Recombinant artificial lectins with oligomannose glycan specificity

Abstract

This disclosure contemplates recombinant oligomannose binding lectins reported herein. In certain embodiments, this disclosure contemplates uses of recombinant oligomannose binding lectins disclosed herein for purification and detection methods. In certain embodiments, this disclosure contemplates uses of recombinant oligomannose binding lectins disclosed herein for treating or preventing viral infections, cancer, or other immunological diseases or conditions. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising recombinant oligomannose binding lectins disclosed herein or nucleic acid or vector encoding the same.

Description

[0001] RECOMBINANT ARTIFICIAL LECTINS WITH OLIGOMANNOSE GLYCAN SPECIFICITY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 538,956 filed September 18, 2023 and U.S. Provisional Application No. 63 / 648,870 filed May 17, 2024. The entirety of each of these applications is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Al 149297 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0007] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 23175PCT.xml. The XML file is 16,674 bytes, was created on September 5, 2024, and is being submitted electronically via the USPTO patent electronic filing system.

[0008] BACKGROUND

[0009] Glycosylation is the process by which specific sugars or groups of sugars (also referred to as carbohydrates or glycans) are linked to lipids or proteins resulting in glycolipids or glycoproteins. Lectins are proteins and glycoproteins that bind specifically to certain carbohydrates. Naturally occurring lectins have a wide range of biological functions such as regulating cell adhesion, immune responses, and protein folding.

[0010] Proteins and antibodies are often modified with complex carbohydrates by biological processes. N-glycans refer to specific glycans added to Asn(N)-X-Ser / Thr sequences within proteins. One prominent example is asparagine (Asn or single letter designation N) found at amino acid position 297 of the constant region (Fc) of human antibodies. Glycosylation of the Fc region is associated with effector functions of the immune system. The structures of N-glycans are typically characterized as being one of three general types: high-mannose type (HM-type), complex type (CT-type), and hybrid type (Hy-type). The N- glycans share a core sugar sequence Man3, i.e., Mana(l-6)-[Mana(l-3)]-ManP(l-4)- GlcNAcP(l-4)-GlcNAcpi-Asn. However, depending on the cellular environment, mature carbohydrate modifications decorate or modify the core sugar sequence which can be classified based on the substitution or modification of Man3, i.e., the a(l,3) and a(l,6) antennae. High- mannose (oligomannose) refers to additional and exclusively mannose substitutions at both the Man3 a(l,3) and a(l,6) antennae, e.g., Man9GlcNAc2-Asn; or exclusively mannose substitutions at the Man3 a(l,6) antenna, e.g., (Man)5(GlcNAc)2-Asn.

[0011] Members of the phylum Bacteroidetes represent the main bacterial phylum of the human large bowel. The genomes of Bacteroidetes contain many different polysaccharide utilization loci (PULs) that encode the apparatus required to utilize as fuel multi-subunit carbohydrates, with each PUL orchestrating the recognition and degradation of a specific glycan into their individual sugar components that otherwise cannot be processed by the host.

[0012] Trastoy et al. report a structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides. Nat Comm, 2020, 11, Article number: 899.

[0013] Trastoy et al. report GH18 endo-P-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans. J Biol Chem, 2021, 297(2): 101011.

[0014] Sato reports high mannose-binding proteins with anti-viral or anti-tumor activity. Yakugaku Zasshi, 2015, 135(11) 1281-1289.

[0015] Mitchell et al. report natural lectins that have antiviral activity. Antiviral Research, 2017, 142, 37-54.

[0016] Park et al. report metastasis of cholangiocarcinoma is promoted by extended high-mannose glycans. PNAS, 2020,117(14):7633-7644.

[0017] Dent et al. report Avaren-Fc lectin-body targeting HCV high-mannose glycans in a human liver chimeric mouse model. Cell Mol Gastroenterol Hepatol, 2021, 11(1): 185-198.

[0018] Lloyd et al. report that lectin H84T BanLec has broad spectrum antiviral activity against human herpesviruses. Sci Rep, 2022, 12, 1641.

[0019] Oh et al. report antitumor activity of a lectin-body targeting cancer-associated high- mannose glycans. Mol Ther, 2022, 30(4): 1523-1535. Boyaval et al. report high-mannose N-glycans as malignant progression markers in early - stage colorectal cancer. Cancers, 2022, 14, 1552.

[0020] See also WO2022 / 187851, WO2018148541, WO2016 / 044397, and WO2015 / 171975.

[0021] References cited herein are not an admission of prior art.

[0022] SUMMARY

[0023] This disclosure reports molecularly engineered artificial lectins with high-specificity and high-affinity binding to high-mannose N-glycans derived from an engineered Endoglycosidase from the human gut symbiont Bacteroides thetaiotaomicron. In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins having mutations at amino acid positions reported herein; also referred to as Bacteroides Artificial Lectins with High-Mannose Specificity (BALMS).

[0024] In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising a D161A and E163A mutation pattern, e.g., comprising the amino acid sequence of FADAW (SEQ ID NO: 2) or DFADAWA (SEQ ID NO: 3) or VDFADAWAE (SEQ ID NO: 4). In certain embodiments, the recombinant oligomannose binding lectin comprises the amino acid sequence of

[0025] MKNLYKILASSIAIFAFCACSQEEMPVNQSDNNQSEVVTRSATGIKNIVYI E VNDINPLNAGSYIMDDAPFFDYVILFAANIRGVGSDATLYNNPNVQYILDHKDTLIKPLQ DKGIKVLLGLLGD H TGLGFANMNSAQTEQFATAVANAVSQYGLDG VDFADAWAE YGRNGYPSGSTGSFSNLITALHNKMPGKTITVFNYGYTSELTGVNSYIDYGIYAFFNSPS WSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGYGAIGYYDLRANNIVSVLNG VAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 1) or variants thereof. In certain embodiments, variants maintain the amino acid glutamic acid (E) at position 52. In certain embodiments, variants maintain the amino acid histidine (H) at position 126.

[0026] In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising a F78A and A79F mutation pattern, e.g., comprising the amino acid sequence of DYVILAFANI (SEQ ID NO: 7), VILAFANIR (SEQ ID NO: 8), DYVILAFANIR (SEQ ID NO: 9), LAFAN (SEQ ID NO: 13), LAFANI (SEQ ID NO: 14), LAFANIR (SEQ ID NO: 15), or ILAFANIR (SEQ ID NO: 16). In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising the amino acid sequence of LAFAN (SEQ ID NO: 13) and FAD AW (SEQ ID NO: 2).

[0027] In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising the D161A and E163A mutation pattern and the F78A and A79F mutation pattern.

[0028] In certain embodiments, the recombinant oligomannose binding lectin comprises the amino acid sequence of

[0029] MKNLYKILASSIAIFAFCACSQEEMPVNQSDNNQSEVVTRSATGIKNIVYI E VN DINPLNAGSYIMDDAPFFDYVIL AF ANIRGVGSDATLYNNPNVQYILDHKDTLIKPLQD KGIKVLLGLLGD H TGLGFANMNSAQTEQFATAVANAVSQYGLDG VDFADAWAE YGRNGYP SGSTGSF SNLITALHNKMPGKTITVFNYGYT SELTGVN S YID YGIYAFFNSP S WSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGYGAIGYYDLRANNIVSVLNG VAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 10) or variants thereof. In certain embodiments, the variants maintain the D161A and E163A mutation pattern and the F78A and A79F mutation pattern. In certain embodiments, variants maintain the amino acid glutamic acid (E) at position 52. In certain embodiments, variants maintain the amino acid alanine (A) at position 78. In certain embodiments, variants maintain the amino acid phenylalanine (F) at position 79. In certain embodiments, variants maintain the amino acid histidine (H) at position 126. In certain embodiments, variants maintain the amino acid alanine (A) at position 161. In certain embodiments, variants maintain the amino acid alanine (A) at position 163. In certain embodiments, the variant has greater than 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10.

[0030] In certain embodiments, this disclosure relates to uses of a recombinant oligomannose binding lectin disclosed herein in the manufacture of therapeutic monoclonal antibodies to selectively purify antibodies with desired N-glycosylation properties. In certain embodiments, this disclosure contemplates uses of recombinant oligomannose binding lectins disclosed herein for purification and detection methods.

[0031] In certain embodiments, this disclosure relates to a recombinant oligomannose binding lectin disclosed herein adsorbed to solid matrix to bind to high-mannose N-glycosylated human IgG antibodies for depletion of said IgG glycoforms from mixtures of high-mannose and complex- type N-glycosylated IgG antibodies common in the manufacture of therapeutic monoclonal antibodies.

[0032] In certain embodiments, this disclosure contemplates uses of recombinant oligomannose binding lectins disclosed herein for treating or preventing viral infections, cancer, or other diseases or conditions.

[0033] In certain embodiments, this disclosure relates to nucleic acids encoding a recombinant oligomannose binding lectin as disclosed herein. In certain embodiments, the nucleic acid encoding a recombinant lectin as disclosed herein is in operable combination with a heterologous promoter.

