Recombinant Protein Variants

Abstract

A modified lectin protein is provided having at least one amino acid modification in an amino acid sequence of SEQ ID NO. 1 or in an amino acid sequence that has at least 60% homology with it. The amino acid modification is selected from one or more of the following: at least one amino acid modification at a carbohydrate-binding site; at least one amino acid modification at the N-terminus; at least one amino acid modification at position 76; or at least one amino acid modification at position 44 or 89. The modified lectin protein does not consist of the amino acid sequence of any of SEQ ID NO. 2 to 4.

Description

RECOMBINANT PROTEIN VARIANTS CROSS REFERENCE TO RELATED APPLICATIONS The application claims the benefit of India's Provisional Application No. 201821032765 filed on August 31, 2018, the full content of which is incorporated by reference in this description. FIELD OF INVENTION The present invention relates to a modified lectin protein and a nucleic acid molecule comprising a nucleic acid sequence encoding the modified lectin protein. The invention further relates to a recombinant vector comprising such a nucleic acid molecule and a transformed host cell comprising the recombinant vector. In addition, the invention relates to a pharmaceutical composition comprising the modified lectin protein and to the detection of a cancer cell, the diagnosis of cancer, and the treatment of cancer in a patient. The present invention further relates to a process for producing a recombinant lectin protein from Sclerotium rolfsii. BACKGROUND OF THE INVENTION Lectins are carbohydrate-binding proteins with high specificity; they are macromolecules that are highly specific for the sugar portions of other molecules. Lectins perform recognition at the cellular and molecular levels and play numerous roles in biological recognition processes involving cells, carbohydrates, and proteins. They are divalent or polyvalent carbohydrate-binding proteins that bind to and precipitate glycoproteins and agglutinate red blood cells. Animal lectins are most often found to aid in cell-cell interactions, while plant lectins are known to protect against potential predators or pathogens. Purified lectins are important in a clinical setting because they are used for blood typing. Some of the glycolipids and glycoproteins of an individual's red blood cells can be identified using lectins. Many lectins are used as biomarkers that indicate the early detection of malignant growth or as inducers of autophagy, while others also exhibit the ability to inhibit cancerous growth through apoptosis. Due to unregulated cell proliferation, some carbohydrate portions are expressed as an antigen on cancer cells. Lectins are used as drug delivery agents in cancer therapy because they bind specifically to malignant tumors. Furthermore, since lectins also modulate cancer-associated pathways, they have potential as both therapeutic and diagnostic agents for cancer. There are various antigens to which lectins bind that have been characterized on the surface of cancer cells; most of these antigens are specific to a particular type of cancer, and the binding of lectins to these antigens can result in the inhibition of cancer growth by inducing apoptosis in cancer cells. Currently, most commercially available lectins are derived from plants and other eukaryotes. nncrzn / i znz / zi / Yl· Sclerotium rolfsii lectin (SRL) is a lectin isolated from the sclerotic bodies of the soil-borne phytopathogenic fungus S. rolfsii. SRL is specific for the Thomsen-Frledenreich (TF) and Tn antigens. The TF antigen is a disaccharide (Galp1^3GalNAc-a-Ser / Thr) that is overexpressed on the cell surface of several different human cancer cells. The Tn antigen is a monosaccharide (GalNAc-α-). Due to its specificity for the TF and Tn antigens, SRL has been shown to bind to human colon cancer, ovarian cancer, and leukemic cells. The crystal structure of SRL has been determined (Leonidas et al., J Mol Biol. 2007 May 11;368(4):1145-61), but experimental validation of the carbohydrate binding sites identified from the crystal structure has not been carried out. While lectins offer many advantages as cancer-fighting tools, they also have several limitations, including a lack of selectivity, inconsistent quality and performance, and difficult scalability in production. Furthermore, plant-derived lectins have often been reported to bind to a variety of different glucan structures and therefore lack the selectivity required for many applications. Additionally, batch-to-batch variability is common when using plant lectins. Product quality depends on both the plant material isolation methodology and the quality of the starting plant material itself. Isolating lectins from natural sources is unreliable because the lectins obtained in this way lack consistency with respect to desirable properties. Further isolation of proteins from natural sources is a costly and difficult process. Techniques used to isolate naturally occurring lectins typically yield very low results, especially if the protein is present only in low concentrations. Furthermore, they are sometimes unable to distinguish between isoforms of the same lectin. Therefore, they are obtained as mixtures, which introduces a wide range of uncertainty. In this respect, the production of recombinant lectins using recombinant DNA (rDNA) techniques has the advantage of providing unique proteins with better and more consistent yields, which can be accurately characterized in a drastically shorter time and are easily scalable.Using recombinant DNA technology, the gene that produces the protein of interest can be transferred to a suitable host. The protein can then be produced and isolated with less time and effort compared to traditional methods. WO 2010 / 095143 describes recombinant lectin variants Rec-2 and Rec-3, which are derived from the naturally occurring SRL sequence by substitution of 3 or 5 amino acids, respectively. The crystal structure of these variants has been reported (Peppa et al., Molecules. 2015 Jun 12;20(6):10848-65). WO 2014 / 203261 describes a recombinant lectin variant derived from the naturally occurring SRL sequence by substituting 12 amino acids. There remains a need for other lectin variants with alternative properties. In particular, it is advantageous for lectins used in cancer diagnosis and as therapeutic agents to be soluble and stable without compromising their specific affinity for malignant tumors / cancer cells. Therefore, there is a need for novel recombinant lectin sequences and efficient methods to produce recombinant lectins that have sufficient expression levels of the nncrzn / iznz / q / Yi transgene in appropriate host cells and that possess solubility and / or stability while also maintaining affinity for malignant cells. The present invention seeks to address one or more of the above needs. SUMMARY OF THE INVENTION According to one aspect of the present invention, a modified lectin protein is provided comprising an amino acid sequence selected from: i) SEQ ID NO. 1; or i) an amino acid sequence having at least 60% homology with i), wherein the amino acid sequence of i) or i) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or i); b) at least one amino acid modification at the N-terminal end of i) or ii), wherein the cleavage of a starter methionine increases compared to the amino acid sequence of i); c) at least one amino acid modification that reduces the formation of dimers of the modified lectin protein compared to a lectin protein of SEQ ID NO. 1; or d) at least one amino acid modification that reduces the oxidation of the modified lectin protein compared to a lectin protein from SEQ ID NO. 1. In some forms, the modified lectin protein does not consist of the amino acid sequence of any of the SEQ ID NOS. 2 to 4. In some additional forms, the modified lectin protein has a cytotoxic effect. According to one aspect of the present invention, a modified lectin protein is provided, wherein the modified lectin protein comprises an amino acid sequence selected from any of: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a. at least one amino acid modification at a carbohydrate binding site of i) or i); or b. at least one amino acid modification at an N-terminal end of i) or ii), c. at least one amino acid modification at position 76; or d. at least one amino acid modification at position 44 or 89, wherein the modified lectin protein does not consist of the amino acid sequence of any of SEQ ID NOS. 2 to 4. According to another aspect of the invention, a modified lectin protein is provided, wherein the modified lectin protein comprises an amino acid sequence selected from any of: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), nfrcezn / Lznz / q / YL wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or i); or b) at least one amino acid modification at the N-terminal end of i) or II), wherein the cleavage of an initiating methionine increases compared to the amino acid sequence of i); c) at least one amino acid modification that reduces the formation of dimers of the modified lectin protein compared to a lectin protein of SEQ ID NO. 1 at position 76; or d) at least one amino acid modification that reduces the oxidation of the modified lectin protein compared to a lectin protein of SEQ ID NO. 1 at position 44 or 89, wherein the modified lectin protein does not consist of the amino acid sequence of any of SEQ ID NOS. 2 to 4. Optionally, a modified lectin protein of the invention has biological activity. In one embodiment, the modified lectin protein comprises an amino acid sequence that has at least 70%, 80%, 90%, 95%, 97% or 99% homology with SEQ ID NO. 1. Still in accordance with another aspect of the present invention, a modified lectin protein is provided comprising an amino acid sequence selected from: i. SEQ ID NO. 1; or ii. an amino acid sequence having at least 60% homology with i), wherein the amino acid substitution is selected from one or more of: a. an amino acid substitution at the primary carbohydrate attachment site, wherein the substituent amino acid is selected from one or more of: i. a nonpolar, polar, acidic or basic amino acid at position 27 and / or at position 28; ii. a nonpolar amino acid at position 47 and / or a polar amino acid at position 48; II. a nonpolar amino acid at position 70, a polar amino acid at position 71 and / or a nonpolar amino acid at position 72; and / or iv. any other amino acid at position 105, b. an amino acid substitution at the secondary carbohydrate attachment site, wherein the substituting amino acid is selected from one or more of: i. a nonpolar amino acid at position 77, a nonpolar amino acid at position 78 and / or a polar amino acid at position 80; ii. any other amino acid at position 101; ii. a nonpolar amino acid at position 112; and / or a polar amino acid at position 114. In another embodiment, the modified lectin protein comprises at least one amino acid modification at a carbohydrate binding site of i) or ii). In some forms, the carbohydrate binding site is a primary and / or secondary carbohydrate binding site. nfrcezn / Lznz / q / Yl· In one embodiment, the primary carbohydrate binding site comprises a position selected from one or more of: 27, 28, 47, 48, 70, 71, 72 and 105 in SEQ ID NO. 1 or a corresponding position in a sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it. In one of these modalities, the position of the amino acid modification is selected from one or more of: i) 27 and / or 28; ii) 47 and / or 48; iii) 70, 71 and / or 72; and / or v) 105. In another embodiment, the secondary carbohydrate binding site comprises a position selected from one or more of: 77, 78, 80, 101, 112 and 114 in SEQ ID NO. 1 or a corresponding position in the sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it. In one