Actinohibin variant polypeptides and related methods

Abstract

Treatment of growth factor receptor-mediated cancers, such as epidermal growth factor receptor (EGFR)-mediated cancers and / or insulin-like growth factor 1 receptor (IGF1R)-mediated cancers, such as lung cancer, such as non-small cell lung cancer, that are sensitive to polypeptides that specifically bind to high mannose-type glycan epitopes are described. Methods are provided for reducing activation of EGFR and / or IGF1R in cancer cells, inhibiting cancer cell migration, treating cancer and / or reducing tumor growth in a subject (e.g., a human patient).

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 139,115, filed January 19, 2021, the entire teachings of which are incorporated herein by reference.

[0002] Incorporating material by reference in an ASCII text file This application incorporates by reference the sequence listing contained in the ASCII text file filed concurrently herewith: a) Filename: 56001008001PCT_Sequence_Listing_ST25.txt, created on December 6, 2021 with a size of 19000 bytes.

[0003] Government support This invention was made with government support under grants 1R21CA216447-01 from the National Institutes of Health (NIH) / National Cancer Institute (NCI), P30-GM106396 from the National Institutes of Health (NIH), and W81XWH-10-2-0082-CLIN2 from the Department of Defense (DOD). The government has certain rights in this invention. [Background technology]

[0004] Changes in protein glycosylation patterns have been shown to be associated with various disease pathologies, including viral infections and cancer. 1,2 One such change observed in several cancer types is a significant increase in the proportion of high-mannose glycans, which are composed of asparagine-linked glycans (N-glycans) that contain five to nine terminal mannose residues. 3,4In normal cells, these glycoforms appear in the endoplasmic reticulum (ER) but are subsequently processed into complex-type glycans by a series of mannosidases and glycosyltransferases in the Golgi apparatus as the nascent glycoprotein passes through the secretory pathway. Thus, high-mannose glycans are generally considered to be "immature" N-glycans that are confined to the ER under normal conditions. 1 However, recent studies based on quantitative mass spectrometry analysis of cancer tissues have demonstrated that this is not always the case. For example, high-mannose glycans were elevated in serum samples from breast cancer patients, which correlated with cancer progression. 5 In an analysis of a large cohort of paired breast cancerous and adjacent non-tumorous tissues, it was found that high mannose glycans (Man8) along with triantennary glycans were dramatically increased in tumor membrane fractions. 6 Increased abundance of high mannose glycans was also observed in colorectal tumor tissues. 7~9 , hepatocellular carcinoma 10,11 , metastatic bile duct carcinoma 12 , lung adenocarcinoma 13 , pancreatic cancer 14 , ovarian cancer 15,16 , prostate cancer 17 , and basal cell carcinoma and squamous cell carcinoma of the skin 18 It has also been observed in the inflammatory bowel disease (IGD) and inflammatory bowel disease (IGF) in mice. Taken together, the abnormal increase in high-mannose glycans on malignant cells may provide a unique biomarker for drug development. Nevertheless, their druggability remains unclear, as few agents exist that can distinguish tumor-associated high-mannose glycans from other glycoforms present on the surface of normal cells. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a critical need to identify and characterize new therapeutic agents that target cancer glycobiomarkers.

[0006] The subject matter disclosed herein is based, in part, on the discovery that the "lectibody" AvFc is selective for oligomannose glycans overrepresented on the surface of cancer cells. Furthermore, AvFc has been found to inhibit cancer-associated targets such as the epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) and to mediate anti-cancer activities, including antibody-dependent cell-mediated cytotoxicity (ADCC). [Means for solving the problem]

[0007] In one aspect, the disclosure provides a method of reducing growth factor receptor activation in a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0008] In some embodiments, the growth factor receptor is a cancer-associated growth factor receptor, hi particular embodiments, the growth factor receptor comprises EGFR, IGF1R, or a combination thereof.

[0009] In another aspect, the disclosure provides a method of inducing ADCC in a cancer cell comprising contacting the cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0010] In another aspect, the disclosure provides a method of inhibiting migration of a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0011] In some embodiments, the cancer cells are cells of a human patient.

[0012] In certain embodiments, the cancer cells are non-small cell lung cancer (NSCLC) cells.

[0013] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope.

[0014] In another aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; The present invention provides a method of treating cancer in a subject in need thereof, comprising:

[0015] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope, where the cancer is characterized by abnormal cell surface accumulation of high mannose glycans.

[0016] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope, where the cancer is mediated by inappropriate activation of a growth factor receptor.

[0017] In another aspect, the disclosure provides a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16.

[0018] In some embodiments, the growth factor receptor comprises EGFR, IGF1R, or a combination thereof.

[0019] In certain embodiments, the cancer is lung cancer.

[0020] In certain embodiments, the cancer is resistant to treatment with an antibody that specifically binds to the growth factor receptor.

[0021] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 90% identical to at least one of the sequences set forth in SEQ ID NOs:1-13.

[0022] In a particular embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9.

[0023] In certain embodiments, the polypeptide further comprises an Fc.

[0024] In a particular embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0025] In another aspect, the disclosure provides a method of reducing growth factor receptor activation in a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0026] In another aspect, the disclosure provides a method of inducing ADCC in a cancer cell comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0027] In another aspect, the disclosure provides a method of inhibiting migration of a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0028] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprises: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) administering or providing for administration to the subject a therapeutically effective amount of the polypeptide if abnormal accumulation is determined to be present in the biological sample; The present invention provides a polypeptide comprising:

[0029] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) administering or providing for administration to the subject a therapeutically effective amount of the polypeptide if abnormal accumulation is determined to be present in the biological sample; Provide for use, including

[0030] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0031] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0032] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering to the subject an effective amount of the polypeptide.

[0033] In another aspect, the disclosure provides the use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0034] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; Provide for use, including

[0035] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; Provide for use, including

[0036] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0037] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0038] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering to the subject an effective amount of the polypeptide.

[0039] In another aspect, the disclosure provides the use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0041] The foregoing will become apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the embodiments. [Brief description of the drawings]

[0042] [Figure 1] Figures 1A-1D show that AvFc recognizes high mannose glycans on cancer cell lines. Figure 1A Binding of AvFc to various cancer cell lines, normal human peripheral blood mononuclear cells (PBMCs), and non-tumorigenic cell lines was assessed by flow cytometry using 0.1, 1, or 10 mg / mL of drug. The percentage of FITC-positive cells is shown as a heat map, with most cell lines reaching saturation at 10 mg / mL. Figure 1B Immunofluorescence was used to visualize binding of AvFc at 1 mg / mL to the non-tumorigenic lung epithelial cell line BEAS-2B and to A549 cells with or without endoglycosidase H (EndoH) treatment. AvFc shows no binding to either BEAS-2B or endoH-treated A549 cells. Figures 1C-1D Flow cytometry of BEAS-2B and A549 after staining with either AvFc or ConA shows that ConA can weakly bind to both BEAS-2B and A549 cells and that endoH digestion of the cells abolishes binding by both lectins. [Diagram 2]Figure 2 shows the inhibition of AvFc binding to cancer cells by HIV-1 gp120 and yeast mannan. A549 lung cancer cells were incubated with AvFc (1 μg / ml) and various concentrations of HIV-1 envelope glycoprotein gp120 and yeast mannan for 30 min at 4°C. Cells were washed and stained with 10 μg / ml goat anti-human IgG FITC for 30 min at 4°C. Cells were then washed and analyzed for binding on a FACS Canto II (BD Biosciences) using a FACSDiva (BD Biosciences). [Diagram 3]Figures 3A-3G depict the identification of putative cancer cell surface binding partners of AvFc. Potential binding partners were isolated using co-immunoprecipitation and identified using mass spectrometry. Figure 3A Silver staining of AvFc and AvFclec-fractions obtained after co-immunoprecipitation. In addition to the band corresponding to AvFc itself (≈77 kDa), there are other species of higher and lower molecular weight, suggesting that AvFc successfully pulled down potential binding partners. Figures 3B-D Co-immunoprecipitation was used to confirm the interaction of AvFc with EGFR / IGF1R isolated from A549 and H460 cells. AvFc interacts with EGFR and IGF1R from both cell lines, but not AvFclec-, as revealed by pull-down with AvFc and subsequent blotting with anti-EGFR or anti-IGFR antibodies. Note that longer exposure times were required for IGF1R in A549 due to low expression of the receptor, resulting in detection of AvFc and AvFclec- in the blot due to cross-reactivity of the detection antibody. When exposure times were extended with H460 IP, AvFc and AvFclec- bands appeared (right panel). Figure 3E Potential binding partners isolated using co-immunoprecipitation and identified using mass spectrometry. Figure 3F AvFc and cetuximab (CTX) binding to monomeric and dimeric EGFR. EGFR monomer and dimer were purified from A549 cells without and with 20 ng / ml EGF treatment for 10 min, respectively. To maintain the dimeric form, EGFR dimer was cross-linked with BS3 linker (Thermo Fisher Scientific, 21580) and filtered through a 200 kDa cutoff filter (Advantec, USY-20). Vesicles were composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (molar ratio 90:10). Uniform size (100 nm) vesicles were prepared by vesicle extrusion. EGFR (monomer) or EGFR (dimer) was inserted into the vesicles (protein:lipid = molar ratio 2000:1) in PBS containing 0.7% CHAPS.After dialysis overnight at 4°C in 2 L of PBS, various concentrations of EGFR proteo-liposomes were applied to ELISA plates coated with 2 μg / ml of anti-EGFR antibody (rabbit, Cell Signaling Technology, D38B1). Plate-captured EGFR proteo-liposomes were detected with 13.7 nM AvFc or CTX and anti-human IgG1-HRP conjugate (SouthernBiotech, 9054-05). Figure 3G Expression levels of EGFR and IGF1R in lung cancer cell lines. EGFR and IGF1R expression was determined by immunoblot analysis using EGFR and IGF1R antibodies. Actin 1 was included as a control. [Figure 4]Figures 4A-4G show that AvFc blocks EGFR and IGF1R signaling. The phosphorylation status of EGFR and IGF1R on A549 cells after treatment with the respective ligands was detected by anti-pEGFR (Y1068) and anti-IGF1R (Y1135 / 1136) antibodies. Figure 4A: Representative immunoblot shows that treatment of A549 cells with 30 nM AvFc and CTX, but not AvFclec-, before the addition of 2 ng / ml EGF, resulted in a decrease in EGFR activation. Figure 4B: Quantification of the immunoblot in panel A. Figure 4C: Representative immunoblot shows that treatment of A549 cells with 30 nM AvFc, but not CTX or AvFclec-, before the addition of 2 ng / ml IGF1, resulted in a decrease in IGF1R activation. Fig. 4D Quantification of immunoblots in panel C. Fig. 4E After incubation of A549 cells with AvFc, CTX, or AvFclec- and then stimulation with a mixture of EGF and IGF1, AKT and MAPK1 phosphorylation was reduced only with AvFc. Fig. 4F Quantification of pAKT / AKT in panel E. Fig. 4G Quantification of pMAPK1 / MAPK1 in panel E. All relative band intensities were measured by ImageJ. Bars represent mean ± SEM (N=3). Group means were compared using unpaired two-tailed Student's t-test for Figs. 4B and 4D or one-way ANOVA with Bonferroni's post-hoc test for Figs. 4F and 4G (*p<0.05, **p<0.01, ***p<0.001). [Diagram 5] Figures 5A-5B show that AvFc inhibits A549 and H460 cell migration. Migration of A549 cells (Figure 5A) or H460 cells (Figure 5B) was measured in 8 μm pore transwells after treatment of cells with 30 nM AvFc, CTX, or AvFclec- and stimulation with EGF or IGF1. Error bars represent the mean ± SEM from three replicates. Groups were analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test (**p<0.01, ***p<0.001). [Figure 6]Figures 6A-6C show the in vitro Fc-mediated anticancer activity of AvFc. Figures 6A-6B FcγRIIIa activation by AvFc, AvFclec-, and CTX on A549 (Figure 6A) or H460 (Figure 6B) cells in a reporter cell-based assay. Representative data from at least two independent experiments are shown. Columns and bars represent the mean ± SEM (n=3). Groups were compared by two-way ANOVA followed by Tukey's multiple comparison test (**p<0.01, ***p<0.001, ****p<0.0001). Figure 6C relates to a human PBMC-based ADCC assay using A549 lung cancer cells. Cells were preincubated with serial dilutions of AvFc or CTX for 30 min in a 37°C / 5% CO2 incubator. PBMC were added to initiate ADCC at optimized effector / target ratios (50:1 for AvFc and 25:1 for CTX). After 6 h incubation in a 37°C / 5% CO2 incubator, cell supernatants were harvested to measure released lactose dehydrogenase and calculate % target cell lysis. Experiments were performed in triplicate and mean ± SEM is shown for each data point. [Figure 7]Figures 7A-7E show the in vivo anticancer activity of AvFc in A549 subcutaneous xenograft challenge model (Figure 7A) and H460 subcutaneous xenograft challenge model (Figure 7B) in SCID mice. Four days after challenge, mice were treated ip with 25 mg / kg AvFc or CTX or vehicle control every other day (Q2D) for a total of six doses, as indicated by the arrows. Animals were monitored until day 26 in the A549 model and until day 16 in the H460 model. Tumor volumes were compared by two-way ANOVA followed by Tukey's multiple comparison test (*p<0.05, **p<0.01, ***p<0.001 between vehicle and AvFc, #p<0.05, ##p<0.01, ###p<0.001 between vehicle and CTX). Figures 7C-7D A549-GFP human lung cancer metastasis model in SCID mice. SCID mice were challenged iv with A549-GFP cells on day 0, followed by ip injection of 25 mg / kg AvFc (n=10) or vehicle control (n=9) every other day, as indicated. Lungs were removed on day 35 and GFP signal intensity in the lungs from each mouse was quantified. Columns and bars represent mean ± SEM, and dots represent individual mice (Figure 7C). GFP images of lungs (four right lobes and a single left lobe) from all animals in the vehicle and AvFc groups are shown in Figure 7D. Bar = 5 cm. Statistical differences between groups were analyzed by Mann-Whitney test. Figure 7E Efficacy of AvFc in the B16F10 melanoma metastasis model. Briefly, animals were injected iv with 250,000 B16F10 cells on day 0. Treatment with 25 mg / kg AvFc or AvFclec-(ip) (a non-glycosylated variant of AvFc) was started on day 0 and continued every other day for a total of six treatments. Animals were euthanized on day 21 and lung nodules were counted. AvFc provided protection against tumor formation, whereas the non-glycosylated variant AvFclec- failed to provide protection, suggesting that binding to high-mannose glycans is required for the anticancer activity of AvFc. [Figure 8]Figures 8A-8E show IHC analysis of AvFc binding to primary human lung (NSCLC) tissues and EGFR. Figures 8A-8B IHC staining with AvFc or biotinylated anti-human IgG secondary antibody alone. Representative staining from lung tissue of patient 117 is shown with hematoxylin as counterstain. Bar = 200 μm (Figure 8A) or 100 μm (Figure 8B). Figure 8C Quantification of AvFc staining for lung tissue from all 10 patients tested using ImageJ. The number of positively stained cells between tumor and matched adjacent tissue was compared using a nonparametric Wilcoxon matched-pairs signed-rank test (**p<0.01). Figure 8D Representative immunoblot analysis of EGFR isolated from NSCLC tumor or matched adjacent tissue samples from 5 patients. EGFR was isolated with anti-EGFR IgG1 using protein A bead precipitation and detected with AvFclec-, AvFc, or CTX. CBB: Coomassie brilliant blue staining. Figure 8E Quantification of EGFR immunoblots using densitometric analysis. Relative binding intensity (tumor:adjacent) to AvFc and CTX is shown. Columns and bars represent mean ± SEM (n = 5) and data were compared using unpaired t-test (*p < 0.05). [Figure 9] Figure 9 shows EGFR and IGF1R expression in primary human lung tissue. IHC staining with anti-EGFR or anti-IGF1R. Representative staining from lung tissue of patient 117 is shown with hematoxylin as counterstain. [Figure 10] Figure 10 shows the effect of mouse serum on the in vitro ADCC activity of AvFc against A549 cells. The assay was performed as described in Example 1, except for the addition of serum from immunized (ADA titer 104-105) or non-immunized animals. In general, the addition of serum caused a small increase in the signal, with ADA-containing serum increasing it the most. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Next, an embodiment of the present invention will be described.