[0034] In certain embodiments, this disclosure relates to vectors and expression systems comprising a nucleic acid encoding a recombinant oligomannose binding lectin as disclosed herein.

[0035] In certain embodiments, this disclosure relates to solid supports conjugated to or linked to a recombinant lectin disclosed herein and uses in methods of purifying a protein, antibody, or other agent in a sample having N-glycans or oligomannose glycans.

[0036] In certain embodiments, this disclosure contemplates a recombinant oligomannose binding lectin disclosed herein fused to a human IgG Fc. In certain embodiments, this disclosure contemplates uses of a recombinant oligomannose binding lectin disclosed herein fused to a human IgG Fc to selectively target aberrant high-mannose glycosylation.

[0037] In certain embodiments, this disclosure contemplates the manufacture and development of therapeutical tools for treatment of diverse types of cancer and for inhibition of viral infections. In certain embodiments, this disclosure contemplates uses of a recombinant oligomannose binding lectin disclosed herein or recombinant oligomannose binding lectin disclosed herein fused to a human IgG Fc in therapeutic applications, e.g., treating cancer, or treating or preventing viral or other microbial infections.

[0038] In certain embodiments, this disclosure contemplates uses of a recombinant oligomannose binding lectin disclosed herein fused to a human IgG Fc to selectively target aberrant high- mannose glycosylation on tumor cells and direct antibody-mediated effector functions to kill said tumor cells.

[0039] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a recombinant oligomannose binding lectin disclosed herein to a subject in need thereof. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a fusion protein comprising a recombinant oligomannose binding lectin disclosed herein and a human IgG Fc to a subject in need thereof.

[0040] In certain embodiments, the recombinant oligomannose binding lectin or the fusion protein is administered in combination with other pharmaceutical agents, anti-cancer agents, anti-viral agents, immune cells, T cells, NK cells, or a hematopoietic stem cell transplant.

[0041] In certain embodiments, this disclosure relates to chimeric antigen receptors wherein the targeting domain comprises a recombinant oligomannose binding lectin disclosed herein. In certain embodiments, this disclosure contemplates uses of recombinant oligomannose binding lectins disclosed herein as the targeting domain in chimeric antigen receptor (CAR) T cells to selectively bind to aberrant high-mannose glycosylation on tumor cells and direct T cell killing of said tumor cells.

[0042] In certain embodiments, this disclosure relates to an immune cell expressing a chimeric antigen receptor with a targeting domain comprising a recombinant oligomannose binding lectin disclosed herein. In certain embodiments, the immune cell is a T cell or a NK cell.

[0043] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of cells, e.g., immune cells, expressing a chimeric antigen receptor with a targeting domain comprising a recombinant oligomannose binding lectin disclosed herein to a subject in need thereof.

[0044] In certain embodiments, this disclosure relates to fusion proteins comprising a recombinant lectin oligomannose binding disclosed herein and a human IgG Fc.

[0045] In certain embodiments, the recombinant lectin or the recombinant oligomannose binding lectin fused to a human IgGl Fc is used to bind oligomannose / high-mannose N-glycans on viral envelope proteins leading to viral neutralization.

[0046] In certain embodiments, this disclosure relates to methods of treating or preventing a viral infection comprising administering an effective amount of a recombinant oligomannose binding lectin or a human IgGl Fc fusion protein disclosed herein to a subject in need thereof.

[0047] In certain embodiments, this disclosure relates to a recombinant oligomannose binding lectin disclosed herein conjugated to a label or a recombinant oligomannose binding lectin disclosed herein fused to a human IgGl Fc or other immunostimulatory domain conjugated to a label. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0048] Figure 1A illustrates Man9GlcNac2 interactions of Bacteroidetes PUL BT1285 modified with mutation D161A and E163A (BALMS) identified by alanine scanning mutagenesis. Further analysis provides E53 and H126 are key residues involved in oligomannose recognition.

[0049] Figure IB shows data indicating residues E52 and H126 are interacting with specific mannoses from the Man9GlcNac substrate.

[0050] Figure 2A shows a low-resolution structure of a complex of two BALMS and Fc with a High-Mannose Fc substrate (Fc-HM). Analytical SEC and analytic ultracentrifugation of BALMS confirmed the stable complex formation between Fc-IgGl and BALMS at 2 to 1 ratio. By Negative- staining TEM reconstruction of BALMS and Fc was obtained in a low-resolution structure of the BALMS Fc complex.

[0051] Figure 2B shows an AlphaFold™ model of chimeric molecule BALMS-Fc indicates the BALMS is fused to the C-term (fused to CH3 domain) and the Fc region of human immunoglobulin G1 (IgGl).

[0052] Figure 2C illustrates diagram of B ALMS-CAR T cell generation.

[0053] Figure 3A illustrates purification of IgG CT antibodies using BALMS affinity column. A 1 : 1 mixture of IgGs CT and IgG EIM were incubated into Nickel affinity column that previously were loaded with excess of BALMS (15 mg).

[0054] Figure 3B shows LC-MS data of IgGs CT (G0 / G1) and IgG HM such as Man8 / Man8, Man9 / Man8, and Man9 / Man9, of the mixture injected and flow through (Ft) that was recovered and analyzed.

[0055] Figure 3C shows data where Ft was incubated with a high-mannose specific endoglycosidase or complex-type specific endoglycosidase before proceeding with LC-MS analysis of glycoform profiles. A wash was also performed with HBS buffer and an elution with imidazole to release BALMS attached to certain IgGs. LC-MS assays was used to analyze the glycoforms on IgGs released from the column.

[0056] Figure 4A shows a scheme of ribosome display selection. Combinatorial library generated by Error-prone PCR was selected in 6 rounds of ribosome display. In every round of selection, library was pre-selected against negative target CT-IgG to secure high specificity of final candidates. Pre-selected library was then selected against specific target HM-IgG. mRNA of the mRNA-ribosome-protein complex recognizing specific target was isolated and reverse transcribed to DNA. DNA library with decreased complexity was then amplified and selected in next rounds of selection. In case of the final, 6th round, library was cloned into the bacterial vector for protein expression.

[0057] Figure 4B shows data indicating F78A and A79F mutations are desirable for improving binding affinity (KD = 44 pM).

[0058] DETAILED DISCUSSION

[0059] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0060] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0061] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0062] An "embodiment" of this disclosure refers to an example, but not necessarily limited to such example. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0063] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0064] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0065] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods.

[0066] A "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

[0067] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0068] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0069] A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide and retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0070] Modifications and changes can be made in the structure of the peptides of this disclosure and still result in a molecule having similar characteristics as the peptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a peptide that defines the biological functional activity of the peptide, certain ammo acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties. Amino acid substitutions are generally based on the relative similarity of the ammo acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala to Gly or Ser), (Arg to Lys), (Asn to Gin or His), (Asp to Glu, Cys, or Ser), (Gin to Asn), (Glu to Asp), (Gly to Ala), (His to Asn, Gin), (Leu to He or Vai), (Lys to Arg), (Met to Leu, Tyr), (Ser to Thr), (Thr to Ser), (Trp to Tyr), (Tyr to Trp or Phe), and (Vai to He or Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a peptide as set forth above. In certain embodiments, peptide variants are those having greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to the peptide of interest. In general, homologous peptides of the present disclosure are characterized as having one or more amino acid substitutions, deletions, and / or additions.

[0071] "Identity," as known in the art, is a relationship between two or more peptide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between peptides as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. In certain embodiments, sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. The term "sample" is used in its broadest sense, in that it has chemical makeup that is physical for analysis, i.e., analyte. In one sense it can refer to a nasal fluid, saliva, cough droplets, or expelled droplets of saliva into the air, e.g., produced by speaking, or other lung fluid blood. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples include bodily fluids, urine, feces, nasal drip, seminal fluid, hair, skin (dead or epithelial layer of skin), finger or toenail clipping, and blood products such as plasma, serum, and the like. Preferably the sample is from a subject and encompass fluids, solids, tissues, and gases.

[0072] Recombinant oligomannose binding lectins

[0073] This disclosure contemplates recombinant oligomannose binding lectins having mutations at amino acid positions reported herein. In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising a D161 A and E163A mutation pattern, e.g., the amino acid sequence of FAD AW (SEQ ID NO: 2) or DFADAWA (SEQ ID NO: 3) or VDFADAWAE (SEQ ID NO: 4).