of these modalities, the position of the amino acid modification is selected from one or more of: i) 77, 78 and / or 80; i) 101; iii) 112 and / or 114. In another form, amino acid modification is an amino acid substitution where a substitute amino acid replaces an original amino acid. In one form, amino acid substitution is a conservative or favorable amino acid substitution. In one embodiment, the amino acid substitution at the primary carbohydrate binding site is selected from one or more of: i) at position 27: a conservative, favorable or unfavorable amino acid, wherein the conservative amino acid is nonpolar or acidic; the favorable one is polar or basic and the unfavorable amino acid is nonpolar; ii) at position 28: a conservative, favorable, neutral or unfavorable amino acid, wherein the conservative amino acid is nonpolar; the favorable one is polar, the neutral one is acidic or basic and the unfavorable amino acid is polar; ii) at position 47: an unfavorable amino acid, which is basic or nonpolar; iv) at position 48: an unfavorable amino acid, which is nonpolar; v) at position 70: an unfavorable amino acid, which is nonpolar; vi) at position 71: an unfavorable amino acid, which is nonpolar; vii) at position 72: an unfavorable amino acid, which is nonpolar; and / or viii) at position 105: a conservative, favorable, neutral or unfavorable amino acid, wherein the conservative amino acid is basic or nonpolar; the favorable one is polar, the neutral one is acidic, basic or polar and / or the unfavorable amino acid is polar, nonpolar or acidic. In another modality, the amino acid substitution at the secondary carbohydrate binding site is selected from one or more of: i) at position 77: an unfavorable amino acid that is nonpolar; nncrzn / i ζηζ / ζι / γ ¡i) at position 78: an unfavorable amino acid that is nonpolar; iii) at position 80: an unfavorable amino acid that is nonpolar; iv) at position 101: a favorable, unfavorable or neutral amino acid, wherein the favorable amino acid is polar or basic, the unfavorable amino acid is nonpolar and the neutral amino acid is nonpolar or acidic; v) at position 112: an unfavorable amino acid that is nonpolar; vi) at position 114: an unfavorable amino acid that is polar. In one embodiment, the modified lectin protein comprises at least one amino acid modification at the N-terminal end of i) or ii), wherein the N-terminal end comprises a position selected from: 1 and / or 2 in SEQ ID NO. 1 or a corresponding position in the sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it. In one embodiment, the amino acid modification is an amino acid substitution at position 1 where the substituent amino acid is neither threonine nor valine. In additional embodiments, the substituent amino acid is selected from alanine, glycine, proline, or serine. Still in additional embodiments, the amino acid modification is an amino acid substitution at position 2 where the substituent amino acid is tryptophan. In some modalities, the cleavage of an initiator methionine increases or decreases compared to a control. In another embodiment, the modification of the amino acid at position 76 is a substitution of the amino acid with a nonpolar amino acid. In some embodiments, the nonpolar amino acid is selected from glycine, valine, or leucine. In one embodiment, the modification of the amino acid at position 44 or 89 is a substitution of the amino acid with a nonpolar amino acid. In some embodiments, the modification of the amino acid is preferably at position 89. In some embodiments, the nonpolar amino acid is selected from leucine, isoleucine, or valine. In another modality, the modified lectin protein is soluble, partially soluble, or insoluble and / or exhibits cytotoxicity. In some modalities, the modified lectin protein has cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a control. In an alternative modality, the modified lectin protein has a percentage of cytotoxicity that is less than 10% of a control, or it has no cytotoxicity. In another alternative modality, the modified lectin protein has a percentage of cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher compared to that of a control. In another form, the modified lectin protein has a length equal to or less than 500, 400, 300, 250, 200 or 150 amino acids. According to another aspect of the present invention, a pharmaceutical composition comprising a modified lectin protein and a pharmaceutically acceptable diluent or excipient, and optionally an additional therapeutic ingredient, is provided. Furthermore, a method of treating cancer in a patient is provided, comprising administering the modified lectin protein or the pharmaceutical composition of the modified lectin protein to the patient. nbcrzn / ι znz / zi / Yl· According to another aspect of the invention, a pharmaceutical composition is provided comprising a modified lectin protein as described above and a pharmaceutically acceptable diluent or excipient, and optionally an additional therapeutic ingredient. According to a further aspect of the invention, a method of treating cancer in a patient is provided comprising administering the modified lectin protein as described above to a patient. In some embodiments, the method comprises administering the pharmaceutical composition described above to a patient. According to another aspect of the present invention, a modified lectin protein or a pharmaceutical composition of a modified lectin protein is provided for use in cancer treatment. Furthermore, a modified lectin protein is provided for use in cancer cell detection, cancer diagnosis, and / or cancer therapy. According to a further aspect of the invention, a modified lectin protein as described above is provided for use in medicine. Alternatively, a pharmaceutical composition as described above is provided for use in medicine. In some embodiments, the modified lectin protein or the pharmaceutical composition as described above is for use in the treatment of cancer. According to a further aspect of the invention, the modified lectin protein as described above is provided when used in the detection of a cancer cell, cancer diagnosis, and / or cancer therapy. According to one aspect of the present invention, a nucleic acid molecule is provided comprising a nucleotide sequence encoding a modified lectin protein, wherein the modified lectin protein comprises an amino acid sequence selected from: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), and wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or ii); b) at least one amino acid modification at the N-terminal end of I) or II); c) at least one amino acid modification at position 76; or d) at least one amino acid modification at position 44 or 89, According to another aspect of the present invention, a nucleic acid molecule is provided comprising a nucleotide sequence encoding a modified lectin protein, wherein the modified lectin protein comprises an amino acid sequence selected from: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), and wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or i); b) at least one amino acid modification at the N-terminal end of i) or ii), wherein the cleavage of a starter methionine increases compared to the amino acid sequence of i); nncrzn / i znz / zi / Yl· c) at least one amino acid modification that reduces the formation of dimers of the modified lectin protein compared to a lectin protein of SEQ ID NO. 1; or d) at least one amino acid modification that reduces the oxidation of the modified lectin protein compared to a lectin protein from SEQ ID NO. 1. According to another aspect of the invention, a nucleic acid molecule comprising a nucleotide sequence encoding a modified lectin protein as described above is provided. In a further aspect of the invention, a recombinant vector comprising an insert of this nucleic acid molecule is provided. In some embodiments, the vector is operationally assembled in a 5' to 3' direction: it comprises a promoter that functions in a host cell; a nucleotide sequence as described above that encodes a modified lectin protein; and a termination signal. In another embodiment, the recombinant vector can be replicated, transcribed, translated, and / or expressed in a single-celled organism. In another aspect of the invention, a transformed host cell comprising the nucleic acid molecule described above is provided. In some embodiments, the host cell is an Escherichia coli bacterium or a yeast cell. According to another aspect of the present invention, a recombinant vector is provided comprising an insert of a nucleic acid molecule, wherein the nucleic acid molecule comprises the nucleotide sequence encoding a modified lectin protein comprising an amino acid sequence selected from: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), and wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or i); b) at least one amino acid modification at the N-terminal end of i) or i); c) at least one amino acid modification at position 76; or d) at least one amino acid modification at position 44 or 89. According to another aspect of the present invention, a recombinant vector is provided comprising an insert of a nucleic acid molecule, wherein the nucleic acid molecule comprises the nucleotide sequence encoding a modified lectin protein comprising an amino acid sequence selected from: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), and wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate binding site of i) or i); b) at least one amino acid modification at the N-terminal end of i) or ii), wherein the cleavage of a starter methionine increases compared to the amino acid sequence of i); nfrcezn / Lznz / q / YL c) at least one amino acid modification that reduces the formation of dimers of the modified lectin protein compared to a lectin protein of SEQ ID NO. 1; or d) at least one amino acid modification that reduces the oxidation of the modified lectin protein compared to a lectin protein from SEQ ID NO. 1. In a final aspect of the invention, a method is provided for producing a recombinant lectin protein from Sclerotium rolfsii comprising: i) cultivate a host cell containing the recombinant vector as described above that encodes a recombinant lectin protein; i) express the recombinant lectin protein; iii) isolate a crude recombinant lectin protein from the culture. BRIEF DESCRIPTION OF THE ACCOMPANYING SEQUENCES • SEQ ID NO. 1: represents the amino acid sequence of the naturally occurring S. rolfsiide lectin. • SEQ ID NO. 2: represents a variant of the amino acid sequence of the S. rolfsii lectin (reported as Rec-2 in WO 2010 / 095143). • SEQ ID NO. 3: represents a variant of the amino acid sequence of the S. rolfsii lectin (reported as Rec-3 in WO 2010 / 095143). • SEQ ID NO. 4: represents a variant of the amino acid sequence of the S. rolfsii lectin (reported in WO 2014 / 203261). DETAILED DESCRIPTION OF THE INVENTION Definitions: The term protein as used in this description refers to a polymer of amino acid residues. The term lectin as used in the present description refers to a carbohydrate-binding protein. The term modified lectin protein as used in the present description refers to a polymer of amino acid residues that has carbohydrate-binding activity and contains at least one amino acid modification. The term amino acid, as used herein, refers to synthetic and naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that have a similar function to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and include proteinogenic amino acids. Naturally occurring amino acids also include those modified after translation in cells. Synthetic amino acids include non-canonical amino acids such as selenocysteine ​​and pyrrolysine. Typically, synthetic amino acids are not proteinogenic. The term amino acid modification, as used herein, refers to the addition, deletion, or