[0044] Some aspects of the present disclosure are described below with reference to examples for illustrative purposes only. It should be understood that many specific details, relationships, and methods are set forth to provide a thorough understanding of the present disclosure. However, one of ordinary skill in the relevant art will readily recognize that the present disclosure can be practiced without one or more of the specific details, or with other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the illustrated ordering of acts or events, as some acts may be performed in different orders and / or in parallel with other acts or events. Moreover, not all illustrated acts, steps, or events may be required to practice a method according to the present disclosure. Many of the techniques and procedures described or referenced herein are well understood and typically employed using conventional methods by those of ordinary skill in the art.

[0045] definition It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] Although any methods and materials similar or equivalent to those described herein can be used in the testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0047] When lists are presented, unless otherwise stated, each individual member of that list and any combination of that list should be understood as a separate embodiment. For example, a list of embodiments presented as "A, B, or C" should be interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0048] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a combination of two or more cells, and the like.

[0049] The conjunction term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within this meaning and thus meets the requirements of the term "and / or" as used herein. The concurrent applicability of two or more options is also understood to be within this meaning and thus meets the requirements of the term "and / or."

[0050] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to include their generally accepted meanings in the patent art: (i) "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude any other unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or ingredient not recited in the claim, and (iii) "consisting essentially of" limits the scope of the claim to the specifically recited materials or steps "and which do not materially affect the basic and novel characteristics" of the claimed invention. Embodiments described with the phrase "comprising" (or its equivalent) also provide as embodiments those described with "consisting of" and "consisting essentially of" independently.

[0051] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is determined or measured, i.e., the limitations of the measurement system. Unless otherwise specified in the examples or elsewhere herein in the context of a particular assay, result, or embodiment, "about" means within one standard deviation or within 5%, whichever is greater, in accordance with convention in the art.

[0052] As used herein, the term "sequence identity" refers to the degree, expressed as a percentage, that two nucleotide sequences or two amino acid sequences have identical residues at the same positions when the sequences are aligned to achieve the maximum level of identity. In sequence alignment and comparison, typically, one sequence is referred to as a reference sequence and is compared to a test sequence. The sequence identity between the reference and test sequences is expressed as the percentage of positions over the entire length of the reference sequence where the reference and test sequences share identical nucleotides or amino acids when the reference and test sequences are aligned to achieve the maximum level of identity. As an example, when the test sequence has identical nucleotides or amino acid residues at 70% of the same positions over the entire length of the reference sequence when aligned to achieve the maximum level of identity, the two sequences are considered to have 70% sequence identity.

[0053] Alignment of sequences for comparison to achieve maximum identity levels can be readily performed by those skilled in the art using appropriate alignment methods or algorithms. In some cases, the alignment may include gaps introduced to provide maximum identity levels. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally, Ausubel et al., Current Protocols in Molecular Biology).

[0054] When using sequence comparison algorithms, test and reference sequences are input into a computer, followed by designating coordinates as necessary, and designating program parameters for the sequence algorithm. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the designated program parameters. A commonly used tool for determining percent sequence identity is the Protein Basic Local Alignment Search Tool (BLASTP), available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (Altschul et al., 1990).

[0055] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" are used interchangeably herein.

[0056] "Prevent," "preventing," "prevention," or "prophylaxis" of a disease or disorder means preventing the disorder from occurring in a subject.

[0057] "Responsive," "responsive," or "likely to respond" means any type of improvement or positive response, whether detectable or undetectable, such as alleviation or amelioration of one or more symptoms, lessening the extent of the disease, a stabilized (i.e., not worsening) state of the disease, preventing the spread of the disease, delaying or slowing the progression of the disease, amelioration or palliation of the disease state, and remission (whether partial or total).

[0058] Next, an embodiment of the present invention will be described.

[0059] Methods of the Disclosure In one aspect, the disclosure provides a method of reducing growth factor receptor activation in a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0060] In another aspect, the disclosure provides a method of reducing growth factor receptor activation in a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0061] In certain embodiments, the growth factor receptor comprises at least one cancer-associated growth factor receptor, for example, 2, 3, 4, 5, or more cancer-associated growth factor receptors.

[0062] In some embodiments, the growth factor receptor comprises at least one tumor-associated growth factor receptor, for example, 2, 3, 4, 5, or more tumor-associated growth factor receptors.

[0063] In some embodiments, the growth factor receptor (e.g., a cancer-associated growth factor receptor) comprises an epidermal growth factor receptor (EGFR). In certain embodiments, the growth factor receptor comprises an insulin-like growth factor 1 receptor (IGF1R). In particular embodiments, the growth factor receptor comprises EGFR and IGF1R. In some embodiments, the growth factor receptor comprises an ErbB family receptor (e.g., EGFR, ErbB2, ErbB3, and / or ErbB4), a fibroblast growth factor receptor (FGFR), IGF1R, IGF2R, or a platelet-derived growth factor receptor (PDGFR), or a combination thereof.

[0064] In some embodiments, the EGFR is human EGFR (also known as HER1 or ErbB1 (Ullrich et al., Nature 309:418-425, 1984) having an amino acid sequence set forth in GenBank Accession Nos. NP_005219, NP_001333826, NP_001333827, NP_001333828, NP_001333829, NP_001333870, NP_005219, NP_958439, NP_958440, and NP_958441, as well as variants thereof (e.g., naturally occurring variants).

[0065] In some embodiments, the IGF1R is human IGF1R (also known as CD221, IGFIR, IGFR, or JTK13) having the amino acid sequence set forth in GenBank Accession No. NP_000866.1, NP_001278787.1, EAX02222.1, XP_016877628.1, XP_016877625.1, XP_016877626.1, XP_016877627.1, or XP_011519818.1, as well as variants thereof (e.g., naturally occurring variants).

[0066] In certain embodiments, activation of a growth factor receptor (e.g., a cancer-associated growth factor receptor such as EGFR and / or IGF1R) is reduced by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, growth factor receptor activation is reduced by about 1-90%, for example, by about 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0067] In another aspect, the disclosure provides a method of inhibiting (e.g., slowing and / or reducing) cancer cell migration, comprising contacting a cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0068] In another aspect, the disclosure provides a method of inhibiting (e.g., slowing and / or reducing) migration of a cancer cell, comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0069] In certain embodiments, cancer cell migration is inhibited (e.g., slowed and / or reduced) by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, cancer cell migration is inhibited by about 1-90%, e.g., by about 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0070] In certain embodiments, cancer cell migration is reduced by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In certain embodiments, cancer cell migration is reduced by about 1-90%, e.g., by about 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0071] In another aspect, the disclosure provides a method of inducing ADCC in a cancer cell comprising contacting the cancer cell with a polypeptide that specifically binds to a high mannose glycan epitope.

[0072] In another aspect, the disclosure provides a method of inducing ADCC in a cancer cell comprising contacting the cancer cell with a polypeptide that specifically binds to a highly glycosylated protein.

[0073] Epitope "Epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes typically consist of chemically active (e.g., polar, non-polar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. In discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space through folding of the protein molecule.

[0074] The term "high mannose glycan" refers to an asparagine-linked glycan (N-glycan) that contains 5-9 terminal mannose residues attached to a chitobiose (GlcNAc2) core. High mannose glycans are formed in the endoplasmic reticulum of eukaryotic cells and are attached to newly synthesized nascent polypeptides that contain an asparagine-X-serine / threonine sequence, where X can be any amino acid except proline. These glycans are then typically processed and matured into complex glycans that contain fewer mannose residues as the nascent polypeptide transits the secretory pathway via the Golgi apparatus. As a result, very little high mannose glycan remains attached to proteins that appear on the surface of healthy normal cells. However, exceptionally high levels of high mannose glycans are often found on cell surface and secreted proteins produced by malignant cells.

[0075] Non-limiting examples of high mannose glycans include Man9GlcNAc2 (Man9), Man8GlcNAc2 (Man8), Man7GlcNAc2 (Man7) Man6GlcNAc2 (Man6), and Man5GlcNAc2 (Man5).

[0076] In some embodiments, the high mannose glycan epitope is cancer associated. In certain embodiments, the high mannose glycan epitope is tumor associated.

[0077] In certain embodiments, the high mannose glycan epitope comprises one or more terminal α1,2-linked mannose residues.

[0078] In certain embodiments, the polypeptide specifically binds to two or more high mannose type glycan epitopes, for example, three, four, five, or more high mannose type glycan epitopes.

[0079] Highly glycosylated proteins In certain embodiments, the hyperglycosylated protein comprises at least about 10 N-glycosylation sites, such as at least about 11, 12, 13, 14, 15, 16, 17, or 18 N-glycosylation sites. In particular embodiments, the hyperglycosylated protein comprises between about 13 and 16 N-glycosylation sites.

[0080] In some embodiments, the polypeptide specifically binds to two or more hyperglycosylated proteins, for example, 2, 3, 4, 5, or more hyperglycosylated proteins.

[0081] In certain embodiments, the hyperglycosylated protein comprises hyperglycosylated agrin, cell cycle regulatory protein 50a, endothelial protein C receptor, epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), integrin alpha-1, integrin alpha-2, integrin alpha-3, integrin beta-5, laminin subunit alpha-5, laminin subunit beta-1, laminin subunit beta-2, low density lipoprotein receptor-related protein 1, neutral amino acid transporter b(0), protocadherin FAT1, solute carrier family 12 member 7, or a combination thereof.