[0074] In certain embodiments, the recombinant oligomannose binding lectin comprises the amino acid sequence of

[0075] MKNLYKILASSIAIFAFCACSQEEMPVNQSDNNQSEVVTRSATGIKNIVYI E VNDI NPLNAGSYIMDDAPFFDYVILFAANIRGVGSDATLYNNPNVQYILDHKDTLIKPLQDKGI KVLLGLLGD H TGLGFANMNSAQTEQFATAVANAVSQYGLDG VDFADAWAE YGRNGYPSGSTGSFSNLITALHNKMPGKTITVFNYGYTSELTGVNSYIDYGIYAFFNSPS WSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGYGAIGYYDLRANNIVSVLNG VAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 1) or variants thereof. In certain embodiments, the variant maintains a D161A and E163A mutation pattern. In certain embodiments, the variant maintains amino acid glutamic acid (E) at position 52. In certain embodiments, the variant maintains amino acid histidine (H) at position 126. In certain embodiments, the variant has greater than 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1.

[0076] In certain embodiments, this disclosure relates to nucleic acids encoding a recombinant oligomannose binding lectin as disclosed herein. In certain embodiments, the nucleic acids encoding a recombinant lectin as disclosed herein is in operable combination with a heterologous promoter. In certain embodiments, the nucleic acid comprises the nucleotide sequence of ATGAAAAACTTGTATAAAATTTTAGCGTCATCTATCGCTATATTTGCATTTTG

[0077] CGCATGCTCACAAGAAGAA (SEQ ID NO: 5) and / or

[0078] ATGCCAGTAAACCAATCAGATAATAATCAATCTGAAGTAGTCACCCGTTCGG CCACAGGTATTAAAAACATCGTTTATATTGAAGTAAATGATATCAATCCTCTTAATG CAGGGTCATATATTATGGATGATGCTCCATTTTTCGATTATGTAATTCTGTTCGCTGC CAACATAAGAGGTGTAGGCTCAGACGCTACTTTATATAACAATCCAAATGTACAGTA CATTTTGGACCACAAAGACACTCTGATTAAGCCTCTTCAAGACAAAGGTATCAAAGT TTTACTCGGTCTATTAGGTGACCATACAGGACTCGGATTCGCAAACATGAATTCAGC TCAGACTGAACAGTTTGCTACCGCCGTTGCCAATGCTGTTAGTCAATATGGATTAGA TGGTGTAGACTTTGCGGACGCGTGGGCTGAATACGGTAGAAATGGTTATCCATCTGG AAGTACAGGTTCTTTCTCTAATTTGATTACTGCGCTCCATAACAAAATGCCAGGTAA GACGATTACTGTATTCAATTATGGCTACACAAGTGAGCTTACAGGAGTTAATAGCTA TATTGATTATGGCATTTACGCATTCTTCAACTCCCCATCGTGGAGTACAGGTTTTGGT ATGCCGAATAGTAAATTTGCACCTTATACCATTAATCTGAATAGTGCTCCGTCTGCT GCTAGTGCTCAACTTTATTCAGGACAAGTTGCAAGTAAAGGTTATGGTGCTATCGGT TACTATGACCTTAGAGCCAACAATATCGTAAGCGTATTGAATGGTGTTGCAAAAGGA GCTTTCAAGTCTACTTGTACTTATGACGGTAATTCTTATCCAAAGAATTATTAA (SEQ ID NO: 6) or variants. In certain embodiments, the nucleic acid is DNA, RNA, or combinations thereof. In certain embodiments, any T is individually and independently at any position substituted with U. In certain embodiments, the variants are synonymous or non-synonymous codons. In certain embodiments, this disclosure relates to double stranded or single stranded nucleic acids or nucleobase polymer optionally comprising a label comprising a sequence that hybridizes to or is the reverse complement of SEQ ID NO: 6 or segment thereof. In certain embodiments, the segment comprises 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more nucleotides or nucleobases and / or the segment comprises less than 10, 20, 25, 30, 40, 50, 100, 200, 300, 400, 500, 600, or 700 nucleotides or nucleobases.

[0079] In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising a D161A and E163A mutation pattern, e.g., the amino acid sequence of FADAW (SEQ ID NO: 2) or DFADAWA (SEQ ID NO: 3) or VDFADAWAE (SEQ ID NO: 4). In certain embodiments, this disclosure relates to recombinant oligomannose binding lectins comprising a F78A and A79F mutation pattern, e g., comprising the amino acid sequence of DYVILAFANI (SEQ ID NO: 7), VILAFANIR (SEQ ID NO: 8), or DYVILAFANIR (SEQ ID NO: 9), LAFAN (SEQ ID NO: 13), LAFANI (SEQ ID NO: 14) LAFANIR (SEQ ID NO: 15), or ILAFANIR (SEQ ID NO: 16). In certain embodiments, the recombinant oligomannose binding lectin comprises the amino acid sequence of

[0080] MKNLYKILASSIAIFAFCACSQEEMPVNQSDNNQSEVVTRSATGIKNIVYI E VN DINPLNAGSYIMDDAPFFDYVIL AF ANIRGVGSDATLYNNPNVQYILDHKDTLIKPLQD KGIKVLLGLLGD H TGLGFANMNSAQTEQFATAVANAVSQYGLDG VDFADAWAE YGRNGYPSGSTGSFSNLITALHNKMPGKTITVFNYGYTSELTGVNSYIDYGIYAFFNSPS WSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGYGAIGYYDLRANNIVSVLNG VAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 10) or variants thereof. In certain embodiments, the variant maintains the D161A and E163A mutation pattern and the F78A and A79F mutation pattern. In certain embodiments, variants maintain the amino acid glutamic acid (E) at position 52. In certain embodiments, variants maintain the amino acid alanine (A) at position 78. In certain embodiments, variants maintain the amino acid phenylalanine (F) at position 79. In certain embodiments, variants maintain the amino acid histidine (H) at position 126. In certain embodiments, variants maintain the amino acid alanine (A) at position 161. In certain embodiments, variants maintain the amino acid alanine (A) at position 163. In certain embodiments, the variant has greater than 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10.

[0081] F78A and A79F, L123I A165I G267E mutation pattern

[0082] TGIKNIVYI E VNDINPLNAGSYIMDDAPFFDYVIL AF ANIRGVGSDATLYNN PNVQYILDHKDTLIKPLQDKGIKVLLGL I GD H TGLGFANMNSAQTEQFATAVANAVS QYGLDGVD FADAW I EYGRNGYPSGSTGSFSNLITALHNKMPGKTITVFNYGYTSE LTGVNSYIDYGIYAFFNSPSWSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGY E Al GYYDLRANNIVSVLNGVAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 11) or variants thereof, wherein the N-terminal T is position 43.

[0083] In certain embodiments, the variant maintains the D161 A and E163A mutation pattern and the F78A and A79F mutation pattern. In certain embodiments, variants maintain the D161A and E163A mutation pattern, e g., amino acid sequence FADAW (SEQ ID NO: 2). In certain embodiments, variants maintain the amino acid glutamic acid (E) at position 52. In certain embodiments, variants maintain the amino acid alanine (A) at position 78. In certain embodiments, variants maintain the amino acid phenylalanine (F) at position 79. In certain embodiments, variants maintain the amino acid Histidine (H) at position 126. In certain embodiments, variants maintain the amino acid alanine (A) at position 161. In certain embodiments, variants maintain the amino acid alanine (A) at position 163. In certain embodiments, the variant has greater than 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11.

[0084] A79V N227Y mutation pattern

[0085] TGIKNIVYI E VNDINPLNAGSYIMDDAPFFDYVILF V ANIRGVGSDATLYNNPNV QYILDHKDTLIKPLQDKGIKVLLGLLGD H TGLGFANMNSAQTEQFATAVANAVSQYG LDGVDFADAWAEYGRNGYPSGSTGSFSNLITALHNKMPGKTITVFNYGYTSELTGVNS YIDYGIYAFFNSPSWSTGFGMPNSKFAPYTINLNSAPSAASAQLYSGQVASKGYGAIGYY DLRA Y NIVSVLNGVAKGAFKSTCTYDGNSYPKNY (SEQ ID NO: 12) or variants thereof, wherein the N-terminal T is position 43.

[0086] In certain embodiments, variants maintain the D161A and E163A mutation pattern. In certain embodiments, variants maintain the amino acid glutamic acid (E) at position 52. In certain embodiments, variants maintain the amino acid valine (V) at position 79. In certain embodiments, variants maintain the amino acid histidine (H) at position 126. In certain embodiments, variants maintain the amino acid tyrosine (Y) at position 227. In certain embodiments, the variant has greater than 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12.

[0087] In certain embodiments, this disclosure relates to vectors and expression systems comprising a nucleic acid encoding a recombinant oligomannose binding lectin as disclosed herein.