substitution of an amino acid at a particular position in an amino acid sequence. In one embodiment, the addition of an amino acid refers to the addition of at least 1, 2, 3, 4, or 5 amino acids at a particular position in an amino acid sequence. The addition, deletion, or substitution processes are carried out according to the present invention or according to methods known to the skilled trades. In one embodiment, amino acid modification, as used herein, refers to one or more modifications selected from: acetylation, nitration, glycation, and / or sulfonation. The term amino acid substitution, as used herein, refers to the substitution of an amino acid at a particular position in an amino acid sequence. The term amino acid substitution encompasses both conservative and non-conservative amino acid substitutions. A conservative amino acid substitution provides a functionally similar amino acid. In other words, the amino acid that replaces the original amino acid (i.e., the substituent amino acid) has similar biochemical properties. A non-conservative substitution provides a functionally different amino acid. In other words, the amino acid that replaces the original amino acid (i.e., the substituent amino acid) has different biochemical properties. In one modality, the amino acid substitution is a favorable amino acid substitution. A favorable amino acid substitution preserves a biological function and / or other property of the modified lectin protein.In another modality, both conservative and favorable amino acid substitutions are based on a multiple or pairwise sequence alignment of lectin proteins. A conservative substitution is the substitution with the amino acid that occurs in the maximum number of natural lectin proteins at the corresponding position, and a favorable substitution is the substitution with the amino acid that occurs in few natural lectin proteins at the corresponding position. Both substitutions are expected to preserve or enhance the cytotoxicity of the modified protein. In one modality, the biological function is a cytotoxic effect as defined below. In another modality, the substituent amino acid is selected as a favorable amino acid substitution based on a multiple or pairwise sequence alignment of lectin proteins, preferably fungal lectin proteins. Amino acids can be grouped according to different biochemical properties. Examples include polar amino acids, nonpolar amino acids, acidic amino acids, and basic amino acids. In one modality, the amino acid used for amino acid modification is at least one selected from the group consisting of, but not limited to: polar, nonpolar, acidic, basic, selenocysteine, pyrrolysine, and non-canonical. The terms homology and homologue, as used herein, refer to two or more referenced entities that share at least partial identity over a given region or portion. Areas, regions, or domains of homology or identity refer to a portion of two or more referenced entities that share homology or are identical. Thus, when two sequences are identical in one or more sequence regions, they share identity in these regions. Substantial homology refers to a molecule that is structurally or functionally conserved such that it has, or is predicted to have, at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or a relevant / corresponding region or portion of the reference molecule with which it shares homology. nncrzn / i znz / zi / Yl· In one approach, the percentage of homology between two sequences is determined using the BLASTP algorithm with predefined parameters (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Specifically, the BLASTP algorithm can be accessed online at the following URL: http: / / blast.ncbi.nlm.nh.gov / Blast.cqi. In an alternative approach, for global sequence alignments, the percentage of homology between two sequences is determined using the EMBOSS Needle algorithm with predefined parameters. Specifically, the EMBOSS Needle algorithm can be accessed online at the following URL: https: / / www.ebi.ac.uk / Tools / psa / emboss needle / . Unless otherwise stated, the term homology is used interchangeably with the term sequence identity in this description. The term "correspondent position," as used herein, refers to an analogous position between two or more sequences. In one modality, the corresponding position is determined by sequence alignment of at least one first and one second sequence. The corresponding position in at least the first and second sequences is aligned in the output of the sequence alignment and can therefore be identified. In one modality, the sequence alignment is a multiple or pairwise sequence alignment. In one modality, the sequence alignment is performed using an algorithm. In one modality, the algorithm is either BLAST or EMBOSS Needle, as discussed previously. The term carbohydrate binding site, as used herein, refers to the amino acid residues in a lectin protein that are involved in the recognition and binding of a carbohydrate structure. In one embodiment, the carbohydrate binding site is involved in the recognition and binding of the TF antigen (a disaccharide; Galpl → 3GalNAc-α-Ser / Thr) and / or the Tn antigen (a monosaccharide; GalNAc-α-). In another embodiment, the carbohydrate binding site encompasses a primary carbohydrate binding site and / or a secondary carbohydrate binding site. The term primary carbohydrate binding site, as used herein, refers to one or more amino acid residues involved in the recognition and binding of a specific carbohydrate structure. In one modality, the specific carbohydrate structure is the TF antigen. In one modality, the amino acid residues constituting the primary carbohydrate binding site are selected from one or more of positions 27, 28, 47, 48, 70, 71, 72, and / or 105 in SEQ ID NO. 1 or a corresponding position in a sequence that has at least 60% homology to it. In one modality, the corresponding position is determined by sequence alignment. The term secondary carbohydrate binding site, as used herein, refers to one or more amino acid residues involved in the recognition and binding of a specific carbohydrate structure. In one embodiment, the specific carbohydrate structure is the Tn antigen. In one embodiment, the amino acid residues constituting the secondary carbohydrate binding site are selected from one or more of positions 77, 78, 80, 101, 112, and / or 114 in SEQ ID NO. 1 or a corresponding position in a sequence that has at least 60% homology nncrzn / i znz / zi / Yl· with it. In one embodiment, the corresponding position is determined by sequence alignment. The term cytotoxic effect, as used herein, refers to a substance that destroys cancer cells or inhibits the growth of cancer cells (i.e., exerts antiproliferative activity). In one modality, the percentage of cytotoxicity of a substance is determined using a Sulforhodamine B (SRB) assay as detailed in Example 5. In one modality, a percentage of cytotoxicity of at least 20%, 30%, 40%, 50%, or 60% as determined by the SRB assay is indicative of a cytotoxic effect. In one modality, the modified lectin protein has a percentage of cytotoxicity that is at least 60%, 70%, 80%, or 90% of that of a control. In one modality, the control is a lectin protein from SEQ ID No. 1, and in another modality, the control is SEQ ID No. 2. The term N-terminal end, as used herein, refers to amino acid residues located toward the beginning of the amino acid sequence (i.e., toward the amino terminus). In one embodiment, the N-terminal end refers to amino acid residues located within the first 10% or 5% of the amino acid sequence. The term "cleavage of an initiator methionine," as used herein, refers to the removal of the N-terminal (initiator) methionine from an amino acid sequence. In one modality, the initiator methionine cleavage is catalyzed by the enzyme methionine aminopeptidase (MAP). In another modality, the initiator methionine cleavage is determined by mass spectrometry or HPLC analysis performed by a person skilled in the technique. The term "initiator methionine cleavage increase," as used herein, refers to an increase in the degree of cleavage of the initiator methionine relative to a control. In one modality, it refers to an increase of at least 5%, 10%, 25%, or 50% in the degree of cleavage of the initiator methionine relative to a control. In one modality, the control is a lectin protein from SEQ ID NO. 1, and in another modality, the control is SEQ ID NO. 2. The term dimer formation, as used herein, refers to the formation of an oligomer containing two monomers that are either identical or non-identical. In one embodiment, dimer formation refers to the production of a dimer containing two lectin proteins, modified according to the present invention (identical or non-identical). In an alternative embodiment, dimer formation refers to the production of a dimer containing a lectin protein modified according to the present invention and an alternative lectin protein, such as one consisting of the sequence in SEQ ID NO. 1. In one embodiment, dimer formation is mediated by a disulfide bond between cysteine ​​residues in each monomer. In another embodiment, the cysteine ​​residue is at position 76 of SEQ ID NO. 1 or a corresponding position in an amino acid sequence having at least 60% homology with it.In one modality, the corresponding position is determined by sequence alignment. In another modality, the level of dimer formation is determined using any of mass spectrometry, size exclusion chromatography, and / or SDSPAGE analysis. The term reduced dimer formation, as used herein, refers to a decrease in the level of dimer formation compared to a control. In one modality, reduced dimer formation refers to a decrease of at least 5%, 10%, 25%, or 50% in the level of dimer formation compared to a control. In one modality, the control is a lectin protein from SEQ ID NO. 1, and in another modality, the control is SEQ ID NO. 2. The term oxidation, as used in this description, refers to a loss of electrons or an increase in oxidation state. In one embodiment, the term oxidation refers to the oxidation of an amino acid residue, examples of which include methionine, cysteine, tryptophan, tyrosine, and / or histidine. In one embodiment, the term oxidation refers to the oxidation of a methionine residue to methionine sulfoxide. In one embodiment, the methionine residue that is susceptible to oxidation is at position 44 or 89 of SEQ ID NO. 1 or a corresponding position in an amino acid sequence that has at least 60% homology to it. In one embodiment, the corresponding position is determined by sequence alignment. In one embodiment, the level of oxidation is determined using mass spectrometry and / or reversed-phase high-performance liquid chromatography (RP-HPLC).Oxidation is believed to affect protein expression and activity. In one modality, the effect of oxidation is determined by the substitution effect of amino acids susceptible to oxidation at position 44 or 89 of SEQ ID NO. 1 on the expression and / or activity of the soluble lectin protein. The term reduced oxidation, as used herein, refers to a decrease in the oxidation level relative to a control. In one modality, reduced oxidation refers to a decrease of at least 5%, 10%, 25%, or 50% in the oxidation level relative to a control. In an alternative modality, reduced oxidation refers to increased expression and / or activity of the soluble lectin protein. In one modality, the control is a lectin protein from SEQ ID NO. 1, and in another modality, the control is SEQ ID NO. 2. The term "soluble," as used herein, refers to the modified lectin protein that is expressed in a soluble or at least partially soluble form. In one modality, the solubility of the modified lectin protein is determined by cell lysis of a host cell expressing the modified lectin protein and subsequent SDS-PAGE analysis of