[0082] In some embodiments, the hyperglycosylated protein comprises EGFR. In certain embodiments, the hyperglycosylated protein comprises IGF1R. In certain embodiments, the hyperglycosylated protein comprises EGFR and IGF1R.

[0083] Polypeptides The terms "polypeptide," "peptide," or "protein" refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A protein, peptide, or polypeptide can contain any suitable L- and / or D-amino acids, including normal α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statins), and unusual amino acids (e.g., citrulline, homocitrulline, homoserine, norleucine, norvaline, ornithine). Amino, carboxyl, and / or other functional groups on the peptide can be free (e.g., unmodified) or protected with suitable protecting groups. Suitable protecting groups for amino and carboxyl groups and methods for adding or removing protecting groups are known in the art and are disclosed, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis," John Wiley and Sons, 1991. Functional groups of proteins, peptides, or polypeptides can also be derivatized (e.g., alkylated) or labeled (e.g., with detectable labels such as fluorogens or haptens) using methods known in the art. Proteins, peptides, or polypeptides can optionally include one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclization modifications), N-methyl-α-amino group substitutions). Additionally, proteins, peptides, or polypeptides can be analogs of known and / or naturally occurring peptides, e.g., peptide analogs having conservative amino acid residue substitutions.

[0084] The term "specifically binding to" or "specifically binds to" refers to a preferential interaction, i.e., a significantly higher binding affinity between an antibody or antigen-binding fragment thereof and its epitope compared to other antigens or amino acid sequences.

[0085] In certain embodiments, the polypeptides are highly selective for malignant cells over non-cancerous or normal healthy cells. For example, flow cytometry analysis showed that the 50% effective binding concentration (EC50) of AvFc was 42 ng / mL and 30 ng / mL EC50 for A549 and H460 human lung cancer cell lines, respectively, while it was >10 μg / mL for BEAS-2B non-tumorigenic lung epithelial cell line. In certain embodiments, the EC50 of the polypeptides of the present disclosure for malignant cells is about 10-60 ng / mL, e.g., about 10-55 ng / mL, 15-55 ng / mL, 15-50 ng / mL, 20-50 ng / mL, 20-45 ng / mL, 25-45 ng / mL, 25-42 ng / mL, or 30-42 ng / mL. In some embodiments, the EC50 of the polypeptides for malignant cells is about 30-42 ng / mL.

[0086] In some embodiments, the polypeptide comprises a wild-type actinohibin amino acid sequence (SEQ ID NO:1) or a variant thereof.

[0087] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one of the sequences set forth in SEQ ID NOs:1-13.

[0088] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 1. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

[0089] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to at least one of the sequences set forth in SEQ ID NOs:2-13.

[0090] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 2. In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0091] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 3. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.

[0092] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 4. In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0093] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 5. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

[0094] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 6. In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6.

[0095] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 7. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7.

[0096] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 8. In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8.

[0097] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 10. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10.

[0098] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 11. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:11.

[0099] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about: 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 12. In certain embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:12.

[0100] In some embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 13. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:13.

[0101] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 9. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9.

[0102] In some embodiments, the polypeptide further comprises a fragment crystallizable domain of an antibody (Fc), a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv).

[0103] In certain embodiments, the polypeptide further comprises a Fab.

[0104] In some embodiments, the polypeptide further comprises an scFv.

[0105] In some embodiments, the polypeptide further comprises an Fc. In certain embodiments, the polypeptide comprises the high mannose glycan-binding (actinomycete-derived oligomannose-binding) lectin Avaren and IgG1 Fc (fragment crystallizable region (Fc) of human immunoglobulin G1) ("lectibody" AvFc). In some embodiments, the polypeptide comprises an amino acid sequence at least about 75% (e.g., at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 16. In particular embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0106] cancer cells In some embodiments, the cancer cell is an in vitro cell.

[0107] In certain embodiments, the cancer cells are ex vivo cells.

[0108] In some embodiments, the cancer cells are cells of a subject (eg, a human patient).

[0109] In certain embodiments, the cancer cells are mammalian cells, such as cells from a dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, or human. In some embodiments, the cancer cells are primate cells. In particular embodiments, the cancer cells are human cells.

[0110] In some embodiments, the cancer cell is a cell of a cholangiocarcinoma (e.g., metastatic cholangiocarcinoma), a blood cancer (e.g., melanoma), a breast cancer (e.g., breast carcinoma), a cervical cancer (e.g., cervical carcinoma), a colon cancer (e.g., colon adenocarcinoma), a colorectal cancer, a liver cancer (e.g., hepatocellular carcinoma), a lung cancer (e.g., a lung adenocarcinoma such as large cell lung carcinoma), an ovarian cancer, a pancreatic cancer, a prostate cancer, or a skin cancer (e.g., a cutaneous basal cell carcinoma or a squamous cell carcinoma).

[0111] In certain embodiments, the cancer cells are lung cancer cells. In certain embodiments, the lung cells are non-small cell lung cancer (NSCLC) cells.

[0112] In some embodiments, the cancer cells are solid tumor cells selected from breast, lung, prostate, colon, bladder, ovarian, renal, gastric, rectal, colorectal, testicular, head and neck, pancreatic, brain, and skin cancer cells.

[0113] In certain embodiments, the cancer cells are blood cancer cells selected from leukemia (e.g., acute leukemia, chronic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma), and multiple myeloma cells.

[0114] In certain embodiments, the cancer cells are characterized by abnormal surface accumulation of high mannose glycans.

[0115] In some embodiments, the cancer cells express a protein with abnormal accumulation of high mannose glycans on their cell surface. In certain embodiments, the protein comprises a growth factor receptor, laminin, integrin, transporter, or a combination thereof. In some embodiments, the protein comprises agrin, cell cycle control protein 50a, endothelial protein C receptor, epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), integrin alpha-1, integrin alpha-2, integrin alpha-3, integrin beta-5, laminin subunit alpha-5, laminin subunit beta-1, laminin subunit beta-2, low density lipoprotein receptor-related protein 1, neutral amino acid transporter b(0), protocadherin FAT1, solute carrier family 12 member 7, or a combination thereof.

[0116] In some embodiments, the protein associated with abnormal accumulation of high mannose glycans comprises EGFR. In certain embodiments, the protein associated with abnormal accumulation of high mannose glycans comprises IGF1R. In particular embodiments, the protein associated with abnormal accumulation of high mannose glycans comprises EGFR and IGF1R.

[0117] In some embodiments, the cancer cells are characterized by one or more tumor-associated glycobiomarkers. In certain embodiments, the cancer cells are characterized by two or more tumor-associated glycobiomarkers.

[0118] In some embodiments, the cancer cells are characterized by inappropriate activation of a growth factor receptor. In some embodiments, the cancer cells are characterized by inappropriate activation of EGFR. In certain embodiments, the cancer cells are characterized by inappropriate activation of IGF1R. In particular embodiments, the cancer cells are characterized by inappropriate activation of EGFR and IGF1R.

[0119] In some embodiments, the cancer cells are resistant to antibodies that specifically bind to growth factor receptors, for example, anti-EGFR antibodies such as cetuximab (CTX).

[0120] Methods of Treating Cancer In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope.

[0121] In another aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; The present invention provides a method of treating cancer in a subject in need thereof, comprising:

[0122] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope, where the cancer is characterized by abnormal cell surface accumulation of high mannose glycans.

[0123] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide that specifically binds to a high mannose glycan epitope, where the cancer is mediated by inappropriate activation of a growth factor receptor.

[0124] In another aspect, the disclosure provides a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16.

[0125] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprises: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) administering or providing for administration to the subject a therapeutically effective amount of the polypeptide if abnormal accumulation is determined to be present in the biological sample; The present invention provides a polypeptide comprising:

[0126] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) administering or providing for administration to the subject a therapeutically effective amount of the polypeptide if abnormal accumulation is determined to be present in the biological sample; Provide for use, including

[0127] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0128] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0129] In another aspect, the disclosure provides a polypeptide for use in treating cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering to the subject an effective amount of the polypeptide.

[0130] In another aspect, the disclosure provides the use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0131] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; Provide for use, including

[0132] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the polypeptide specifically binding to a high mannose glycan epitope, the method comprising: a) providing a biological sample from the subject; b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; and c) if abnormal accumulation is determined to be present in the biological sample, administering or providing for administration to the subject a therapeutically effective amount of a polypeptide that specifically binds to a high mannose glycan epitope; Provide for use, including

[0133] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0134] In another aspect, the disclosure provides a use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being characterized by abnormal cell surface accumulation of high mannose glycans, the polypeptide specifically binds to a high mannose glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0135] In another aspect, the disclosure provides a use of a polypeptide in the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering to the subject an effective amount of the polypeptide.

[0136] In another aspect, the disclosure provides the use of a polypeptide in the manufacture of a pharmaceutical agent for the treatment of cancer in a subject in need thereof, the cancer being mediated by inappropriate activation of a growth factor receptor, the polypeptide specifically binds to a high mannose type glycan epitope, and the method comprising administering an effective amount of the polypeptide to the subject.

[0137] composition In some embodiments, the polypeptides described herein are provided in a composition, such as a pharmaceutical composition.

[0138] In some embodiments, the composition (e.g., a pharmaceutical composition) further comprises one or more pharma- ceutically acceptable carriers, excipients, stabilizers, diluents, or isotonicity agents (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable pharma- ceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Non-limiting examples of pharma- ceutically acceptable carriers, excipients, stabilizers, diluents, or isotonicity agents include buffers (e.g., phosphate, citrate, histidine), antioxidants (e.g., ascorbic acid or methionine), preservatives, proteins (e.g., serum albumin, gelatin, or immunoglobulins), hydrophilic polymers, amino acids, carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrins), chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes), non-ionic surfactants (e.g., Tween), PLURONICS™, and polyethylene glycol (PEG).

[0139] In some embodiments, the compositions (e.g., pharmaceutical compositions) of the present disclosure are formulated for a suitable administration schedule and route. Non-limiting examples of administration routes include oral, rectal, mucosal, intravenous, intramuscular, subcutaneous, topical, etc. In some embodiments, the compositions (e.g., pharmaceutical compositions) of the present disclosure are stored in the form of an aqueous solution or a dried formulation (e.g., lyophilized).

[0140] In some embodiments, the composition is formulated to be administered by infusion (e.g., intravenous infusion) or injection (e.g., intramuscular, subcutaneous, intraperitoneal, or intratumoral injection). In certain embodiments, the composition is formulated to be administered by intravenous infusion. In some embodiments, the composition is formulated to be administered by intramuscular injection. In particular embodiments, the composition is formulated to be administered by subcutaneous injection. In some embodiments, the composition is formulated to be administered by intraperitoneal injection. In certain embodiments, the composition is formulated to be administered by intratumoral injection.

[0141] In some embodiments, the compositions are formulated to be administered with one or more additional therapeutic agents as a combination therapy. Non-limiting examples of the one or more additional therapeutic agents include chimeric antigen receptor (CAR)-expressing T cells (CAR-T cells), CAR-expressing natural killer cells (CAR-NK cells), CAR-expressing macrophages (CAR-M cells), chemotherapeutic agents, immune checkpoint inhibitors, T cell redirectors, radiation therapy, surgery, and standard of care agents.

[0142] A pharmaceutical composition, meaning the product resulting from combining a polypeptide that specifically binds to a high mannose glycan epitope with one or more additional therapeutic agents, includes both immobilized and non-immobilized combinations.

[0143] "Fixed combination" refers to a single pharmaceutical composition comprising two or more compounds, for example, a polypeptide that specifically binds to a high mannose glycan epitope and one or more additional therapeutic agents, administered simultaneously in the form of a single entity or dosage. In some embodiments, a pharmaceutical composition comprising a polypeptide that specifically binds to a high mannose glycan epitope and one or more additional therapeutic agents is provided as a fixed combination.

[0144] "Non-fixed combination" refers to separate pharmaceutical compositions each comprising one or more compounds, e.g., a polypeptide that specifically binds to a high mannose glycan epitope and one or more additional therapeutic agents, either administered simultaneously, in parallel, or sequentially without specific intervening time restrictions, such administration providing effective levels of the two or more compounds in the subject. In some embodiments, a pharmaceutical composition comprising a polypeptide that specifically binds to a high mannose glycan epitope and one or more additional therapeutic agents is provided as a non-fixed combination.

