaw. After the last thaw, 250 U of Benzonase was added for 10 minutes at room temperature to digest the DNA and the final protein concentration was measured (protocol adapted from Rudolph et al 1999. Anal. Biochem. 269, 66-71).

[00101] Pre-incubation with both recombinant full-length SNX9 (Origene, catalog number TP302822) and truncated SNX9 (amino acids 1-362; Proteintech, catalog number Ag8446) concentration- dependently enhanced IMM20065 binding to the surface of Huh7 hepatocellular carcinoma cells. Similar enhancement of binding was also observed when recombinant full-length SNX9 were used at lower concentrations (Fig. 12). In addition, pre-incubation of IMM20065 with freeze-thaw lysate from Huh7 cells enhanced binding of the antibody to the surface of Huh7 cells. With precedence for SNX9 dimerization in the literature (Bendris and Schmid, Trends Cell Biol. 2017 Mar;27(3):189-200), these data suggest that the presence of SNX9 in the tumor microenvironment, resulting from secretion, cell lysis, or other forms of mislocalization, could enhance IMM20065 binding to the surface of cancer cells.

[00102] Example 7. IMM20065 internalizes into antigen-positive cancer cell lines. To evaluate whether IMM20065 can be internalized into SNX9-positive cancer cells, IMM20065 was tested via both standard immunofluorescence (IF; Fig. 13) and pH-sensitive fluorescent imaging on the IncuCyte® Live Cell Analysis System (Satorius; Fig. 14). For standard IF, Huh7 hepatocellular carcinoma cancer cells were plated on glass coverslips in 24-well plates overnight. Test antibodies were diluted in complete culture medium, added to cells at a final concentration of 100 μg/mL, and incubated for 1, 2, or 4 hours. Cells were then washed, fixed, permeabilized, washed again, and stained with anti-EGFR antibody (Cell Signaling, catalog number D38B1) overnight at 4°C. Cells were then washed, incubated with secondary antibodies and Hoescht stain, washed again, mounted with mounting solution (50% v/v glycerol, 50% 0.1 M NaHCO₃ pH 7.4), and imaged using a fluorescent microscope. Images of cells incubated 1, 2, and 4 hours with IMM20065 show time-dependent internalization of IMM20065 antibody, especially after 4 hours of incubation. Antigen-dependent internalization into cancer cells supports the use of an anti- SNX9 antibody as an ADC therapeutic.

[00103] For pH-sensitive fluorescent imaging on the IncuCyte®, cells were plated in 96-well black clear-bottom plates overnight at 37°C and 5% CO₂. IMM20065 and the corresponding isotype control were pre-incubated for 5 minutes at room temperature with Zenon pHrodo iFL Human IgG labeling reagent (ThermoFisher, catalog number Z25612) and then added to the pre-plated cells at a final concentration of 10 μg/mL. Cell confluence and internalization-dependent fluorescence was monitored on the IncuCyte® for 48 hours. IMM20065 demonstrated internalization into antigen-positive Huh7 hepatocellular carcinoma cells, and to a lesser extent OE19 oesophageal carcinoma cells, over isotype control antibody.

Table 1. IMM20065 and a-SNX9 control antibody (15721-1-AP) binding to cancer and normal cell lines

M W

Table 3. SPR Quantification of IMM20065 binding to human SNX9

Numbers in parenthese represent the standard error for a given data set extracted from the 1:1 fitting program

Table 4. Comparison of IMM20065 binding to human SNX9 versus related family members

Numbers in parentheses represent experimental standard deviation based upon the results from the various data sets.

Claims

What is claimed:

1. An isolated antibody, or antigen-binding fragment thereof, comprising a variable heavy chain (VHC) and a variable light chain (VLC), wherein:

A) the VHC comprises an amino acid that shares at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 7; and

B) The VLC comprises an amino acid that shares at least 90% homology with the amino acid sequence corresponding to SEQ ID NO: 10.

2. The antibody, or antigen-binding fragment of claim 1. comprising at least one of the complementary determining region (CDR) loops H1, H2, H3, L1, L2, and L3, wherein: the H1 loop amino acid sequence comprises SEQ. ID NO: 15; the H2 loop amino acid sequence comprises SEQ ID NO: 16; the H3 loop amino acid sequence comprises SEQ. ID NO: 17; the L1 loop amino acid sequence comprises SEQ ID NO: IS; the 12 loop amino acid sequence comprises SEQ. ID NO: 19; and the L3 loop amino acid sequence comprises SEQ ID NO: 20.

3. The antibody, or antigen-binding fragment of claim 1 or 2, wherein the antibody, or antigen-binding fragment, binds specifically to any one of Sorting nexin ("SNX")9, SNX18, and SNX33.

4. The antibody, or antigen-binding fragment of claim 1 or 2, wherein the antibody, or antigen-binding fragment, binds specifically to any one of SNX9, SNX18, and SNX33, wherein the SNX9, SNX18, and SNX33 are components of a multi-protein complex,

5. The antibody, or antigen-binding fragment of any one of claims 1-4, wherein the antibody, or antigen-binding fragment partially or fully blocks, inhibits, or neutralizes a biological activity of one or more of SNX9, SNX18, and SNX33.