the lysis supernatant and pellet. The presence of the modified lectin protein in the lysis supernatant indicates that it is soluble. The presence of the modified lectin protein in both the lysis supernatant and the pellet indicates that it is partially soluble. In another modality, the term "soluble," as used herein, refers to the modified lectin protein that does not form inclusion bodies. Using the method described above, the presence of the modified lectin protein in the pellet indicates that it is expressed as inclusion bodies. The term nucleic acid molecule, as used herein, refers to a polymer of multiple nucleotides. Nucleic acid molecules may comprise naturally occurring nucleic acids or they may comprise synthetic nucleic acids. In one embodiment, the nucleic acid molecule is DNA or a derivative thereof. In an alternative embodiment, the nucleic acid molecule is RNA or a derivative thereof. nncrzn / i znz / zi / Yl· The term nucleotide as used in the present description refers to naturally occurring nucleotides and synthetic nucleotide analogues that are recognized by cellular enzymes. In the search to develop new lectins with altered and / or improved physicochemical properties and / or biological activity, the inventors of the present invention developed various lectin variants, with modification of the naturally occurring lectin sequence in active and non-active sites. In one embodiment, the present invention provides recombinant lectin variants derived from the naturally occurring lectin sequence that exhibit altered properties; preferably, they exhibit specificity for certain sugar chains uniquely found in certain cancer cells and / or improved solubility and / or stability compared to the naturally occurring protein. These recombinant lectins are obtained by deliberately modifying the naturally occurring lectin. In one embodiment of the present invention, the naturally occurring lectin is derived from the group consisting of, but not limited to, fungi and plants. Typically, the naturally occurring lectin is derived from a soilborne phytopathogenic fungus. In an illustrative embodiment of the present invention, the phytopathogenic fungus is S. rolfsii.It is preferred that recombinant lectins derived from the amino acid sequence of the naturally occurring lectin have specificity towards the Tn antigen and / or the TF antigen and, therefore, bind to human colon cancer, ovarian cancer and leukemic cells. In general terms, the present invention relates to a modified lectin protein comprising an amino acid sequence selected from SEQ ID NO. 1 or an amino acid sequence having at least 60% homology with it, wherein the modified lectin protein comprises at least one amino acid modification in SEQ ID NO. 1 or in the amino acid sequence having at least 60% homology with it. The sequence in SEQ ID NO. 1 corresponds to the naturally occurring S. rolfsii lectin sequence (as reported in WO 2010 / 095143). The amino acid modification is selected from one or more of: an amino acid modification at a carbohydrate-binding site of SEQ ID NO. 1 or a sequence having at least 60% homology with it; an amino acid modification at the N-terminal end of SEQ ID NO. 1; or a modified amino acid at the N-terminal end of SEQ ID NO. 1.1 or a sequence that has at least 60% homology with this; an amino acid modification that reduces the formation of dimers of the modified lectin protein; and / or an amino acid modification that reduces oxidation. It is preferred that the modified lectin protein not consist of the amino acid sequence of any of SEQ ID NO. 2 (as reported in WO 2010 / 095143 as the recombinant variant Rec-2), SEQ ID NO. 3 (as reported in WO 2010 / 095143 as the recombinant variant Rec-3), or SEQ ID NO. 4 (as reported in WO 2014 / 203261). SEQ IDs 2 through 4 are examples of amino acid sequences that have at least 60% homology with SEQ ID NO. 1. In particular, SEQ IDs 2, 3, and 4 have homology of 97.9%, 96.5%, and 91.5% with SEQ ID NO. 1, respectively (as determined using the EMBOSS Needle). Carbohydrate binding site nfrcezn / Lznz / q / Yl· In a first embodiment of the present invention, the carbohydrate binding site is a primary carbohydrate binding site comprising amino acid positions 27, 28, 47, 48, 70, 71, 72, and 105 in SEQ ID NO. 1 (as reported in Leonidas et al., 2007) or the corresponding positions of a sequence having at least 60% homology with SEQ ID NO. 1. The primary carbohydrate binding site exhibits specificity for the TF (Galp1^3GalNAc-a-Ser / / Thr) antigen expressed on the surface of cancer cells. In the first embodiment, the modified lectin protein contains an amino acid substitution at one or more of the positions of the primary binding site. In one embodiment, the modified lectin protein contains an amino acid substitution at one or more positions selected from: 27 and / or 28; 47 and / or 48; 70, 71 and / or 72; and / or 105. In a second embodiment of the present invention, the carbohydrate binding site is a secondary carbohydrate binding site comprising amino acid positions 77, 78, 80, 101, 112, and 114 in SEQ ID NO. 1 (as reported in Leonidas et al., 2007) or the corresponding positions in a sequence having at least 60% homology with SEQ ID NO. 1. The secondary carbohydrate binding site exhibits specificity for the Tn antigen (GalNAc-α-). In the second embodiment, the modified lectin protein contains an amino acid substitution at one or more positions of the secondary binding site. In one embodiment, the modified lectin protein contains an amino acid substitution at one or more positions selected from: 77, 78, and / or 80; 101; and / or 112 and / or 114. It is preferred that an amino acid substitution according to the first and / or second modality be a conservative or favorable amino acid substitution. A conservative amino acid substitution refers to the substituting amino acid (i.e., the one that replaces the original amino acid) having biochemical properties similar to those of the original amino acid. In one modality, a polar amino acid is substituted for a different polar amino acid; a nonpolar amino acid is substituted for a different nonpolar amino acid; an acidic amino acid is substituted for a different acidic amino acid; or a basic amino acid is substituted for a different basic amino acid. Furthermore, it refers to the amino acid substitution that occurs in the maximum number of natural lectin proteins at the corresponding positions based on a multiple or pairwise alignment of lectin protein sequences.In one embodiment, the modified lectin protein contains a favorable amino acid substitution such that the modified lectin protein retains a biological function and / or other property of the modified lectin protein. In a preferred embodiment, the modified lectin protein contains a favorable amino acid substitution such that the modified lectin protein retains a cytotoxic effect and / or is soluble. In one embodiment, an amino acid residue is selected for a favorable amino acid substitution based on multiple or pairwise sequence alignment of natural lectin proteins, preferably fungal lectin proteins. The favorable substitution is the amino acid substitution that occurs in few natural lectin proteins at the corresponding positions.Without wishing to adhere strictly to theory, it is believed that selecting an amino acid residue that is present in a corresponding position in a homologous sequence and including it in the modified lectin protein is more likely to maintain a biological function and / or other property (such as cytotoxic effect and / or solubility) of the modified lectin protein. In an alternative modality, an amino acid substitution according to the first and / or second modality is a non-conservative or unfavorable amino acid substitution. A non-conservative or unfavorable amino acid substitution refers to the substituting amino acid (i.e., the one that replaces the original amino acid) having biochemical properties that are different from those of the original amino acid. For example, a polar amino acid is substituted for a non-polar amino acid or vice versa. A non-conservative or unfavorable amino acid substitution also refers to the substitution with an amino acid that is not present at the corresponding positions in other natural lectin proteins when aligned in pairs or in multiple sequences. In one modality, the non-conservative or unfavorable amino acid substitution alters the cytotoxic effect and / or the solubility of the modified lectin protein compared to a control.In one modality, the altered cytotoxic effect and / or solubility is determined with respect to the lectin protein of SEQ ID NO. 1 and in another modality the cytotoxic effect and / or solubility is determined with respect to the lectin protein of SEQ ID NO. 2. In one embodiment, the substituent amino acid at the primary carbohydrate binding site is selected from one or more of: a. in position 27: conservative, favorable or unfavorable amino acid, where the conservative amino acid is nonpolar or acidic; the favorable one is polar or basic and the unfavorable amino acid is nonpolar; b. at position 28: conservative, favorable, neutral or unfavorable amino acid, where the conservative amino acid is nonpolar; the favorable one is polar, the neutral one is acidic or basic and the unfavorable amino acid is polar; c. in position 47: unfavorable amino acid, which is basic or nonpolar; d. in position 48: unfavorable amino acid, which is nonpolar; e. at position 70: unfavorable amino acid, which is nonpolar; f. in position 71: unfavorable amino acid, which is nonpolar; g. at position 72: unfavorable amino acid, which is nonpolar; and / or h. at position 105: conservative, favorable, neutral or unfavorable amino acid, where the conservative amino acid is basic or nonpolar; the favorable one is polar, the neutral one is acidic, basic or polar and / or the unfavorable amino acid is polar, nonpolar or acidic. In particular, the substituent amino acid at the primary carbohydrate binding site is selected from one or more of: a. glycine (Y27G), tryptophan (Y27W), phenylalanine (Y27F), glutamic acid (Y27E) or histidine (Y27H) at position 27; and / or glycine (A28G), tryptophan (A28W), serine (A28S), aspartic acid (A28D) or histidine (A28H) at position 28; b. leucine at position 47 (S47L); and / or tryptophan at position 48 (G48W); c. isoleucine at position 70 (H70I); tryptophan at position 71 (N71W); and / or glycine at position 72 (Y72G); and d. phenylalanine (R105F), glutamine (R105Q), glutamic acid (R105E), leucine (R105L), lysine (R105K), alanine (R105A), serine (R105S), valine (R105V), isoleucine (R105I), proline (R105P), methionine (R105M), glycine (R105G), threonine (R105T), tyrosine (R105Y), tryptophan (R105W), asparagine (R105N), cysteine ​​(R105C), aspartic acid (R105D) or histidine (R105H) at position 105. In one embodiment, the substituent amino acid at the secondary carbohydrate attachment site is selected from one or more of: a. at position 77: an unfavorable amino acid that is nonpolar; nfrcezn / Lznz / q / Yl· b. at position 78: an unfavorable amino acid that is nonpolar; c. at position 80: an unfavorable amino acid that is nonpolar; d. at position 101: a favorable, unfavorable or neutral amino acid, wherein the favorable amino acid is polar or basic, the unfavorable amino acid is nonpolar and the neutral amino acid is nonpolar or acidic; e. at position 112: an unfavorable amino acid that is nonpolar; f. in position 114: an unfavorable amino acid that is polar. In particular, the substituent amino acid at the secondary carbohydrate attachment site is selected from one or more of: a. phenylalanine at position 77 (D77F), glycine at position 78 (I78G) and tryptophan at position 80 (T80W); b. phenylalanine (R101F), glutamine (R101Q), methionine (R101M), glutamic acid (R101E); and lysine (R101K) at position 101; c. glycine at position 112 (Y112G) and / or asparagine at position 114 (V114N). It is preferred that the modified lectin protein containing an amino acid substitution according to the first and / or second modality have a cytotoxic effect. In one modality, the cytotoxic effect is determined using an SRB assay. In a particularly preferred modality, the modified lectin protein has a percentage of cytotoxicity that is greater than or equal to that of a control. In one modality, the control is a lectin protein from SEQ ID NO. 1. In an alternative modality, the control is a lectin protein other than SEQ ID NO. 1. In one modality, the percentage of cytotoxicity of the modified lectin protein represents a 20% increase compared to that of the control. In a preferred modality, this represents a 45% increase.In alternative formulations, the percentage of cytotoxicity of the modified lectin protein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher compared to that of the control. Furthermore, it is preferred that the modified lectin protein containing an amino acid substitution, according to the first and / or second formulation, be soluble or partially soluble. Modification at the N-terminal end In a third embodiment of the present invention, the modified lectin protein comprises an amino acid substitution at position 1 and / or 2 of SEQ ID NO. 1 or a corresponding position in a sequence having at least 60% homology with it. It is preferred that the substituting amino acid (i.e., the one replacing the original amino acid) at position 1 is not valine or threonine. In particular, it is preferred that the substituting amino acid at position 1 have a small side chain. Preferably, the substituting amino acid is selected from one of: alanine, glycine, proline, or serine. In a further embodiment, the substituting amino acid at position 2 is tryptophan. In an alternative embodiment, the substituting amino acid at position 2 is a different nonpolar amino acid. In one embodiment, the modified lectin protein contains the amino acid substitutions as defined above at positions 1 and 2 of SEQ ID NO.1 or the corresponding positions of a sequence that has at least 60% homology with it. In a preferred embodiment, the substituent amino acids at positions 1 and 2 are alanine and tryptophan, respectively. nncrzn / i znz / zi / Yl· It is preferred that the amino acid sequence of the lectin protein modified according to the third modality increase the cleavage of an N-terminal (initiator) methionine compared to a control. In one modality, the control is an amino acid sequence from SEQ ID NO. 1, and in another modality, the control is from SEQ ID NO. 2. In one modality, the cleavage of the initiator methionine is catalyzed by the enzyme methionine aminopeptidase (MAP). The degree of cleavage of the initiator methionine is determined using a method known to a person skilled in the art, preferably by mass spectrometry or high-performance liquid chromatography (HPLC). Without wishing to adhere strictly to theory, it is believed that the degree of cleavage of the initiator methionine by MAP is affected by the amino acid residue at the first and / or second position after the initiator methionine (e.g., position 1 and / or 2 of SEQ ID NO. 1).In particular, an amino acid sequence comprising an amino acid residue with a small side chain in the first position after the initiator methionine is believed to increase the degree of cleavage of the initiator methionine (as discussed above). Furthermore, it is preferred that the lectin protein modified according to the third modality be soluble and / or have a cytotoxic effect. Most preferably, the modified lectin protein is soluble and has a cytotoxic effect. In one modality, the cytotoxic effect is determined using an SRB assay. In one modality, the modified lectin protein exhibits a percentage of cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a control. In another modality, the modified lectin protein has a percentage of cytotoxicity that is less than 10% of a control, or has no cytotoxicity. In a further modality, the percentage of cytotoxicity is at least 60%, 70%, 80%, or 90% of that of a lectin protein of SEQ ID No. 1. In one variant of the third embodiment, the amino acid substitution at the N-terminus is at a position other than position 1 and / or 2 of SEQ ID NO. 1 or the corresponding position in a sequence that has at least 60% homology with it. In one embodiment, the modified lectin protein contains an amino acid substitution in the first 10% or 5% of the amino acid sequence (other than positions 1 and / or 2). Reduced dimer formation The naturally occurring S. rolfsiide protein has been reported to exist as a monomer under acidic conditions and form a dimer at neutral or basic pH (Leonidas et al., 2007). Without adhering strictly to theory, it is believed that the cysteine ​​residue at position 76 of SEQ ID NO. 1 mediates dimer formation through the formation of a disulfide bond. In certain formulations, it is preferable to reduce dimer formation so that only one form of the protein (i.e., the monomeric form) is present. Therefore, in a fourth embodiment of the present invention, the modified lectin protein contains an amino acid substitution at position 76 of SEQ ID NO. 1 or a corresponding position in a sequence having at least 60% homology with it, such that the amino acid residue at that position is no longer cysteine. In a preferred embodiment, the substituent amino acid (i.e., the one that replaces the original amino acid) at position 76 is glycine. In an alternative embodiment, the substituent amino acid at position 76 is a different nonpolar amino acid residue. In one embodiment, the substituent amino acid is selected based on the sequence alignment of fungal lectin protein nfrcezn / Lznz / q / Yl· as reported in Figure 6 of Leonidas et al., 2007. Therefore, in one embodiment, the nonpolar substituent amino acid is selected from either valine or leucine. The modified lectin protein containing the amino acid substitution according to the fourth modality exhibits reduced dimer formation compared to a lectin protein of SEQ ID No. 1. In one modality, the level of dimer formation is determined using mass spectrometry. In an alternative modality, the level of dimer formation is determined using size exclusion chromatography or SDS-PAGE analysis. It is preferred that the lectin protein modified according to the fourth modality be soluble and / or have a cytotoxic effect. More preferably, the modified lectin protein is soluble and has a cytotoxic effect. In one modality, the cytotoxic effect is determined using an SRB assay. In one modality, the modified lectin protein exhibits a percentage of cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a control.In one modality, the modified lectin protein has a cytotoxicity percentage that is less than 10⁷% of a control, or no cytotoxicity. In a further modality, the cytotoxicity percentage is at least 60⁷%, 80⁷%, or 90⁷% that of a control. In one modality, the control is a lectin protein from SEQ ID NO. 1, and in another modality, the control is SEQ ID NO. 2. In one variant of the fourth embodiment, the amino acid substitution that reduces dimer formation is at a position other than position 76. For example, without wishing to adhere strictly to the theory, in one embodiment, alternative bonds other than a disulfide bond contribute to dimer formation, and an alternative amino acid substitution is used to disrupt the formation of these bonds and thus reduce dimer formation. Reduced oxidation In a fifth embodiment of the present invention, the modified lectin protein contains an amino acid substitution at position 89 of SEQ ID NO. 1 or a corresponding position of an amino acid sequence having at least 60° homology with it. In a further embodiment, the modified lectin protein contains an amino acid substitution at position 44 and / or 89 of SEQ ID NO. 1 or a corresponding position of an amino acid sequence having at least 60° homology with it. In a preferred embodiment, the substituent amino acid (i.e., the one that replaces the original amino acid) at position 89 is valine. In an alternative embodiment, the substituent amino acid at position 89 is a different nonpolar amino acid residue. In one embodiment, the substituent amino acid is selected based on the alignment of fungal lectin protein sequences as reported in Figure 6 of Leonidas et al., 2007.Therefore, in one embodiment, a nonpolar amino acid residue is selected from either leucine or isoleucine. In a further embodiment, the modified lectin protein contains an amino acid substitution at position 44 as defined above for position 89. In one embodiment, the modified lectin protein contains an amino acid substitution at both positions 44 and 89 as defined above. Without adhering to any specific theory, it is believed that the methionine residues at positions 44 and / or 89 of SEQ ID NO. 1 are susceptible to oxidation, resulting in the formation of methionine sulfoxide (nncrzn / i znz / zi / Yl·) at these positions. The oxidation of the methionine residues to methionine sulfoxide has the potential to alter the biological activity of the lectin. Therefore, in certain modality, it is preferable to reduce the oxidation of the lectin protein. Accordingly, the modified lectin protein containing the amino acid substitution as defined in modality 5 exhibits reduced oxidation compared to a lectin protein of SEQ ID NO. 1. In one modality, the level of oxidation is determined using mass spectrometry. In an alternative modality, the level of oxidation is determined using RP-HPLC.Furthermore, it is preferred that the lectin protein modified according to the fifth modality be soluble and / or have a cytotoxic effect. Most preferably, the modified lectin protein is soluble and has a cytotoxic effect. In one modality, the cytotoxic effect is determined using an SRB assay. In one modality, the modified lectin protein exhibits a percentage of cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a control. In another modality, the modified lectin protein has a percentage of cytotoxicity that is less than 10% of a control, or has no cytotoxicity. In a further modality, the percentage of cytotoxicity is at least 60%, 70%, or 90% of that of a control. In one modality, the control is a lectin protein from SEQ ID NO. 1, and in another modality, the control is SEQ ID NO. 2. In one variant of the fifth modality, the amino acid substitution that reduces oxidation is located at a position other than position 44 and / or 89. For example, without adhering strictly to the theory, in one modality, alternative amino acid residues such as cysteine, tryptophan, tyrosine, and histidine amino acid residues contribute to the oxidation of the lectin protein. Therefore, an alternative amino acid substitution is employed to limit oxidation at these sites and thus reduce the overall oxidation of the protein. Additional features In the first five embodiments described above, the amino acid modification is an amino acid substitution. However, in a variant of any of the first five embodiments, the amino acid modification is a modification other than an amino acid substitution. In one variant embodiment, the amino acid modification is the addition or deletion of an amino acid at a particular position in the amino acid sequence. In another embodiment, the amino acid addition is the addition of at least 1, 2, 3, 4, or 5 amino acids at a particular position in an amino acid sequence. In the first five embodiments described above, the modified lectin protein is preferably designated to have a cytotoxic effect. In an additional embodiment relevant to any of the first five embodiments, the modified lectin has an additional biological function (besides having a cytotoxic effect).In one modality, the biological function is related to the specificity of the lectin protein modified by an antigen. In the first through fifth embodiments described above, the modified lectin protein may contain an amino acid modification in an amino acid sequence that has at least 60% homology with SEQ ID NO. 1. In an additional embodiment that is relevant to any of the first through fifth embodiments, the amino acid sequence has at least 70%, 75%, 80%, or 85% homology with the amino acid sequence of SEQ ID NO. 1. It is particularly preferred that the amino acid sequence nncrzn / i znz / zi / Yl· have at least 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO. 1. It should be understood that the modified lectin protein may contain a combination or a plurality of any of the amino acid modifications described above in relation to the first through fifth modalities. In a further embodiment of the present invention, a pharmaceutical composition is provided comprising a modified lectin protein as described above. The pharmaceutical composition further comprises a pharmaceutically acceptable diluent or excipient. Illustrative diluents and excipients include sterile water, physiological saline solution, and phosphate buffer. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic ingredient. Additional details of additional components of the pharmaceutical composition can be found in Remington's Pharmaceutical Sciences and US Pharmacopoeia, 1984, Mack Publishing Company, Easton, PA, USA. During use, the modified lectin protein, as described above (hereinafter, the drug), is administered to a patient requiring treatment. In one modality, an appropriate dose of the drug is 0.1 to 1 mg / kg. In one modality, the patient suffers from cancer. In one modality, the cancer is selected from one of the following: ovarian cancer, leukemia, and / or colon cancer. In principle, any mode of administration of the drug may be used. In one modality, the drug is administered by one of the following methods: injection, spray, or inhalation. In one modality, the modified lectin protein, as described above, is used in the detection of a cancer cell. In one modality, the modified lectin protein as described above is used in a diagnostic method; preferably, in a cancer diagnostic method. In a further embodiment, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a modified lectin protein as described above. In one embodiment, the nucleic acid molecule includes any change in the nucleotide sequence including, but not limited to, substitution, deletion, and / or addition. It should be noted that, due to the degeneracy of the genetic code, nucleic acid molecules encoding a particular modified lectin variant can have a range of nucleotide sequences. For example, all codons GCA, GCC, GCG, and GCT encode the amino acid alanine. The nucleic acid molecules can be DNA, RNA, or derivatives of either. In one embodiment, the present invention relates to a recombinant DNA molecule comprising a vector. Preferably, the vector is a plasmid or a viral vector. In one embodiment, a recombinant vector is provided comprising a nucleic acid molecule insert comprising a nucleotide sequence encoding a modified lectin as described above. In a further embodiment, the recombinant vector is an expression vector and operatively comprises, in the 5' to 3' direction: a promoter functional in a host cell; a structural nucleic acid sequence nfrcezn / Lznz / q / Yl· encoding a modified lectin protein as described above; and a termination signal. In a further embodiment of the present invention, a process is provided for producing a recombinant lectin protein from Sclerotium rolfsii, and in particular, a modified lectin protein as described above. In one embodiment, the cloned nucleotide sequences encode modified lectin proteins that are close to the amino acid sequence of naturally occurring lectins but provide alternative properties. Alternatively, the nucleotide sequences encoding the modified lectin variants can be synthesized using chemical or recombinant means and expressed in a suitable host to obtain the recombinant proteins. Suitable host cells include prokaryotic cells and both lower and higher eukaryotic cells. The introduction of the recombinant molecule into the host cells can be effected using methods known in the art.In an illustrative embodiment of the present invention, the suitable host is a microbial cell. In a preferred embodiment, the microbial cell is selected from the group consisting of, but not limited to, a yeast cell, Escherichia coli, an insect cell line, or a mammalian cell line. The recombinant proteins as described above can be obtained by isolation as an expression product from a recombinant host. The recombinant proteins of the present invention, in one embodiment, are purified by conventional techniques, typically conventional erythrocyte methods. In another illustrative embodiment of the present invention, the molecular mass of the recombinant lectins, as determined by SDS-PAGE, is approximately 16,000 Da. In yet another illustrative embodiment of the present invention, the recombinant lectins may possess blood group specificity, typically for human blood groups. In yet another illustrative embodiment of the present invention, the recombinant proteins may have a unique ability to recognize the TF antigen and its cryptic forms. Therefore, although this invention has been described with reference to illustrative embodiments, this description is not intended to be interpreted in a limiting manner. Several modifications of the illustrative embodiments, as well as other embodiments of the invention, will be evident to those skilled in the art with reference to this description. It is therefore contemplated that the present invention will cover any such modifications or embodiments that fall within the true scope of the invention. EXAMPLES Example 1: Site-directed mutagenesis The amino acid sequence of the naturally occurring S. rolfsii lectin sequence (SEQ ID NO. 1) was modified at the different specific positions described below in Table 1 by site-directed mutagenesis. A plasmid was extracted from E. coli BL21 DE3 cells, encoding the amino acid sequence of the naturally occurring SRL sequence cloned into a pET20b vector, and used as a template. A 50 µL PCR reaction was set up containing 10 µL of 5X Q5 nncrzn / i znz / zi / Yl· Reaction Buffer, 1 µL of 10 mM dNTP, 2.5 µL of 10 µM Forward Primer, 2.5 µL of 10 µM Reverse Primer, 0.5 µL of Q5 high fidelity DNA polymerase enzyme and 20 ng of total template volume, to be completed to 50 µL with distilled water. The gene of interest was amplified using PCR with an initial denaturation step at 98 °C for 30 seconds, followed by 35 amplification cycles consisting of a denaturation step at 98 °C for 30 seconds, an annealing step at 55 °C for 30 seconds, and an extension step at 72 °C for 30 seconds. Finally, an additional extension step was performed at 72 °C for 5 minutes. All PCR reactions were performed using a Mastercycler Pro. The PCR products were analyzed on 1.2% agarose gels containing ethidium bromide (EtBr). Table 1: List of primers used for the construction of different lectin variants derived from the naturally occurring SRL sequence: nncczn / i ζηζ / ζι / γ a. Clone variants for efficient cleavage of the initiator methionine Clone variant Change Direct primer Reverse primer ULLB-0005 / 001 T1S ATATACATATGAGCTATAAAATTACCG TATGCTAGTTATTGC TCAGCGGT ULLB-0005 / 002 T1A ATATACATATGGCGTATAAAATTACCG ULLB-0005 / 003 T1P ATATACATATGCCGTATAAAATTACCG ULLB-0005 / 004 T1G ATATACATATGGGCTATAAAATTACCG ULLB-0005 / 005 T1A, Y2W ATATACATATGGCGTGGAAAATTACCG b. Clone variants to alter the primary carbohydrate binding site Clone variant Change Forward primer Reverse primer ULLB-0005 / 008 Y27G, A28W GTGTGGAAAGGCTGGAATGGCGGTAC GTACCGCCATTCCAGCCTTTCCACAC ULLB-0005 / 009 S47L, G48W GATGGGTGGTCTGTGGACCAGCGG CCGCTGGTCCACAGACCACCCATC ULLB-0005 / 010 H70I, N71W, Y72G CCTTTGGTGTGATTTGGGGCAAACGGT GGTG CACCAGCGTTTGCCCCCAAATCACACCA AAGG ULLB-0005 / 011 R105F CGAAGAAGCGTTTGAACGCCAG CTGGCGTTCAAACGCTTCTTCG c. Clone variants to alter the secondary carbohydrate binding site Clone variant Change Forward primer Reverse primer ULLB-0005 / 012 D77F, I78G, T80W CTGGTGTTTTGGCGTGTGGAACCTGGC AGCGGATGAAAC CAGGTTCCACACGCCAAAACACCAGCG TTTATAATTATGC ULLB-0005 / 013 R101F GTCAGAAAAACTTTGAAGAAGCGC GCGCTTCTTCAAAG IIIII UI GAC ULLB-0005 / 014 Y112G, V114N GGCCAGAACAAAAATGCGAAAGGCCGT AAC GTTCTGGCCGTTACTCAGCTGGCGTTC d. Clone variants for the prevention of dimer formation and protein oxidation Clone variant Direct primer change Reverse primer ULLB-0005 / 015 C76G CGCTGGGGCGATATTGTGACC GGTCACAATATCGCCCCAGCG ULLB-0005 / 016 M89V GAAACCGGCGTGGTTTATTAATCAG CTGATTAATAACCACGCCGGTTTC nfrcezn / Lznz / q / YL Example 2: Restrictive digestion PCR products (obtained from example 1) or plasmids were digested with the restriction enzymes Ndel and BamHI, using 500 ng of PCR product / plasmid, 1 μL of 10x CutSmart buffer, and 1-2 units of Ndel and 1-2 units of BamHI, to a final volume of 10 μL. The reaction was incubated at 37°C for 45 minutes to 1 hour, and the digestion results were observed on a 1.2% agarose gel containing ethidium bromide (EtBr). Next, the DNA was extracted and purified from agarose gels. Example 3: Ligation of the pET vector and confirmation of transformants by colony PCR The ligation reaction with the pET vector was performed using a mixture consisting of 100 ng of DNA sample (restriction enzyme digested product from Example 2), 50 ng of digested pET vector, 1 µL of 10X T4 DNA ligase buffer, and 1 µL of T4 DNA ligase enzyme, to a final volume of 10 µL. This reaction was carried out at 22 °C for 1 hour. The ligation mixture was transformed into competent E. cali DH5a cells using the heat shock method. The cells were then seeded on LA / Kanamycin plates and incubated overnight at 37 °C. The transformants were then PCR-assayed to verify the integrity and absence of insert damage using forward and reverse pET primers under the following PCR conditions. The PCR program included an initial denaturation step at 95 °C for 10 minutes, followed by 35 amplification cycles consisting of a denaturation step at 95 °C for 30 seconds, an annealing step at 55 °C for 30 seconds, and an extension step at 72 °C for 45 seconds. Finally, an additional extension step was performed at 72 °C for 10 minutes.The PCR reaction consisted of a bacterial colony (DNA template), 10 μM of pET forward primer, 10 μM of pET reverse primer, 5 μL of EconoTaq PLUS GREEN 2X Master Mix (Lucigen), and distilled water to a final volume of 10 μL. The PCR products were analyzed on a 1.2% agarose gel containing EtBr. Example 4: Plasmid DNA extraction and expression analysis Positive transformants were inoculated into LB / Kanamycin liquid cultures, and plasmid DNA was subsequently extracted from E. coli. All constructs prepared in this work, with inserts in pET vectors, were confirmed by sequencing. The pET27b vector, containing each modified nucleotide sequence encoding a lectin variant, was transformed into E. coli BL 21DE3 GOLD cells. Positive clones were selected by expression analysis in self-induction medium. The level and size of recombinant lectin expression were confirmed by SDS-PAGE analysis. Glycerol pools from positive clones were prepared and maintained at -80 °C. The glycerol stock solution (40 µL) was inoculated into 50 mL of LB broth (containing 20 pg / mL of kanamycin) and incubated at 37°C at 140 rpm for 16 hours. The 1% culture was inoculated into 200 mL of production medium comprising 1% (w / v) yeast extract, 1.2% (w / v) dextrose, 0.3% (w / v) KH₂PO₄, 1.25% (w / v) K₂HPO₄, 0.5% (w / v) (NH₄)₂SO₄, 0.05% (w / v) NaCl, 0.1% (w / v) MgSO₄·7H₂O, and 0.1% (v / v) trace metal solution. Kanamycin was added to a final concentration of 20 µg / mL. The flasks were incubated at 37 °C and 140 rpm. When the culture DOe⁻ reached 1.5, the temperature was reduced to 18 °C and the culture was