[0145] In some embodiments, a polypeptide (e.g., AvFc) is systemically administered to a subject at about 10-50 mg / kg, e.g., about 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg, or about 10-45 mg / kg, 15-45 mg / kg, 15-40 mg / kg, 20-40 mg / kg, 20-35 mg / kg, 25-35 mg / kg, or 25-30 mg / kg. In certain embodiments, a polypeptide (e.g., AvFc) is systemically administered to a subject at about 10-50 mg / kg about every 2-7 days (e.g., about every 2, 3, 4, 5, 6, or 7 days) for about 2-10 weeks (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or about 2-9, 3-9, 3-8, 4-8, 4-7, 5-7, or 5-6 weeks). In particular embodiments, a polypeptide (e.g., AvFc) is systemically administered to a subject at about 25 mg / kg every other day (Q2D) for 14 or 20 days (for a total of 8 or 11 doses, respectively). In some embodiments, a polypeptide (e.g., AvFc) is systemically administered to a subject at about 10-50 mg / kg every 7 days (Q7D) for 1-2 months.

[0146] cancer "Cancer" refers to an abnormal growth of cells that tend to proliferate uncontrollably and, in some cases, to metastasize (spread) to other areas of the patient's body.

[0147] In some embodiments, the cancer is cholangiocarcinoma (e.g., metastatic cholangiocarcinoma), blood cancer (e.g., melanoma), breast cancer (e.g., breast carcinoma), cervical cancer (e.g., cervical carcinoma), colon cancer (e.g., colon adenocarcinoma), colorectal cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., lung adenocarcinoma such as large cell lung carcinoma), ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer (e.g., cutaneous basal cell carcinoma or squamous cell carcinoma).

[0148] In certain embodiments, the cancer is lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

[0149] In some embodiments, the cancer is a solid tumor, e.g., breast cancer, lung cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, renal cancer, gastric cancer, colon cancer, rectal cancer, testicular cancer, head and / or neck cancer, pancreatic cancer, brain cancer, or skin cancer.

[0150] In other embodiments, the cancer is a hematological cancer, such as leukemia, lymphoma, or myeloma. Hematological cancers treatable according to the methods described herein include leukemia (e.g., acute leukemia, chronic leukemia), lymphoma (e.g., B-cell lymphoma, T-cell lymphoma), and multiple myeloma.

[0151] In certain embodiments, the cancer is characterized by abnormal cell surface accumulation of high mannose glycans. In some embodiments, the cell surface high mannose glycans on cancer cells of the present disclosure are about 2-10 times higher than on normal cells.

[0152] In some embodiments, the cancer is characterized by cell surface expression of a protein with abnormal accumulation of high mannose glycans. In certain embodiments, the protein comprises a growth factor receptor, laminin, integrin, transporter, or a combination thereof. In some embodiments, the protein comprises agrin, cell cycle control protein 50a, endothelial protein C receptor, epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), integrin alpha-1, integrin alpha-2, integrin alpha-3, integrin beta-5, laminin subunit alpha-5, laminin subunit beta-1, laminin subunit beta-2, low density lipoprotein receptor-related protein 1, neutral amino acid transporter b(0), protocadherin FAT1, solute carrier family 12 member 7, or a combination thereof. In certain embodiments, the protein with abnormal accumulation of high mannose glycans comprises EGFR, IGF1R, or both. In some embodiments, the protein associated with abnormal accumulation of high mannose glycans comprises EGFR. In certain embodiments, the protein associated with abnormal accumulation of high mannose glycans comprises IGF1R.

[0153] In some embodiments, the cancer is characterized by one or more tumor-associated glycobiomarkers. In certain embodiments, the cancer is characterized by two or more tumor-associated glycobiomarkers.

[0154] In some embodiments, the cancer is mediated by inappropriate activation of a growth factor receptor. In some embodiments, the growth factor receptor comprises EGFR. In certain embodiments, the growth factor receptor comprises IGF1R. In particular embodiments, the growth factor receptor comprises EGFR and IGF1R.

[0155] subject The term "subject" means an animal (e.g., a mammal). In some embodiments, the subject is a mammal. In certain embodiments, the subject is a mammal selected from the group consisting of dog, cat, mouse, rat, hamster, guinea pig, horse, pig, sheep, cow, chimpanzee, macaque, cynomolgus monkey, and human. In some embodiments, the subject is a primate. In particular embodiments, the subject is a human.

[0156] The term "subject in need" refers to a mammalian subject, preferably a human, diagnosed with or suspected of having a disease (e.g., cancer, such as lung cancer), to which a polypeptide according to the methods of the invention will or has been administered. A "subject in need" includes subjects who already have an unwanted physiological change or disease as well as subjects susceptible to developing a physiological change or disease.

[0157] Diagnosis may be performed by any method or technique known in the art. One of skill in the art will appreciate that subjects treated in accordance with the present disclosure may have been subjected to standard testing or may have been identified without testing as being at risk due to the presence of one or more risk factors associated with the disease or condition.

[0158] In some embodiments, the subject is an adult patient. In certain embodiments, the subject is a juvenile patient. In particular embodiments, the subject is a pediatric patient.

[0159] In some embodiments, the subject is between 18 and 75 years of age. In certain embodiments, the subject is 40 years of age or older, e.g., at least 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 years of age.

[0160] In certain embodiments, the subject is 18 years of age or older, e.g., 18-40 years of age or younger, 18-45 years of age or younger, 18-50 years of age or younger, 18-55 years of age or younger, 18-60 years of age or younger, 18-65 years of age or younger, 18-70 years of age or younger, 18-75 years of age or younger, 40-75 years of age or younger, 45-75 years of age or younger, 50-75 years of age or younger, 55-75 years of age or younger, 60-75 years of age or younger, 65-75 years of age or younger, 60-75 years of age or younger, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, or 75 years of age or older.

[0161] In some embodiments, the subject is 18 years of age or younger, e.g., 0-18 years of age, 0-12 years of age, 0-16 years of age, 0-17 years of age, 2-12 years of age, 2-16 years of age, 2-17 years of age, 2-18 years of age, 3-12 years of age, 3-16 years of age, 3-17 years of age, 3-18 years of age, 4-12 years of age, 4-16 years of age, 4-17 years of age, 4-18 years of age, 6-12 years of age, 6-16 years of age, 6-17 years of age, 6-18 years of age, 9-12 years of age, 9-16 years of age, 9-17 years of age, 9-18 years of age, 12-16 years of age, 12-17 years of age, or 12-18 years of age.

[0162] In some embodiments, the subject is 12 years of age or older.

[0163] In certain embodiments, the subject is 2 years of age or older, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years of age or older. In some embodiments, the subject is 4 years of age or older. In some embodiments, the subject is 5 years of age or older. In some embodiments, the subject is 6 years of age or older.

[0164] In some embodiments, the subject has been diagnosed with cancer (e.g., lung cancer) for at least about 1 month, e.g., at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 30 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0165] In certain embodiments, the subject is newly diagnosed with cancer (e.g., lung cancer). "Newly diagnosed" refers to a subject who has been diagnosed with cancer (e.g., lung cancer) but has not yet received treatment for the cancer.

[0166] In certain embodiments, the subject is treatment naive.

[0167] In some embodiments, the subject has received one or more prior anti-cancer therapies. In certain embodiments, the one or more prior anti-cancer therapies include one or more chemotherapeutic agents, checkpoint inhibitors, targeted anti-cancer therapies, or kinase inhibitors, or any combination thereof.

[0168] In certain embodiments, the subject is relapsing or resistant to treatment with one or more previous anticancer therapies. "Refractory" refers to disease that does not respond to treatment. Refractory disease may be resistant to treatment before or at the start of treatment, or refractory disease may become resistant at the time of treatment. "Relapse" refers to the return of a disease or signs and symptoms of a disease after a period of improvement following previous treatment with a therapeutic agent.

[0169] In some embodiments, the subject is resistant to treatment with an antibody that specifically binds to a growth factor receptor, for example an anti-EGFR antibody such as cetuximab (CTX).

[0170] treatment "Treating," "treating," or "treatment" of a disease or disorder, such as cancer, means achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who previously had symptoms of the disorder.

[0171] A "therapeutically effective amount," "effective amount," or "effective dosage" is an amount effective, at the dosage and for the period of time required, to achieve a desired therapeutic result (e.g., treating, curing, inhibiting, reversing a physiological response or pathology, etc.). The full therapeutic effect does not necessarily occur with a single administration, but may occur only after a series of administrations. Thus, a therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount may vary with factors such as the disease state, age, sex, and weight of the mammal, the mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual.

[0172] In some embodiments, a therapeutically effective amount of a polypeptide comprises: a) reducing activation of growth factor receptors; b) inhibition of cancer cell migration; c) Induction of cytotoxicity, or d) a slowing of tumor growth, or Combinations of the above is sufficient.

[0173] In some embodiments, the therapeutically effective amount of the polypeptide is sufficient to reduce activation of a growth factor receptor (e.g., EGFR, IGF1R, or both). In certain embodiments, the growth factor receptor comprises a cancer-promoting growth factor receptor (e.g., a tumor-promoting growth factor receptor). In particular embodiments, the growth factor receptor comprises two or more cancer-promoting growth factor receptors.

[0174] In certain embodiments, a therapeutically effective amount of the polypeptide is sufficient to reduce activation of EGFR. In some embodiments, a therapeutically effective amount of the polypeptide is sufficient to reduce activation of IGF1R. In particular embodiments, a therapeutically effective amount of the polypeptide is sufficient to reduce activation of EGFR and IGF1R by their respective ligands.

[0175] In certain embodiments, activation of a growth factor receptor (e.g., EGFR, IGF1R, or both) is reduced by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, growth factor receptor activation is reduced by about 1-90%, e.g., reduced by about 1-90%, e.g., reduced by about 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0176] In some embodiments, a therapeutically effective amount of the polypeptide is sufficient to inhibit (eg, slow or reduce) cancer cell migration.

[0177] In certain embodiments, a therapeutically effective amount of the polypeptide is sufficient to slow or inhibit cancer cell migration mediated by activation of EGFR. In some embodiments, a therapeutically effective amount of the polypeptide is sufficient to slow or inhibit cancer cell migration mediated by activation of IGF1R. In particular embodiments, a therapeutically effective amount of the polypeptide is sufficient to slow or inhibit cancer cell migration mediated by activation of EGFR and IGF1R.

[0178] In certain embodiments, cancer cell migration is reduced by at least about 10%, e.g., at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In certain embodiments, cancer cell migration is reduced by about 1-90%, e.g., by about 1-85%, 5-85%, 5-80%, 10-80%, 10-75%, 15-75%, 15-70%, 20-70%, 20-65%, 25-65%, 25-60%, 30-60%, 30-55%, 35-55%, 35-50%, or 40-50%.

[0179] In some embodiments, a therapeutically effective amount of the polypeptide is sufficient to induce cytotoxicity, hi certain embodiments, the cytotoxicity comprises one or more Fc-mediated cytotoxicity (ADCC).

[0180] In some embodiments, the therapeutically effective amount of the polypeptide is sufficient to slow tumor growth.

[0181] diagnosis In certain embodiments, the treatment method further comprises determining whether a biological sample from a subject in need thereof is characterized by high mannose-type glycan epitopes.

[0182] "Diagnosing" or "diagnosis" refers to a method of determining whether a subject is afflicted with a given disease or condition, or is likely to develop a given disease or condition in the future, or is likely to respond to a treatment for a previously diagnosed disease or condition, i.e., a method of stratifying a patient population based on the likelihood of responding to a treatment. Diagnosis is typically performed by a physician based on general guidelines for the disease being diagnosed or other criteria that suggest that a subject is likely to respond to a particular treatment.

[0183] In some embodiments, the method comprises the steps of: a) providing a biological sample from the subject; and b) determining the presence or absence of abnormal accumulation of high mannose glycan epitopes in the biological sample; Further includes:

[0184] "Biological sample" refers to a fluid collection, cell, or tissue isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids, such as blood, serum, and serous fluid, plasma, lymphatic fluid, urine, saliva, cyst fluid, tear drops, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, fluids of the pleural, pericardial, peritoneal, abdominal, and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media, lavage fluids, tissue biopsies, tumor tissue biopsies, tumor tissue samples, fine needle aspirates, surgically resected tissues, organ cultures, or cell cultures.

[0185] In certain embodiments, the biological sample is a blood sample, hi some embodiments, the biological sample is a tumor tissue biopsy.

[0186] In certain embodiments, the one or more proteins include a growth factor receptor, laminin, integrin, transporter, or a combination thereof. In some embodiments, the protein includes agrin, cell cycle control protein 50a, endothelial protein C receptor, epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), integrin alpha-1, integrin alpha-2, integrin alpha-3, integrin beta-5, laminin subunit alpha-5, laminin subunit beta-1, laminin subunit beta-2, low density lipoprotein receptor-related protein 1, neutral amino acid transporter b(0), protocadherin FAT1, solute carrier family 12 member 7, or a combination thereof. In particular embodiments, the one or more proteins include EGFR, IGF1R, or both. In some embodiments, the one or more proteins include EGFR. In certain embodiments, the one or more proteins include IGF1R.

