TEM8 monoclonal antibodies and their use

Abstract

Disclosed are monoclonal antibodies and antigen binding fragments thereof that specifically bind to the extracellular domain of Tumor Endothelial Marker 8 (TEM8). In some examples, the antibodies and antigen binding fragments are useful for methods of detecting and treating pathogenic angiogenesis. In other examples, the antibodies and antigen binding fragments are useful for methods of detecting and treating cancer. In further examples, the antibodies and antigen binding fragments are useful for methods of treating cardiovascular disease, such as myocardial infarction.

Description

4239-112153-02TEM8 MONOCLONAL ANTIBODIES AND THEIR USECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Application No. 63 / 750,425, filed January 28, 2025, which is incorporated by reference in its entirety.FIELD

[0002] This application relates to the fields of cancer and cardiovascular disease, particularly to antibodies and antigen binding fragments that specifically bind Tumor Endothelial Marker 8 (TEM8) and their use to treat cancer and cardiovascular disease.ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant No. ZIA BC 010578, awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0004] The electronic sequence listing, submitted herewith as an XML file named 4239-112153-02.xml (104,539 bytes), created on January 26, 2026, is herein incorporated by reference in its entirety.BACKGROUND

[0005] Tumor Endothelial Marker 8 (TEM8), also known as Anthrax Toxin Receptor 1 (ANTXR1), is a single pass, cell surface glycoprotein originally identified based on its overexpression in the endothelial cells that line the tumor vasculature of human colorectal cancer. TEM8 also functions as a cell surface receptor for Anthrax toxin, and shares 58% amino acid identity with CMG2 (also known as ANTXR2), a second receptor for Anthrax toxin protein. Unlike VEGF, VEGFRs, and many other key angiogenesis regulators, TEM8 is not required for developmental angiogenesis, wound healing, or normal physiological angiogenesis of the corpus luteum. TEM8 is up-regulated on tumor vessels of various tumor types in both mice and humans, and, in some tumors, is also expressed by the tumor cells.

[0006] A need exists for chemotherapeutic agents that target TEM8, and for high affinity antibodies that specifically bind TEM8 on the cell surface.4239-112153-02SUMMARY

[0007] Isolated monoclonal antibodies and antigen binding fragments thereof that specifically bind to the extracellular domain of TEM8 are provided herein. The disclosed monoclonal antibodies and antigen binding fragments are useful, for example, for the treatment and inhibition of TEM8-associated cancer or cardiovascular disease.

[0008] In some implementations, the antigen or antigen binding fragment comprises a heavy chain variable (VH) region and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL set forth as:a) SEQ ID NOs: 9 and 10 (YL9);b) SEQ ID NOs: 15 and 16 (YK2);c) SEQ ID NOs: 23 and 24 (YK3);d) SEQ ID NOs: 29 and 30 (YK7); ore) SEQ ID NOs: 37 and 38 (YL51); andwherein the monoclonal antibody or antigen binding fragment thereof specifically binds to the TEM8 extracellular domain.

[0009] In some implementations, disclosed are nucleic acid molecules encoding these antibodies and antigen binding fragments, vectors including these nucleic acid molecules, and host cells including these vectors.

[0010] Pharmaceutical compositions including the antibodies, antigen binding fragments, nucleic acid molecules, and vectors are also disclosed. In more implementations, disclosed is the use of these pharmaceutical compositions to treat a TEM8-associated cancer, or TEM8-associated pathological angiogenesis (such as macular degeneration due to pathological angiogenesis), or cardiovascular disease (such as myocardial infarction) in a subject.

[0011] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several implementations which proceeds with reference to the accompanying figures.BRIEF DESCRIPTION OF THE FIGURES

[0012] FIG.1. TEM8 antibodies (full IgGs) bind TEM8 protein with high affinity. ELISA was used to measure antibody binding to TEM8. Human TEM8-ECD (extracellular domain) protein was coated into the wells of a 96-well ELISA plate. IgGs were then added at concentrations from 5xl0-6to 50 nM with 1:10 dilutions. Bound IgGs were detected with4239-112153-02goat anti -human Fc HRP. YK2, YK7, YL9 and YL51 had subnanomolar affinities, while YK3 had lower affinity. The antibodies recognize the mouse TEM8 protein with similar affinities.

[0013] FIG. 2. YL9 blocks the interaction between TEM8 and PA (protective antigen produced by anthracis bacillus)). TEM8 can bind the protective antigen (PA) subunit of anthrax toxin protein. PA protein was coated onto ELISA 96-well plates. TEM8-ECD-myc (extracellular domain tagged with the myc epitope) protein was added to wells at 3, 6, 12, 24 nM alone or with the IgGs indicated. In the wells with IgGs, each IgG is present at 20 nM constantly. Binding of mTEM8 to PA was detected with HRP-anti-myc secondary antibodies. YL9 could efficiently block TEM8-PA interaction.

[0014] FIGs. 3A and 3B. (FIG. 3A) Arginine 88 (R88) and glutamic acid 125 (E125) of human TEM8 are critical sites for YL9 recognition. TEM8 full length protein carrying either multiple amino acid mutations (Mut 4) or a single amino acid mutation (Mut 27-39) were expressed on CHO-PR230 cells (a TEM8 negative cell line). TEM8 expression on the cell surface was detected by flow cytometry using either m825 (a control anti-TEM8 antibody) or YL9. Note that YL9 bound robustly to all TEM8 mutants, except for mutant 4, 28 and 33 (partially). Changing R88 to alanine completely abolished the binding of YL9 to TEM8, while mutation of E125 to alanine reduced the binding. (FIG. 3B) Antibody docking predicts K94 and R88 at the respective interface between L2 and YL9 and the extracellular domain of TEM8.

[0015] FIGs. 4A-4D. Anti-TEM8 antibodies block cardiac fibroblast contraction. (FIG. 4A) Mouse cardiac fibroblasts, derived from TEM8 wildtype (WT) or knockout (KO) mice, maintained in complete medium, were placed into medium supplemented with low-serum (0.5% FBS) for 16 hours. In the same medium cells were mixed with collagen I solution. TGF-β (10ng / ml) was added to the cells / collagen mixture with or without the TEM8 antibodies - YL9 or YK2 (at 20 pg / ml). The mixture was plated in an untreated 24-well plate and allowed to solidify at 37°C, 5% CO2 for 2 hours. After supplementing the wells with an additional 500 pl of the appropriate medium, the cells / gel mixture was gently detached from the bottom of each well and cultured for 48 hours, during which time the contraction occurred. The image in each well was captured with an inverted microscope. (FIG. 4B) Gel contraction was quantified by measuring the surface area of each cell / gel mixture and using the formula: 1 - (surface area of the cells / gel) / (total area of the well). Note: TGF pi activated contraction depends on TEM8 function. YL9 and YK2 anti-TEM8 antibodies4239-112153-02significantly blocked the gel contraction. TEM8 contraction was completely blocked in TEM8 KO cardiac fibroblasts. (FIG. 4C) Immunoblot comparing T8Abl (L2) and T8Ab2 (YL9) activity on TGFP signaling in TEM8 WT mouse CF (mCF). TEM8 KO CF were used as a control. (FIG. 4D) The effect of TEM8 Abs on TGF0 signaling in human primary CF (hCF). For immunoblots, H3 antibody served as a loading control.

[0016] FIG. 5A. YL9 anti-TEM8 antibody inhibits UACC tumor growth. Human UACC melanoma cells (2 million per mouse) mixed with Matrigel were implanted subcutaneously on the flank of athymic nude mice. When the tumor size reached about 50 mm3, mice were randomized into two groups and treated with vehicle (PBS, control) or 20 mg / kg YL9 (3 times per week). Tumors were measured with a digital caliper, and tumor volumes were calculated using the formula LxW2x0.5. YL9 inhibited the tumor growth by over 90%.Antibody treatment did not alter body weights or cause any other signs of toxicity. n=l 1 (vehicle) or n=7 (YL9) mice / group.

[0017] FIG. 5B. YL9 anti-TEM8 antibody inhibits HepG2 HCC tumor growth. Human HepG2 HCC tumor cells (2 million per mouse) mixed with Matrigel were implanted subcutaneously on the flank of athymic nude mice. When the tumor size reached about 50 mm3, mice were randomized into two groups and treated with vehicle (PBS, control) or 10 mg / kg YL9 (3 times per week). Tumors were measured with a digital caliper, and tumor volumes were calculated using the formula LxW2x0.5. YL9 inhibited the tumor growth by over 70%. Antibody treatment did not alter body weights or cause any other signs of toxicity. n=10 (vehicle) or n=10 (YL9) mice / group.SEQUENCES

[0018] The nucleic and amino acid sequences provided herein are shown using standard letter abbreviations. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. See Example 4 for additional detail on sequences. In the accompanying sequence listing:

[0019] SEQ ID NOs: 1-6 are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the YL9 antibody according to the IMGT numbering system.

[0020] SEQ ID NOs: 7 and 8 are the heavy and light chain variable regions of the YL9 antibody.

[0021] SEQ ID NOs: 9-14 are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the YK2 antibody according to the IMGT numbering system.4239-112153-02

[0022] SEQ ID NOs: 15 and 16 are the heavy and light chain variable regions of the YK2 antibody.

[0023] SEQ ID NOs: 17-22 are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the YK3 antibody according to the IMGT numbering system.

[0024] SEQ ID NOs: 23 and 24 are the heavy and light chain variable regions of the YK3 antibody.

[0025] SEQ ID NOs: 25, 10, 26-27, 13, and 28 are HCDR1. HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the YK7 antibody according to the IMGT numbering system.

[0026] SEQ ID NOs: 29 and 30 are the heavy and light chain variable regions of the YK7 antibody.

[0027] SEQ ID NOs: 31-36 are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2. and LCDR3 of the YL51 antibody according to the IMGT numbering system.

[0028] SEQ ID NOs: 37 and 38 are the heavy and light chain variable regions of the YL51 antibody.

[0029] SEQ ID NO: 39 is the amino acid sequence of TEM8 protein.

[0030] SEQ ID NOs: 40 and 41 are amino acid and nucleotide sequences for a YL9-based ScFv.

[0031] SEQ ID NOs: 42 and 43 are amino acid and nucleotide sequences for a YK2 -based ScFv.

[0032] SEQ ID NOs: 44 and 45 are amino acid and nucleotide sequences for a YK3 -based ScFv.

[0033] SEQ ID NOs: 46 and 47 are amino acid and nucleotide sequences for a YK7 -based ScFv.

[0034] SEQ ID NOs: 48 and 49 are amino acid and nucleotide sequences for a YL51 -based ScFv.

[0035] SEQ ID NOs: 50-101 are CDR sequences for the YL9, YK2, YK3, YK7, and YL51 antibodies based on the Kabat and Chothia numbering systems.

[0036] SEQ ID NOs: 102-115 are sections of the TEM8 protein showing alanine mutations.DETAILED DESCRIPTIONI. Summary of Terms

[0037] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the4239-112153-02singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various implementations, the following explanations of terms are provided:

[0038] About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0039] Administration: The introduction of an agent, such as a disclosed antibody, into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravascular, the agent (such as antibody) is administered by introducing the composition into a blood vessel of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

[0040] Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

[0041] Angiogenesis: A biological process leading to the generation of new blood vessels through sprouting or growth from pre-existing blood vessels. The process involves the migration and proliferation of endothelial cells from preexisting vessels. Angiogenesis occurs during pre- and post-natal development, and in the adult. Angiogenesis occurs during the normal cycle of the female reproductive system, wound healing, and during pathological processes such as cancer, where it is essential for the growth of solid tumors.

[0042] Anthrax: An acute disease caused by the bacterium Bacillus anthracis, and in particular the toxin it produces. Anthrax toxin is a mixture of three protein components: (i) protective antigen (PA), (ii) edema factor (EF), and (iii) lethal factor (LF). Cellular entry of Anthrax toxin requires PA binding to one of its two cell-surface receptors, ANTXR1 (also known as TEM8) or ANTXR2 (also known as CMG2 receptor), on the host cell (see, for4239-112153-02example, Van der Goot and Young, Mol. Aspects Med., 30(6):406-412, 2009; Moayeri and Leppla, Curr Opin Microbiol 7(1): 19-24, 2004).

[0043] Anthrax protective antigen (PA): The protein secreted by Bacillus anthracis that forms the Anthrax toxin with edema factor (EF) and lethal factor (LF). Cellular entry of Anthrax toxin requires PA binding to one of its two cell-surface receptors, ANTXR1 (also known as TEM8) or ANTXR2 (also known as CMG2 receptor), on the host cell (see, for example, Van der Goot and Young, Mol. Aspects Med., 30(6):406-412, 2009; Moayeri and Leppla, Curr Opin Microbiol 7(1 ): 19-24, 2004). After protease cleavage, PA binds to the two toxic enzymes (EF and LF) and mediates their transportation into the cytosol where they exert their pathogenic effects (Bradley et al., Nature 414:225, 2001). The smaller cleaved 63 kD PA remnant (PA63) oligomerizes, exposing a second binding domain and binds to either EF, an 89 kD protein, to form edema toxin, or LF, a 90 kD protein, to form lethal toxin (LeTx) (Leppla et al., Salisbury Med. Bull. Suppl. 68:41-43, 1990), and the complex is internalized into the cell where it enters the endosomal system (Singh et al., Infect. Immun. C1A^>53, 1999; Friedlander, J. Biol. Chem. 261:7123, 1986). From these endosomes, the PA63channel enables translocation of LF and EF to the cytosol by a pH- and voltagedependent mechanism (Zhao et al., J. Biol. Chem., 270:18626, 1995). In some embodiments, the TEM8 specific antibodies or conjugates including TEM8 specific antibodies disclosed herein are capable of blocking PA binding to TEM8. In one example, PA includes an amino acid sequence set forth in GENBANK® Accession No. AAF86457, as accessed on September 19, 2013.