[0088] A “nucleic acid” refers to a DNA- or RNA-molecule and is used synonymous with polynucleotide. Wherever herein reference is made to a nucleic acid or nucleic acid sequence encoding a particular protein and / or peptide, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences allowing in a suitable host, e.g., a human being, its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the peptide. As used herein, a "nucleotide" is given its ordinary meaning as used in the art, i.e., a molecule comprising a sugar moiety, a phosphate group, and a base (usually nitrogenous). Typically, the nucleotide comprises one or more bases connected to a sugar-phosphate backbone (a base connected only to a sugar moiety, without the phosphate group, is a "nucleoside"). The sugars within the nucleotide can be, for example, ribose sugars (a "ribonucleic acid," or "RNA"), or deoxyribose sugars (a "deoxyribonucleic acid," or "DNA"). In some cases, the polymer can comprise both ribose and deoxyribose sugars. Examples of bases include, but not limited to, the naturally occurring bases (e.g., adenosine or "A," thymidine or "T," guanosine or "G," cytidine or "C," or uridine or "U"). In some cases, the polymer can also comprise nucleoside analogs (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitorpyrrole, 4-methylindole,

[0089] 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine,

[0090] 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine,

[0091] 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, N-methyladenosine, 2-amino-6-chloropurine, 3 -methyl adenosine, 5-propynylcytidine, 5-propynyluridine, 5 -bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,

[0092] 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, etc., chemically or biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'- fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides arabinose, hexose, etc.), modified phosphate moieties (e g., phosphorothioates or 5’-N- phosphoramidite linkages), and / or other naturally and non- naturally occurring bases substitutable into the polymer, including substituted and unsubstituted aromatic moieties. A nucleic acid sequence may be composed of DNA nucleotides, RNA nucleotides or a combination of both types.

[0093] "Amino acid sequence" is defined as a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified, or composed of synthetic amino acids. The terms "protein" and "peptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. As used herein, where "amino acid sequence" is recited herein to refer to an amino acid sequence of a protein molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. Furthermore, unless the context demands otherwise, the term "peptide" and "polypeptide" and "protein" are used interchangeably to refer to amino acids in which the amino acid residues are linked by covalent peptide bonds or alternatively (where post-translational processing has removed an internal segment) by covalent disulfide bonds, etc. The ammo acid chains can be of any length and comprise at least three amino acids, they can include domains of proteins or full-length proteins. Unless otherwise stated the terms peptide, polypeptide, and protein also encompass various modified forms thereof, including but not limited to glycosylated forms, phosphorylated forms, etc.

[0094] The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of’ in reference to a peptide having an amino acid sequence refers to a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the C- terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the N-terminus may be connected to additional amino acids, e g., as part of a larger peptide.

[0095] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0096] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state. The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0097] Protein "expression systems" refer to in vivo (e.g., host cell) and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfecting with a DNA or mRNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Proteins may be recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids, and nucleotides. In the presence of an expression vector, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): el41, both hereby incorporated by reference in their entirety.

[0098] A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-51phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5 -fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p- galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP- glucose:galactose-l -phosphate uridyltransferasel (galT), feedback-insensitive a subunit of anthranilate synthase (0ASA1D), 2-deoxyglucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).

[0099] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as F18,35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0100] In certain embodiments, the disclosure relates to recombinant polypeptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

[0101] In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a polypeptide disclosed herein or chimeric protein thereof.

[0102] In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

[0103] In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0104] The term "fusion" when used in reference to a polypeptide refers to the expression product of two or more coding sequences obtained from different sources such that they do not exist together in a natural environment, that have been cloned together and that, after translation, act as a single polypeptide sequence. Fusion polypeptides are also referred to as "hybrid" polypeptides. The coding sequences include those obtained from the same or from different species of organisms.

[0105] As used herein, the term “conjugated” refers to linking molecular entities through covalent bonds, or by other specific binding interactions, such as due to hydrogen bonding or other van der Walls forces. The force to break a covalent bond is high, e.g., about 1500 pN for a carbon-to- carbon bond. The force to break a combination of strong protein interactions is typically a magnitude less, e g., biotin to streptavidin is about 150 pN. Thus, a skilled artisan would understand that conjugation must be strong enough to restrict the breaking of bonds in order to implement the intended results. In certain embodiments, the term conjugated is intended to include linking molecular entities that do not break unless exposed to a force of about greater than about 5, 10, 25, 50, 75, 100, 125, or 150 pN depending on the context.

[0106] A "linking group" refers to any variety of molecular arrangements that can be used to bridge or conjugate molecular moieties together. An example formula may be -Rn- wherein R is selected individually and independently at each occurrence as: -CRnRn-, -CHRn-, -CH-, -C-, -CH2-, -C(OH)Rn, -C(0H)(0H)-, -C(0H)H, -C(Hal)Rn-, -C(Hal)(Hal)-, -C(Hal)H-, -C(N3)Rn-, -C(CN)Rn-, -C(CN)(CN)-, -C(CN)H-, -C(N3)(N3)-, -C(N3)H-, -O-, -S-, -N-, -NH-, -NRn-, -(C=O)-, -(C=NH)-, -(C=S)-, -(C=CH2)-, which may contain single, double, or triple bonds individually and independently between the R groups. If an R is branched with an Rn it may be terminated with a group such as -CH3, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N3, -CN, or -Hal, or two branched Rs may form an aromatic or non-aromatic cyclic structure. It is contemplated that in certain instances, the total Rs or “n” may be less than 100 or 50 or 25 or 10. Examples of linking groups include bridging alkyl groups, alkoxyalkyl, polyethylene glycols, amides, esters, and aromatic groups.

[0107] As used herein, the term “surface” refers to the outside part of an object. The area is typically of greater than about one hundred square nanometers, one square micrometer, or more than one square millimeter. Examples of contemplated surfaces are on a particle, bead, wafer, array, well, microscope slide, transparent or opaque glass, polymer, or metal, or the bottom of a zero-mode waveguide. A “zero-mode waveguide (ZMW)” refers to a confined structure or chamber located in an opening, e.g., hole, of a metal film deposited on a transparent substrate. See Levene et al., Science, 2003, 299:682-686. Pharmaceutical Compositions

[0108] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising a recombinant oligomannose binding lectin, nucleic acid, or vector as reported herein. The pharmaceutical compositions provided herein may generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.

[0109] In certain embodiments, the nucleic acid or recombinant vector encodes a recombinant immunoglobulin endoglycosidase disclosed herein having at least one open reading frame that can be translated by a cell or an organism provided with the nucleic acid, DNA, RNA, or mRNA. If more than one protein is translated, the proteins can be expressed in one vector / nucleic acid or in multiple (a plurality of) separate nucleic acids / vectors wherein the product of translation is a recombinant oligomannose binding lectin disclosed herein. The product may also be a fusion protein composed of more than one recombinant oligomannose binding lectin disclosed herein, e.g., a fusion protein that has two or more recombinant oligomannose binding lectins, wherein recombinant oligomannose binding lectins are optionally linked by self-cleaving linker sequences.

[0110] In certain embodiments, nucleic acid, DNA, RNA, or mRNA may be designed to have two (bicistronic) or more (multicistronic) open reading frames (ORF). An open reading frame in this context is a sequence including a start codon that can be used as a location to start translation of the encoded nucleic acid into a recombinant immunoglobulin endoglycosidase. Translation of such nucleic acid(s) yields two (bicistronic) or more (multicistronic) distinct translation products / proteins (provided the ORFs are not identical). For expression in eukaryotes such nucleic acids may comprise an internal ribosomal entry site (IRES) sequence which allows for expression of two or more proteins on a single nucleic acid molecule.

[0111] In certain embodiments, the recombinant oligomannose binding lectin, nucleic acid, or recombinant vector may be administered naked without being associated with any further vehicle. In certain embodiments, the recombinant oligomannose binding lectin, nucleic acid, or recombinant vector may be administered in a pharmaceutical composition having a pharmaceutically acceptable excipient selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0112] In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0113] In certain embodiment, the pharmaceutically acceptable excipient is a cationic or polycationic compound and / or with a polymeric carrier. In certain embodiments, the recombinant oligomannose binding lectin, nucleic acid, or recombinant vector are in a pharmaceutical composition associated with or complexed with a cationic or polycationic compound or a polymeric carrier. In certain embodiments, the cationic or polycationic compound is protamine, spermine, spermidine, poly-L-lysine (PLL), poly-histidine, or poly-arginine, cationic polysaccharides, such as chitosan, polybrene, cationic polymers, polyethyleneimine (PEI), homo- and co-polymers of lactic acid and glycolic acid, or polymethylmethacrylate.

[0114] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, albumin, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, citric acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, hydrophilic polymers such as polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylene polyoxypropylene block polymers, polyethylene glycol and antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine. In advantageous embodiments, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol, or glycine.

[0115] The disclosure also encompasses products obtainable by further processing of a liquid formulation, such as a frozen, lyophilized or spray-dried product. Upon reconstitution, these solid products can become liquid formulations as described herein (but are not limited thereto). In its broadest sense, therefore, the term "formulation" encompasses both liquid and solid formulations. However, solid formulations are understood as derivable from the liquid formulations (e.g. by freezing, freeze-drying or spray-drying), and hence have various characteristics that are defined by the features specified for liquid formulations herein.

[0116] In certain embodiments, the formulations are isotonic in relation to human blood. Isotonic solutions possess the same osmotic pressure as blood plasma, and so can be intravenously infused into a subject without changing the osmotic pressure of the blood plasma.