[0187] Embodiment 1. A method for reducing activation of epidermal growth factor receptor (EGFR) or insulin-like growth factor 1 receptor (IGF1R) or both in cancer cells, the method comprising contacting the cancer cells with an effective amount of a polypeptide comprising an actinohibin variant.

[0188] 2. The method of item 1, wherein the polypeptide reduces activation of EGFR in cancer cells.

[0189] 3. The method according to item 1 or 2, wherein the polypeptide reduces activation of IGF1R in cancer cells.

[0190] 4. A method of inhibiting cancer cell migration, the method comprising contacting a cancer cell with an effective amount of a polypeptide comprising an actinohibin variant.

[0191] 5. The method according to any one of items 1 to 4, wherein the cancer cells are non-small cell lung cancer (NSCLC) cells.

[0192] 6. The method according to any one of items 1 to 5, wherein the cancer cells are resistant to anti-EGFR antibodies.

[0193] 7. A method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide comprising an actinohibin variant, wherein the cancer is epidermal growth factor receptor (eGFR) mediated or insulin-like growth factor 1 receptor (IGF1R) mediated or a combination thereof.

[0194] 8. The method according to item 7, wherein the cancer is mediated by EGFR.

[0195] 9. The method according to item 7 or 8, wherein the cancer is mediated by IGF1R.

[0196] 10. The method according to any one of items 7 to 9, wherein the cancer is lung cancer.

[0197] 11. The method of item 10, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0198] 12. The method according to any one of items 7 to 11, wherein the cancer is resistant to an anti-EGFR antibody.

[0199] 13. The method according to any one of items 1 to 12, wherein the actinohibin variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, and optionally the actinohibin variant comprises the amino acid sequence shown in SEQ ID NO: 9.

[0200] 14. The method according to any one of items 1 to 13, wherein the polypeptide further comprises an antibody fragment crystallizable domain (Fc), and optionally the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 16.

[0201] 15. The method according to any one of items 1 to 13, wherein the polypeptide further comprises an antigen-binding fragment domain of an antibody (Fab) or a single chain variable fragment of an antibody (scFv). EXAMPLES

[0202] Aberrant protein glycosylation is a hallmark of cancer, yet few drugs currently available target cancer glycobiomarkers. We have demonstrated that a translational fusion protein "lectibody" ("lectibody" AvFc), consisting of the high mannose glycan-binding (actinomycete-derived oligomannose-binding) lectin Avalen and IgG1 Fc (crystallizable fragment region (Fc) of human immunoglobulin G1), selectively recognizes a range of cell lines derived from various cancers, including lung, breast, colon, and hematological cancers, at nanomolar concentrations. We characterized the binding of AvFc to non-small cell lung cancer (NSCLC) cell lines A549 and H460. Co-immunoprecipitation proteomic analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the common targets of lectibody in these cells. AvFc blocked activation (e.g., phosphorylation and downstream signaling) of EGFR and IGF1R by their respective ligands in A549 cells. In addition, AvFc inhibited migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxicity in SCID mice and significantly delayed A549 and H460 tumor growth. Immunohistochemical analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially bound to tumors over adjacent non-tumor tissues. These results suggest that AvFc induces anti-cancer activity through Fc-mediated effector functions along with inhibition of tumor-promoting growth factor receptors. These findings provide evidence that the increased abundance of high-mannose glycans in the glycocalyx of cancer cells may be a druggable target and that AvFc provides a new tool to probe and target this tumor-associated glycobiomarker.

[0203] Few agents are available that can distinguish tumor-associated high-mannose glycans from other glycoforms present on the surface of normal cells, and their druggability remains unclear.

[0204] Previously, an antibody-like "lectibody" molecule called Avalen-Fc (AvFc) was created that is composed of the oligomannose-specific Avalen lectin and the crystallizable fragment domain (Fc) of human IgG1. 19 . Avalen is an engineered variant of the actinomycete antiviral lectin actinohibin with amino acid substitutions that improve solubility and productivity 20,21 AvFc neutralized the infectivity of multiple HIV strains and Hepatitis C virus at nanomolar concentrations through high-affinity binding to clustered mannose-rich glycans on the envelope glycoproteins. 19,22 Additionally, lectibodies exhibited antibody-dependent cell-mediated viral inhibition against HIV-infected peripheral blood mononuclear cells (PBMCs) through their ability to interact with activating Fcγ receptors, such as FcγRI and FcγRIIIa. Preliminary safety studies in mice and rhesus monkeys showed that systemic administration of AvFc did not induce any discernible toxicity. 19 Furthermore, systemic administration of AvFc at 25 mg / kg every other day (Q2D) for 14 or 20 days (a total of 8 or 11 doses, respectively) completely protected against HCV challenge in a chimeric human liver mouse model without causing hepatotoxicity or any other significant adverse effects. 22 These results support the use of AvFc in novel therapeutic strategies to target high mannose glycans that may appear on the cell surface at high density under pathological conditions.

[0205] In light of the growing evidence of abnormal overexpression of high mannose glycans on neoplastic cells, it is hypothesized that AvFc may efficiently recognize these mannose-rich glycans on the surface of cancer cells and exert antitumor activity. To address this hypothesis, Examples 2-7 examine the ability of lectibodies to target cancer using human non-small cell lung cancer (NSCLC) cell lines, human NSCLC, and murine xenograft models of primary human NSCLC tissue sections. The results provide support for novel anticancer strategies that target tumor-associated high mannose glycans.

[0206] Example 1. Materials and Methods Human Lung Tissue De-identified post-operative human lung cancer tissues and matched adjacent tissues were obtained from the University of Louisville Hospital (Louisville, KY). The pathological type of each tumor was determined to be NSCLC. Written informed consent was provided by all participants, and the study protocol was approved by the University of Louisville Human Subjects Protection Program (Study #18.1240). Discrimination between tumor and adjacent tissue was performed by the surgeon at the time of tissue removal, and tissues were immediately frozen in liquid nitrogen at the time of surgery and stored at -80°C.

[0207] Animal Housing and Care Nine-week-old female Prkdc mice were cultured in a temperature-controlled environment with a 12-h alternating light / dark cycle and free access to standard food and water. scid 1. / SzJ(SCID) mice (The Jackson Laboratory, Bar Harbor, ME) were bred. Investigators were not blinded to sample administration. All experimental procedures were approved by the University of Louisville Institutional Animal Care and Use Committee.

[0208] reagent Antibodies specific for EGFR (D38B1), phospho-EGFR (Y1068), IGF1R (D23H3), phospho-IGF1R (Y1131), AKT, phospho-AKT (S473), MAPK1, and phospho-MAPK1 (ERK1 / 2) were purchased from Cell Signaling Technology (Danver, MA). EGF and IGF1 were purchased from Thermo Fisher Scientific (Waltham, MA). CTX was obtained from the University of Louisville Hospital Pharmacy.

[0209] cell culture All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and authenticated by the supplier. Cells were grown according to ATCC recommendations, with quality assured based on viability and morphological examination, and were routinely screened and tested at low passage number for mycoplasma using a commercially available PCR-based kit (ATCC, Manassas, VA). Specifically, unless otherwise stated, A549 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and H460 cells were grown in RPMI 1640 supplemented with 10% FBS and 1% penicillin / streptomycin.

[0210] Generation of AvFc AvFc and AvFc lec- was generated using a transient plant expression vector in Nicotiana benthamiana as previously described. 19 Briefly, 4-week-old plants were transformed with a magnICON® vector containing the gene for AvFc by agroinfiltration and incubated for 1 week. At that time, leaf tissue was homogenized in NaPi buffer pH 7.4, clarified by centrifugation, and then subjected to fast protein liquid chromatography on an AKTA pure system (GE Healthcare Life Sciences, Chicago, IL) using Protein A as the first chromatography step and ceramic hydroxyapatite (CHT) as the clean-up step. Endotoxins were removed from the purified protein using Triton X-114 phase separation, and the protein was subsequently concentrated using a 10 kDa MWCO centrifuge filter and sterilized with a 0.2 μm filter. Purity was assessed by SDS-PAGE, with AvFc appearing as a band of approximately 77 kDa under non-reducing conditions.

[0211] Flow cytometric analysis of AvFc binding to human cells Cancer cell lines were harvested and incubated with various concentrations of AvFc (0.1, 1, and 10 μg / mL) in culture medium for 30 min on ice and washed three times with DPBS. Cells were then incubated with goat F(ab')2 anti-human IgG Fc-FITC antibody (Abcam, Cambridge, MA) for 30 min on ice in the dark. After an additional three washes with DPBS, cells were fixed with 1% formalin for 15 min on ice. 10,000 events / sample were counted and FITC was detected using FlowJo. + Data were acquired on a FACSCalibur flow cytometer (BD BioSciences, San Jose, CA) by determining the percentage of cells that were immunized with the non-glycosylated AvFc mutant as a negative control. lec- Analyses were performed in triplicate.

[0212] Immunofluorescence 1,000 A549 cells or 10,000 BEAS-2B cells / chamber were seeded on Lab-Tek II chamber slides (Thermo Fisher Scientific, Waltham, MA) and incubated for 24 hours. After washing with PBS, cells were fixed with 4% formalin in PBS for 20 minutes at room temperature. After incubation with 0.2% Triton X-100 in PBS for 15 minutes at room temperature, human Fc Block™ (BD, San Jose, CA) was added to the cells and incubated for an additional 10 minutes. Cells were then blocked with 3% BSA-PBS for 30 minutes at room temperature and then incubated with 250 units of endoglycosidase H for 1 hour at 37°C according to the manufacturer's protocol (New England Biolabs, Ipswich, MA). The cells were then stained with 10 μg / ml AvFc for 3 hours at room temperature, washed with PBS, and then stained with a 1:40 dilution of anti-human IgG-FITC (Sigma, Mendota Heights, Minn.) for 1 hour at room temperature. The cells were then mounted with a coverslip using fluorescent mounting medium with DAPI (VECTASHIELD®, Burlingame, Calif.). Slides were analyzed by fluorescence confocal microscopy (ZEISS LSM 880).

[0213] Co-immunoprecipitation 1×10 6 A549 or H460 cells were seeded in 10 cm2 plates (Corning, Tewksbury, MA) and incubated in growth medium for 24 h. Cells were washed with PBS, and cell lysates were prepared in T-PER buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with protease / phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). After centrifugation at 13,000 × g for 10 min at 4 °C, the supernatant was mixed with 4 μg AvFc or AvFc-lec. After 24 h of incubation at 4 °C, 10 μl of Protein A beads (Santa Cruz, Dallas, TX) were added. After an additional 2 h of incubation at 4 °C, the mixture was washed with T-PER buffer and immunoblot analysis was performed.

[0214] EGFR isolation Tissue homogenates were prepared with silicon beads and a Precellys® 24 homogenizer (Bertin, Rockville, Mass.) in T-PER buffer (Thermo Fisher Scientific, Waltham, Mass.) containing a protease inhibitor cocktail (Sigma, Mendota Heights, Minn.). Debris was removed by centrifugation at 13,000×g for 10 min at 4° C. The supernatant was incubated with 4 μg of anti-EGFR IgG1 (D38B1) (Cell Signaling Technology, Danver, MA) and 20 μg of Protein A beads (Santa Cruz, Dallas, TX) for 4 hours at 4° C. The mixture was washed with T-PER buffer.

[0215] Immunoblot analysis SDS-PAGE and membrane transfer cassettes were purchased from Thermo Fisher Scientific (Waltham, MA). Protein samples were run on 10% Bolt Bis-Tris Plus gels using NuPAGE MES SDS running buffer (Thermo Fisher Scientific, Waltham, MA). Transfer to PVDF membranes in NuPAGE transfer buffer was performed at 10 V overnight at 4 °C. Membranes were then incubated with 3% BSA in TBST for 2 h and anti-EGFR, anti-IGF1R, anti-human Fc, or AvFc in TBST supplemented with 1% BSA overnight at 4 °C. HRP-tagged secondary antibodies (anti-rabbit IgG, Santa Cruz, Dallas, TX; anti-human IgG, SouthernBiotech, Birmingham, AL) were used for protein detection, and membrane images were taken using an Amersham Imager 600 (GE Healthcare Life Sciences, Chicago, IL).

[0216] Proteomic analysis Co-immunoprecipitation followed by mass spectrometry was used to identify potential cell surface binding partners of AvFc. Co-immunoprecipitation in this case was performed using the Pierce Co-immunoprecipitation Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Briefly, to reduce non-specific interactions, whole cell lysates of A549 and H460 cells were pre-cleared with control agarose resin and then incubated with 100 μg of AvFc or AvFc covalently attached to agarose resin. lec- The mixture was co-incubated with 1 M Tris base for 2 h at 4° C. Bound proteins were then eluted with a low pH buffer and neutralized with 1 M Tris base for mass spectrometry analysis.