[0044] Anti-angiogenic agent: A molecule that decreases or reduces angiogenesis, for example, a molecule that decreases pathological angiogenesis. In some examples, antibodies that specifically bind TEM8 or conjugates including such antibodies are anti-angiogenic agents that decrease pathological angiogenesis. Additional anti-angiogenic agents include, but are not limited to, vascular endothelial growth factor (VEGF) antibodies (e.g., bevacizumab) and vascular endothelial growth factor receptor (VEGFR) antibodies or small molecules (such as DMXAA (also known as Vadimezan or 5,6-Dimethyl-9-oxo-9H-xanthen-4-yl)-acetic acid.

[0045] Antibody and Antigen Binding Fragment: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as the TEM8 extracellular domain. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal4239-112153-02antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.

[0046] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dübel (Eds.), Antibody Engineering, Vols. 1-2, 2nded., Springer-Verlag, 2010).

[0047] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0048] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0049] Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda ( ) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0050] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen.

[0051] References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “V ” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0052] The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity -determining regions” or “CD Rs” (see, e.g., Kabat et4239-112153-02al., Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U. S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0053] The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U. S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27 ( l):55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0054] In some implementations, a disclosed antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications, such as the “LS” mutations that increase antibody half-life, or the “LALA-PG” mutations that prevent binding to Fc-gamma receptors.

[0055] A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal4239-112153-02antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014.)

[0056] A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one implementation, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

[0057] A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0058] A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some implementations, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1stEd. New York:4239-112153-02Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005: Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0059] A “bispecific antibody” is a recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.

[0060] Antibody-drug conjugate (ADC): A molecule that includes an antibody (or antigenbinding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with ADCs include anti-microtubule agents (such as maytansinoids, auristatin E and auristatin F) and interstrand crosslinking agents e.g., pyrrolobenzodiazepines; PDBs).

[0061] Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having TEM8-associated cancer.

[0062] Breast cancer: A neoplastic tumor of breast tissue that is or has potential to be malignant. The most common type of breast cancer is breast carcinoma, such as ductal carcinoma. Ductal carcinoma in situ is a non-invasive neoplastic condition of the ducts. Lobular carcinoma is not an invasive disease but is an indicator that a carcinoma may develop. Infiltrating (malignant) carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA, IIIB, and IV).

[0063] Carcinoma: A malignant tumor including transformed epithelial cells. Non-limiting examples of carcinomas include adenocarcinoma, squamous cell carcinoma, anaplastic carcinoma and large and small cell carcinoma. In some examples, a carcinoma is a breast carcinoma, colorectal carcinoma, lung carcinoma or melanoma.

[0064] Cardiac Ejection Fraction (EF): the percentage of blood volume ejected in each cardiac cycle and is a representation of left ventricle (LV) systolic performance. It is4239-112153-02calculated from the end-diastolic and end-systolic volumes of the left ventricle. The formula for calculating EF is: EF% = ((LV v,d-LV v,s) / LV v,d)*100.

[0065] Cardiovascular disease (CVD): A group of diseases that includes, but is not limited to, atherosclerosis, coronary artery disease (CAD), angina pectoris (commonly known as "angina"), thrombosis, ischemic heart disease, coronary insufficiency, peripheral vascular disease, myocardial infarction, cerebrovascular disease (such as stroke), transient ischemic attack, arteriolosclerosis, small vessel disease, elevated cholesterol, intermittent claudication, hypertension, Heart Failure with preserved Ejection Fraction (HFpEF).

[0066] Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nded., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds FLT3 used in combination with a radioactive or chemical compound.

[0067] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigenbinding portion (such as a single domain antibody or scFv) and a signaling domain, such as a signaling domain from a T cell receptor (e.g. CD3Q. Typically, CARs are comprised of an antigen-binding moiety, a transmembrane domain and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine -based activation motif (IT AM), such as CD3ζ or FcεRIγ. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and / or DAP10. A “chimeric antigen receptor T cell” is a T cell expressing a CAR, and has antigen specificity determined by the antibody-derived targeting domain of the CAR.

[0068] Collagen: The collagen family includes at least 15 types of collagen, which are major components of the extracellular matrix. Type I collagen is the principal collagen found in4239-112153-02skin and bone and consists of two type I al collagen subunits (COL1 Al) and one type I a2 collagen (COL1A2) subunit.

[0069] Colorectal cancer: A neoplastic tumor of colon, rectum or anus tissue that is or has the potential to be malignant. The main types of colorectal cancer include colorectal carcinomas such as adenocarcinoma and squamous cell carcinoma. Infiltrating (malignant) carcinoma of the colon can be divided into stages (I, II, III and IV).

[0070] Conditions sufficient to form an immune complex: Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a delectably greater degree than, and / or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014, for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

[0071] The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scans, radiography, and affinity chromatography.

[0072] Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one implementation, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to the TEM8 extracellular domain covalently linked to an effector molecule, such as a detectable label. The linkage can be by chemical or recombinant means. In one implementation, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule.4239-112153-02Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”

[0073] Conservative variants: “Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, a TEM8-specific antibody can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for TEM8. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0074] Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some implementations less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

[0075] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:1) Alanine (A), Serine (S), Threonine (T);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0076] Non-conservati ve substitutions are those that reduce an activity or function of the antibody, such as the ability to specifically bind to TEM8. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

[0077] Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.4239-112153-02

[0078] Control: A reference standard. In some implementations, the control is a negative control, such as sample obtained from a healthy patient not having a TEM8-associated cancer. In other implementations, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with a TEM8-associated cancer. In still other implementations, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

[0079] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

[0080] Cytotoxic agent: Any drug or compound that kills cells.

[0081] Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to the cells intended to be targeted, as opposed to the cells of the rest of an organism. In one embodiment, in contrast, the term “toxicity” refers to toxicity of an immunotoxin to cells other than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term “animal toxicity” refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.

[0082] E / e’ Ratio (e / e’): The ratio of early diastolic mitral inflow velocity to early diastolic mitral annulus velocity. Used for the evaluation of LV filling pressure, and it has been used as a marker to diagnose diastolic heart failure.

[0083] Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0084] Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the4239-112153-02second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination).Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0085] Detecting: To identify the existence, presence, or fact of something.

[0086] Effector molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted, or a detectable marker. Effector molecules can include, for example, polypeptides and small molecules. Some effector molecules may have or produce more than one desired effect.

[0087] Engineered T-Cell Receptor: A modified T-cell receptor that includes a binding domain, such as an scFv, that specifically binds to a tumor associate antigen and targets a T-cell expressing the engineered T-cell receptor to relevant tumor tissue. Non-limiting examples of types of engineered T-cell receptors for use in cancer therapy include Chimeric Antigen Receptors (CARs) (see, e.g., June and Sadelain, NEJM, 279:64-73, 2018; Sterner & Sterner, Blood Cancer J, 11:69, 2021), Synthetic T cell Receptor and Antigen Receptors (STARs) including costimulatory STARs (see, e.g., Wand et al., Am. J. Hematol., 97:992-1004, 2022), T cell receptor fusion constructs (TRuCs) (see, e.g., Baeuerle et al., Nature Communications, 10: 2087, 2019), and HLA-independent TCRs (HIT) (see, e.g., Mansilla-Soto et al., Nat. Med., 28:345-352, 2022).

[0088] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on the extracellular domain of TEM8.

[0089] Expression: Transcription or translation of a nucleic acid sequence. For example, an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become4239-112153-02mRNA. An encoding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0090] Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0091] Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0092] A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

[0093] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc4239-112153-02region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to EU numbering. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.

[0094] Fibrosis: The formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue.

[0095] Fractional Shortening (FS): the percentage change in left ventricle (LV) cavity dimensions in each cardiac cycle. It is calculated from the left ventricular internal dimension end-diastolic and left ventricular internal dimension end-systolic of the left ventricle. The formula for calculating FS is: FS% = ((LVID,d-LVID,s) / LVID,d)*100.

[0096] Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originated from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as an scFv, is expressed in a cell, such as a mammalian cell.Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0097] Host cell: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell’’ is used.

[0098] IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype comprises IgAi and IgA2. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C-terminus called the tail piece (tp). Polymeric4239-112153-02IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.

[0099] IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgG1, IgG2, IgG3, and IgG4.

[0100] Immune complex: The binding of antibody or antigen binding fragment (such as a scFv) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

[0101] Inhibiting or Treating a Disease: A therapeutic intervention (for example, administration of a therapeutically effective amount of an antibody that specifically binds TEM8) that reduces a sign or symptom of a disease or pathological condition related to a disease (such as TEM8-associated cancer, or pathological angiogenesis, or myocardial ischemia, or myocardial infarction). Treatment can also induce remission or cure of a condition. In particular examples, treatment includes preventing the progression of a disease, for example inhibiting cardiac fibrosis caused as a result of a myocardial infarction.Prevention does not require the disease be eliminated or halted, it is sufficient, for example, to slow the progression of the disease.

[0102] Reducing a sign or symptom of a disease or pathological condition related to a disease, refers to any observable beneficial effect of the treatment. Reducing a sign or symptom associated with myocardial ischemia (such as cardiac fibrosis) can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject (such delayed tissue scarring) a reduction in severity of some or all clinical symptoms of the disease (such as reduced chest discomfort), a slower progression of the disease (for example by prolonging the life of a subject with myocardial ischemia), a reduction in the number of relapses of the disease, or by an improvement in the overall health or well-being of the subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0103] Ischemia / Reperfusion Injury: In addition to the immediate injury that occurs during deprivation of blood flow, ischemic / reperfusion injury involves tissue injury that occurs after4239-112153-02blood flow is restored. Current understanding is that much of this injury is caused by chemical products and free radicals released into the ischemic tissues.

[0104] When a tissue is subjected to ischemia, a sequence of chemical events is initiated that may ultimately lead to cellular dysfunction and necrosis. If ischemia is ended by the restoration of blood flow, a second series of injurious events ensue, producing additional injury. Thus, whenever there is a transient decrease or interruption of blood flow in a subject, the resultant injury involves two components — the direct injury occurring during the ischemic interval and the indirect or reperfusion injury that follows. When there is a long duration of ischemia, the direct ischemic damage, resulting from hypoxia, is predominant. For relatively short duration ischemia, the indirect or reperfusion mediated damage becomes increasingly important. In some instances, the injury produced by reperfusion can be more severe than the injury induced by ischemia per se. This pattern of relative contribution of injury from direct and indirect mechanisms has been shown to occur in all organs.

[0105] Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0106] Kabat position: A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. (Sequences of Proteins of Immunological Interest, 5thEdition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

[0107] LALA-PG: Mutations in the constant region of an IgG antibody at L234A, L235A, and P329A, with numbering according to EU convention. In some examples making the LALA-PG substitutions disrupts Fc receptor binding. In some examples the IgG antibody is murine IgG2a. In other examples the IgG antibody is human IgGl. See Lo et al., J. Bio. Chem. (2017) 292:3900-08; Saunders Front. Immuno. (2019) 10:1296.4239-112153-02

[0108] Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0109] The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0110] Lung cancer: A neoplastic tumor of lung tissue that is or has the potential to be malignant. The main types of lung cancer are lung carcinomas: adenocarcinoma, small cell carcinoma, squamous cell carcinoma, or non-small cell carcinoma. Lung cancer is typically staged from I to IV; other classifications are also used, for example small-cell lung carcinoma can be classified as limited stage if it is confined to one half of the chest and within the scope of a single radiotherapy field; otherwise, it is extensive stage.

[0111] Myocardial Infarction (MI): An event that occurs when blood stops flowing properly to part of the heart and the heart muscle is injured due to inadequate oxygen delivery. The most common triggering event is the disruption of an atherosclerotic plaque in an epicardial coronary artery, which leads to a clotting cascade, sometimes resulting in total occlusion of the artery. If impaired blood flow to the heart lasts long enough, it triggers a process called the ischemic cascade; the heart cells in the territory of the occluded coronary artery die, chiefly through necrosis.

[0112] Myocardial Ischemia: A decrease in the blood supply to the heart, for example, caused by constriction or obstruction of one or more blood vessels due to atherosclerosis. Myocardial ischemia can lead to direct ischemic injury of heart tissue due to cell death caused by reduced oxygen supply. Myocardial ischemia sometimes results from vasoconstriction or thrombosis or embolism. Myocardial ischemia can occur acutely, as during surgery, or from trauma to tissue incurred in accidents, injuries and war settings, for instance. It can also occur sub-acutely, as found in atherosclerotic peripheral vascular disease, where progressive narrowing of blood vessels leads to inadequate blood flow to the heart (and other tissues and organs. Myocardial ischemia can be caused by a myocardial infarction.4239-112153-02

[0113] Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize or invade surrounding tissue is referred to as “benign.” A tumor that does metastasize or invade the surrounding tissue is referred to as “malignant.” Cancer is neoplastic growth that is malignant or has the potential to become malignant.

[0114] Tumors of the same tissue type are primary tumors originating in a particular organ (such as colon, skin, breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumors of different sub-types. For examples, lung carcinomas can be divided into an adenocarcinoma, small cell, squamous cell, or non-small cell tumors.

[0115] Examples of solid tumors, such as sarcomas (connective tissue cancer) and carcinomas (epithelial cell cancer), include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

[0116] Nucleic acid (molecule or sequence): A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA or RNA. The nucleic acid can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (such as A, T / U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides.

[0117] “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0118] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the4239-112153-02biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0119] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0120] Pathological angiogenesis: Angiogenesis that is medically undesired or harmful to a subject, such as angiogenesis associated with a tumor or the generation of blood vessels in or surrounding a tumor. Tumor vasculature can be distinct from normal vasculature in that several genes can be differentially expressed in tumor-associated blood vessels (St. Croix et al., Science, 289, 1197-1202, 2000). One of these genes, tumor endothelial marker 8 (TEM 8), is upregulated in the vasculature of malignant solid tumors, with limited expression in healthy tissues. Other examples of pathological angiogenesis include comeal or retinal angiogenesis (as in a corneal transplant or the retina of a subject with macular degeneration or diabetes).

[0121] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

[0122] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional4239-112153-02non-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, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some implementations, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

[0123] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some implementations, the polypeptide is a disclosed antibody or a fragment thereof.