incubated for an additional 1 hour. The culture was then induced with 0.25 mM IPTG and incubated at 18 °C for 20 hours. Culture samples before and after induction were analyzed for protein expression and solubility by SDS-PAGE.The culture broth was centrifuged at 9000 rpm for 15 minutes at 15 °C. The resulting pellet was suspended in lysis buffer (25 mM Tris, 1 mM EDTA, pH 8.0). The cells were homogenized under high pressure at 18,000 psi (124,100 kPa). The lysate was clarified using 0.1-micrometer hollow fiber pre-equilibrated with lysis buffer. The clarified protein solution was subjected to ion exchange chromatography to purify the recombinant lectin. Example 5: Biological assay - Antiproliferative activity of purified lectin against the ovarian cancer cell line (PA-1) The antiproliferative activity of purified recombinant lectin variants was determined using a sulforhodamine B (SRB) assay. Lectin proteins are thought to exert a cytotoxic effect on the PA-1 ovarian cancer cell line by binding to TF / Tn antigens; therefore, the assay can also provide information on lectin protein specificity. Antiproliferative activity further indicates lectin protein stability, as the protein conformation is thought to be maintained to sustain activity. The assay measured total biomass by staining cellular proteins with SRB. SRB is a bright pink aminoxanthene dye that can form an electrostatic complex with basic amino acid residues of trichloroacetic acid-fixed cell proteins under mildly acidic conditions. It can dissociate under mildly basic conditions and can be solubilized for measurement.It has been widely used for detecting drug toxicity against different types of cancerous and non-cancerous cell lines. The cells were briefly washed, fixed, and stained with the dye. The incorporated dye was then released from the cells using a Tris base solution. The amount of incorporated dye released from the stained cells was directly proportional to the cell biomass and could be measured to indicate the degree of cytotoxicity caused by the test material. The cytotoxicity of the cells was monitored / measured when the cells were in the logarithmic growth phase. Tests were performed in a final volume of 200 µL and included a 200 µL control sample of cell-free medium to be used as a blank for absorbance readings. Dilutions of the assay were performed in serum-free medium to reduce background, and a 10-fold dilution of the desired concentration was prepared. On day one, cells were seeded using an initial trypsinization step, counted using the trypan blue method in a Neuebauer chamber, and seeded into the wells of a 96-well flat-bottom plate (dark-walled plate) at a density of 5000 cells / well. On day two, after overnight incubation, the plate medium was filled to 180 pL / well, and the cells were then treated with 20 µL of each test element at concentrations ranging from 2.5 to 80 pg / ml, so that the total volume in each well was 200 µL. The plates were incubated from day two to day four. On day five, the cells were treated with SRB. The plates were then examined under a microscope under sterile conditions. The cells were fixed by gently layering 50 µL of a 50% trichloroacetic acid (TCA) solution (cold) on top of the growth medium. The plates were not moved after the fixation step to avoid cell displacement, which would result in inaccuracies. These plates were then incubated for 1 hour at 4°C and subsequently washed with purified water four times to remove excess fixative and serum proteins. The plates were air-dried. Some of these plates were stored at room temperature for later use. The plates were then stained with SRB dye by adding 0.4% SRB dye solution (50 µL) to cover the culture surface of the wells, followed by incubation for 30 minutes at 28°C. The stain was then removed by decanting and subsequently rinsed with a washing solution (1% acetic acid). The plates were washed in 5 wash cycles until all unexposed stain was removed. The plates were then air-dried until no moisture was visible. For solubilization, 200 µL of SRB solubilization buffer (Tris 10 mm), equal to the original volume of the well, were added to the plate, followed by incubation at 28 °C for 5 minutes. The plate was then gently shaken for 5 to 10 minutes to dissolve the dye, and the absorbance was measured at 580 nm. Example 6: Amino acid modification of the naturally occurring sequence of S. rolfsii lectin The naturally occurring lectin sequence was modified to alter the physicochemical properties as described below. a) Improvement in the efficiency of initiator methionine cleavage: The high expression rate of recombinant proteins in E. coli limits the cleavage of the N-terminal methionine (initiator methionine) by the enzyme methionine aminopeptidase (MAP), resulting in a mixture of protein containing Met-lectin and Met-free lectin. An important factor affecting the cleavage of the initiator methionine is the amino acid sequence following the methionine residue. MAP cleaves all proteins with small side chains at the second amino acid position (i.e., the first amino acid residue after the initiator methionine). Proteins with valine or threonine residues following the initiator methionine are cleaved much less efficiently by MAP than those with alanine, glycine, proline, or serine at this position.Clones were constructed by replacing the first amino acid (Threonine) and the second (Tyrosine) at the N-terminus of the naturally occurring lectin sequence nncrzn / i znz / zi / Yl· with four different amino acids (Table 2) to verify the effect on the cleavage of the initiator methionine, solubility, specificity, and biological activity. The solubility, specificity, and biological activity of the lectin variants were not affected by the modifications. The recombinant lectin was purified from five variants of the naturally occurring lectin sequence engineered for efficient methionine cleavage at the shaker flask level. All variants of the naturally occurring lectin sequence, in which threonine at position 1 was replaced by alanine, glycine, serine, and proline, showed similar soluble expression compared to the control. The biological activity of all variants was also similar to that of the control. Therefore, changing the first and / or second amino acid in the lectin sequence did not affect expression or biological activity compared to the control. b) Prevention of dimer formation and protein oxidation: The cysteine ​​residues present in the S. rolfsii lectin monomer are thought to contribute to dimer formation via disulfide bonding and may therefore affect the lectin's specificity and biological activity. The methionine residue in the S. rolfsii lectin is thought to be prone to oxidation during the purification process. The cysteine ​​and methionine residues at positions 76 and 89 were replaced by amino acid substitution to prevent dimer formation and protein oxidation, respectively. The ULLB-0005 / 015 variant of the naturally occurring lectin sequence, where the cysteine ​​at position 76 was replaced with glycine to prevent dimer formation, expressed recombinant lectin in a soluble form without affecting biological activity compared to the control.Similarly, the ULLB-0005 / 016 variant of the naturally occurring lectin sequence, in which methionine at position 89 was replaced with valine to prevent protein oxidation, was expressed in soluble form without affecting biological activity compared to the control. Therefore, replacing cysteine ​​and methionine at positions 76 and 89 with glycine and valine, respectively, does not affect the expression, solubility, specificity, or biological activity of the lectin. nncczn / i znz / zi / Yl· Table 2. Summary of clones engineered for efficient cleavage of the initiator methionine, prevention of dimer formation, and prevention of protein oxidation Changes made for efficient cleavage of the initiator methionine Clone Variant Sequence Change Theoretical Molecular Weight (Dalton) Theoretical Isoelectric Point Expression Biological Assay (PA-1) (% Cytotoxicity) Methionine Content (Met) (% Abundance) Control TYKIT....... 16044.73 6.47 Soluble 31.6 - 58 7.31 ULLB- 0005 / 001 SYKIT...... 16029.70 6.49 Soluble 35.13 1.99 ULLB- 0005 / 002 AYKIT...... 16013.70 6.57 Soluble 33.47 9.32 ULLB- 0005 / 003 PYKIT........ 16039.74 6.61 Soluble 47.2 6.99 ULLB- 0005 / 004 GYKIT........ 15999.66 6.57 Soluble 45 1.12 ULLB- 0005 / 005 AWKIT........ 16036.74 6.57 Soluble 34.29 15.6 i. Changes made to prevent dimer formation and protein oxidation Clone Variant Sequence Change Theoretical Molecular Weight (Dalton) theoretical pl Expression Biological Assay (PA-1) (% cytotoxicity) ULLB-0005 / 015 C76G 15997.64 6.47 Soluble 30 ULLB-0005 / 016 M89V 16011.67 6.47 Soluble 34 (Control) NA NA 6.47 Soluble 31.6-58 nncrzn / iznz / q / Yi The naturally occurring lectin sequence was also modified to alter biological activity as described below. c) Modification of the primary carbohydrate binding site. The primary carbohydrate binding site shows specificity for the TF (Gaipi^3GalNAc-aSer / / Thr) antigen expressed on cancer cells. The amino acids at the primary carbohydrate binding sites were altered, and the modified lectin proteins were investigated for solubility, specificity, and biological activity. Regarding the effect on specificity and biological activity, the cytotoxicity of the modified lectin proteins was evaluated against an ovarian cancer cell line (PA-1). The amino acid modifications made at the primary carbohydrate binding site are illustrated in Table 3. The ULLB-0005 / 008 (Y27G and A28W) and ULLB0005 / 011 (R105F) variants of the naturally occurring lectin sequence expressed recombinant lectin in a soluble form; however, biological activity was lost.While the ULLB-0005 / 010 variant (H701, N71W, and Y72G) was expressed as partially soluble, and its cytotoxicity was higher than that of the recombinant lectin in the ULLB-0005 / 009 variant (S47L and G48W), it was expressed as inclusion bodies. Therefore, the change in the primary carbohydrate binding site affects both the solubility and the biological activity of the lectin. The data from this example demonstrate that amino acid residues at positions 27, 28, 47, 48, 70, 71, 72, and 105 define the primary carbohydrate binding site. It was concluded that the primary carbohydrate binding site is involved in binding to the TF antigen present on the surface of cancer cells. Table 3. Summary of clones engineered to alter primary carbohydrate binding sites Clone variant Change in amino acid sequence Theoretical molecular weight Theoretical isoelectric point Expression Biological assay (PA-1) (% cytotoxicity) ULLB-0005 / 008 Y27G, A28W 16052.74 6.47 Soluble No activity ULLB-0005 / 009 S47L, G48W 16198.97 6.47 IB formation No activity ULLB0005 / 010 H70I, N71W, Y72G 15985.73 6.38 Partially soluble 52 ULLB-0005 / 011 R105F 16034.72 6.16 Soluble No activity Control Not applicable 16044.73 6.47 Soluble 31.6 to 58 d) Modification of the secondary carbohydrate binding site: The secondary carbohydrate binding site involved in binding to GalNAc-α- (Tn antigen) was modified. The amino acids at the secondary carbohydrate binding sites were altered, and the modified lectin proteins were investigated for solubility, specificity, and biological activity. The effect on specificity and biological activity against the ovarian cancer cell line (PA-1) was evaluated. The amino acid modification performed at the secondary carbohydrate binding site is shown in Table 4 and affects the solubility, specificity, and / or biological activity of the recombinant lectin. Secondary binding sites were selected from 77, 78, 80, 101, 112, or 114 and modified to substitute D, I, T, R, Y, and V with F, G, W, F, G, and N, respectively, to prepare a number of novel variants from the naturally occurring lectin sequence. The ULLB-0005 / 012 (D77F, I78G, and