[0217] Following reduction and alkylation with 100 mM dithiothreitol and 50 mM iodoacetamide, protein samples were digested with trypsin (1:50 ratio) using a filter-assisted sample preparation approach. Tryptic digests (0.5 mg) were separated using a Proxeon EASY n-LC (Thermo-Fisher Scientific) UHPLC system and a Dionex (Sunnyvale, CA) 2 cm Acclaim PepMap 100 trap and a 15 cm Dionex Acclaim PepMap RSLC (C18, 2 μm, 100A) separation column. The eluate was introduced into an LTQ-Orbitrap ELITE (Thermo-Fisher Scientific) using a Nanospray Flex source and MS2 data collected in a data-dependent manner with the Top20 rapid CID method. All MS1 data were acquired using Fourier transform ion cyclotron resonance MS at 240,000 resolution, and MS2 data was acquired using a linear ion trap. MSn data were searched using Proteome Discoverer 1.4 (Thermo Scientific) with Sequest HT (SageN) and Mascot version 4.0 (Matrix Science) with a decoy database search strategy against the UniProt Knowledgbase Homo sapiens reference proteome. Searches were performed with a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 50 ppm. Search data result files were imported into Scaffold version 4.3.4 (Proteome Software Inc.) and filtered with a mass error filter of 2 ppm and removal of decoy hits to control for a false discovery rate of <1.0% in PeptideProphet and ProteinProphet (Institute for Systems Biology). Peptide and protein analyses were accepted >95.0% of the time by the PeptideProphet or ProteinProphet algorithms. Comparison of protein abundance between sample sets was performed in Scaffold using the intensity-based absolute quantification (iBAQ) method.AvFc binds to AvFc. lec- The results were further refined using Gene Ontology (GO) terms to extract the most abundant membrane receptors, transporters, and adhesion molecules that are not bound by .

[0218] Transwell migration assay Inserts of 8-μm-pore transwell plates (VWR International, Radnor, PA) were cultured with 1 × 10 cells in 200 μl of serum-free growth medium. 5 A549 or H460 cells were seeded. These cells were co-incubated with 30 nM AvFc or CTX for 2 h at 37° C. Growth medium supplemented with 20% FBS was then added to the outer wells, and EGF or IGF-1 was added to the transwell inserts at a final concentration of 2 ng / mL. After 6 h, the number of migrated cells was determined by trypsinization and trypan blue staining (Thermo Fisher Scientific, Waltham, MA).

[0219] ADCC reporter assay Antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed by ADCC Reporter Bioassay (Promega, Madison, WI) according to the manufacturer's protocol. Each sample was tested in triplicate. Briefly, three NSCLC cell lines were used as target cells, each of which was incubated with 10,000 4 Cells / well were seeded into milky white 96-well flat-bottom culture plates (Corning, Tewksbury, MA) and incubated at 37°C and 5% CO2. After 24 hours, various concentrations of AvFc, AvFc lec-, or CTX was added to target cells at a ratio of 15:1 along with Jurkat NFAT-luc FcγRIIIa expressing cell line (Jur-Fcr_RIIIa; Promega, Madison, WI). FcγRIIIa signaling activates NFAT transcription factors and induces expression of firefly luciferase via an NFAT-responsive promoter. After 24 hours of co-culture, firefly luciferase activity was measured on a Synergy HT luminometer (BioTeck, Winooski, VT) using the Britelite Plus Reporter Gene Assay System (Perkin Elmer, Waltham, MA). Jur-FcγRIIIa cells co-cultured with target cells in the absence of antibody provided a no-antibody control luciferase production level, which was subtracted from the actual signal to obtain antibody-specific activation in relative light units (RLU). Background was determined by taking the average of wells with only target cells. Fold induction was calculated using the following formula: Induction magnification = (RLU induced -RLU background ) / (RLU noAbcontrol -RLU background ) The calculation was carried out using

[0220] ADCC assay with primary human PBMC effector cells Similar to the reporter assay, plated A549 target cells were preincubated with serial dilutions of AvFc or CTX for 30 min at 37° C. PBMCs were added at a ratio of 50:1 for AvFc and 25:1 for CTX to initiate ADCC. After 6 h of incubation in a 37° C. incubator, cell supernatants were harvested, released lactose dehydrogenase was measured, and % target cell lysis was calculated relative to a drug-free control. Each sample was tested in triplicate.

[0221] Subcutaneous lung cancer xenograft challenge model 1 × 10 7 A549 cells or 5 × 10 5Mice were then randomly organized into three groups and treated with vehicle (n=10), 25 mg / kg AvFc (n=10), and 25 mg / kg CTX (n=10). Vehicle treatment consisted of AvFc formulation buffer (30 mM histidine pH 7.4, 100 mM sucrose, 100 mM NaCl). Treatments were administered ip 4, 6, 8, 10, 12, and 14 days after the formation of palpable lesions. Body weights and tumor volumes were measured every other day after treatment. Animals were euthanized 26 days after A549 challenge and 16 days after H460 challenge.

[0222] A549 lung metastasis model A549 cells expressing GFP were grown to a given confluency in growth medium. After harvesting, 2 × 10 6 Cells were injected iv into 9-week-old female SCID mice. Mice were then randomly organized into two groups and treated with vehicle (n=10) and 25 mg / kg AvFc (n=10). IP treatments were administered on days 0, 2, 4, 6, 8, and 10. Mice were weighed every other day after treatment. Finally, animals were euthanized at 28 days and lungs were surgically removed and fixed in 10% formalin. To detect GFP signals, each lung lobe was separated and GFP signals were detected with an Amersham Imager 600 (GE Healthcare Life Sciences, Chicago, IL).

[0223] immunohistochemistry Immunohistochemical staining was performed on frozen sections of frozen tissue from lung patients undergoing surgery. Staining was performed with VECTASTAIN® Elite® ABC HRP Kit (Peroxidase, Standard) (Vector Labs, Burlingame, CA). 8 μm tissue sections were placed on positively charged slides (VWR International, Radnor, PA) and air dried. Sections were then incubated in methanol diluted 3% H2O2 at room temperature for 10 min and washed with Tris-buffered saline supplemented with 0.01% Triton-X100 (TBST). Avidin / biotin blocking was performed using a blocking kit for 15 min at room temperature (Abcam, Cambridge, MA), and then Fc receptors were blocked with Fc blocking solution for 10 min at room temperature (BD, San Jose, CA). Sections were further blocked with 3% goat serum in TBST for 30 min at room temperature. To stain the tissue, AvFc at 0.5 μg / ml in TBST supplemented with 1% goat serum was added for 30 min at room temperature, followed by incubation with biotinylated anti-human IgG in TBST with 1% goat serum for 30 min at room temperature as well (Vector Labs, Burlingame, CA). ABC solution was then added for 30 min at room temperature (Vector Labs, Burlingame, CA), and DAB staining solution was applied according to the manufacturer's protocol (Vector Labs, Burlingame, CA). Between each step, sections were washed three times with TBST. Counterstaining was performed with hematoxylin. Sections were sequentially dehydrated with 95% ethanol, 100% ethanol, and CitriSolv (Decon lab, King of Prussia, PA). Images were taken with an Olympus CKX41 microscope (Olympus, Tokyo, Japan) equipped with a UPlanFL 10× / 0.30 lens.

[0224] statistical analysis Group means and standard errors were derived from values ​​obtained in three individual replicates, and assays were performed independently at least twice unless otherwise noted. For all data, outliers were determined by statistical analysis using the Grubbs test (p<0.05) and excluded from further analysis. Statistical significance was analyzed by one- or two-way analysis of variance (ANOVA) with Bonferroni post-hoc test or Wilcoxon matched-pairs signed-rank test as indicated in figure legends using GraphPad Prism 5 (San Diego, CA). Differences were considered statistically significant when p<0.05.

[0225] Example 2. AvFc selectively recognizes various cancer cell lines. Given that high mannose glycans are elevated in various neoplastic cells and tissues5-8,10,13-17, we tested whether AvFc could effectively recognize cancer cells. Flow cytometric analysis showed that lectibodies bound to a range of human cancer cell lines derived from breast, lung, colon, blood, cervical, and prostate tumors at nanomolar concentrations. Nanomolar concentrations (0.1-10 μg / mL) of AvFc exhibited discernible binding to most of the 27 cancer cell lines tested, although with varying degrees of efficiency. MDA-MB-231 breast carcinoma, A549 lung adenocarcinoma, H460 large cell lung carcinoma, HT-29 colon adenocarcinoma, SK-MEL-2 melanoma, and HeLa cervical carcinoma cell lines were among those most predominantly recognized by lectibodies, even at the lowest concentration analyzed (i.e., 0.1 μg / mL or 1.3 nM) (Figure 1A). In contrast, AvFc poorly recognized normal human PBMCs and nontumorigenic cell lines, including MCF10 mammary epithelial cells and BEAS-2B lung epithelial cells, and showed negligible binding to uncommon cancer cell lines, including MDA-MB-468 breast carcinoma, Raji Burkitt's lymphoma, and SU-DHL-4 B cell lymphoma cells (Figures 1A-1D).

[0226] Endoglycosidase H (EndoH), which specifically cleaves high-mannose glycans 23When A549 cells were treated with lectibody 1, AvFc binding to the cell line was almost completely abolished (Fig. 1B-1D). Furthermore, lectibody binding to A549 cells was inhibited by yeast mannan and HIV-1 envelope glycoprotein gp120 in a dose-dependent manner (Fig. 2). These results demonstrate that AvFc interaction with cancer cells is mediated through the high mannose-binding activity of the lectin domain of lectibodies. The mannose-binding lectin concanavalin A (ConA) also strongly recognized A549 cells, and similar to AvFc, this interaction was disrupted by EndoH digestion of cell surface glycans. However, unlike AvFc, ConA exhibited a relatively weak but appreciable interaction with nontumorigenic BEAS-2B cells (Fig. 1C-1D), highlighting distinct glycan recognition mechanisms between lectibodies and canonical legume lectins.

[0227] These analyses suggest that AvFc has a higher selectivity for malignant cells than for non-cancerous or normal healthy cells, since AvFc did not show any significant binding to the non-tumorigenic epithelial cell lines MCF10a and BEAS-2B, human PBMCs (Figures 1A-1D), or primary mesenteric lymph node cells isolated from rhesus monkeys. 19 The specificity of AvFc for high mannose glycans has been previously demonstrated by glycan array analysis using over 600 mammalian N-glycans (see Figure 2B in Hamorsky et al.). 19 , strongly suggesting an abnormal accumulation of high-mannose glycans on the surface of cancer cells. Similar results regarding selectivity for cancer-associated high-mannose glycans were previously reported for the IgM monoclonal antibody TM10, isolated from mice immunized with FasL-expressing B16F10 murine melanoma cells. 37Similar to AvFc, the epitope of TM10 appears to be a cluster of high mannose glycans, particularly Man9, and the antibody recognized human melanoma, prostate, ovarian, and breast cancer cells, with no apparent surface binding to non-transformed cells. However, unlike AvFc, TM10 showed little anticancer activity in vivo or in vitro. Given that antibodies of the IgM isotype typically have poor tissue penetration, short biological half-lives, and lack Fc-mediated effector functions, we attributed the lack of therapeutic efficacy to the specific isotype of the TM10 antibody. 37 Therefore, although selectivity for cancer cells appears to be similar between AvFc and TM10, the presence of the Fc region from IgG1 is a major differentiator for AvFc, a molecular design that offers a significant advantage as a potential anticancer agent.

[0228] These findings add to a growing body of evidence suggesting that a high proportion of high-mannose glycans represents a unique property of cancer cell glycocalyx. Unlike other conventional mannose-binding lectins such as ConA, AvFc preferentially recognizes clusters or groups of high-mannose glycans that contain terminal α1,2-linked mannose residues. 19,22 Such dense mannose-rich structures are rare in the glycocalyx of normal cells, as exemplified by data showing that AvFc has no ability to recognize the non-tumorigenic BEAS-2B bronchial epithelial cell line, whereas ConA showed significant binding (Figures 1C-1D). ConA interacts with both internal and external α-D-mannosyl and α-D-glucosyl residues and has four carbohydrate-binding sites. 38Because of this, it is more "promiscuous" than AvFc and has the ability to recognize a wider range of glycoforms. Furthermore, additional data suggest that AvFc has low affinity for individual glycans and glycoproteins with a small number of glycans, but high affinity for high mannose-rich glycoproteins such as HIV gp120 (not shown). This means that there is a threshold level of high mannose glycans that must be in close proximity for AvFc to bind with any appreciable affinity, and non-cancerous cells simply cannot reach this threshold. The data herein support the view that AvFc is superior to conventional mannose-specific lectins in terms of selectivity for tumor-associated high mannose glycans.