[0124] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein is in its natural environment within a cell. In one implementation, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0125] Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several implementations, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or4239-112153-02eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

[0126] Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the percentage identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs and variants of a VL or a VH of an antibody that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest.

[0127] Any suitable method may be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U. S. A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(l):237-244, 1988; Higgins and Sharp, Bioinformatics, 5(2): 151-3, 1989; Corpet, Nucleic Acids Res. 16(22): 10881- 10890, 1988; Huang et al. Bioinformatics, 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994., Altschul et al,, J. Mol. Biol, 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol, Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

[0128] Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

[0129] Skin cancer: A neoplastic tumor of skin tissue that is or has the potential to be malignant. Melanoma is a skin cancer of transformed melanocytes (cells that make the4239-112153-02pigment melanin). Melanocytes are found primary in the skin, but are also present in the bowel and eye. Melanoma in the skin includes superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or can be amelanotic. Similarly, any subtype may show desmoplasia (dense fibrous reaction with neurotropism), which is a marker of aggressive behavior and a tendency for local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma and uveal melanoma. Melanoma is staged from I to IV.

[0130] Specifically bind: When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as the TEM8 extracellular domain) and does not bind in a significant amount to other proteins present in the sample or subject. A limited degree of non-specific interaction may occur between an antibody (such as an antibody that specifically binds to the TEM8 extracellular domain) and a non-target (such as a cell that does not express TEM8). Specific binding can be determined by methods known in the art. See Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014, for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti vity.

[0131] With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a KD of less than about 10’7Molar, such as less than about 10sMolar, 10'9, or even less than about 10’10Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide-ligand interaction or an antibody-antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0132] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like. In an example, a subject is a human.

[0133] Therapeutically effective amount: The amount of an agent (such as a TEM8 specific antibody) that alone, or together with one or more additional agents, induces the desired response, such as, for example treatment of TEM8-associated cancer, or pathological angiogenesis, myocardial ischemia, or myocardial infarction, in a subject. When administered to a subject, a dosage will generally be used that will achieve target tissue4239-112153-02concentrations that has been shown to achieve a desired in vitro effect. Ideally, a therapeutically effective amount provides a therapeutic effect without causing a substantial cytotoxic effect in the subject.

[0134] A therapeutically effective amount of a TEM8-specific antibody or antigen binding fragment as described herein that is administered to a human or veterinary subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject. A therapeutically effective amount can be determined by varying the dosage and measuring the resulting therapeutic response, such as improved cardiac blood flow or cardiac ejection. Therapeutically effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays. The disclosed agents can be administered in a single dose, or in several doses, as needed to obtain the desired response. However, the therapeutically effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.

[0135] Toxin: An effector molecule that induces cytotoxicity when it contacts a cell.Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, auristatins (such as monomethyl auristatin E (MMAE; see for example, Francisco et al., Blood, 102: 1458-1465, 2003)) and monomethyl auristatin F (MMAF; see, for example, Doronina et al., BioConjugate Chem., 17: 114-124, 2006), maytansinoids (such as DM1; see, for example, Phillips et al., Cancer Res., 68:9280-9290, 2008), Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.

[0136] Tumor Endothelial Marker 8 (TEM8): Also known as Anthrax Toxin Receptor 1 (ANTXR1), TEM8 is a highly conserved, integrin-like, single-pass transmembrane receptor that was originally discovered based on its overexpression in tumor-associated vasculature and was subsequently found to also be widely overexpressed in cancer-associated fibroblasts (CAFs) (Szot et al., J. Clin. Invest. (2018) 128:2927-43; Carson-Walter et al., Cancer Res. (2001) 61: 6649-55; Nanda et al., Cancer Res. (2004) 64:817-20; St Croix et al., Science (2000) 289:1197-1202). Unlike vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and many other key angiogenesis regulators, TEM8 is not required for4239-112153-02developmental angiogenesis, wound healing, or normal physiological angiogenesis of the corpus luteum (St Croix et al., Science, 289(5482): 1197-1202, 2000; Nanda et al., Cancer Res., 64(3):817-820, 2004). TEM8 is expressed at low levels in normal adult tissues and has been reported to be induced in vivo following ischemia insult (Chaudhary, et al., Cancer-Cell (2012) 21:212-26; Anderson, et al., PLoS One (2016) ll:e0146586). In cell culture TEM8 is a stress induced molecule that increases in vascular cells and CAFs in response to serum and growth factor deprivation (Chaudhary, et al., Cancer Cell (2012) 21:212-26, Hsu, et al., Nature Communications (2022) 13(l):7078. doi: 10.1038 / s41467 -022-34643-5). TEM8 also functions as a cell-surface receptor for Anthrax toxin, and shares 58% amino acid identify with CMG2 (also known as ANTXR2), which is a second receptor for Anthrax toxin protein (Scobie et al., Proc. Natl. Acad. Sci. U. S. A., 100(9):5170-5174, 2003).

[0137] TEM8 protein sequence is known (see, for example, GENBANK® Accession No. NP_115584.1, incorporated by reference herein as present in the database on October 21, 2022). Additionally, exemplary nucleic acid sequences encoding TEM8 protein are known (see, for example, GENBANK® Accession No. NM_032208.2, incorporated by reference herein as present in the database on October 21, 2022; see also for example, GENBANK® Accession No. NM_032208.3 incorporated by reference herein as present in the database on October 21, 2022). In one example, TEM8 is a polypeptide having an amino acid sequence set forth as SEQ ID NO: 39. The TEM8 extracellular domain is the portion of TEM8 that extends into the extracellular space, approximately amino acids 28-320 of SEQ ID NO: 39.> SEQ ID NO: 39 (TEM8 protein) MATAERRALGIGFQWLSLATLVLICAGQGGRREDGGPACYGGFDLYFILDKSGSVLHHWNEIYYFVEQLAHKFIS PQLRMSF IVFSTRGTTLMKLTEDREQIRQGLEELQKVLPGGDTYMHEGFERASEQI YYENRQGYRTASVI IALTD GELHEDLFFYSEREANRSRDLGAIVYCVGVKDFNETQLARIADSKDHVFPVNDGFQALQGI IHS ILKKSCIE ILA AEPSTICAGESFQWVRGNGFRHARNVDRVLCSFKINDSVTLNEKPFSVEDTYLLCPAPILKEVGMKAALQVSMN DGLSF ISSSVI ITTTHCSDGS ILAIALLILFLLLALALLWWFWPLCCTVI IKEVPPPPAEESEEEDDDGLPKKKW PTVDASYYGGRGVGGIKRMEVRWGEKGSTEEGAKLEKAKNARVKMPEQEYEFPEPRNLNNNMRRPSSPRKWYSPI KGKLDALWVLLRKGYDRVSVMRPQPGDTGRCINFTRVKNNQPAKYPLNNAYHTSSPPPAPIYTPPPPAPHCPPPP PSAPTPP IPSPPSTLPPPPQAPPPNRAPPPSRPPPRPSV

[0138] TEM8-associated cancer: A cancer that overexpresses TEM8 and / or contains a tumor microenvironment with vascular endothelial cells overexpressing TEM8. TEM8-associated cancers include, but are not limited to, colorectal, skin, lung, breast, prostate, or head and neck cancer.

[0139] Tumor microenvironment: The cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the4239-112153-02extracellular matrix (ECM), including stromal cells. Tumors can influence the microenvironment by releasing extracellular signals, promoting pathological angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells, such as in immuno-editing.

[0140] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0141] Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and / or protein) DNA, a naked or packaged RNA, a subcomponent of a vims or bacterium or other microorganism that may be replication-incompetent, or a vims or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a constmct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some implementations, a viral vector comprises a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment that specifically binds to the TEM8 extracellular domain. In some implementations, the viral vector can be an adeno-associated vims (AAV) vector.

[0142] Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.IL Description of Several Implementations

[0143] Isolated monoclonal antibodies and antigen binding fragments thereof that specifically bind to the extracellular domain of TEM8 are provided. The antibodies and antigen binding fragments can be fully human. In several embodiments, the antibodies and antigen binding fragments can neutralize a biological function or property of TEM8 protein in vivo, including, but not limited to, a reduction and / or inhibition of pathological4239-112153-02angiogenesis, a reduction and / or inhibition of tumor growth, a reduction or inhibition of tumor metastasis, and / or a reduction or inhibition in fibrosis following onset of ischemic conditions.

[0144] In several embodiments, the antibodies and antigen binding fragments can be used to treat TEM8-associated cancer, TEM8-associatred pathological angiogenesis, and / or cardiovascular disease, such as myocardial infarction. Also disclosed herein are compositions including the antibodies and antigen binding fragments and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies or antigen binding fragments, expression vectors including these nucleic acids, and isolated host cells that express the nucleic acids are also provided.

[0145] Compositions comprising the monoclonal antibodies specific for TEM8 can be used for research, diagnostic and therapeutic purposes. For example, the monoclonal antibodies can be used to diagnose or treat a subject having pathogenic angiogenesis.A. Monoclonal Antibodies that Specifically Bind to TEM8 and Antigen Binding Fragments Thereof

[0146] The discussion of monoclonal antibodies below refers to isolated monoclonal antibodies that include heavy and / or light chain variable domains (or antigen binding fragments thereof) comprising a CDR1, CDR2, and / or CDR3 with reference to the IMGT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequence and the CDRs of the heavy and light chain of the disclosed monoclonal antibody according to the IMGT numbering scheme are provided in the listing of sequences, but these are exemplary only.

[0147] In some implementations, a monoclonal antibody is provided that comprises the heavy and light chain CDRs of any one of the antibodies described herein. In some implementation, a monoclonal antibody is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein.1. Exemplary monoclonal antibodies and antigen binding fragments

[0148] Antibody sequences, including the amino acid sequences of variable regions and CDRs (according to the IMGT, Kabat, and Chothia numbering schemes) of the heavy and light chains of exemplary monoclonal antibodies that bind to the TEM8 extracellular domain are shown in Example 4. Further description is provided below.4239-112153-02Monoclonal antibody YL9

[0149] In some implementations, the antibody or antigen binding fragment is based on or derived from the YL9 antibody, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment may be used to treat TEM8-associated cancer, TEM8-associated pathological angiogenesis, and / or cardiovascular disease.

[0150] In some examples, the antibody or antigen binding fragment comprises a VHand a VLcomprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the YL9 antibody, and specifically binds to the extracellular domain of TEM8.

[0151] In some implementations, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 8, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to the extracellular domain of TEM8. In additional implementations, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 8 and 9, respectively, and specifically binds to the extracellular domain of TEM8.

[0152] In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 1, 2, and 3, respectively, and / or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 4, 5, and 6, respectively, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 1, 2, and 3, respectively, a V comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 4, 5, and 6 respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and the antibody or4239-112153-02antigen binding fragment specifically binds to the extracellular domain of TEM8. In this implementation, variations due to sequence identify fall outside the CDRs.

[0153] In some implementations, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 8, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 8 and 9, respectively, and specifically binds to the extracellular domain of TEM8.Monoclonal antibody YK2

[0154] In some implementations, the antibody or antigen binding fragment is based on or derived from the YK2 antibody, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment may be used to treat TEM8-associated cancer, TEM8-associated pathological angiogenesis, and / or cardiovascular disease.

[0155] In some examples, the antibody or antigen binding fragment comprises a VHand a VLcomprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the YK2 antibody, and specifically binds to the extracellular domain of TEM8.

[0156] In some implementations, the antibody or antigen binding fragment comprises a VHcomprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 15, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 16, and specifically binds to the extracellular domain of TEM8. In additional implementations, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 15 and 16, respectively, and specifically binds to the extracellular domain of TEM8.4239-112153-02

[0157] In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 9, 10, and 11, respectively, and / or a VLcomprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 12, 13, and 14, respectively, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 9, 10, and 11, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 12, 13, and 14 respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 16, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16, and the antibody or antigen binding fragment specifically binds to the extracellular domain of TEM8. In this implementation, variations due to sequence identify fall outside the CDRs.

[0158] In some implementations, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 15, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 16, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 15 and 16, respectively, and specifically binds to the extracellular domain of TEM8.Monoclonal antibody YK3

[0159] In some implementations, the antibody or antigen binding fragment is based on or derived from the YK3 antibody, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment may be used to treat TEM8-associated cancer, TEM8-associated pathological angiogenesis, and / or cardiovascular disease.

[0160] In some examples, the antibody or antigen binding fragment comprises a VHand a VLcomprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the YK3 antibody, and specifically binds to the extracellular domain of TEM8.

[0161] In some implementations, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least4239-112153-0297%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 23, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VLcomprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 24, and specifically binds to the extracellular domain of TEM8. In additional implementations, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 23 and 24, respectively, and specifically binds to the extracellular domain of TEM8.

[0162] In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 17, 18, and 19, respectively, and / or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 20, 21, and 22, respectively, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 17, 18, and 19, respectively, a V comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 20, 21, and 22 respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 23, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 24, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24, and the antibody or antigen binding fragment specifically binds to the extracellular domain of TEM8. In this implementation, variations due to sequence identify fall outside the CDRs.

[0163] In some implementations, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 23, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 24, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 23 and 24, respectively, and specifically binds to the extracellular domain of TEM8.4239-112153-02Monoclonal antibody YK7

[0164] In some implementations, the antibody or antigen binding fragment is based on or derived from the YL7 antibody, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment may be used to treat TEM8-associated cancer, TEM8-associated pathological angiogenesis, and / or cardiovascular disease.

[0165] In some examples, the antibody or antigen binding fragment comprises a VHand a VLcomprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the YL7 antibody, and specifically binds to the extracellular domain of TEM8.

[0166] In some implementations, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 30, and specifically binds to the extracellular domain of TEM8. In additional implementations, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 29 and 30, respectively, and specifically binds to the extracellular domain of TEM8.

[0167] In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 25, 10, and 26, respectively, and / or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 27, 13, and 28, respectively, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 25, 10, and 26, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 27, 13, and 28, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 30, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 30, and the4239-112153-02antibody or antigen binding fragment specifically binds to the extracellular domain of TEM8. In this implementation, variations due to sequence identify fall outside the CDRs.