[0117] In certain embodiments, the pharmaceutical composition is administered either systemically or locally, i.e., parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other path of administration. The mode of administration, the dose and the number of administrations can be optimized.

[0118] In certain embodiments, this disclosure relates to kits containing a recombinant oligomannose binding lectin and materials useful for the treatment, or prevention of diseases or conditions. In certain embodiments, the kit comprises a container, a product label and a package insert. Suitable containers include, for example, bottles, vials, syringes, and boxes containing the same. The containers may be of a variety of materials, e.g., glass, plastic, or cardboard. The container holds the composition which is effective in treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Typically, at least one active agent in the kit is a recombinant oligomannose binding lectin, nucleic acid, or recombinant vector as disclosed herein. The product label on, or associated with, the container indicates that the composition is used for treating the condition of choice. In certain embodiments, the kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as a phosphate buffer saline or a citrate buffered saline. It may further include other materials desirable from a user or commercial standpoint, including other buffers, diluents, filters, needles, and syringes. In certain embodiments, a dosage unit form can be, e.g., in the format of a prefdled syringe, an ampoule, cartridge or a vial.

[0119] In certain embodiments, this disclosure relates to kits or articles of manufacture, comprising a recombinant oligomannose binding lectin, nucleic acid, or recombinant vector as disclosed herein and instructions for use by, e.g., the subject or a healthcare professional. The kits or articles of manufacture may include a container, vial, or a syringe containing the formulation as described herein. Preferably, the container, vial, or syringe is composed of glass, plastic, or a polymeric material. The syringe, ampoule, cartridge, or vial can be manufactured of any suitable material, such as glass or plastic and may include rubber materials, such as rubber stoppers for vials and rubber plungers and rubber seals for syringes and cartridges.

[0120] An artificial lectin with high-affinity and high-specificity for high-mannose N-glycans derived from an endoglycosidase of the gut symbiont Bacteroides thetaiotaomicron (B. thetaiotaomicron)

[0121] Members of the phylum Bacteroidetes represent the main bacterial phylum of the human large bowel. The genomes of Bacteroidetes contain many different polysaccharide utilization loci (PULs) that encode the apparatus required to utilize as fuel multi-subunit carbohydrates, with each PUL orchestrating the recognition and degradation of a specific glycan into their individual sugar components that otherwise cannot be processed by the host. The gene products of PULs harbor enzymes that are contemplated to target specific glycans. B. thetaiotaomicron species encodes two high-mannose mammalian N-glycan (HMNG) depolymerizing system (in PUL 16, BT1285).

[0122] High-mannose-specific enzymes are secreted to initiate the high-mannose N-glycan degradation pathway and hydrolyze the oligosaccharide from its polypeptide chain on the cell surface. BT1285 was identified as one of the two endo-P-N-acetylglucosaminidases (ENGases) that specifically catalyzes the hydrolysis of the pi— 4 linkage between the first two GlcNAc residues of high-mannose N-glycans but is not able to hydrolyze complex-type (CT-type) N- glycans.

[0123] In order to engineer a Bacteroides Artificial Lectin with High-Mannose Specificity (BALMS) from an active ENGase enzyme, a double mutant inactive version of ENGase BT1285 was constructed, in which the acid / base residues that participate in the hydrolysis of the glycosidic bond were both mutated to alanine (BT1285 D161A-E163A). To determine the binding affinities of BT1285 to high-mannose N-glycans, surface plasma resonance (SPR) assays were performed using CM5 and Protein A sensor chips. Dissociation constants (KDs) were calculated for each interaction when possible.

[0124] Table 1 shows binding affinities and thermostability of BALMS - BT1285 D161A-E163A to different glycoproteins

[0125] As shown in table 1, the data confirms the specificity of BT1285 D161A-E163A to high- mannose N-glycan substrates, whereas it was unable to bind to complex-type N-glycans on IgGl . Additionally, these assays showed thatBT1285 D161A-E163A binds to oligomannose glycans on RNAseB (i.e., Man5 and Man6) and high-mannose N-glycosylated IgGl (i.e., Man8 and Man9), showing a binding affinity to high-mannose N-glycans in the low nanomolar range (4 nM to 8 nM). In addition, a 5-fold higher affinity of BALMS was observed for IgGl-Man9 than for IgGl- Man5, suggesting that BT1285 D161A-E163A engages at least some of the additional four mannose residues found in Man9. Importantly, the slow dissociation rate (koff = 4.378E-4 1 / s) and affinity (KD = 4nM to 8 nM) of BT1285 D161A-E163A against all high-mannose N-glycans are comparable to the binding of certain lectins but have not been described for any ENGase or glycosyl hydrolase. Accordingly, BT1285 D161A-E163A is believed to be the first BALMS (Bacteroides Artificial Lectin with High-Mannose Specificity) as an oligomannose N-glycan binding molecule.

[0126] Using differential scanning fluorimetry (DSF), the thermostability of BALMS were analyzed at different pHs. BALMS were highly thermostable, showing melting temperatures between 56 and 63 degrees Centigrade, in a broad range of pHs (from 2 to 9). Notably, the highest Tms were registered at physiological conditions. Alanine scanning mutagenesis

[0127] Mutagenesis of residues (E52A, N54A, D55A, N81A, N94A, H126A, E166A, R169A), which decorate the P-barrel core of the enzyme and contact the Man9GlcNAc2 N-linked glycan substrate, was performed. The ability of the BT1285 to process the N-linked HM glycans on two substrates, RNaseB and HM-IgG was determined. Single point mutations E52A and H126A substantially reduced the hydrolytic activities of these mutant enzymes, relative to wild type, for RNaseB and IgG substrates at 37 C and pH 7.4 (Figure IB). Completely abolishing hydrolytic activity of BT1285 was achieved by making a double mutant in residues involved in the substrate recognition (E52A-H126A), indicating that these two residues are essential for the high-mannose substrate recognition.

[0128] Evaluation of the crystal structure indicated that the 03 of Man (-4) makes hydrogen bond with Hl 26, and the 02 of the central Man (-2) and the 06 atom of Man (-6) make hydrogen bonds with E52 (Figure 1A). Collectively, the mutational analysis of the BT1285 residues that contact the high-mannose N-glycan indicated that the interactions with the mannose from the core of N- glycans, Man (-2) and antenna 1 were important for glycan recognition while those with antenna 2 were not relevant for substate recognition.

[0129] SPR experiments and analysis were performed to determine how H126A mutation affects binding affinity of BALMS to HM-glycans. BT1285, H126A showed lower affinity (one thousand times lower: from low nM range to a few uM) than BALMS to RNAseB, IgG-Man9 and IgGMan5. These results confirmed the importance of Hisl26 for binding to the oligomannosidic substrate.

[0130] Considering the high affinity between BALMS and high mannose linked IgGl (HM-IgGl), experiments indicating interactions through different biochemical and biophysical techniques were performed such as size exclusion chromatography (SEC), SEC -multi -wavelength light scattering (MALS), SEC-small angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), Negative-staining transmission electron microscopy (TEM) and cryo-EM. Analytical SEC was performed using Fc or IgG-HM or IgG-CT incubated with BT1285(D161A-E163A)). The purification of the BT1285(D161A-E163A)-substrate complex by size exclusion chromatography (SEC) was homogeneous (i.e., one defined peak of the complex) with Fc or IgG-HM but there was no complex formation with IgG-CT. SDS-PAGE analysis of the fractions obtained indicated the formation of a complex of the BALMS-Fc as a 2: 1 complex (around 110 kDa), which was confirmed by analytical ultracentrifugation (AUC), SEC-MALS, SEC-SAXS, negative-staining TEM and cryo-EM.

[0131] Crystallographic analysis and alanine scanning mutagenesis reveal the key residues involved in high-mannose N-glycan recognition of BALMS.

[0132] Structural basis by which BALMS specifically recognize high-mannose N-glycans were established at the molecular level by solving X-ray crystals structures of BT1285 D161A-E163A in complex with its substrate, Man9GlcNAc2 at 1.9 A resolution. The HM hydrolyzing enzymes preferentially recognize the a(l,6) antenna lb and 1 a of high-mannose N-glycans but not the a(l,3) antenna 2. Molecular docking calculations of a CT-type N-glycan into BT1285 revealed that the a(l,6) antenna would make clashes with two different loops and cannot be accommodated into the binding grooves due to steric hindrance.

[0133] Use of BALMS for affinity purification of HM-IgG from CT-IgG to ensure high quality production of mAbs

[0134] In certain embodiments, this disclosure relates to methods of purifying a protein or other agent in a sample having N-glycans or oligomannose glycans comprising contacting a solid support comprising a recombinant oligomannose binding lectin disclosed herein with a sample having N-glycans or oligomannose glycosylated protein or other agent providing oligomannose glycosylated protein or other agent linked to the solid support. In certain embodiments, methods further comprise separating the sample from the solid support providing purified oligomannose glycosylated proteins or other agents linked to the solid support and optionally purified non- oligomannose glycosylated proteins or other agent in the separated sample. In certain embodiments, methods further comprise detaching and isolating the purified oligomannose glycosylated protein or other agent linked to the solid support. In certain embodiments, methods further comprise detecting, measuring, or quantifying oligomannose glycosylated protein or other agent or non-oligomannose glycosylated protein or other agent.