6. The antibody, or antigen-binding fragment of claim 5, wherein binding of the antibody at the surface of a cell is enhanced in the presence of SNX9, SNX18, or SNX33.

7. The antibody, or antigen-binding fragment of claim 6, wherein the SNX9, SNX18, or SNX33 is released or otherwise derived from tumor cells.

8. The antibody, or antigen-binding fragment of ciaim 7, wherein the tumor cells have undergone apoptosis.

9. The antibody, or antigen-binding fragment of ciaim 7 or 8, wherein the tumor cells are chemotherapeutic targets.

10. The antibody, or antigen-binding fragment of ciaim 5, wherein binding of the antibody, or antigen- binding fragment to SNX9, SNX18, or SNX33 is followed by internalization of the antibody into a cell.

11. The antibody, or antigen-binding fragment of claim 10. wherein the cell is a tumor cell.

12. The antibody, or antigen-binding fragment of any one of claims 7-9, and 11, wherein the tumor cell is a hepatocellular carcinoma (HCC), glioblastoma (GBM), lung adenocarcinoma (LUAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIRP). .

13. The antibody or antigen-binding fragment of claim 5, wherein the SNX9, SNX18, and SNX33 are present in a body fluid.

14. The antibody or antigen-binding fragment of claim 13, wherein the body fluid is blood or a blood derivative.

15. The antibody or antigen-binding fragment of claim 14, wherein the blood derivative is plasma or serum.

16. The antibody or antigen-binding fragment of any one of claims 3-5, wherein the SNX9, SNX18, and SNX33 are associated with an extracellular matrix ("ECM"), or ECM protein.

17. The antibody or antigen-binding fragment of claim 16, wherein the SNX9, SNX18, and SNX33 are present in a tumor micro-environment.

18, The antigen-binding fragment of any one of claims 1-17, wherein the antigen-binding fragment is an isolated variable heavy (VH) single domain monoclonal antibody.

19. The antigen-binding fragment of any one of claims 1-17, wherein the antigen-binding fragment is a single chain (sc)Fv -Fc fragment.

20. The antigen -binding fragment of any one of ciaims 1-17, wherein the isolated antigen -binding fragment comprises an Fv, scFv, Fab, F(ab')2, or Fab' fragment, diabody, or any fragment whose half-life has been increased.

21. The antibody or antigen-binding fragment of any one of the claims 1-20, wherein the antibody or antigen-binding fragment comprises a CH3 scaffold, comprising at least one modification of the wild-type amino acid sequence of the CHS domain derived from an immunoglobulin Fc region.

22. The antibody or antigen-binding fragment of any one of claims 1-21, wherein the antibody or antigen-binding fragment is monoclonal.

23. The antibody or antigen-binding fragment of any one of claims 1-22, wherein the antibody or antigen-binding fragment is human, humanized, or bi-specific.

24. A method of inhibiting tumor growth or metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or an antigen- binding fragment of any of claims 1-23, wherein the antibody or an antigen-binding fragment partially or fully blocks, inhibits, or neutralizes a biological activity of one or more of the SNX9, SNX18, and SNX33.

25. The method of claim 24, wherein the tumor growth or metastasis inhibited is by a (HCC), glioblastoma (GBM), lung adenocarcinoma (I.UAC), lung squamous cell carcinoma (LUSC), head and neck cancer (HNSC), esophageal (ESCA), bile duct cancer (cholangiocarcinoma, CHOL), thyroid cancer (THCA), renal clear cell cancer (KIRC), and renal papillary cell carcinoma (KIR P).

26. The method of inhibiting tumor growth or metastasis according to claim 24 or 25, wherein the partially, or fully blocking, inhibiting, or neutralizing a biological activity of one or more of the SNX9, SNX18, and SNX33 are inhibits angiogenesis that supports tumor growth or metastasis, or both, in the subject.

27. A method for preventing, treating, or ameliorating diseases related to aberrant angiogenesis, comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or an antigen-binding fragment of any of claims 1-26.

28. The method for preventing, treating, or ameliorating diseases related to aberrant angiogenesis according to claim 27, wherein the partially, or fully blocking, inhibiting, or neutralizing a biological activity of one or more of the SNX9, SNX18, and SNX33 inhibits angiogenesis in the subject.

29. The method of claim 27 or 28, wherein the diseases related to aberrant angiogenesis are diabetic retinopathy, macular degeneration, non-exudative or exudative age-related macular degeneration and psoriasis.

30. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the VHC according to claim 1.

31. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the VLC according to claim 1.

32. The isolated nucleic acid molecule of claim 30, wherein the nucleic acid sequence encoding the VHC is operably linked to a promoter.

33. The isolated nucleic acid molecule of claim 31, wherein the nucleic acid sequence encoding the VLC is operably linked to a promoter.

34. An expression vector comprising the isolated nucleic acid molecule of claim 32.

35. An expression vector comprising the isolated nucleic acid molecule of claim 33,

36. An isolated host cell transformed with the nucleic acid molecule or expression vector of claim 32.

37. An isolated host cell transformed with the nucleic acid molecule or expression vector of claim 33.