[0168] In some implementations, the antibody or antigen binding fragment comprises a VHcomprising the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 30, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VHand a VLcomprising the amino acid sequences set forth as SEQ ID NOs: 29 and 30, respectively, and specifically binds to the extracellular domain of TEM8.Monoclonal antibody YL51

[0169] In some implementations, the antibody or antigen binding fragment is based on or derived from the YL51 antibody, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment may be used to treat TEM8-associated cancer, TEM8-associated pathological angiogenesis, and / or cardiovascular disease.

[0170] In some examples, the antibody or antigen binding fragment comprises a VHand a VLcomprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the YL51 antibody, and specifically binds to the extracellular domain of TEM8.

[0171] In some implementations, the antibody or antigen binding fragment comprises a VHcomprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 38, and specifically binds to the extracellular domain of TEM8. In additional implementations, the antibody or antigen binding fragment comprises a VHand a VLindependently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 37 and 38, respectively, and specifically binds to the extracellular domain of TEM8.4239-112153-02

[0172] In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 31, 32, and 33, respectively, and / or a VLcomprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 31, 32, and 33, respectively, a VLcomprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 38, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 38, and the antibody or antigen binding fragment specifically binds to the extracellular domain of TEM8. In this implementation, variations due to sequence identify fall outside the CDRs.

[0173] In some implementations, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to the extracellular domain of TEM8. In more implementations, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 38, and specifically binds to the extracellular domain of TEM8. In some implementations, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 37 and 38, respectively, and specifically binds to the extracellular domain of TEM8.2. Additional Description of Antibodies and Antigen Binding Fragments

[0174] An antibody or antigen binding fragment of the antibodies disclosed herein can be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen binding fragment can include any suitable framework region, such as (but not limited to) a human framework region from another source, or an optimized framework region. Alternatively, a heterologous framework region, such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies.

[0175] The antibody can be of any isotype. The antibody can be, for example, an IgA, IgM or an IgG antibody, such as IgG1, IgG2, IgG3, or IgG4. The class of an antibody that specifically binds to TEM8 protein can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic4239-112153-02acid molecule encoding VL or VH is then operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved, for example, using a vector or nucleic acid molecule that comprises a CLor CHchain. For example, an antibody that specifically binds the TEM8 protein, that was originally IgG may be class switched to an IgA. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG2, IgGs, or IgG4.

[0176] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers and so on.

[0177] The antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or antigen binding fragment is derivatized such that the binding to the TEM8 protein is not affected adversely by the derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).a. Binding affinity

[0178] In several implementations, the antibody or antigen binding fragment specifically binds the TEM8 protein with an affinity (e.g., measured by KD) of no more than 1.0 x 10'7M, no more than 1.0 x 10"8M, no more than 5.0 x 10"8M, no more than 1.0 x 10"9M, no more than 5.0 x 10'9M, no more than 1.0 x 10'10M, no more than 5.0 x 10’10M, or no more than 1.0 x 1011M. KD can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293(4)1865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg / ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (NUNC™ Catalog #269620), 100 pM or 26 pM [125I]-antigen are mixed with serial dilutions of a Fab of interest4239-112153-02(e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57(20):4593-4599, 1997). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pl / well of scintillant (MICROS CINT™-20; PerkinElmer) is added, and the plates are counted on a TOPCOUNT™ gamma counter (PerkinElmer) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[0179] In another assay, KD can be measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N. J.) at 25° C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg / ml (~0.2 pM) before injection at a flow rate of 5 l / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C at a flow rate of approximately 25 l / min.Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106M-1s-1by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. Affinity can also be measured by high throughput SPR using the Carterra LSA.4239-112153-02b. Antigen Binding Fragments

[0180] Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a heavy chain and VLand specifically bind TEM8 protein. These antibody fragments retain the ability to selectively bind with the antigen and are “antigen-binding” fragments. Non-limiting examples of such fragments include:(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, 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;(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain;(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;(4) Fv, a genetically engineered fragment containing the VHand VLexpressed as two chains; and(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012,doi: 10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (VH-domain-linker domain-VL-domain; VL-domain-linker domain-VH-domain) may be used.(6) A dimer of a single chain antibody (SCFV2), defined as a dimer of a scFV. This has also been termed a “miniantibody.”

[0181] Any suitable method of producing the above-discussed antigen binding fragments may be used. Non-limiting examples are provided in Harlow and Lane, Antibodies: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, New York, 2013.

[0182] Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment.Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted4239-112153-02F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.

[0183] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.c. Variants

[0184] In some implementations, amino acid sequence variants of the antibodies provided herein are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0185] In some implementations, antibody variants having one or more amino acid substitutions are provided. In a non-limiting example, sites of interest for substitutional mutagenesis include the framework regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, increased effectiveness for treating TEM8-associated cancer, decreased immunogenicity, or improved antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).

[0186] The variants typically retain amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield.

[0187] In some implementations, an antibody or antigen binding fragment is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.4239-112153-02

[0188] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(1):26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some implementations, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.

[0189] In one implementation, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol., 336(5):1239-1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5):614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; US Pat. Appl. No. US 2003 / 0157108 and WO 2004 / 056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng., 87(5): 614-622, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003 / 085107).4239-112153-02

[0190] Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.) U. S. Pat. No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0191] In several implementations, the constant region of the antibody comprises one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum halflife of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several implementations, the antibody comprises an amino acid substitution that increases binding to the FcRn. Several such substitutions are known, such as substitutions at IgG constant regions T250Q and M428E (see, e.g., Hinton et al., J Immunol., 176(1):346-356, 2006); M428E and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2): 157- 159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18( 12): 1759- 1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen binding fragments can be linked to or comprise a Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions (EU numbering). The M428L and N434S substitutions (EU numbering) are equivalent to M459L and N465S substitutions (Kabat numbering). Exemplary sequences of an IgGl constant region containing the M428L and N434S substitutions are provided herein, which can be paired with an appropriate light chain constant region, and appropriate VH and VL regions as provided herein to generate a monoclonal antibody. As used herein, reference to an antibody with the “LS” substitution (or similar language) indicates that the antibody heavy chain is an IgG with M428L and N434S substitutions.

[0192] In some implementations, the constant region of the antibody comprises one or more amino acid substitutions to optimize ADCC. ADCC is mediated primarily through a set of closely related Fey receptors. In some implementations, the antibody comprises one or more amino acid substitutions that increase binding to FcyRIIIa. Several such substitutions are4239-112153-02known, such as substitutions at IgG constant regions S239D and I332E (see, e.g., Lazar et al, Proc. Natl., Acad. Sci. U. S. A., 103(11):4005-4010, 2006): and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U. S. A., 103(11):4005-4010, 2006).

[0193] In further examples, the LALA-PG mutations (L234A, L235A and P329G) are introduced, which block binding to Fey receptors.

[0194] In some examples, the antibodies, or an antigen binding fragment thereof is modified such that it is directly cytotoxic to infected cells, or uses natural defenses such as complement, ADCC, or phagocytosis by macrophages.

[0195] In some implementations, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be detemrined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.B. Chimeric Antigen Receptors (CARs)

[0196] The disclosed monoclonal antibodies can be used to produce CARs (also known as chimeric T cell receptors, artificial T cell receptors or chimeric immunoreceptors) and / or cytotoxic T lymphocytes (CTLs) engineered to express CARs. Generally, CARs include a binding moiety, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many instances,4239-112153-02the binding moiety is an antigen binding fragment of a monoclonal antibody, such as a scFv or single-domain antibody. The spacer / hinge region typically includes sequences from IgG subclasses, such as IgGl, IgG4, IgD and CD8 domains. The transmembrane domain can be can derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8 or CD28. Several different endodomains have been used to generate CARs. For example, the endodomain can consist of a signaling chain having an ITAM, such as CD3ζ or FceRIγ. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27 and / or DAP10.

[0197] CTLs expressing CARs can be used to target a specific cell type, such as an TEM8-associated cancer. Thus, the monoclonal antibodies disclosed herein can be used to engineer CTLs that express a CAR containing the TEM8-specific monoclonal antibody (for example, an scFv or a VH single-domain antibody), thereby targeting the engineered CTLs to TEM8-expressing cells, such as TEM8 -expressing cells associated with colorectal, skin, lung, breast, prostate, or head and neck cancer. Engineered T cells have previously been used for adoptive therapy for some types of cancer (see, for example, Park et al., Mol Ther 15(4):825-833, 2007). The use of T cells expressing CARs is more universal than standard CTL-based immunotherapy because CTLs expressing CARs are HLA unrestricted and can therefore be used for any patient having a tumor that expresses the target antigen.

[0198] Accordingly, provided herein are CARs that include a TEM8-specific antibody or antigen binding fragment thereof.

[0199] Also provided are isolated nucleic acid molecules and vectors encoding the CARs, and host cells, such as CTLs, expressing the CARs. CTLs expressing CARs comprised of a TEM8-specific monoclonal antibody or antigen binding fragment thereof can be used for the treatment of TEM8-associated cancers. In some embodiments herein, the CAR is a bispecific CAR.

[0200] In some instances, it is desirable to regulate the activation and expansion of CAR-expressing T cells after they have been infused into a patient. Several strategies have been developed to module CAR-expressing T cells in vivo, including the use of antibody -based switches that mediate interactions between CAR-expressing T cells and targeted tumors cells, as described by Rodgers et al. (Proc Natl Acad Sci USA 113(4): E459-E468, 2016, which is incorporated herein by reference). The antibody -based switches are comprised of a tumor antigen-specific antibody that has been grafted with a peptide neo-epitope (PNE). Switchable4239-112153-02CAR T (sCAR-T) cells are designed to specifically bind the PNE. Since the sCAR-T cells do not bind endogenous antigens, the presence of the switch is required for its activation.

[0201] Thus, provided herein are antibody-based switches that include a TEM8-specific monoclonal antibody or antigen binding fragment disclosed herein fused to a heterologous peptide, such as a PNE. In some embodiments, the heterologous peptide is not endogenous to humans (for example, it is a peptide that is not found in the human proteome). In some examples, the heterologous peptide is about 8 amino acids to about 20 amino acids in length, such about 10 to about 18 amino acids in length, such as about 12 to about 16 amino acids in length, such as about 14 amino acids in length. In particular examples, the heterologous peptide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.C. Immunoconjugates

[0202] The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody. A therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule. One of skill in the art will appreciate that therapeutic agents can include various drugs such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as125I,32P,14C,3H and35S and other labels, target moieties and ligands.

[0203] The choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell). Conversely, where it is desired to invoke a non-lethal biological response, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0204] With the therapeutic agents and antibodies described herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0205] Effector molecules can be linked to an antibody of interest using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means4239-112153-02may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids 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.

[0206] 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 the linker to release the effector molecule from the antibody 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.

[0207] In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0208] The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).4239-112153-02

[0209] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.

[0210] The antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP). An antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

[0211] An antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary4239-112153-02antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0212] An antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides:3H,14C,13N,35S,90Y,99Tc,111In,125I,131I.

[0213] An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.

[0214] Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U. S. Patent Nos.5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U. S. Patent No. 5,602,095). As used herein " Pseudomonas exotoxin" refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264:14256-14261, 1989).

[0215] PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell.Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U. S. Patent Nos. 4,892,827; 5,512,658: 5,602,095: 5,608,039; 5,821,238; and 5,854,044; U. S. Patent Application Publication No.2015 / 0099707; PCT Publication Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al.. J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333: C1-C6, 1997.4239-112153-02

[0216] Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to PE-LR, PE-6X, PE-8X, PE-LR / 6X and PE-LR / 8X (see, for example, Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al.. Proc Natl Acad Sci USA 105(32): 11311 - 11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954 and WO 2011 / 032022, which are herein incorporated by reference).

[0217] In some examples, the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other examples, the PE is a variant designated PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other examples, the PE variant is PE with reducing immunogenicity. In yet other examples, the PE is a variant designated PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0218] Modification of PE may occur in any previously described variant, including cytotoxic fragments of PE (for example, PE38, PE-LR and PE-LR / 8M). Modified PEs may include any substitution(s), such as for one or more amino acid residues within one or more T-cell epitopes and / or B cell epitopes of PE, or deletion of one or more T-cell and / or B-cell epitopes (see, for example, U. S. Patent Application Publication No. 2015 / 0099707).

[0219] Contemplated forms of PE also include deimmunized forms of PE, for example versions with domain II deleted (for example, PE24). Deimmunized forms of PE are described in, for example, PCT Publication Nos. WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0220] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing the tumor or viral antigen on their surface. Thus, an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing cell-surface antigen. This can be done for therapeutic, diagnostic or research purposes. Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.

[0221] Alternatively, the molecule linked to an antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety4239-112153-02that is preferably shielded 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. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0222] Antibodies described herein can also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example,3H,125I,35S,14C, or32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.

[0223] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.D. Antibody-Drug Conjugates (ADCs)

[0224] ADCs are compounds comprised of a tumor antigen-specific antibody (or antigenbinding fragment thereof) and a drug, typically a cytotoxic agent, such as an anti -microtubule agent or cross-linking agent. Because ADCs are capable of specifically targeting cancer cells, the drug can be much more potent than agents used for standard chemotherapy. The most common cytotoxic drugs currently used with ADCs have an IC50 that is 100- to 1000-fold more potent than conventional chemotherapeutic agents. Common cytotoxic drugs include anti-microtubule agents, such as maytansinoids and auristatins (such as auristatin E and auristatin F). Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PDBs), which covalently bind the minor groove of DNA to form interstrand crosslinks. In many instances, ADCs comprise a 1:2 to 1:4 ratio of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0225] The antibody and drug can be linked by a cleavable or non-cleavable linker.However, in some instances, it is desirable to have a linker that is stable in the circulation to prevent systemic release of the cytotoxic drug that could result in significant off-target4239-112153-02toxicity. Non-cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, digestion of the antibody by lysosomal proteases results in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0226] One method for site-specific and stable conjugation of a drug to a monoclonal antibody is via glycan engineering. Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant β1,4-galactosyltransferase enzyme (Y289L-Gal-T1; U. S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162, herein incorporated by reference), 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical handle for conjugation.