[0135] In certain embodiments, this disclosure relates to methods of purifying antibodies in a sample having oligomannose glycans comprising contacting a solid support comprising a recombinant oligomannose binding lectin disclosed herein with a sample of oligomannose glycosylated antibodies providing oligomannose glycosylated antibodies linked to the solid support. In certain embodiments, methods further comprise separating the sample from the solid support providing purified oligomannose glycosylated antibodies linked to the solid support and purified non-oligomannose glycosylated antibodies in the separated sample. In certain embodiments, methods further comprise detaching and isolating the purified oligomannose glycosylated antibodies linked to the solid support. In certain embodiments, methods further comprise detecting, measuring, or quantifying oligomannose glycosylated antibodies or non- oligomannose glycosylated antibodies.

[0136] Experiments to test purification of IgG CT antibodies were performed using a BALMS affinity column (Figure 3A). A nickel affinity column was loaded with an excess of BALMS. After exhaustive washes with HBS buffer, a 1 : 1 molar ratio mixture of IgGs CT and IgG HM was injected into that column. Then, the Flow through (Ft) was recovered for LC-MS analysis, confirming the presence of CT N-glycans on IgGs in this fraction. Then, the Ft was incubated with an endoglycosidase High-Mannose specific or Complex-type specific endoglycosidases before proceeding with LC-MS analysis of glycoform profiles, revealing that effectively CT-IgGs were purified from a mixture of IgGs. HM-IgG species were not detected. Subsequently, mainly CT- IgGs were removed from the column after wash with HBS buffer. HM-IgG were finally removed from the column together with BALMS after stringent elution with imidazole containing buffer (Figure 3C).

[0137] BALMS are useful for high mannose lectin affinity chromatography to separate / select complex-type N-glycan containing antibodies from mixtures of antibodies or other glycoproteins containing high-mannose and hybrid-type N-glycans during the therapeutic mAb manufacturing process. BALMS provide an affinity purification method to substantially remove high-mannose and hybrid-type N-glycosylated mAbs from complex mixtures that could be performed in the manufacturing process for therapeutic mAbs. Separation of high mannose and hybrid-type glycans from complex-type glycans containing antibodies is desirable to ensure high quality and higher glycan homogeneity in mAb production.

[0138] A BALMS-Sepharose column is useful for efficient separation methods. Accordingly, contemplated are columns prepacked with BALMS coupled to Sepharose™, spherical agarose size exclusion chromatography base matrix (silica particles or agarose [D-galactose-3, 6-Anhydro-L- galactose) particles e.g., in the size range of about 2 pm to 12 pm in diameter] and used for coupling affinity ligands to the matrix, or a monolithic BALMS affinity column. High quality purified BALMS are covalently attached to the monolith column through the primary amine groups of BALMS (containing Lys residues) using an NHS ester-activated crosslinker. The glycan binding site is protected during the conjugation process, so the BALMS activity is well maintained. Polymeric monolithic BALMS affinity columns are contemplated using a syringe (within a syringe), a peristaltic pump, or a liquid chromatography system for convenient separation of high mannose containing glycoproteins and glycolipids. Additionally, polyacrylic acid (PAA) brushes functionalized FeiCL magnetic nanoparticles for conjugation with Ni2+-NTA (N, N-Bis(carboxymethyl)-L-lysine) are contemplated to enhance the correct oriented binding of His-tagged BALMS.

[0139] In certain embodiments, this disclosure relates to solid supports conjugated to or linked to a recombinant oligomannose binding lectin disclosed herein. In certain embodiments, this disclosure contemplates recombinant oligomannose binding lectins disclosed herein adsorbed to solid matrix e.g., Sepharose™ (a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed) to bind to high-mannose N-glycosylated human IgG antibodies for depletion of said IgG glycoforms from mixtures of high-mannose and complex-type N- glycosylated IgG antibodies typically found in the manufacture of therapeutic monoclonal antibodies.

[0140] In certain embodiments, this disclosure relates to methods of purifying a protein, antibody, or other agent in a sample having N-glycans or oligomannose glycans comprising contacting a solid support comprising a recombinant oligomannose binding lectin disclosed herein with a sample having N-glycans or oligomannose glycosylated protein, antibody, or other agent providing oligomannose glycosylated protein or other agent linked to the solid support.

[0141] In certain embodiments, methods further comprise separating the sample from the solid support providing purified oligomannose glycosylated proteins or other agents linked to the solid support and optionally purified non-oligomannose glycosylated protein, antibody, or other agent in the separated sample. In certain embodiments, methods further comprise detaching and isolating the purified oligomannose glycosylated protein, antibody, or other agent linked to the solid support. In certain embodiments, methods further comprise detecting, measuring, or quantifying oligomannose glycosylated protein, antibody, or other agent or non-oligomannose glycosylated protein, antibody, or other agent. Use of BALMS for treating cancer

[0142] Correlations between high-mannose N-glycan levels on tumor cells with cancer progression and metastasis have been reported. Collectively, this aberrant protein N-glycosylation constitutes a hallmark of cancer and the particular increase of high-mannose N-glycans on malignant cells provides for a unique glycan-specific biomarker for drug development. There are currently very few agents that can distinguish tumor-associated high-mannose N-glycans from other forms present on the surface of a normal cell.

[0143] In certain embodiments, this disclosure contemplates using a recombinant oligomannose binding lectin herein conjugated to a label. In certain embodiments, this disclosure relates to methods of using a labeled recombinant oligomannose binding lectin disclosed herein for detecting cancerous biomolecules, viruses, cells, or tissues comprising contacting a labeled recombinant oligomannose binding lectin disclosed herein and a sample containing or suspected of containing a cancerous cell, virus, or tissue associated with high-mannose expression providing a labeled biomolecule cell, virus, or tissue. In certain embodiments, methods further comprise detecting, measuring, or quantifying the labeled biomolecule, cell, virus, or tissue.

[0144] Abnormal glycan signatures are present on malignant cells as well as on the adjacent supporting tumor-associated stroma. Hence, targeting high-mannose N-glycans are contemplated to both directly destroy malignant cells and disrupt the protective tumor microenvironment. Thus, oligomannose specific lectins reported herein are useful for targeting the tumor glycome.

[0145] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a recombinant oligomannose binding lectin disclosed herein to a subject in need thereof. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a fusion protein comprising a recombinant oligomannose binding lectin disclosed herein and a human IgG Fc to a subject in need thereof.

[0146] "Cancer" refers to any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and can have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.

[0147] The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and mast cells; the lymphoid cell line produces B, T, NK, and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0148] In certain embodiments, the cancer has cells or tissues that express high mannose glycans such as colorectal cancer, hepatocellular carcinoma, metastatic cholangiocarcinoma, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, pancreatic cancer, ovarian cancer, prostate cancer, skin cancer, and skin basal cell carcinoma and squamous cell carcinoma.

[0149] Also contemplated are malignancies located in the blood, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, uterus, and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS- related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0150] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a recombinant oligomannose binding lectin disclosed herein in combination with another anti-cancer agent to a subj ect in need thereof. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a fusion protein comprising a recombinant oligomannose binding lectin disclosed herein and a human IgG Fc in combination with another anti-cancer agent to a subject in need thereof. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a CAR expressing cell with a targeting domain comprising a recombinant oligomannose binding lectin disclosed herein in combination with another anti-cancer agent to a subject in need thereof.

[0151] A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfdzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, fdgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5- fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0152] In certain embodiments, the oligomannose binding lectin disclosed herein is conjugated to a therapeutic agent, anti-cancer agent, or cytotoxic agent for uses in treating disease or conditions such as cancer. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various antitumor or anticancer agents, or growth inhibiting agent. A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or in vivo. Examples of growth inhibitory agents include agents that block cell cycle progression, such as agents that induce G1 arrest and M-phase arrest. Such inhibitory agents include, for example, the vinca alkaloids (vincristine, vinorelbine and vinblastine), paclitaxel, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin, tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and cytarabine.

[0153] Chimeric antigen receptor (CAR) T cell therapy has been employed for the treatment of hematologic malignancies but has not been as effective toward solid tumors, due to the scarcity of tumor specific antigens and the complex tumor microenvironment. Cell therapies for solid tumors are thwarted by the hostile tumor microenvironment and by heterogeneous expression of tumor target antigens. Thus, in certain embodiments, it is contemplated that recombinant oligomannose binding lectins reported herein are useful to overcome antigenic heterogeneity and stromal barriers present in solid tumors particularly in the context of CAR T cell therapies that target sugar residues that are a hallmark of both malignant and associated stromal cells. In certain embodiments, this disclosure contemplates the incorporation of gly can-targeting with recombinant oligomannose lectins disclosed herein attached to a chimeric antigen receptor that recognizes high-mannose N- glycan within T cells, thereby extending their activity to include both malignant cells and their supporting stromal cells, disrupting the tumor microenvironment that otherwise diminishes the activity of cellular therapies against solid tumors.