[0229] Example 3. AvFc binds to EGFR and IGF1R and blocks their signaling. We continued our study using two representative NCSLC cell lines, A549 and H460 cells, which showed high AvFc binding by flow cytometry analysis (Figure 1A), with half-maximal effective concentrations of approximately 42 ng / mL for A549 and 30 ng / mL for H460. To identify the molecular targets of AvFc in these cells, we performed a 10-fold increase in AvFc or AvFc binding activity. lec- A pull-down assay was employed using protein A beads conjugated to lectibody 1 (the latter being a variant of lectibody lacking high mannose binding activity). 22 Binding partners were isolated from A549 and H460 cell lysates and identified using mass spectrometry. lec-Unique proteins were revealed in the fractions bound to AvFc that were not isolated by ELISA (Figure 3A). Proteomic analysis of these fractions showed that AvFc recognized a number of molecules found on the cell surface and in the extracellular matrix (Table 2), many of which were shared between the two NSCLC cell lines. These included laminins, integrins, transporters, and growth factor receptors (Table 1). We focused on two major growth factor receptors, namely the epidermal growth factor receptor (EGFR) and the insulin-like growth factor 1 receptor (IGF1R), because they are known to play a pivotal role in cancer progression in NSCLC. 24~26 To validate the proteomic results, we performed co-immunoprecipitation immunoblot analysis, the results of which confirmed the interaction of AvFc with these receptors in both A549 and H460 cells (Figure 3B-D). EGFR and IGF1R are dimerized upon ligand binding and phosphorylated to trigger pro-survival signaling cascades. 27~30 We investigated whether AvFc can suppress the occurrence of signal transduction by these receptors. lec- A549 cells were treated with EGF or IGF1 after preincubation in serum-free medium with either EGFR or the FDA-approved anti-EGFR monoclonal antibody cetuximab (CTX), and the phosphorylation status of their respective receptors was analyzed by immunoblot (Figures 4A-4D). AvFc blocked activation of both EGFR and IGF1R, as demonstrated by the reduction in band intensity of the phosphorylated forms of these receptors (pEGFR and pIGF1R), whereas AvFc lec-Results suggest that AvFc did not block the activation of EGFR. In contrast, CTX blocked the activation of EGFR as effectively as AvFc, whereas an EGFR-specific monoclonal antibody failed to inhibit IGF1R. To test whether AvFc could block these receptors simultaneously, inhibition of major downstream signaling pathways shared between EGFR and IGF1R, specifically the AKT and MAPK pathways involved in cell invasion, proliferation, and drug resistance, was assessed after treatment of A549 cells with a mixture of EGF and IGF1. 31~34 Immunoblot analysis showed that AvFc significantly attenuated the phosphorylation of AKT and MAPK kinase 1 (MAPK1) in the presence of EGF and IGF1, whereas CTX and AvFc significantly attenuated the phosphorylation of AKT and MAPK kinase 1 (MAPK1) in the presence of EGF and IGF1. lec- was shown to be unable to inhibit either (Figures 4E to 4G).

[0230] [Table 1]

[0231] Binding of AvFc to EGFR and IGF1R was confirmed by a non-glycosylated mutant AvFc that did not recognize either receptor. lec- Based on the use of the lectin activity of AvFc (Figures 3A-3G, 4A-4G, and 8A-8D), lec- The lack of high mannose binding activity of was also clearly demonstrated (see Figure 5 of Dent et al. 22 The presence of high mannose glycans on the cell surface of EGFR in human cancers was previously demonstrated by Johns et al. 55 Similarly, high mannose-binding Pseudomonas fluorescent lectin was shown to bind to cell surface EGFR in MKN28 gastric cancer cells by Sato et al. 56 Hasegawa et al. demonstrated that protein D and concanavalin A, known as high mannose binders, recognize EGFR in A549 cells. 57Taken together, the data and previous studies strongly suggest that EGFR and IGF1R in A549 and H460 carry high mannose glycans.

[0232] Considering that changes in N-glycosylation modifications may result in any glycoproteins in cancer cells globally, it is not surprising that proteomic analysis revealed a large number of proteins recognized by AvFc in A549 and H460 cells (Table 1). It is intriguing that EGFR and IGF1R were among the common cell surface glycoproteins targeted by AvFc in these NSCLC cells (Figures 3A-3D). These receptors, which are often overexpressed and strongly associated with NSCLC cancer progression, 24~26 Results suggest that EGFR and IGF1R present dense high-mannose glycans on cancer cells. In fact, they are both highly glycosylated, containing around 13 and 16 N-glycosylation sites, respectively (UniProtKB: P00533 and P08069). Also, because the activated forms of EGFR and IGF1R ectodomains exist as dimers, 39~41 Thus, it is plausible that AvFc exhibits high affinity for these receptors. AvFc was found to have a higher affinity for EGFR dimers than for EGFR monomers (Figure 3F), suggesting that high expression and activation of EGFR on the surface of some cancer cells may increase local high mannose glycan concentrations and promote AvFc binding and antitumor activity. One consequence of AvFc binding to EGFR and IGF1R was the blockade of their activation and subsequent downstream signaling, as demonstrated by data showing that AvFc inhibited phosphorylation of both receptors to the same extent as AKT and MAPK1 upon stimulation with their respective ligands in A549 cells (Figures 4A-4G).

[0233] The cell lines tested were chosen to represent a broad selection of commercially available cancer cell lines, without being based on their EGFR or IGF1R status. Flow cytometric binding studies (Figures 1A-1D) were performed prior to examining potential glycoprotein binding partners on the cell surface (Figures 3A-3D). The primary binding mechanism of AvFc to cancer cells is due to the overabundance of high mannose-type glycans, which are likely present on many, if not all, glycoproteins passing through the endomembrane secretory pathway in cancer cells. Thus, although EGFR and IGF1R appear to contribute significantly to the overall AvFc binding levels in some cell lines (e.g., A549 and H460), it is believed that overall binding to cancer cell lines does not always depend on these receptors. This view is supported by proteomic analyses that showed several other AvFc targets other than EGFR and IGF1R (Tables 1 and 2).

[0234] We analyzed the levels of EGFR and IGF1R in some of the lung cancer cell lines (Figure 3G). A549 and H460 showed the highest levels of EGFR and IGF1R, respectively, but the levels of these receptors were not significantly different in other cell lines, including H1437, which showed the next highest AvFc binding after A549 and H460 (Figure 1A). This suggests that the correlation between EGFR / IGF1R expression levels and AvFc binding appears to be weak, if at all. The data also suggest that other glycoprotein targets (e.g., those identified by proteomic analysis) contributed to the recognition of AvFc by cancer cells. Considering that the binding activity of AvFc does not depend solely on specific receptors, this study highlights the versatility of targeting high mannose glycans as a novel anticancer strategy.

[0235] Example 4. AvFc inhibits migration of A549 and H460 cells. These results suggest that AvFc effectively binds to both EGFR and IGF1R on the cell surface, thereby intercepting their ligands and preventing receptor activation and subsequent AKT and MAPK signaling. Since AKT and MAPK signaling pathways are involved in migration, 35,36 To investigate the effect of AvFc on cell migration using A549 and H460 cells, a transwell culture system was used in which cells were co-incubated with rectibody and then treated with EGF or IGF1 in serum-free medium. These cells were then seeded into transwells and cells in the bottom chamber were quantified after 6 hours. As shown in Figures 5A-5B, AvFc significantly blocked migration of both cell lines, regardless of which growth factor the cells were stimulated with, whereas AvFc significantly blocked migration of both cell lines. lec- In contrast, CTX only effectively inhibited the migration of EGF-treated A549 cells, but not when the cells were treated with IGF1 (Fig. 5A). In addition, CTX failed to show any effect on the migration of H460 cells, even when the cells were stimulated with EGF (Fig. 5B).

[0236] Example 5. AvFc induces ADCC against cancer cells. Next, we investigated the consequences of AvFc binding to cancer cells from a different angle, i.e., whether the Fc region of lectibody can direct antibody-dependent cell-mediated cytotoxicity (ADCC). Since ADCC is mainly mediated by NK cells expressing FcγRIIIa, we assessed the Fc-mediated activity of AvFc against A549 and H460 cells using an in vitro assay based on FcγRIIIa-activated luciferase expression. As shown in Figures 6A-6B, AvFc activated FcγRIIIa in a dose-dependent manner. In contrast, AvFc lec-failed to activate FcγRIIIa at all concentrations tested, demonstrating that activity is dependent on AvFc binding to high mannose glycans on cancer cells. Notably, AvFc showed significantly higher efficacy than CTX against A549 cells at the top three concentrations tested (0.08, 0.40, and 2.00 μM). Moreover, AvFc exhibited notable activity against H460 (up to more than 30-fold above baseline), whereas CTX was ineffective against large cell lung carcinoma cell lines (Figures 6A-6B). To confirm the ability of AvFc to induce Fc-mediated cell killing activity, a canonical ADCC assay was performed using human PBMC effector cells and A549 cells as targets. As shown in Figure 6C, the lectibody exhibited dose-dependent cell lysis activity against A549 cells, although it is noteworthy that the efficacy of the lectibody was nearly twice that of CTX (approximately 80% for AvFc vs. approximately 40% for CTX).

[0237] Example 6. AvFc exhibits anti-tumor effects in murine A549 and H460 xenograft models. Prkdc challenged A549 and H460 xenografts implanted in the posterior left flank scid The antitumor effect of AvFc was evaluated in / SzJ(SCID) mice. Intraperitoneal treatment with 25 mg / kg AvFc or CTX was started 4 days after tumor challenge and continued every 2 days for a total of 6 times. AvFc treatment significantly reduced A549 (Figure 7A) and H460 (Figure 7B) tumor growth compared to vehicle control. In contrast, CTX showed similar efficacy as AvFc against A549 tumors but failed to show any effect on H460 tumor growth. To further evaluate the antitumor effect of AvFc, mice were challenged intravenously with A549-GFP and subsequently treated with the same dosing regimen as in the flank tumor model. AvFc significantly inhibited the growth of A549-GFP cells in the lungs compared to vehicle control, as shown by fluorescence imaging of lungs isolated 18 days after the last dose (Figures 7C-7D). Taken together, these data clearly demonstrated that AvFc has the ability to induce antitumor activity in vivo.

[0238] To further elucidate the potential antitumor mechanism of AvFc action, ADCC activity was measured in both reporter cell-based and human PBMC-based assays (Figures 6A-6C). In both assay formats, AvFc induced strong ADCC responses by effectively activating FcγRIIIa on the surface of engineered Jurkat cells (which express luciferase in response to activation) and PBMCs. In contrast, CTX had no activity against H460 cells and only moderate activity against A549, which was inferior to AvFc, in both reporter cell-based and PBMC-based assays. This was consistent with the results of the 100% ADCC assays in which AvFc was inhibited by Prkdc challenged with A549 and H460 xenografts. scid Consistent with the antitumor effects of AvFc seen in A549 / SzJ(SCID) mice (Figures 7A-7D), AvFc treatment slowed the growth of both xenografts, whereas CTX was only effective against A549. Collectively, these results suggest that the superior antitumor activity of AvFc in these models is due to binding and inhibition of multiple cell surface receptors in addition to more potent ADCC activity.

[0239] Example 7. AvFc preferentially binds to human NSCLC tumor tissue Immunohistochemistry (IHC) was used to examine the binding of AvFc to primary tumors and adjacent tissues isolated from human NSCLC patients. Compared with adjacent tissues, AvFc binding was more evident in NSCLC tumors (Figures 8A-8B), suggesting that lectibody has the ability to distinguish their differential glycosylation patterns. Among matched-pair tissues from 10 NSCLC patients analyzed, 7 showed significantly higher AvFc binding in tumors than in adjacent tissues (Figure 8C). Considering that EGFR was one of the major molecular targets of AvFc in A549 and H460 (Figures 3A-3D), it was hypothesized that the tumor selectivity of AvFc found in lung tissues of NSCLC patients could be partially attributed to the receptor. Therefore, we used co-immunoprecipitation to enrich EGFR from tumor and adjacent tissue lysates from five NSCLC patients, and then coimmunoprecipitation was performed with AvFc, CTX, or AvFc. lec- The lectibody was detected by Western blot using AvFc (samples no. 151, 117, 448, 234, 155). Representative images of tissue samples from patient 117 are shown in FIG. 8D, and the relative band intensities between tumor EGFR and its counterpart from adjacent tissues are shown in FIG. 8E. AvFc reacted with both tumor and adjacent tissue EGFR, but showed a stronger signal in the former (approximately 2-fold on average, FIG. 8E), suggesting that the lectibody had a higher affinity for tumor-derived EGFR. In contrast, CTX showed bands of similar intensity for tumor and adjacent tissue EGFR in all five tissues tested (FIGS. 8D-8E). AvFc lec- failed to probe the receptor from both tumor and adjacent tissues. Collectively, these results suggest that AvFc has preferential binding to NSCLC tumor-derived EGFR over normal lung tissue, but CTX was unable to distinguish between them.