[0227] The oligosaccharide chain attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-Gl) or completely degalactosylated (IgG-GO). Treatment of a monoclonal antibody with β1,4-galactosidase converts the antibody to the IgG-GO glycoform. The mutant β1,4-galactosyltransferase enzyme is capable of transferring 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-Gl and IgG-GO glycoforms. The chemical handle on the transferred sugar enables conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0228] Provided herein are ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) TEM8. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent and / or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, a PDB, an auristatin, a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0229] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al., J Am4239-112153-02Chem Soc, 87:5793-5795, 1965; Leimgruber et al., J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs, both naturally-occurring and synthetic analogues, have been reported (Gerratana, Med Res Rev 32(2):254-293, 2012; and U. S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As one example, PDB dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010 / 0203007). Exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US 2011 / 0256157; and WO 2011 / 130598).

[0230] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U. S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U. S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U. S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0231] In some embodiments, the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U. S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin- 10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer (U. S. Patent No.5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine -phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine -norephedrine), 5-benzoyl valeric acid-AE ester (AEVB), and other auristatins (see, for example, U. S.Publication No. 2013 / 0129753).4239-112153-02

[0232] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998).Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U. S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0233] In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can operate to kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and / or interactions of the drug molecules with the cell membrane. Nonlimiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11 (4): 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0234] In some embodiments, the ADC can further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC. In some embodiments, ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.

[0235] In some examples, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Exemplary linkers with such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0236] In some examples, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, a heteroatom of4239-112153-02the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

[0237] In some examples, the linker is a cleavable linker, which facilitates release of the drug. Examples of cleavable linkers include acid-labile linkers (for example, comprising hydrazone), protease-sensitive linkers (for example, peptidase-sensitive), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; U. S. Patent No.5,208,020).

[0238] The ADCs disclosed herein can be used for the treatment of a TEM8-associated cancer alone or in combination with another therapeutic agent and / or in combination with any standard therapy for the treatment of cancer (such as surgical resection of the tumor, chemotherapy or radiation therapy).E. Multi-specific Antibodies

[0239] Multi-specific antibodies are recombinant proteins comprised of antigen-binding fragments of two or more different monoclonal antibodies. For example, bispecific antibodies are comprised of antigen-binding fragments of two different monoclonal antibodies. Thus, bispecific antibodies bind two different antigens and trispecific antibodies bind three different antigens. Multi-specific antibodies can be used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and at least one tumor antigen. The TEM8-specific monoclonal antibodies disclosed herein can be used to generate multi-specific (such as bispecific or trispecific) antibodies that target both TEM8and CTLs, or target both TEM8and NK cells, thereby providing a means to treat TEM8-expressing cancers.

[0240] Bi-specific T-cell engagers (BiTEs) are a type of bispecific monoclonal antibody that are fusions of a first single-chain variable fragment (scFv) that targets a tumor antigen and a second scFv that binds T cells, such as bind CD3 on T cells. In some embodiments herein, one of the binding moieties of the BiTE (such as one of the scFv molecules) is specific for TEM8.

[0241] Bi-specific killer cell engagers (BiKEs) are a type of bispecific monoclonal antibody that are fusions of a first scFv that targets a tumor antigen and a second scFv that binds a NK cell activating receptor, such as CD 16.4239-112153-02

[0242] Provided herein are multi-specific, such as trispecific or bispecific, monoclonal antibodies comprising a TEM8-specific monoclonal antibody. In some embodiments, the multi-specific monoclonal antibody further comprises a monoclonal antibody, or antigenbinding fragment thereof, that specifically binds a component of the T cell receptor, such as CD3. In other embodiments, the multi-specific monoclonal antibody further comprises a monoclonal antibody, or antigen-binding fragment thereof, that specifically binds a NK cell activating receptor, such as CD 16, Ly49, or CD94. Also provided are isolated nucleic acid molecules and vectors encoding the multi-specific antibodies, and host cells comprising the nucleic acid molecules or vectors. Multi-specific antibodies comprising a TEM8-specific antibody can be used for the treatment of cancers that express TEM8. Thus, provided herein are methods of treating a subject with cancer by selecting a subject with a cancer that expresses TEM8, and administering to the subject a therapeutically effective amount of the TEM8-targeting multi-specific antibody.F. Antibody-Nanoparticle Conjugates

[0243] The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic agents or other anti-cancer agents directly to tumor cells via binding of the antibody to a tumor specific antigen (e.g. TEM8) expressed on the surface of tumor cells. The use of nanoparticles reduces off-target side effects and can also improve drug bioavailability and reduce the dose of a drug required to achieve a therapeutic effect. Nanoparticle formulations can be tailored to suit the drug that is to be carried or encapsulated within the nanoparticle. For example, hydrophobic molecules can be incorporated inside the core of a nanoparticle, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell. Examples of nanoparticles include, but at not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, niosomes, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3): 381 -394, 2011).

[0244] Liposomes are currently one of the most common types of nanoparticles used for drug delivery. An antibody conjugated to a liposome is often referred to as an “immunoliposome.” The liposomal component of an immunoliposome is typically a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, the phospholipids are composed of a hydrophilic head group and two hydrophobic chains to enable encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from the circulation via macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size and charge of the liposome can be modulated.4239-112153-02The surface of the liposome may also be modified, such as with a glycolipid or sialic acid. For example, the inclusion of polyethylene glycol (PEG) significantly increases circulation half-life. Liposomes for use as drug delivery agents, including for preparation of immunoliposomes, have been described in the art (see, for example, Paszko and Senge, Cun- Med Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U. S. Patent Application Publication Nos. 2011 / 0268655; 2010 / 0329981).

[0245] Niosomes are non-ionic surfactant-based vesicles having a structure similar to liposomes. The membranes of niosomes are composed only of nonionic surfactants, such as polyglyceryl-alkyl ethers or N-palmitoylglucosamine. Niosomes range from small, unilamellar to large, multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and increase bioavailability of encapsulated drugs.

[0246] Dendrimers include a range of branched polymer complexes. These nanoparticles are water-soluble, biocompatible and are sufficiently non-immunogenic for human use.Generally, dendrimers consist of an initiator core, surrounded by a layer of a selected polymer that is grafted to the core, forming a branched macromolecular complex.Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents and chemotherapeutic agents.

[0247] Polymeric micelles are composed of aggregates of amphiphilic co-polymers (consisting of both hydrophilic and hydrophobic monomer units) assembled into hydrophobic cores, surrounded by a corona of hydrophilic polymeric chains exposed to the aqueous environment. In many cases, the polymers used to prepare polymeric micelles are heterobifunctional copolymers composed of a hydrophilic block of PEG, poly(vinyl pyrrolidone) and hydrophobic poly(L-lactide) or poly(L-lysine) that forms the particle core. Polymeric micelles can be used to carry drugs that have poor solubility. These nanoparticles have been used to encapsulate a number of anti -cancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0248] Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The formulation selected typically depends on the solubility of the therapeutic agent to be carried / encapsulated: poorly water-soluble drugs are more readily encapsulated within a nanospheres, while water-soluble and labile drugs, such as DNA and proteins, are more4239-112153-02readily encapsulated within nanocapsules. The polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkylcyanoacrylates), poly(lactic acid) (PLA), poly(glycolic acids) (PGA), and poly(D, L-lactic-co-glycolic acid) (PLGA).

[0249] Antibodies, including scFv and single-domain antibodies, can be conjugated to a suitable nanoparticle according to standard methods known in the art. For example, conjugation can be either covalent or non-covalent. In some embodiments in which the nanoparticle is a liposome, the antibody is attached to a sterically stabilized, long circulation liposome via a PEG chain. Coupling of antibodies or antibody fragments to a liposome can also involve thioester bonds, for example by reaction of thiols and maleimide groups. Crosslinking agents can be used to create sulfhydryl groups for attachment of antibodies to nanoparticles (Paszko and Senge, CurrMed Chem 19(31)5239-5277, 2012).G. Polynucleotides and Expression

[0250] Nucleic acid molecules (for example, cDNA or RNA molecules) encoding the amino acid sequences of antibodies, antigen binding fragments, and conjugates described herein (e.g., that specifically bind TEM8) are provided. Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several implementations, nucleic acid molecules can encode the VH, the VL, or both the VH and VL (for example in a bicistronic expression vector) of a disclosed antibody or antigen binding fragment. In several implementations, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment.

[0251] The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids which differ in sequence, but which encode the same antibody sequence or a conjugate or fusion protein including the VL and / or VH of the antibody.

[0252] Nucleic acid molecules encoding the antibodies, antigen binding fragments, and conjugates that specifically bind TEM8 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.4239-112153-02

[0253] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4thed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0254] Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3 SR).

[0255] The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be expressed as individual proteins including the VH and / or VL. (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Methods of expressing and purifying antibodies and antigen binding fragments are known and further described herein (see, e.g., Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011). An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a VH and VL, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.

[0256] To create a scFv the VH- and VL-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single -chain protein, with the VL and VH domains joined by the flexible linker (see, e.g., Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. U. S. A., 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2nded., Springer-Verlag, 2010; Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

[0257] The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to TEM8 and another antigen. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains.4239-112153-02

[0258] One or more DNA sequences encoding the antibodies, antigen binding fragments, or conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

[0259] The expression of nucleic acids encoding the antibodies and antigen binding fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter and a human T cell lymphotrophic virus promoter (HTLV)-l. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (z.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

[0260] To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription / translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and / or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and / or acceptor sequences (for example, CMV and / or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the4239-112153-02cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, GPt, neo, and hyg genes.

[0261] Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.

[0262] Once expressed, the antibodies, antigen binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylatically, the polypeptides should be substantially free of endotoxin.

[0263] Methods for expression of antibodies, antigen binding fragments, and conjugates, and / or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory’ Manual, New York: Cold Spring Harbor Laboratory Press, 2009.H. Compositions and Methods of UseI. Pharmaceutical Compositions

[0264] Compositions are provided that include one or more of the disclosed antibodies or antigen binding fragments that specifically bind TEM8 in a carrier (such as a pharmaceutically acceptable carrier). The compositions can be prepared in unit dosage forms for administration to a subject. The compositions can be formulated for systemic (such as intravenous) or local (such as pericardial) administration. In one example, the antibody that specifically binds TEM8 or an antigen binding fragment thereof is formulated for parenteral administration, such as intravenous administration. Compositions including an antibody or antigen binding fragment as disclosed herein are of use, for example, for the treatment of cardiovascular disease or myocardial infarction.4239-112153-02

[0265] The compositions for administration can include a solution of the antibody or antigen binding fragment dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions 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, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody or antigen binding fragment or conjugate 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.

[0266] A typical composition for intravenous administration comprises about 0.01 to about 30 mg / kg of antibody or antigen binding fragment per subject per day. Any suitable method may be used for preparing administrable compositions; non-limiting examples are provided in such publications as Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013. In some aspects, the composition can be a liquid formulation including one or more antibodies, antigen binding fragments (such as an antibody or antigen binding fragment that specifically binds to TEM8), in a concentration range from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 1 mg / ml to about 10 mg / ml.

[0267] Antibodies or an antigen binding fragment thereof, or a nucleic acid encoding such molecules, can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution, or an antigen binding fragment or a nucleic acid encoding such antibodies or antigen binding fragments, can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg / kg of body weight. Considerable experience is available in the art in the administration of antibody dmgs, which have been marketed in the U. S. since the approval of Rituximab in 1997. Antibodies, antigen binding fragments, or a nucleic acid encoding such molecules, can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, 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 be4239-112153-02infused 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.

[0268] Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 µm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 µm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 µm in diameter and are administered subcutaneously or intramuscularly.

[0269] Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug.

[0270] In some embodiments, long-term administration is utilized, for example by using a continuous release pump. In further embodiments, prolonged administration is used, for example by administering a composition including the antibody or antigen binding fragment thereof that specifically binds to the TEM8 extracellular domain on the cell surface in a controlled release formulation.

[0271] In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U. S. Patent No. 5,643,578, U. S. Patent No. 5,593,972 and U. S. Patent No. 5,817,637. U. S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof. In some aspects, a disclosed4239-112153-02antibody or antigen binding fragment is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005).

[0272] In several aspects, a subject can be administered an effective amount of an AAV viral vector that comprises one or more nucleic acid molecules encoding a disclosed antibody or antigen binding fragment. The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody or antigen binding fragment, and administration of the effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antibody or antigen binding fragment in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015.

[0273] In one aspect, a nucleic acid encoding a disclosed antibody or antigen binding fragment thereof is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

[0274] Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 µg / kg to about 50 mg / kg, and typically are about 0.005 mg / kg to about 5 mg / kg (see, e.g., U. S. Patent No. 5,589,466).2. Methods of Treating Cancer

[0275] The antibodies, compositions, CARs (and CTLs expressing CARs), ADCs, multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes and immunoconjugates disclosed herein can be administered to treat a TEM8-associated cancer, for example to slow or inhibit the pathogenesis and / or metastasis of a TEM8- associated cancer. In these applications, a therapeutically effective amount of a composition 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 associated with TEM8 expression (such as TEM8-positive pathological angiogenesis associated with the cancer), such as breast, colorectal, lung or skin cancer.

[0276] Provided herein is a method of treating a TEM8- associated cancer in a subject by administering to the subject a therapeutically effective amount of a TEM8- associated4239-112153-02antibody, immunoconjugate, CAR (e.g. a CTL expressing a CAR), ADC, multi-specific (such as bispecific or trispecific) antibody, antibody -nanoparticle conjugate, immunoliposome or composition disclosed herein. Also provided herein is a method of inhibiting metastasis of a TEM8-associated cancer in a subject by administering to the subject a therapeutically effective amount of a TEM8-specific antibody, immunoconjugate, CAR (e.g. a CTL expressing a CAR), ADC, multi-specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. In some embodiments, the TEM8-associated cancer is breast, colorectal, lung or skin cancer.