[0154] Increased abundance of high-mannose N-glycans is observed in colorectal tumor tissues, hepatocellular carcinoma, metastatic cholangiocarcinoma, lung adenocarcinoma, pancreatic cancer, ovarian cancer, breast cancer, prostate cancer and skin basal cell carcinoma and squamous cell carcinoma.

[0155] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a CAR cell expressing a targeting domain of a recombinant oligomannose binding lectin disclosed herein to a subject in need thereof. In certain embodiments, the caner is colorectal cancer, hepatocellular carcinoma, metastatic cholangiocarcinoma, lung adenocarcinoma, pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, and skin basal cell carcinoma and squamous cell carcinoma.

[0156] In certain embodiments, this disclosure contemplates uses of a recombinant lectin disclosed herein fused to the human IgGl Fc region to selectively target aberrant high-mannose glycosylation on tumor cells and direct antibody-mediated effector functions to kill said tumor cells. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering to a subject in need thereof an effective amount of a fusion protein comprising a recombinant lectin disclosed herein and a human IgG Fc.

[0157] In certain embodiments, the recombinant lectin or the fusion protein is administered in combination with immune cells, T cells, NK cells, or a hematopoietic stem cell transplant.

[0158] In certain embodiments, this disclosure contemplates uses of recombinant lectin sequences disclosed herein as the targeting domain in CAR T cells to selectively bind to aberrant high- mannose glycosylation on tumor cells and direct T cell killing of said tumor cells.

[0159] In certain embodiments, this disclosure relates to chimeric antigen receptors wherein the targeting domain comprises a recombinant oligomannose binding lectin sequence disclosed herein.

[0160] In certain embodiments, this disclosure relates to an immune cell expressing a chimeric antigen receptor with a targeting domain comprising a recombinant oligomannose binding lectin disclosed herein. In certain embodiments, the immune cell is a T cell or a NK cell.

[0161] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of an immune cell expressing a chimeric antigen receptor with a targeting domain comprising a recombinant oligomannose binding lectin disclosed herein to a subject in need thereof.

[0162] In certain embodiments, this disclosure relates to fusion proteins comprising a recombinant oligomannose binding lectin disclosed herein and a human IgG Fc. Amino acids 1 to 350 are approximately an extracellular domain and amino acids 351 to 371 are approximately a transmembrane domain. Human IgGl Fc is commercially available as amino acids Glu 99 to Lys 330 of Accession # P01857-1. The fragment crystallizable region (Fc region) is the tail region of an antibody. Segments of the Fc region interact with receptors referred to as “Fc receptors” (often cell surface receptors). The Fc region is involved in activating the immune system by interactions with Fc receptors. As used herein, a "chimeric antigen receptor" or "CAR" refers to a protein receptor, which introduces an antigen specificity, via an antigen binding domain, onto cells (immune cells) to which it is expressed (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the T cell activity (e.g. lytic capacity and self-renewal) of T cells. A CAR typically includes an extracellular antigen-binding domain (ectodomain), a transmembrane domain and an intracellular signaling domain. The intracellular signaling domain generally contains at least one immunoreceptor tyrosine-based activation motif (ITAM) signaling domain, e.g., derived from CD3zeta, and optionally at least one costimulatory signaling domain, e.g., derived from CD28 or 4-1BB.

[0163] In order to improve the ability of immune cells to kill cancerous cells, T cells can be isolated from the blood of a patient and genetically altered to express chimeric antigen receptors (CARs) to specifically target proteins expressed on the surface of cancerous cells or other targets and stimulate an immune response. When put back into the patient, the cells attack the cancerous cells. Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177): 177ra38.

[0164] T cells and other immune cells can be purified and isolated from blood or bone marrow. For example, T cells can be collected via apheresis, a process that withdraws blood from the body and removes one or more blood components (such as plasma, platelets, or other white blood cells). The T cells are exposed to a recombinant vector, such as a lentiviral vector, that infects the cells in a way that a recombinant chimeric antigen receptor (CAR) protein is produced and presented in the cell membrane.

[0165] Before and / or after infecting the isolated cells with the recombinant vector, the cells may be induced to replicate. The genetically modified T cells may be expanded by growing cells in the laboratory until there are sufficient number of them. Optionally, these CAR T cells are frozen. The modified cells are then administered back to the patient. Various T cell subsets, as well as T cell progenitors and other immune cells such as natural killer (NK) cells, macrophages, or monocytes can also be targeted with a CAR.

[0166] The targeting sequence is typically connected to intracellular domains by a hinge / transmembrane region, commonly derived from CD8 or IgG4. The intracellular domains may contain co- stimulatory domains such as CD80, CD86, 4-1 BBL, OX40L and CD70 and / or CD28 linked to the cytoplasmic signaling domain of CD3zeta. See Sadelain et al. The basic principles of chimeric antigen receptor (CAR) design, Cancer Discov. 2013, 3(4): 388-398.

[0167] Peripheral blood mononuclear cells (PBMCs) may be isolated by leukapheresis. T cells can be enriched by mononuclear cells counter-flow elutriation and expanded by addition of anti- CD3 / CD28 antibody coated paramagnetic beads for activation of T cells. CD3 is expressed on T cells as it is associated with the T cells receptor (TCR). The majority of TCR are made up of alpha beta chains (alpha beta T-cells). Alpha beta T-cells typically become double-positive intermediates (CD4+CD8+) which mature into single-positive (CD4+CD8-) T helper cells or (CD4-CD8+) cytotoxic T cells. T helper cells interact with antigen presenting dendritic cells and B cells.

[0168] T cells may be isolated and separated from a human sample (blood or PBMCs or bone marrow) based on the expression of alpha beta T cells receptor (TCR), gamma delta T cells receptor, CD2, CD3, CD4, CD8, CD4 and CD8, NK1.1, CD4 and CD25 and other combinations based on positive or negative selection. In certain embodiments, the immune cells are CD8+, CD4+, alpha beta T cells, delta gamma T cells, natural killer cells and / or double-negative alpha beta T cells, macrophages, NK T cells, and cells derived from umbilical cord blood, bone marrow, or peripheral blood from human samples.

[0169] Use of BALMS fused to CAR-T-cells and / or fused to Fc, as anticancer therapeutic reagent targeting glycoproteins with high-mannose N-glycans on the surface of solid tumors

[0170] There is evidence that aberrant overexpression of high-mannose N-glycans occurs in neoplastic cells. It is contemplated that BALMS are able to recognize these mannose-rich glycans on the surface of cancer cells and thereby exhibit antitumor activity and constitute an anticancer strategy targeting tumor-associated high-mannose glycans. The incorporation of glycan-targeting BALMS attached to a chimeric antigen receptor on the surface of T cells extends their activity to include both malignant cells and their supporting stromal cells, disrupting the tumor microenvironment that otherwise diminishes the activity of cellular therapies against solid tumors.

[0171] As an example of BALMS-CAR T cell generation, one extracts peripheral blood mononuclear cells (PBMCs) from healthy human donors to purify T cells. One clones BALMS into the SFG (retroviral backbone) vector expressing the IgG4 short hinge, CD28 transmembrane domain, 41BBzeta costimulatory domain, and CD3zeta signaling domain. One transfects the isolated T cells with BALMS-CAR for retroviral transduction. One measures transduction efficiency of T cells by flow cytometry after staining with a rabbit anti-BALMS antibody and then stained with fluorescent goat anti-rabbit IgG.

[0172] In certain embodiments, this disclosure contemplates the use of a chimeric BALMS-Fc molecule as a tool to probe and target the tumor-associated high-mannose N-glycan biomarker, in order to selectively recognize cancer cells and exert antitumor activity through inhibition of HM- containing glycoproteins on the cell surface and by immune activation via Fc receptors, i.e., the use of BALMS-Fc as an anticancer agent.

[0173] BALMS fused to Fc as a potential antiviral reagent targeting viral surface glycoproteins high-mannose N-glycans

[0174] Oligomannose binding lectins disclosed herein are contemplated for use as biologic therapeutics targeting virus-associated high-mannose N-glycans. By bind to high-mannose N- glycans on the viral envelope the agent is able to block attachment and virus entry as well as downstream steps in the virus life cycle, thus arresting virus spread without affecting normal human cells. Based on prior studies and the presence of N-linked glycans on viral envelopes, it is contemplated that BALMS to have antiviral activity against a broad-spectrum of RNA and DNA viruses at nanomolar concentrations through high-affinity binding to high-mannose N-glycans clustered on the envelope glycoproteins of individual viruses (i.e., the glycan shield).