[0240] The ability of AvFc to inhibit both EGFR and IGF1R has important therapeutic implications, since there are currently no FDA-approved anti-cancer drugs that can simultaneously block these receptors. The FDA-approved anti-EGFR antibody therapy CTX, used in this study as a reference, was only able to block EGFR, but not IGF1R, in A549 (Figures 4A-4G and 5A-5B), and, in stark contrast to AvFc, failed to exert any antitumor effect on the H460 cell line, known to be resistant to CTX (Figures 5A-5B, 6A-6B, and 7A-7C). 42 The remarkable in vivo antitumor efficacy observed with AvFc is most likely due to its effective binding to the widespread and highly accumulated high mannose glycans on the surface of cancer cells, and in our ongoing studies, AvFc showed a similar antitumor effect in protecting against B16F10 melanoma lung metastasis, whereas the non-glycobinding mutant AvFc lec- failed to show any effect (Fig. 7E). Despite the fact that colorectal cancer patients show a significant improvement in response rates, overall survival and progression-free survival after CTX treatment in NSCLC patients, CTX response has not been conclusive in clinical trials. 43~45 IGF1R may be involved in resistance mechanisms of CTX and other EGFR-targeting drugs46-49. Because these two receptors share similar downstream signaling pathways (PI3K / AKT / MAPK / NF-κB), IGF1R can bypass EGFR inhibition, whereas their cooperation may promote tumor growth and progression4. 8~50 Overexpression of both EGFR and IGF1R was observed in 24.8% of 125 surgical NSCLC patients, and one study demonstrated that high coexpression of EGFR and IGF1R was a significant prognostic factor for poorer disease-free survival. 50 .

[0241] The selectivity of AvFc was evaluated in 10 pairs of tumor and adjacent lung tissues from NSCLC patients (Figures 8A-8D). Overall, AvFc interacted preferentially with tumor tissues and was capable of distinguishing between tumor and adjacent tissues despite low levels of background AvFc staining in the latter. High mannose expression levels in tissues likely depend on the stage of cancer development. For example, IHC analysis showed that tumors expressed more EGFR and IGF1R than adjacent normal tissues (Figure 9), supporting higher binding of AvFc to tumors. The selective interaction of AvFc with primary NSCLC cells in this analysis demonstrates the utility of AvFc beyond animal models and suggests that it may be usefully targeted to tumors in NSCLC patients.

[0242] Aberrant glycosylation has long been recognized as a hallmark of cancer. Nevertheless, the development of therapeutic agents targeting cancer-associated glycans has been slow. Examples 2-7 show the development of AvFc, a lectibody specific for high-mannose glycans. 19 We demonstrated that AvFc can recognize multiple human cancer cell lines derived from various cancer types. Using two NSCLC cell lines, A549 and H460, we evaluated the therapeutic significance of AvFc interaction with cancer cells, demonstrating that lectibody can block activation of EGFR and IGF1R and cell migration upon stimulation with their respective ligands, induce ADCC activity, and significantly delay xenograft tumor growth in SCID mice. Furthermore, IHC analysis showed that AvFc preferentially binds to primary human NSCLC tumors over isolated adjacent non-tumor lung tissue. To the best of our knowledge, this is the first report demonstrating the antitumor effect of an agent targeting cancer-associated high mannose glycans.

[0243] These findings support the view that high mannose glycans constitute cancer glycobiomarkers. First of all, it remains elusive how or why high mannose glycans become overrepresented on the surface of cancer cells. A few cellular mechanisms have been identified that allow the accumulation of immature glycans in tumors, including stress-independent activation of X-box binding protein 1 (XBP1), misregulation of N-acetylglucosaminyltransferases, and downregulation of α-mannosidase I and mannosyl(α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT1). 11,12,51,52 For example, stress-independent activation of XBP1 was found to increase the levels of high mannose glycans in HEK293 and HeLa cells by affecting mannosidase expression, while reducing GlcNAc sialylation and bibranching. 51 A significant decrease in α-mannosidase I expression was found in cholangiocarcinoma cells and was associated with an increase in mannose-rich glycans and a more advanced metastatic phenotype. 12 The use of kifunensine, a small molecule inhibitor of α-mannosidase I, also increased the high mannose glycan content and produced similar results. 12 Takayama et al. showed that increased high-mannose glycan expression detected in surgical specimens of hepatocellular carcinoma was associated with decreased expression of MGAT1, the major glycosyltransferase that converts high-mannose glycans to complex or hybrid N-glycans. Meanwhile, high-mannose glycans in the helical domain of transferrin receptor protein 1 appear to trigger structural changes that improve noncovalent interaction energies, resulting in enhanced cell migration in metastatic cholangiocarcinoma 12 Recent publications assessing the effects of high mannose glycans on bone marrow-derived mesenchymal stromal cells have provided evidence that these glycans modify the physical and structural properties of the cells themselves, reducing their size and increasing their motility, which may in part explain the greater metastatic potential seen in other cell lines. 53Considering the growing body of evidence showing a close association between high mannose glycan abundance on cancer cells and increased cell migration and metastatic potential, it is of great clinical significance to clarify the causes and processes leading to high mannose overexpression in cancer and to probe the function of the tumor microenvironment and metastasis. In this context, AvFc may provide a valuable tool in probing and monitoring high mannose glycan accumulation on the cell surface to facilitate such studies.

[0244] Taken together, Examples 2-7 demonstrate that the lectibody AvFc, which targets high mannose glycans, can selectively recognize cancer cells and exert antitumor activity, likely through a combination of growth factor receptor inhibition and immune activation via Fc receptors. These findings suggest that the increased abundance of high mannose glycans in cancer, such as those expressed on EGFR and IGF1R, presents a druggable target present in NSCLC, and that AvFc provides a new tool to probe and target tumor-associated high mannose glycan biomarkers.

[0245] Immunogenicity and toxicity of drug candidates are critical parts of drug development. Previous studies have shown that AvFc did not cause any significant toxicity in rats or rhesus monkeys after a single dose or in mice after repeated doses. 19 Additionally, a recent publication also showed that repeated systemic administration of up to 11 doses at 25 mg / kg over a 20-day period did not result in any significant toxicity while protecting against hepatitis C virus challenge in human liver chimeric mice. 22 .

[0246] Regarding immunogenicity, AvFc was produced in a glycoengineered plant host lacking plant-specific N-glycoforms (K1DFX-P2 N. benthamiana), as previously described, thus avoiding potential immunogenicity due to plant-specific glycans. 19,54AvFc induced an anti-drug antibody (ADA) response in mice, which was likely due (at least in part) to the human IgG Fc domain. In vitro ADCC assays showed that pooled mouse sera containing AvFc ADA failed to inhibit (and even slightly increased) the ADCC activity of AvFc against A549 cells, suggesting that ADA may not have a significant effect, if any, on the antitumor activity of lectibodies (Figure 10).

[0247] array SEQ ID NO:1 is the amino acid sequence of a wild-type actinohibin polypeptide. [ka]

[0248] SEQ ID NO:2 is the amino acid sequence of an actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 1. ASGTIRNAETGRLLDSNYDGAVYTLPANGGSYQRWTGPGDGTVRNAETGRLLDSNYDGAVYTLPANGGSYQKWTGPGDGTIQNAETGRLLDSNYDGAVYTLPANGGSYQKWTG (SEQ ID NO: 2)

[0249] SEQ ID NO:3 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as a variant. ASGTIRNAETGRCLDSNYDGAVYTLPCNGGSYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYDGAVYTLPCNGGSYQKWTG (SEQ ID NO: 3)

[0250] SEQ ID NO:4 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 3. [ka]

[0251] SEQ ID NO:5 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 4. [ka]

[0252] SEQ ID NO:6 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 5. ASGTIRNAETGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 6)

[0253] SEQ ID NO:7 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 6. ASGTIRNAETGRCLDSNYDGNVYTLPCNGGSYQRWTGPGDGTVRNAETGRCLDSNYDGNVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYDGNVYTLPCNGGSYQKWTG (SEQ ID NO: 7)

[0254] SEQ ID NO:8 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 7. ASGTIRNAQTGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 8)

[0255] SEQ ID NO:9 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 8 or avalen (an actinohibin variant expressed in Nicotiana). ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 9)

[0256] SEQ ID NO:10 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 9. ASGTIRNAQTGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 10)

[0257] SEQ ID NO:11 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 10. ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAQTGRCLDSNYDGAVYTLPCNGGSYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 11)

[0258] SEQ ID NO:12 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 11. ASGTIRNAQTGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAQTGRLLDSNYNGNVYTLPANGGNYQKWTGPGDGTIQNAQTGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 12)

[0259] SEQ ID NO:13 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 12. ASGTIRNAETGRLLDSNYNGNVYTLPANGGNYQRWTGPGDGTVRNAETGRLLDSNYNGNVYTLPANGGNYQKWTGPGDGTIQNAETGRVLDSNYNGNVYTLPANGGNYQKWTG (SEQ ID NO: 13)

[0260] SEQ ID NO:14 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 13. ASGTIRNAETGRCLDSNYNGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYNGNVYTLPCNGGNYQKWTGPGDGTIQNAETGRCLDSNYNGNVYTLPCNGGNYQKWTG (SEQ ID NO: 14)

[0261] SEQ ID NO:15 is the amino acid sequence of another actinohibin variant polypeptide made in accordance with the presently disclosed subject matter and designated herein as variant 14. ASGTIRNAETGRCLDSNYDGNVYTLPCNGGNYQRWTGPGDGTVRNAETGRCLDSNYDGNVYTLPCNGGNYQKWTGPGDGTIQNAETGRCLDSNYDGNVYTLPCNGGNYQKWTG (SEQ ID NO: 15)

[0262] SEQ ID NO: 16 is an amino acid sequence comprising the actinohibin variant polypeptide of SEQ ID NO: 9 (variant 8) fused via a linker polypeptide to an amino acid sequence comprising the crystallizable fragment (Fc) region of immunoglobulin (Ig)G, referred to herein as AvFc. [ka]

[0263]

Table 2

[0264]

Table 3

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[0266] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0267] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made herein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

1. A composition for reducing activation of a cancer-associated growth factor receptor in a cancer cell, comprising a polypeptide that specifically binds to a high mannose type glycan epitope.

2. a) the polypeptide is in an amount sufficient to reduce activation of the cancer-associated growth factor receptor in the cancer cells by about 10-90%, and / or The composition of claim 1, wherein the cancer-associated growth factor receptor comprises at least one tumor-associated growth factor receptor.

3. 2. The composition of claim 1, wherein the cancer-associated growth factor receptor comprises epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), or a combination thereof.

4. The composition of claim 1 , wherein the cancer cells are cells of a human patient.

5. The composition of claim 1 , wherein the cancer cells are lung cancer cells.

6. The composition of claim 1 , wherein the cancer cells are non-small cell lung cancer (NSCLC) cells.

7. A pharmaceutical composition for use in a method of treating cancer in a subject in need thereof, said composition comprising a polypeptide that specifically binds to a high mannose glycan epitope, said method comprising: a) the cancer is characterized by abnormal cell surface accumulation of high mannose glycans, and / or b) when cancer is mediated by inappropriate activation of growth factor receptors; administering to a subject an effective amount of said pharmaceutical composition.

8. 8. The pharmaceutical composition of claim 7, wherein the growth factor receptor comprises epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), or a combination thereof.

9. The pharmaceutical composition of claim 7, wherein the cancer is lung cancer.

10. The pharmaceutical composition of claim 9, wherein the lung cancer is non-small cell lung cancer (NSCLC).

11. The method of claim 7, wherein the cancer is resistant to treatment with an antibody that specifically binds to a growth factor receptor.

12. The pharmaceutical composition of claim 11 , wherein the subject is resistant to treatment with an antibody that specifically binds to the epidermal growth factor receptor (EGFR).

13. The therapeutically effective amount of the pharmaceutical composition provides, in the subject, a) reducing the activation of growth factor receptors; b) inhibition of cancer cell migration; c) Induction of cytotoxicity, or d) slowing of tumor growth; or a combination thereof, The pharmaceutical composition of claim 7, wherein the

14. The composition of any one of claims 1 to 13, wherein the polypeptide comprises an amino acid sequence that is at least about 90% identical to at least one of the sequences set forth in SEQ ID NOs: 1 to 13.

15. The composition of claim 14 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

9.

16. The composition of any one of claims 1 to 13, wherein the polypeptide further comprises a fragment crystallizable domain of an antibody (Fc), a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv).

17. The composition of claim 16 , wherein the polypeptide further comprises an Fc.

18. The composition of any one of claims 1 to 13, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO:16.

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