[0277] A therapeutically effective amount of a TEM8-specific monoclonal antibody, CAR (e.g. a CTL expressing a CAR), ADC, multi-specific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the antibody-based composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

[0278] Administration of the TEM8-specific antibodies, CARs, ADCs, immunoconjugates, multi-specific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes and compositions disclosed herein 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, compositions and immunoconjugates disclosed herein. Exemplary anticancer 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, 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.

[0279] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0280] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.4239-112153-02

[0281] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0282] Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

[0283] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0284] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK& F 106528, and TNF (tumor necrosis factor; Genentech).

[0285] Another common treatment for some types of cancer is surgical treatment, for example surgical resection of a metastatic tumor. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.3. Methods of Treating Pathological Angiogenesis

[0286] In some embodiments, administration of a therapeutically effective amount of an antibody or antigen binding fragment that specifically binds TEM8 or conjugate thereof or4239-112153-02CAR T cell expressing an antigen binding fragment that specifically binds TEM8 decreases pathological angiogenesis, such as pathological angiogenesis that occurs with macular degeneration. Thus, a subject can be selected for treatment that has, is suspected of having or is at risk of developing macular or corneal degeneration due to pathological angiogenesis.

[0287] In some examples, the antibodies, antigen binding fragments, compositions and conjugates disclosed herein can be administered to a subject to decrease pathological angiogenesis in the subject, to treat corneal or retinal degeneration. In these applications, a therapeutically effective amount of an antibody or antigen binding fragment that specifically binds TEM8 or composition is administered to a subject in an amount and under conditions sufficient to form an immune complex with TEM8, thereby treating the pathological angiogenesis. Examples of suitable subjects include those diagnosed with or suspecting of having corneal or retinal degeneration due to pathological angiogenesis.

[0288] The therapeutically effective amount will depend upon the severity of the disease and the general state of the patient’s health. A therapeutically effective amount is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one embodiment, a therapeutically effective amount is the amount necessary to inhibit pathological angiogenesis, or the amount that is effective at reducing a sign or a symptom of the pathological angiogenesis. The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which prevent or reduce pathological angiogenesis.

[0289] Subjects that can benefit from the disclosed methods include human and veterinary subjects. Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has pathological angiogenesis. The presence of pathological angiogenesis indicates that the pathological angiogenesis can be treated using the methods provided herein.

[0290] Any method of administration can be used for the disclosed antibodies, antigen binding fragments, compositions and additional agents, including local and systemic administration. For example topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, intraretinal, and subcutaneous administration can be used. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Methods of administration include injection for which the antibodies, antigen4239-112153-02binding fragments, or compositions are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes. In some examples, the antibodies, antigen binding fragments, or compositions is applied as an eye drop topically to the cornea, or intravitreally into the eye.4. Methods of Treating Cardiovascular Disease

[0291] Methods are also provided for treating, reducing the risk of, or preventing, cardiovascular disease, such as progressive heart failure caused by myocardial infarction, high blood pressure and other forms of heart disease in a subject. The disclosed methods comprise administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds to the TEM8 extracellular domain on the cell surface as described herein.

[0292] In some aspects, the method inhibits the progression of cardiovascular disease. In further aspects, the method inhibits cardiac fibrosis or development of cardiac fibrosis in the patient following myocardial infarction. In additional aspects, the antibody or antigen binding fragment is administered following myocardial infarction, such as within one day, one week, or one month, of myocardial infarction.

[0293] In some aspects, formation of an immune complex between TEM8 and the antibody or antigen binding fragment treats the myocardial ischemia by reducing or preventing infiltration of immune cells into affected myocardium, activation of myofibroblasts, or maladaptive chronic regeneration and inflammation.

[0294] In some aspects, the TEM8-specific antibody or antigen binding fragment inhibits a biological function or property of TEM8 protein in vivo, including, but not limited to, blocking the interaction between TEM8 and collagen. In some aspects the collagen is type I collagen. In other aspects the collagen is type VI collagen. In several aspects administering antibodies or antigen binding fragments that block the interaction between TEM8 and collagen prevents or treats myocardial ischemia by reducing direct ischemic injury, ischemia, or reperfusion injury.

[0295] In these applications, a therapeutically effective amount of an antibody or antigen binding fragment that specifically binds TEM8 or composition is administered to a subject in an amount and under conditions sufficient to form an immune complex with TEM8, thereby inhibiting the progression of cardiovascular disease, the progression of cardiac fibrosis, or4239-112153-02inhibiting a sign or symptom of cardiovascular disease, myocardial infarction, and / or myocardial ischemia.

[0296] In one example, a desired response is to treat or slow progressive heart failure in a subject, for example to increase blood flow to cardiac tissues in the subject, or to inhibit the progression of or reduce cardiac fibrosis in the subject.

[0297] In one example, a desired response is to treat myocardial ischemia in a subject, for example to increase blood flow to cardiac tissues in the subject, or to inhibit the progression of or reduce cardiac fibrosis in the subject.

[0298] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment decreases a sign or symptom of cardiovascular disease, myocardial infarction, and / or myocardial ischemia in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0299] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment increases cardiac ejection fraction in the subject with reduced cardiac ejection fraction due to cardiovascular disease, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0300] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment reduces progressive decrease in cardiac ejection fraction following myocardial infarction in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0301] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment reduces cardiac fibrosis in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0302] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment increases blood flow to cardiac tissues in the4239-112153-02subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0303] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment inhibits the progression of cardiovascular disease in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0304] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment inhibits the progression of cardiac fibrosis in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment.

[0305] In some aspects, administration of the therapeutically effective amount of the TEM8-specific antibody or antigen binding fragment reduces Heart Failure with preserved Ejection Fraction (HFpEF) in the subject, for example, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% as compared to a response in the absence of or prior to administration of the TEM8-specific antibody or antigen binding fragment. In a non-limiting example, the effect of treatment on Heart Failure with preserved Ejection Fraction (HFpEF) can be assessed in an animal model, such as described in Schiattarella et al., Nature, 568:351-356, 2019.

[0306] Subjects that can benefit from the disclosed methods include human and veterinary subjects. Examples of suitable subjects include those diagnosed with or suspecting of having cardiovascular disease (for example a subject that recently has a myocardial infarction, or a subject with coronary heart disease, or deep vein thrombosis). Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has cardiovascular disease, has had a myocardial infarction, or is at risk of a myocardial infarction. In some examples a subject is selected because they exhibit signs or symptoms of myocardial ischemia, cardiovascular disease, or myocardial infarction.

[0307] The therapeutically effective amount will depend upon the severity of the disease and the general state of the patient’s health. A therapeutically effective amount is that which4239-112153-02provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one example, a therapeutically effective amount is the amount necessary to reduce progressive decrease in cardiac ejection fraction following myocardial infarction in the subject. In another example a therapeutically effective amount is the amount necessary to inhibit the progression of cardiovascular disease in the subject, or the amount effective at reducing a sign or symptom of cardiovascular disease. The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which slow the progression of cardiovascular disease, such as that slow the progression of cardiac fibrosis caused by myocardial infarction.

[0308] Any suitable mode of administration can be used, including local and systemic administration. For example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used.

[0309] In one example, the mode of administration is pericardial administration, for example, pericardial administration of a controlled release formulation including the TEM8-specific antibody or antigen binding fragment. For example, the controlled release formulation can be a solid, semi-solid, or encapsulated liquid, which can be physically placed into the pericardial space. In some examples, the TEM8-specific antibody or antigen binding fragment is physically placed in the pericardial space by an instrument, such as a catheter or needle that is advanced transthoracically or intravascularly, or transmyocardially into the pericardial space.

[0310] The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic). In cases in which more than one agent or composition is being administered, one or more routes of administration may be used; for example, an anticoagulant may be administered orally and an antibody or antigen binding fragment or composition may be administered intravenously. Methods of administration include injection for which the antibodies, antigen binding fragments, or compositions are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes. In some examples, sustained intra-cardiac (or near-cardiac) release of the pharmaceutical preparation that includes a therapeutically effective amount of the antibody or antigen binding fragment may be beneficial.4239-112153-025. Methods of Detection and Diagnosis

[0311] Methods are also provided for the detection of the expression of TEM8 in vitro or in vivo. In one example, expression of TEM8 is detected in a biological sample, and can be used to detect the presence of a cell with cell-surface expression of TEM8 in the sample. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also 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. The method of detection can include contacting a cell or sample, or administering to a subject, an antibody or antigen binding fragment that specifically binds to TEM8, or conjugate there of (e.g. a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment.

[0312] One embodiment provides a method of determining if a subject has TEM8-associated cancer, or TEM8-associate pathological angiogenesis by contacting a sample from the subject with a monoclonal antibody (or conjugate) disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0313] Another embodiment provides a method of confirming a diagnosis of TEM8-associated cancer in a subject by contacting a sample from a subject diagnosed with cancer with a monoclonal antibody (or conjugate) disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0314] In one embodiment, the antibody or antigen binding fragment is directly labeled with a detectable marker. In another embodiment, the antibody that binds TEM8 (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that binds the first antibody is utilized for detection. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.

[0315] Suitable labels for the antibody, antigen binding fragment or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of4239-112153-02suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non -limiting exemplary radioactive labels include123I,131I,35S or3H.III. EXAMPLES

[0316] The following examples are provided to illustrate particular features of certain implementations, but the scope of the claims should not be limited to those features exemplified.Example 1Identification of TEM8 Antibodies

[0317] This example illustrates the isolation and characterization of a panel of fully human IgG anti-TEM8 antibodies from a human antibody library. The selection strategy involved serial panning of the libraries on both TEM8-transfected mammalian cells and purified recombinant TEM8-ED protein derived from mammalian cells, and resulted in the identification of five high-affinity TEM8 antibodies, termed YL9, YK2, YK3, YK7, and YL51.

[0318] Five fully human IgG monoclonal antibodies, YL9, YK2, YK3, YK6, and YL51, were identified from a human naive phage display scFv library by sorting, screening, and affinity maturation against human and mouse TEM8. The TEM8 antibodies were identified from a human scFv naive antibody phage display library which was constructed from using the total RNA of bone marrow collected from 56 healthy donors and PBMCs from 8 healthy donors. Both IgG and IgM VH repertoires were amplified with designed primers and joined with the kappa and lambda variable regions to make single chain fragments. The combined final library had approximate 5x1010independent clones.Heavy and Light chain CDRs were defined by IMGT, Kabat, and Chothia. Sequences of the heavy and light chain variable regions, as well as the heavy and light chain CDRs of the identified antibodies are provided in Example 4, below.

[0319] For the phage library selections, purified fusion proteins of human or mouse TEM8 ectodomain fused to alkaline phosphatase (hTEM8-AP or mTEM8-AP) were4239-112153-02labeled with biotin and used for panning of the human naive scFv phage library. Briefly, amplified phage (~1012plaque-forming unit) was preabsorbed with MyOne streptavidin T1 beads and biotin labelled AP protein. The phage was then incubated with 4 pg of biotin-TEM8-AP for 2 h. Specific phages were captured with fresh streptavidin beads. After extensive washes of the beads with PBS+0.05% Tween 20, the bound phages were eluted with 50 mM acetic acid and the eluted phages were immediately neutralized with 1 M Tris, pH 9.0. The eluted phages were rescued by incubation with exponentially growing TGI bacteria and helper phage. Pannings were repeated for two more times with lower amount of the TEM8 protein and more stringent washes at the latter two rounds. Four hundred individual colonies were picked from the last round of panning and rescued with helper phage for screening. Clones showing positive reactivity to TEM8 protein but negative reactivity with AP alone were selected for further characterization.

[0320] The YK2, YK3, YK7, YL9, and YL51 were converted to human IgGl according to standard methods [(see, e.g., Zhu et al., J Virol. 2006 Jan;80(2):891-9)] and a stable CHO producer cell line was generated. The expressed TEM8 IgG was collected from culture supernatants grown in serum free medium and purified by Protein A chromatography. Antibody preparations used for in vivo studies possessed < 5% aggregates and endotoxin levels were below 1 EU / mg.

[0321] Screening assays were performed to confirm YK2, YK3, YK7, YL9, and YL51 binding affinity to both human and mouse TEM8 (FIG. 1). Human TEM8-ECD (extracellular domain) protein was coated onto the wells of a 96-well ELISA plate. YK2, YK3, YK7, YL9, and YL51 in the IgGl format were added at concentrations from 5 x 10’6to 50 nM with 1:10 dilutions. Bound antibodies were detected using goat anti -human Fc HRP. YK2, YK7, YL9 and YL51 had subnanomolar affinities, while YK3 had lower affinity. The assay was repeated using mouse TEM8-ECD (extracellular domain) protein and showed the antibodies bound with similar affinities.

[0322] Screening assays were also performed to confirm YK7, YL9, and YL51 inhibition of protective antigen (PA) subunit of anthrax toxin to TEM8 (FIG. 2). TEM8 protein is known to bind the PA subunit of anthrax toxin protein, a multi-functional virulence factor produced by Bacillus anthracis. TEM8 antibodies have been shown to neutralize the cytotoxic effects of anthrax toxin by binding to blocking PA from interacting with TEM8. Protective antigen (PA) produced by anthracis bacillus was coated onto the wells of a 96-well ELISA plate. Mouse TEM8-ECD-myc (extracellular domain tagged with myc epitope) protein was added to the wells at concentrations of 3, 6, 12, or 24 nM either4239-112153-02alone or with 20 nM of one of YK7, YL9, or YL51 in the IgGl format. HRP-anti-myc secondary antibodies were used to detect antibody binding to PA. The YL9 antibody efficiently blocked PA binding to TEM8 protein.