[0175] In certain embodiments, this disclosure contemplates a chimeric molecule comprised of the BALMS fused to the N-term (fused to CH2) and / or C-term (fused to CH3 domain) and the Fc region of human immunoglobulin G1 (IgGl), BALMS-Fc. This chimeric construction is contemplated for use to exhibit antibody-dependent cell-mediated virus inhibition against certain virus-infected human cells via its capacity to interact with activating Fc g receptors such as FcgRI, FcgRIIa and FcgRIIIa. Hence, the BALMS-Fc is contemplated to inhibit a wide range of virus strains and elicit Fc-mediated cytotoxicity to virus-infected human cells. Contemplated are uses of BALMS-Fc as a therapeutic molecule targeting high-mannose N-glycans on the cell surface in high densities under pathological conditions. Contemplated are variants with high binding affinity at low (picomolar) concentrations, desirable pharmacological profiles, deimmunization and Fc modifications for extended serum half-life and enhanced ADCC activity. High-mannose N-linked glycans also accumulate on the envelope proteins of many viruses, including human immunodeficiency virus (HIV), human herpesviruses varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and herpes simplex virus 1 (HSV-1), hepatitis C virus (HCV), influenza A and B viruses (IAV, IBV), Ebola virus, and SARS-CoV-2 virus. This aberrantly enriched “glycan shield” and the increase in abundance of high-mannose N-glycans therein has been observed in certain types of viruses, which can efficiently evade the human immune system, such as HIV. It is contemplated that recombinant oligomannose binding lectins disclosed herein are useful to bind glycoproteins on the viral envelope in order to facilitate treatment methods.

[0176] In certain embodiments, the recombinant lectin or the recombinant lectin fused to the human IgGl Fc region is used to bind oligomannose / high-mannose N-glycans on viral envelope proteins leading to viral neutralization.

[0177] In certain embodiments, this disclosure relates to methods of treating or preventing a viral infection comprising administering an effective amount of a recombinant lectin or a human IgGl Fc region fusion protein disclosed herein to a subject in need thereof. In certain embodiments, the virus is human immunodeficiency virus (HIV), human herpesviruses varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and herpes simplex virus 1 (HSV-1), hepatitis C virus (HCV), influenza A and B viruses (IAV, IBV), Ebola virus, and SARS-CoV-2 virus.

[0178] In certain embodiments, the recombinant lectin or a human IgGl Fc region fusion protein disclosed herein is administered in combination another anti-viral agent such as abacavir, galidesivir, ganciclovir, vidarabine, acyclovir, adefovir, amantadine, ampligen, amprenavir, atazanavir, atripla, baloxavir marboxil, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, combivir, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscamet, ibacitabine, ibalizumab, idoxuridine, imiquimod, imunovir, indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, norvir, oseltamivir, penciclovir, peramivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, fumarate tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, zidovudine, or combinations thereof.

[0179] Second generation BT1285i variants with increased affinity to high mannose (HM) substrates

[0180] Experiments were performed to improve the binding affinity of the BT1285i inactivated endoglycosidase (i.e., containing the D161 A and El 63 A mutations) to HM N-glycan substrates by ribosome display directed evolution. Random mutations were introduced into the DNA sequence of the BT1285i inactivated endoglycosidase by error-prone PCR to create a combinatorial library of BT1285i mutant variants. BT1285i variants with high affinity to HM-glycosylated IgG (HM- IgG) were selected from this combinatorial library by six rounds of ribosome display selection. In each round, a non-specific pre-selection step against complex type IgG (CT-IgG) was introduced before specific selection to HM-IgG in order to secure a final specificity of selected variants only to HM-IgG (Figure 4A). After selection, the specificity of the selected variants to HM-IgG were confirmed by ELISA and compared these estimated affinities to BT1285i.

[0181] Candidate variants (Figure 4B) were chosen based on our ELISA results for quantitative determination of binding to HM-IgG by surface plasmon resonance (SPR) analysis. One variant, F78A-A79F-L123LA165I-G267E, exhibited a 22-fold increase in affinity relative to BT1285i. Computational analysis suggested that only residues 78 and 79 of the variant were important for interaction with the HM glycan. Accordingly, an F78A-A79F mutant variant of BT1285i was created. SPR analysis confirmed that its affinity to HM-IgG was increased 72-fold relative to BT1285i.

Claims

CLAIMS1. A recombinant oligomannose binding lectin comprising the amino acid sequence of FAD AW (SEQ ID NO: 2).

2. The recombinant lectin of claim 1 comprising SEQ ID NO: 1 or variant thereof wherein the variant maintains the amino acid E at position 52 and the amino acid H at position 126.

3. A nucleic acid encoding a recombinant lectin of claim 1.

4. A vector comprising a nucleic acid of claim 3.

5. An expression system comprising a nucleic acid or vector of claims 3 or 4.

6. The expression system of claim 5 which is a somatic cell.

7. A chimeric antigen receptor wherein the targeting domain comprises a recombinant lectin of claim 1.

8. An immune cell expressing a chimeric antigen receptor of claim 7.

9. The immune cell of claim 8, which is a T cell or a NK cell.

10. A method of treating cancer comprising administering an effective amount of an immune cell of claim 8 to a subject in need thereof.

11. A fusion protein comprising the recombinant lectin of claim 1 and a human IgG Fc region.

12. A method of treating cancer comprising administering an effective amount of a fusion protein of claim 11 to a subject in need thereof.

13. The method of claim 12, wherein the fusion protein is administered in combination with immune cells, T cells, NK cells, or a hematopoietic stem cell transplant.

14. A method of treating cancer comprising administering an effective amount of an oligomannose binding lectin of claim 1 to a subject in need thereof.

15. A method of treating a viral infection comprising administering an effective amount of a fusion protein of claim 11 to a subject in need thereof.

16. A method of treating a viral infection comprising administering an effective amount of a fusion protein of claim 11 to a subject in need thereof.

17. A method of treating a viral infection comprising administering an effective amount of an oligomannose binding lectin of claim 1 to a subject in need thereof.

18. A solid support linked to an oligomannose binding lectin of claim 1.

19. A method of purifying recombinant antibodies comprising contacting a solid support of claim 18 with a sample of oligomannose glycosylated recombinant antibodies providing oligomannose glycosylated recombinant antibodies linked to the solid support.

20. The method of claim 19, further comprising separating the sample from the solid support providing purified oligomannose glycosylated recombinant antibodies linked to the solid support and purified non-oligomannose glycosylated recombinant antibodies in the separated sample.

21. The method of claim 20, further comprising detaching and isolating the purified oligomannose glycosylated recombinant antibodies.

22. A recombinant oligomannose binding lectin comprising the amino acid sequence of and LAFAN (SEQ ID NO: 13) and FAD AW (SEQ ID NO: 2).

23. The recombinant lectin of claim 22 comprising SEQ ID NO: 10 or variant thereof wherein the variant that maintains the amino acid A at position 78 and the amino acid F at position 79 and amino acid A at position 161 and amino acid A at position 163 and the amino acid E at position 52 and the amino acid H at position 126.

24. A nucleic acid encoding a recombinant lectin of claim 23.

25. A vector comprising a nucleic acid of claim 24.26 An expression system comprising a nucleic acid or vector of claims 24 or 25.

27. The expression system of claim 26 which is a somatic cell.

28. A chimeric antigen receptor wherein the targeting domain comprises a recombinant lectin of claim 22.

29. An immune cell expressing a chimeric antigen receptor of claim 28.

30. The immune cell of claim 29, which is a T cell or a NK cell.

31. A method of treating cancer comprising administering an effective amount of an immune cell of claim 29 to a subject in need thereof.

32. A fusion protein comprising the recombinant lectin of claim 22 and a human IgG Fc region.

33. A method of treating cancer comprising administering an effective amount of a fusion protein of claim 32 to a subject in need thereof.

34. The method of claim 33, wherein the fusion protein is administered in combination with immune cells, T cells, NK cells, or a hematopoietic stem cell transplant.

35. A method of treating cancer comprising administering an effective amount of an oligomannose binding lectin of claim 22 to a subject in need thereof.

36. A method of treating a viral infection comprising administering an effective amount of a fusion protein of claim 32 to a subject in need thereof.

37. A method of treating a viral infection comprising administering an effective amount of a fusion protein of claim 32 to a subject in need thereof.

38. A method of treating a viral infection comprising administering an effective amount of an oligomannose binding lectin of claim 22 to a subject in need thereof.

39. A solid support linked to an oligomannose binding lectin of claim 22.

40. A method of purifying recombinant antibodies comprising contacting a solid support of claim 39 with a sample of oligomannose glycosylated recombinant antibodies providing oligomannose glycosylated recombinant antibodies linked to the solid support.

41. The method of claim 40, further comprising separating the sample from the solid support providing purified oligomannose glycosylated recombinant antibodies linked to the solid support and purified non-oligomannose glycosylated recombinant antibodies in the separated sample.

42. The method of claim 41, further comprising detaching and isolating the purified oligomannose glycosylated recombinant antibodies.

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