[0323] Binding of the YL9 antibody to TEM8 was further characterized through epitope mapping (FIG. 3A). Full-length TEM8 proteins carrying either multiple amino acid mutations (Mut 4) or a single amino acid mutation (Mut 27-39) were expressed on TEM8-negative CHO-PR230 cells (a TEM8 negative cell line). TEM8 expression on the cell surface was detected by flow cytometry. Cells were trypsinized, rinsed, and resuspended in 200 pl ice-cold PBS containing 0.5% BSA (PBSA). Either 1 pg of the YL9 antibody or 1 pg of the control anti-TEM8 antibody (m830) was added to 2 million cells for a one-hour incubation on ice. After three washes with PBSA, a secondary antibody with fluorescent label was added for a 30-minute incubation on ice before 3 additional washes with PBSA and analysis using a BD LSRFortessa apparatus. Data was analyzed using FlowJo software (vl0.8.1). The YL9 antibody bound robustly to all TEM8 mutants, except mutant 4, 28, and 33 (partially). Mutant 28 carried the single mutation of arginine (R88) to alanine and mutant 33 carried the single mutation of glutamic acid (E125) to alanine. A mutation of R88 to alanine completely abolished YL9 binding to TEM8, while a mutation of El 25 to alanine reduced the binding. In contrast, the m830 antibody was unaffected by these mutations and, when the L2 antibody was assessed on the same set of mutants, binding was disrupted for the Mutant 4 construct and the Mutant 29 (K94A) construct, but not for Mutant 28 (R88A). Antibody docking prediction further indicated that L2 and YL9 bound a similar but not identical region on the surface of the TEM8 extracellular domain (FIG. 3B).Example 2Anti-TEM8 Antibodies Block Cardiac Fibroblast Contraction

[0324] Prior studies demonstrated that TEM8 expression levels increase dramatically in ischemic cardiac tissue, and that treatment with TEM8 antibodies following ischemic events improves cardiac function.

[0325] In both mice and humans, myocardial infarction caused by Left Anterior Descending (LAD) occlusion prompts an acute-phase inflammatory response characterized by infiltration of immune cells and activation of myofibroblasts. During the chronic repair phase, both humans and mice experience continuous collagen turnover resulting in excess accumulation of remodeled collagen (i.e. fibrosis). Over time,4239-112153-02chronic, maladaptive regeneration and inflammation causes buildup of fibrosis which can lead to progressive cardiac degeneration, and eventually, failure.

[0326] Labeling following LAD ligation assay in mice (a model of myocardial infarction) shows significantly increased TEM8 expression in the cardiac scar tissue four weeks postligation, and low or undetectable TEM8 expression in the unaffected tissue. Further, using LAD ligation assays in TEM8 wildtype (WT) and knockout (KO) mice it was found that both TEM8-KO and WT mice showed a significant decrease in fractional shortening (FS%) and ejection fraction (EF%) at 1 day post-LAD ligation. However, at 28 days post-LAD ligation, EF% and FS% in WT mice continued to decrease, whereas TEM8 KO mice showed significant improvement in EF% and FS% at 28 days compared to 1 day post-op. Thus, TEM8 KO mice showed significantly improved cardiac function compared to TEM8 WT mice at 28 days post-MI. The reduction in EF% and FS% observed in TEM8 WT mice was reversed by administration of the TEM8 mAb L2 one day post LAD ligation. Similar improvements in cardiac function based on measurements of systolic and diastolic function in mice treated with L2 mAb were also observed.

[0327] To confirm that the newly identified TEM8 antibodies are also active for treatment of cardiac ischemia, the YL9 antibody was assessed in a cardiac cell gel contraction assay (FIGs. 4A and 4B). The TGF-P pathway plays an essential role in cardiac fibrosis and is known to induce robust collagen gel contraction. A collagen gel contraction assay was used to measure the ability of TGF-β to induce cardiac fibroblast remodeling of collagen 1 matrices in TEM8 functionally deficient conditions. Mouse cardiac fibroblasts were derived from TEM8 wildtype (WT) or knockout (KO) mice and maintained in complete medium. Mouse cardiac fibroblasts were placed into medium supplemented with low-serum (0.6% FBS) for 16 hours and subsequently mixed with Collagen 1 solution. TGF-β (10ng / ml) was added to the mixture with or without 20 pg / ml of either TEM8 antibody YL9 or YK2. The mixture was plated in an untreated 24-well plate and allowed to solidify at 37 °C, 5% CO2 for 2 hours. The wells were then supplemented with an additional 500 p.1 of the appropriate medium. The cell / gel mixture was gently detached from the bottom of each well and cultured for 48 hours, during which time the contraction occurred. Exemplary images of each well captured with an inverted microscope are shown in FIG. 4A. The total area (TA) of the well and surface area (SA) of each cell / gel mixture were measured. Gel contraction was quantified using4239-112153-02the formula 1-SA / TA. FIG. 4B provides a summary of results. TEM8 WT cells not treated with TGF-β showed -8% contraction and TEM8 KO cells were unresponsive to TGF-β (-6% contraction). TEM8 WT cells treated with TGF-β showed -65% contraction, but when in the presence of YL9 or YK2, contraction decreased to -40% and -45%, respectively. TGF-β activated contraction requires TEM8 function and YL9 and YK2 are capable of significantly reducing TGF-β activated contraction. To evaluate the role of ANTXR1 in TGFβ signaling in primary cardiac fibroblasts (CF), both L2 (T8Abl) and YL9 (T8Ab2) were assessed and shown to block TGFβ signaling in mouse CFs (FIG.4C) and human CFs (FIG. 4D), resulting in decreased levels of TGFBR1, SMAD2 / 3, and YAP. Taken together, these results show the role of TEM8 signaling in CFs and the efficacy of TEM8 mAb for blocking that signaling for treatment of cardiac ischemiaExample 3Anti-TEM8 Antibody Blocks Human UACC Tumor Growth

[0328] Prior studies demonstrated that TEM8 expression levels increase dramatically in tumor-infiltrating vasculature, and that treatment with TEM8 antibodies inhibits tumor-induced angiogenesis and provides broad antitumor activity. See, e.g., Chaudhary et al., “TEM8 / ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types,” Cancer Cell, 21(2):212-26, 2012.

[0329] This example shows that the YL9 antibody inhibits tumor growth in a mouse model. Human UACC-62 (UACC) melanoma cells were obtained from the DCTD Tumor Repository at NCI (Frederick, MD) and cultured in RPMI 1640 supplemented with 10% FBS. Tumor cells were mixed with Matrigel and implanted subcutaneously on the flank of athymic nude mice (2 million per mouse). Tumors were measured with a digital caliper over time. Tumor volumes were calculated using the formula LxW2x0.5 and presented as a mean + / - SE. When tumor size reached about 50 mm3, the mice were randomized into two groups and treated three times weekly with either vehicle control (PBS; n=l 1) or 20 mg / kg YL9 (n=7). At 60 days post-implantation, tumors were surgically removed, measured, and body weight was compared to that of preimplantation. As shown in FIG. 5A, YL9 treatment inhibited tumor growth by over 90% during the course of the experiment. Antibody treatment did not alter body weights or cause any other signs of toxicity.

[0330] Similar experiments were performed with human hepatocellular carcinoma tumors. HepG2 HCC cells were mixed with Matrigel and implanted subcutaneously on4239-112153-02the flank of athymic nude mice (2 million per mouse). Tumors were measured with a digital caliper over time. Tumor volumes were calculated using the formula LxW2x0.5 and presented as a mean + / - SE. When tumor size reached about 50 mm3, the mice were randomized into two groups and treated three times weekly with either vehicle control (PBS; n=10) or 10 mg / kg YL9 (n=10). At 60 days post -implantation, tumors were surgically removed, measured, and body weight was compared to that of preimplantation. As shown in FIG. 5B, YL9 treatment significantly inhibited tumor growth during the course of the experiment. Antibody treatment did not alter body weights or cause any other signs of toxicity.Example 4Antibody Sequences

[0331] The nucleic and amino acid sequences provided herein are shown using standard letter abbreviations. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. > YL9YL9 CDR sequencesIMGT SI Kabat SI Chothia SI HCDR1 GFTFSTYT 1 TYTMG 50 GFTFSTY 55 HCDR2 INKSGGDT 2 DINKSGGDTYYADSVKG 51 NKSGGD 56 HCDR3 AKASISGSAY 3 ASISGSAYDY 52 ASISGSAYDY 57 DY LCDR1 DLGFKR 4 GGNDLGFKRVH 53 GGNDLGFKRVH 58 LCDR2 YDS 5 YDSDRPS 54 YDSDRPS 59 LCDR3 QVWDSGTDHV 6 QVWDSGTDHW 6 QVWDSGTDHW 6VYL9 VH (SEQ ID NO: 7 ) QVQLVESGGGLTQPGGSLRLSCAASGFTFSTYTMGWVRQAPGKGLEWVSDINKSGGDTYYADSVKGRFTISRDNS KNTLSLQMNSLRAEDTAVYHCAKAS ISGSAYDYWGQGILVTVSS YL9 VL (SEQ ID NO: 8 ) NFMLTQPPSVSMAPGTTARITCGGNDLGFKRVHWYQQKAGQAPVLVISYDSDRPSGIPERFSGSNSGDTATLTIS RVEAGDEADYYCQVWDSGTDHWFGGGTKVTVLG YL9 ScFv protein and nucleic acid sequence QVQLVESGGGLTQPGGSLRLSCAASGFTFSTYTMGWVRQAPGKGLEWVSDINKSGGDTYYADSVKGRF TISRDNSKNTLSLQMNSLRAEDTAVYHCAKASISGSAYDYWGQGILVTVSSGGGGSGGGGSGGGGSNF MLTQPPSVSMAPGTTARITCGGNDLGFKRVHWYQQKAGQAPVLVISYDSDRPSGIPERFSGSNSGDTA TLTISRVEAGDEADYYCQVWDSGTDHWFGGGTKVTVLG (SEQ ID NO: 40 ) caggtgcagctggtggagtctgggggaggcttgacacagccgggggggtccctgagactctcctgcgc agcct ctggattcaccttt gcacct taccatgggttgggtccgccaggctccagggaaggggctgg agtgggt ct cagatattaataaaagtggtggtgacacat ctacgcagactccgtgaagggccggtt c accatctccagagacaattccaagaacacgctgtctctgcaaatgaacagcctgagagccgaggacac4239-112153-02agccgtatat cactgtgcgaaagcctcaatttcggggagtgcctatgactactggggccagggaatct tggtcaccgt ctcctcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcgaatttt atgctg ctcagccaccctcagtgtcaatggccccaggaacgacggcccggattacctgtgggggaaa cgaccttggatttaaacgtgtgcactggtaccagcagaaggcaggccaggcccctgt cctggtcatct cttatgacagtgaccggccctcagggatccctgagcgattctctggctccaactctggggacacggcc accctgaccatcagccgggtcgaagccggggatgaggccgact tt ctgtcaggtgtgggat gtgg tactgat catgtggtattcggcggggggaccaaggt caccgtcctaggccaggccggccaccaccacc accaccacggcgactacaaggacgatgacgataa (SEQ ID NO: 41 )> YK2YK2 CDR sequences (with SEQ ID NOs (SI ) shown)IMGT SI Kabat SI Chothia SI HCDR1 GYTFTSYS 9 SYSIN 60 GYTFTSY 65 HCDR2 IRPHNGNT 10 LIRPHNGNTNYAQKFQG 61 RPHNGN 66 HCDR3 ARDPGGFDH 11 DPGGFDH 62 DPGGFDH 67 LCDR1 QSISGW 12 RASQSISGWLA 63 RASQSISGWLA 68 LCDR2 QAS 13 QASSLQS 64 QASSLQS 69LCDR3 LQDFNFPRT 14 LQDFNFPRT 14 LQDFNFPRT 14 YK2 VH (SEQ ID NO: 15)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYS INWVRQAPGQGLEWMGLIRPHNGNTNYAQKFQGRVTMTTDTS TSTAYMELKSLRYDDTAVYYCARDPGGFDHWGQGTLVTVSS YK2 VL (SEQ ID NO: 16) DIVMTQSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIYQASSLQSGVPSRFSGSGSDTDFTLTI NSLQPEDFATYYCLQDFNFPRTFGQGTKVEIKG YK2 ScFv protein and nucleic acid sequence EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYSINWVRQAPGQGLEWMGLIRPHNGNTNYAQKFQGRV TMTTDTSTSTAYMELKSLRYDDTAVYYCARDPGGFDHWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMT QSPSTLSASVGDRVTITCRASQSISGWLAWYQQKPGKAPKLLIYQASSLQSGVPSRFSGSGSDTDFTL TINSLQPEDFATYYCLQDFNFPRTFGQGTKVEIKG (SEQ ID NO: 42 )gaggtt cagctggtacagtctggagctgaggtgaagaagcctggggcctcagtgaaggtct cctgcaa ggcttctggttacacctttaccagctacagt tcaactgggtgcgacaggcccctggacaagggcttg agtggatgggattaat caggcct cacaatggcaacacaaactatgcacagaagtt ccagggcagagt c accatgaccacagacacgtccacgagcacagcatacatggagctgaagagcctgagatatgacgacac ggccgtgt tt ctgtgcgagagatccgggggggttcgaccactggggccagggaaccctggtcaccg tct ctt caggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacat cgtgatgacc cagtctccttccaccctctctgcatctgt ggagacagagtcaccatcacttgccgggccagtcagag t ttagtggctggctggcctggtatcagcagaaaccagggaaagcccctaaactcctgatct tcagg cgtctagtttacaaagtggagtcccatcaaggttcagcggcagtggatctgacacagatttcactctc accat ca cagcctgcagcctgaagattttgcaactt ttactgt ctacaagatttcaatttccctcg tacttttggccaggggaccaaggtggagatcaaaggccaggccggccaccaccaccaccaccacggcg actacaaggacgatgacgataagggctag (SEQ ID NO: 43 )> YK3YK3 CDR sequences (with SEQ ID NOs (SI ) shown)IMGT SI Kabat SI Chothia SI HCDR1 GYTFTTQA 17 TQAMH 70 GYTFTTQ 75 HCDR2 INPGNDNT 18 WINPGNDNTKYSQKFQD 71 NPGNDN 76 HCDR3 ARDRHWSYDY 19 DRHWSYDY 72 DRHWSYDY 77 LCDR1 QNIATY 20 RASQNIATYLN 73 RASQNIATYLN 78 LCDR2 AAS 21 AASSLQS 74 AASSLQS 79LCDR3 QQSYTTPT 22 QQSYTTPT 22 QQSYTTPT 224239-112153-02YK3 VH (SEQ ID NO: 23) QVQLVQSGAEVKKLGASVKVSCKASGYTFTTQAMHWVRQAPGQGLEWMGWINPGNDNTKYSQKFQDRVTITRDTS ASTAYMELSSLRSEDTAVYYCARDRHWSYDYWGQGTLVTVSS YK3 VL (SEQ ID NO: 24 ) DIQLTQSPSSLSASVGDRVTITCRASQNIATYLNWYQQKPGRAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYTTPTFGQGTRLEIKG YK3 ScFv protein and nucleic acid sequence QVQLVQSGAEVKKLGASVKVSCKASGYTFTTQAMHWVRQAPGQGLEWMGWINPGNDNTKYSQKFQDRV TITRDTSASTAYMELSSLRSEDTAVYYCARDRHWSYDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQL TQSPSSLSASVGDRVTITCRASQNIATYLNWYQQKPGRAPKLLIHAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQSYTTPTFGQGTRLEIKG (SEQ ID NO: 44 )caggt ccagctggtgcaat ct ggggctgaggtgaagaagcttggggcctcagtgaaagttt cctgcaa ggctt ctggatacaccttcactacccaagctatgcattgggtgcgccaggcccccggacaaggt cttg agtggatgggatggat caaccctggcaatgat acacaaaatatt cacagaagtt ccaggacagagt c accattacaagggacacatccgcgagcacagcct catggagctgagcagcctgagatctgaggacac ggctgtttattactgtgcgagagaccgacactggtcctatgactactggggccagggaaccctggtca ccgt ct cct caggt ggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatccagttg acccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtca aaatattgccacctatttaaattggtatcaacagaaaccagggagagcccctaagct cctgat ccatg ctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcaca ctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagagtt cact cccc caccttcggccaagggacacgactggagattaaaggccaggccggccaccaccaccaccaccacggcg actacaaggacgatgacgataagggctag (SEQ ID NO: 45 )> YK7YK7 CDR sequences (with SEQ ID NOs (SI ) shown)IMGT SI Kabat SI Chothia SI HCDR1 GYTFTSYA 25 SYAMN 80 GYTFTSY 85 HCDR2 IRPHNGNT 10 LIRPHNGNTNYAQKFQG 81 RPHNGN 86 HCDR3 ARDPGGFDP 26 DPGGFDP 82 DPGGFDP 87 LCDR1 QSISSW 27 RASQSISSWLA 83 RASQSISSWLA 88 LCDR2 QAS 13 QASRLHS 84 QASRLHS 89LCDR3 QQYDSSPIT 28 QQYDSSPIT 28 QQYDSSPIT 28 YK7 VH (SEQ ID NO: 29) QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGLIRPHNGNTNYAQKFQGRVTMTTDTS TSTAYMELKSLRYDDTAVYYCARDPGGFDPWGQGTLVTVSS YK7 VL (SEQ ID NO: 30 )D IQMTQSPSTLSASVGDRVTI SCRASQS I SSWLAWYQQRPGKAPKIL I YQASRLHSGVPSRFSGSGSGTDFTLTI SSLQPDDLATYYCQQYDSSPITFGQGTRLEIKG YK7 ScFv protein and nucleic acid sequence QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGLIRPHNGNTNYAQKFQGRV TMTTDTSTSTAYMELKSLRYDDTAVYYCARDPGGFDPWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSTLSASVGDRVTISCRASQSISSWLAWYQQRPGKAPKILIYQASRLHSGVPSRFSGSGSGTDFTL TISSLQPDDLATYYCQQYDSSPITFGQGTRLEIKG (SEQ ID NO: 46 )caggt ccagctggtgcaatctgggtctgagttgaagaagcctggggcctcagtgaaggttt cctgcaa ggcttctggatacaccttcact gctatgctatgaattgggtgcgacaggcccctggacaagggcttg agtggatgggattaat caggcct cacaatggcaacacaaact tgcacagaagtt ccagggcagagt c accatgaccacagacacgtccacgagcacagcatacatggagctgaagagcctgagatatgacgacac4239-112153-02ggccgtgtattactgtgcgagagatccgggggggtt cgacccctggggccagggaaccctggtcaccg tctcctcaggtggaggcggttcaggcggaggtggcagcggcggtggcggatcggacatccagatgacc cagtctccttccaccctgtctgcatctgt ggagacagagtcaccatctcttgccgggccagtcagag tattagtagctggttggcctggtatcagcagagaccagggaaagcccctaaaat cctgat ctat cagg cgt cccgt 11 acacagt ggggt cccat caaggt t cagcggcagt ggat ct gggacagact t cact ct c accatcagcagcctgcagcctgatgatttagctacttattactgccaacaatatgatagttctccgat caccttcggccaagggacacgactggagattaaaggccaggccggccaccaccaccaccaccacggcg actacaaggacgatgacgataanggctag (SEQ ID NO: 47 )> YL51YL51 CDR sequencesIMGT SI Kabat SI Chothia SI HCDR1 GYTFTSYG 31 SYGIS 90 GYTFTSY 96 HCDR2 I SAYNGNT 32 WI SAYNGNTNYAQKLQG 91 SAYNGN 97 HCDR3 ARDFLYNWNY 33 DFLYNWNYAERGVGRFDP 92 DFLYNWNYAERGVGR 98 AERGVGRFDP FDP LCDR1 SSNIGAGYD 34 TGSSSNIGAGYDVH 93 TGSSSNIGAGYDVH 99 LCDR2 GNS 35 GNSNRPS 94 GNSNRPS 100LCDR3 QSYDSSLSGS 36 QSYDSSLSGSNV 95 QSYDSSLSGSNV 101 YL51 VH (SEQ ID NO: 37 ) QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTS TSTAYMELRSLRSDDTAVYYCARDFLYNWNYAERGVGRFDPWGQGTLVTVSS YL51 VL (SEQ ID NO: 38 ) QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASL AITGLQAEDEADYYCQSYDSSLSGSNVFGTGTKVTVLG YL51 ScFv protein and nucleic acid sequence QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRV TMTTDTSTSTAYMELRSLRSDDTAVYYCARDFLYNWNYAERGVGRFDPWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPD RFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGSNVFGTGTKVTVLG (SEQ ID NO: 48) caaatgcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaa ggctt ctggttacacctttaccagctatggtat cagctgggtgcgacaggcccctggacaagggcttg agt ggat gggat ggat cagcgcttacaatggtaacacaaactatgcacagaagct ccagggcagagt c accatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacac ggccgtgtattactgtgcgagagattt cttgtataactggaactacgcggaaaggggggtggggaggt tcgacccctggggccagggaaccctggtcaccgt ct cttcaggtggaggcggtt caggcggaggtggc agcggcggtggcggatcgcagtctgtcgtgacgcagccgccctcagtgtctggggccccagggcagag ggt caccatct cctgcact gggagcagct ccaacat cggggcaggt tat gat gtacactggt ac cage agcttccaggaacagcccccaaactcctcatct tggtaacagcaatcggccctcaggggtccctgac cgattctctggctccaagtctggcacctcagcctccctggccatcactgggctccaggctgaggatga ggctgattattactgccagt cctatgacagcagcctgagtggtt egaatgtett cggaactgggacca aggtcaccgt cctaggccaggccggccaccacc ccaccaccacggcgactacaaggacgatgacgat a (SEQ ID NO: 49 )

[0332] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described implementations. We claim all such modifications and variations that fall within the scope and spirit of the claims below

Claims

4239-112153-02It is claimed:

1. An isolated monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL set forth as:a) SEQ ID NOs: 9 and 10 (YL9);b) SEQ ID NOs: 15 and 16 (YK2);c) SEQ ID NOs: 23 and 24 (YK3);d) SEQ ID NOs: 29 and 30 (YK7); ore) SEQ ID NOs: 37 and 38 (YL51); andwherein the monoclonal antibody or antigen binding fragment thereof specifically binds to the extracellular domain of Tumor Endothelial Marker 8 (TEM8).

2. The monoclonal antibody or antigen binding fragment of claim 1, wherein the CDR positions of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are determined by IMGT, Kabat, or Chothia.

3. The monoclonal antibody or antigen binding fragment of any one of the prior claims, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 respectively comprise amino acid sequences set forth as:a) SEQ ID NOs: 1, 2, 3, 4, 5, and 6;b) SEQ ID NOs: 9, 10, 11, 12, 13, and 14;c) SEQ ID NOs: 17, 18, 19, 20, 21, and 22;d) SEQ ID NOs: 25, 10, 26, 27, 13, and 28; ore) SEQ ID NOs: 31, 32, 33, 34, 35, and 36.

4. The monoclonal antibody or antigen binding fragment of any one of the prior claims, wherein the framework region of the VH and VL respectively comprise at least 90% sequence identity to amino acid sequences set forth as:a) SEQ ID NOs: 9 and 10;b) SEQ ID NOs: 15 and 16;c) SEQ ID NOs: 23 and 24;d) SEQ ID NOs: 29 and 30; or4239-112153-02e) SEQ ID NOs: 37 and 38.

5. The monoclonal antibody or antigen binding fragment of any one of the prior claims, wherein the framework region comprises a human framework region.

6. The monoclonal antibody or antigen binding fragment of any one of the prior claims, wherein the VH and VL respectively comprise amino acid sequences set forth as: a) SEQ ID NOs: 9 and 10;b) SEQ ID NOs: 15 and 16;c) SEQ ID NOs: 23 and 24;d) SEQ ID NOs: 29 and 30; ore) SEQ ID NOs: 37 and 38.

7. The antibody of any one of the prior claims, wherein the antibody comprises a human constant domain.

8. The antibody of any one of the prior claims, wherein the antibody is a human antibody.

9. The antibody of any one of the prior claims, wherein the antibody comprises an IgG.

10. The antibody of any one of the prior claims, wherein the antibody comprises a recombinant constant domain that increases the half-life of the antibody.

11. The antibody of any one of the prior claims, wherein the antibody comprises a recombinant constant domain that disrupts Fcreceptor binding.

12. The antibody of claim 11, wherein the recombinant constant domain comprises a LALA-PG mutation according to the EU numbering system.

13. The antigen binding fragment of any one of claims 1-6.4239-112153-0214. The antigen binding fragment of claim 13, wherein the antigen binding fragment comprises a Fv, Fab, F(ab’)2, scFV, or a SCFV2 fragment.

15. An engineered T-cell receptor, comprising the antibody or antigen binding fragment of any one of claims 1-14.

16. The engineered T-cell receptor of claim 15, wherein the engineered T-cell receptor is a Chimeric Antigen Receptor (CAR), a Synthetic T cell Receptor and Antigen Receptor (STAR), a costimulatory STAR, a T-cell receptor fusion construct (TRuC), or an HLA-independent TCR (HIT).

17. A multispecific antibody comprising the antibody or antigen binding fragment of any one of claims 1-16 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

18. A fusion protein comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-16 and a heterologous protein or peptide.

19. An immunoconjugate comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-16 and an effector molecule.

20. An antibody-drug conjugate, comprising the antibody or antigen binding fragment of any one of claims 1-16.

21. An antibody-nanoparticle conjugate, comprising a nanoparticle conjugated to the monoclonal antibody or antigen-binding fragment of any one of claims 1-16.

22. An isolated nucleic acid molecule encoding the antibody, the antigen binding fragment, the engineered T-cell receptor, the multispecific antibody, the fusion protein, or the VH or VL of the antibody, of any one of claims 1-18.

23. The nucleic acid molecule of claim 22, operably linked to a promoter.

24. A vector comprising the nucleic acid molecule of claim 22 or claim 23.4239-112153-0225. A host cell comprising the nucleic acid molecule or vector of any one of claims 22-24.

26. The host cell of claim 25, wherein the host cell is a T cell.

27. A pharmaceutical composition comprising the antibody, antigen binding fragment, the engineered T-cell receptor, the multispecific antibody, the fusion protein, immunoconjugate, antibody-drug conjugate, antibody nanoparticle conjugate, nucleic acid molecule, or vector, of any one of the prior claims; anda pharmaceutically acceptable carrier.

28. A method of producing an antibody or antigen binding fragment that specifically binds to a TEM8 protein, comprising:expressing one or more nucleic acid molecules encoding the antibody or antigen binding fragment of any one of claims 1-16 in a host cell; andpurifying the antibody or antigen binding fragment.

29. A method of treating or inhibiting a TEM8-positive cancer in a subject, comprising administering to a subject in need thereof an effective amount of the antibody, antigen binding fragment, the engineered T-cell receptor, the multispecific antibody, the fusion protein, immunoconjugate, antibody-drug conjugate, antibody nanoparticle conjugate, nucleic acid molecule, vector, host cell, or pharmaceutical composition of any one of claims 1-27.

30. The method of claim 29, wherein the cancer is colorectal, skin, lung, breast, prostate, or head and neck cancer.

31. The method of claim 29 or claim 30, wherein the cancer comprises a tumor microenvironment comprising increased cell surface expression of TEM8.

32. A method of treating cardiovascular disease in a subject, comprising administering to a subject with or at risk of cardiovascular disease a therapeutically effective amount of the antibody, antigen binding fragment, multispecific antibody, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-27.4239-112153-0233. The method of claim 32, wherein the cardiovascular disease is ischemic heart disease, progressive heart failure, heart disease caused by atherosclerosis, coronary artery disease, myocardial ischemia, myocardial infarction, hypertension, cardiomyopathy, and / or Heart Failure with preserved Ejection Fraction (HFpEF).

34. Use of an antibody, antigen binding fragment, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-27 to block ANTXR1 binding to protective antigen (PA).

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