T80W) and ULLB-0005 / 014 (Y112G and V114N) variants, which were engineered to alter the secondary carbohydrate binding site, expressed recombinant lectin in the form of inclusion bodies and partially soluble bodies, respectively. An unfavorable amino acid substitution in the ULLB-0005 / 012 variant may contribute to the insoluble expression of this protein. The ULLB-0005 / 013 variant showed loss of antiproliferative activity against the PA-1 cell line, while the ULLB-0005 / 014 variant showed similar antiproliferative activity compared to the control clone against the PA-1 cell line.The data from this example demonstrate that amino acid residues at positions 77, 78, 80, 101, 112, and 114 define the secondary carbohydrate binding site. It was concluded that modification of the secondary carbohydrate binding site affects both the solubility and the biological activity of the lectin. Table 4. Summary of clones engineered to alter secondary carbohydrate binding sites Clone variant Sequence change Theoretical molecular weight Theoretical isoelectric point Expression Biological assay (PA-1) (% cytotoxicity) ULLB-0005 / 012 D77F, I78G, T80W 16104.82 6.90 IB formation Not performed ULLB-0005 / 013 R101F 16034.72 6.16 Soluble No activity ULLB-0005 / 014 Y112G, V114N 15952.58 6.47 Partially soluble 42.3 Control Not applicable 16044.73 6.47 Soluble 31.6 - 58 nncczn / i ζηζ / ζι / γ Example 7: New site-directed mutagenesis Similar to Example 1, the amino acid sequence of the lectin sequence variant, SEQ ID NO. 2, was modified at the different specific positions as mentioned below in Table 5 of this description by site-directed mutagenesis. Table 5: List of primers used for the construction of different lectin variants derived from the lectin sequence variant, SEQ ID NO. 2. a. Clone variants to alter the primary carbohydrate binding site Variante del clon Cambio Cebador directo Cebador inverso ULLB-0005 / 026 Y27W GTGTGGAAATGGGCGAATGGC GCCATTCGCCCATTTCACAC ULLB-0005 / 027 Y27F GTGTGGAAATTTGCGAATGGC GCCATTCGCAAATTTCCACAC ULLB-0005 / 028 Y27E GTGTGGAAAGGAAGCGAATGGC GCCATTCGCTTCTTCCACAC ULLB-0005 / 029 Y27H GTGTGGAAACATGCGAATGGC GCCATTCGCATGTTTCCACAC ULLB-0005 / 030 A28S GGAAATATAGCAATGGCGGTACC GGTACCGCCATTGCTATATTTC ULLB-0005 / 031 A28G GGAAATATGGCAATGGCGGTACC GGTACCGCCATTGCCATATTTCC ULLB-0005 / 032 A28D GGAAATATGATAATGGCGGTACC GGTACCGCCATTATCATATTTCC ULLB-0005 / 033 A28H GGAAATATCATAATGGCGGTACC GGTACCGCCATTATGATATTTCC ULLB-0005 / 018 R105Q GAAGAAGCGCAGGAACGCCAG CTGGCGTTCCTGCGCTTCTTC ULLB-0005 / 019 R105E GAAGAAGCGGAAAGCGCAG CTGGCGTTCCGCTTCTTC ULLB-0005 / 020 R105L GAAGAAGCGCTGGAACGCCAG CTGGCGTTCCAGCGCTTCTTC ULLB-0005 / 021 R105K GAAGAAGCGAAAAGCGCAG CTGGCGTTCCTGCGTTCTTC ULLB-0005 / 034 R105A GAAGAAGCGGCGGAACGCCAG CTGGCGTTCCGCGTTCTTC ULLB-0005 / 035 R105S GAAGAAGCGAGCGAACGCCAG CTGGCGTTCCGCTCGTCTTC ULLB-0005 / 036 R105VGAAGAAGCGGTGGAACGCCAG CTGGCGTTCCAGCGCTTCTTC ULLB-0005 / 037 R105I GAAGAAGCGATTGAACGCCAG CTGGCGTTCAATCGCTTCTTC ULLB-0005 / 038 R105P GAAGAAGCGGCGGAACGCCAG CTGGCGTTCCGGCGCTTCTTC Variante del clon Cambio Cebador directo Cebador inverso ULLB-0005 / 039 R105M GAAGAAGCGATGGAAGCCCAG CTGGCGTTCCATCGCTTCTTC ULLB-0005 / 040 R105G GAAGAAGCGGGCGAACGCCAG CTGGCGTTCGCCCGCTTCTTC ULLB-0005 / 041 R105T GAAGAAGCGACCGAACGCCAG CTGGCGTTCGGTCGCTTCTTC ULLB-0005 / 042 R105Y GAAGAAGCGTATGAAGCCCAG CTGGCGTTCATACGCTTCTTC ULLB-0005 / 043 R105W GAAGAAGCGTGGGAACGCCAG CTGGCGTTCCACGCTTCTTC ULLB-0005 / 044 R105N GAAGAAGCGAACGAACGCCA CTGGCGTTCGTTCGCTTTTC ULLB-0005 / 045 R105C GAAGAAGCGTGGGAACGCCAG CTGGCGTTCGCACGCTTCTTC ULLB-0005 / 046 R105D GAAGAAGCGGATGAAGCCCAG CTGGCGTTCATCCGCTTCTTC ULLB-0005 / 047 R105H GAAGAAGCGCATGAACGCCAG CTGGCGTTCATGCGCTTCTTC nncrzn / LZnZ / q / ΥΙΛΙ b. Variantes de clones para alterar el sitio de union del carbohidrado Secundario Clone variant Change Direct primer Reverse primer ULLB-0005 / 022 R101Q CAGAAAAACCAGGAAGAAGCGC GCGCI ICI ICCIGGI III ICIG ULLB-0005 / 023 R101M CAGAAAAACATGGAAGAAGCGC GCGCTTCTTCCATG III IICTG ULLB-0005 / 024 R101E CAGAAAAACGAAGAAGAAGCGC GCGCI ICI ICI ICGI III ICIG ULLB-0005 / 025 R101K CAGAAAAACAAAGAAGAGGCGC GCGCTTCTTCTTTGTTTTTCTG Example 8: Amino acid modification of the lectin sequence variant, the SEQ ID NO: 2. The sequence of natural origin of lectin was modified to alter the physico-chemical properties as described below. a. Modification of the primary carbohydrate binding site: The primary carbohydrate binding site shows specificity for the TF antigen (Galpl >3GalNAc-aSer / / Thr) expressed on cancer cells. The amino acids at the primary carbohydrate binding sites were altered, and the modified lectin proteins were investigated for solubility, specificity, and biological activity. Regarding the effect on specificity and biological activity, the cytotoxicity of the modified lectin proteins was evaluated against an ovarian cancer cell line (PA-1). The amino acid modifications made at the primary carbohydrate binding site are illustrated in Table 6. The naturally occurring lectin sequence variants, ULLB-0005 / 028 (Y27E), ULLB-0005 / 032 (A28D), ULLB-0005 / 018 (R105Q), ULLB-0005 / 035 (R105S), ULLB-0005 / 043 (R105W), and ULLB-0005 / 045 (R105C), expressed recombinant lectin in soluble form; however, biological activity was lost.The variants ULLB-0005 / 021 (R105K) and ULLB-0005 / 047 (R105H) were expressed in soluble form, and the cytotoxicity of these variants was considerably higher than that of the other variants. This may be due to the basic nature of the substituted amino acid. Similarly, the conservative substitution at A28 with nonpolar glargine (A28G - ULLB-0005 / 031) was also expressed in soluble form, and the cytotoxicity of this variant was considerably higher than that of the other variants. The variants ULLB-0005 / 019 (R105E) and LJLLB-0005 / 046 (R105D) were expressed as inclusion bodies. It is possible that the substitution of R105 with acidic amino acids contributed to the insoluble expression of this protein. The variants ULLB-0005 / 034 (R105A), ULLB-0005 / 037 (R105I), ULLB-0005 / 038 (R105P) and ULLB-0005 / 041 (R105T) were expressed in soluble form and were therefore unaffected compared to the control.Therefore, changes in the primary carbohydrate binding site affect both the solubility and biological activity of the lectin. Data from this example demonstrate that amino acid residues at positions 27, 28, and 105 define the primary carbohydrate binding site. It was concluded that the primary carbohydrate binding site is involved in binding to the TF antigen present on the surface of cancer cells. nfrcezn / Lznz / q / Yl· Table 6. Summary of clones designed to alter the primary carbohydrate binding sites nfrcezn / Lznz / q / γΐΛΐ Clone variant Change in amino acid sequence Theoretical molecular weight Theoretical isoelectric point Expression Biological assay (PA-1) (% cytotoxicity) ULLB-0005 / 026 Y27W 16066.77 6.47 Soluble 40 ULLB-0005 / 027 Y27F 16027.73 6.47 Soluble 50 ULLB-0005 / 028 Y27E 16009.67 6.17 Soluble No activity ULLB-0005 / 029 Y27H 16017.69 6.55 Soluble 26 ULLB-0005 / 030 A28S 16059.73 6.47 Soluble 43 ULLB-0005 / 031 A28G 16029.70 6.47 Soluble 57 ULLB-0005 / 032 A28D 16087.74 6.17 Soluble No activity ULLB-0005 / 033 A28H 16109.79 6.55 Soluble 49 ULLB-0005 / 018 R105Q 16015.67 6.16 Soluble No activity ULLB-0005 / 019 R105E 16016.66 5.91 Insoluble No activity ULLB-0005 / 020 R105L 16000.70 6.16 Soluble 25.38 ULLB-0005 / 021 R105K 16015.72 6.47 Soluble 53.99 ULLB-0005 / 034 R105A 15958.62 6.16 Soluble 39.29 ULLB-0005 / 035 R105S 15974.62 6.16 Soluble No activity ULLB-0005 / 036 R105V 15986.67 6.16 Soluble 31.2 ULLB-0005 / 037 R105I 16000.70 6.16 Soluble 37.89 ULLB-0005 / 038 R105P 15984.66 6.16 Soluble 42.28 ULLB-0005 / 039 R105M 16018.73 6.16 Soluble 54.48 ULLB-0005 / 040 R105G 15944.59 6.16 Soluble ND ULLB-0005 / 041 R105T 15988.65 6.16 Soluble 37.08 ULLB-0005 / 042 R105Y 16050.72 6.16 Soluble 52.5 ULLB-0005 / 043 R105W 16073.75 6.16 Soluble No activity ULLB-0005 / 044 R105N 16001.64 6.16 Soluble Not determined ULLB-0005 / 045 R105C 15990.68 6.16 Soluble No activity ULLB-0005 / 046 R105D 16002.63 5.90 Insoluble No activity ULLB-0005 / 047 R105H 16024.68 6.26 Soluble 52 Control Not applicable 16044.73 6.47 Soluble 31.6 to 58. Modification of the secondary carbohydrate binding site: The secondary carbohydrate binding site involved in binding to GalNAc-α- (Tn antigen) was modified. The amino acids at the secondary carbohydrate binding sites were altered, and the modified lectin proteins were investigated for solubility, specificity, and biological activity. The effect on specificity and biological activity against the ovarian cancer cell line (PA-1) was evaluated. The amino acid modification at the secondary carbohydrate binding site is shown in Table 7 and affects the solubility, specificity, and / or biological activity of the recombinant lectin. Secondary binding site 101 was modified by substituting R with Q, M, E, and K to prepare numerous novel variants from the naturally occurring lectin sequence.Favorable and neutral substitution at position 101 leads to the soluble expression of the variants ULLB-0005 / 022 (R101Q), ULLB0005 / 023 (R101M), ULLB-0005 / 024 (R101E), and ULLB-0005 / 025 (R101K), which exhibited similar antiproliferative activity compared to the control clone against the PA-1 cell line. Data from this example demonstrate that amino acid residues at position 101 define the secondary carbohydrate binding site. It was concluded that modification of the secondary carbohydrate binding site leads to the soluble expression of recombinant lectin and exhibits biological activity comparable to that of the control. nncczn / iznz / q / Yi Table 7. Summary of clones engineered to alter secondary carbohydrate binding sites Clone variant Change in sequence Theoretical molecular weight Theoretical isoelectric point Expression Biological assay (PA- 1) (% of cytotoxicity) ULLB-0005 / 022 R101Q 16015.67 6.16 Soluble 44.26 ULLB-0005 / 023 R101M 16018.73 6.16 Soluble 32.18 ULLB-0005 / 024 R101E 16016.66 5.91 Soluble 44.21 ULLB-0005 / 025 R101K 16015.72 6.47 Soluble 38.35 Control Does Not Apply 16044.73 6.47 Soluble 31.6-5 SUMMARY OF SEQUENCES SEQ ID NO. 1: TYKITVRVYQTNPNAFFHPVEKTVWKYANGGTWTITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGV HNYKRWCDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYEVKNAKGRNFEIVYTEAEGNDLHANLI IG SEQ ID NO. 2: TYKITVRVYQTNPDAFFHPVEKTVWKYANGGTWTITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGV HNYKRWCDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYQVKNAKGRNFQIVYTEAEGNDLHANLI IG SEQ ID NO. 3: VYKITVRVYQTNPDAFFHPVEKTVWKYANGGTWSITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGV HNYKRWCDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYQVKNAKGRNFQIVYTEAEGNDLHANLI IG SEQ ID NO. 4: VYKITVRVYQTNPDAFFHPVEKTVWKYADGGTWSITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGV HDYKRWCDIVTDLAADETGMVINQEYYSEKDREEARERQNSNYEVKDAKGRNFEIVYTEAEGNDLHADLII G This description refers to multiple approaches to the process, equipment, and systems that constitute this unique and integrated invention, and it is understood that many changes and modifications to the described embodiment can be made without departing from the scope and spirit of the invention. The accompanying examples illustrate specific approaches to the invention. These approaches are described in sufficient detail to enable those skilled in the art to implement the invention, and it should be understood that a person skilled in the art may make modifications to the various approaches described. Where the methods and steps described above indicate certain events occurring in a specific order, those skilled in the art will recognize that the order of certain steps may be modified and that such modifications are in accordance with the principles of the invention. Furthermore, certain steps may be performed simultaneously in a parallel process where possible, or they may be performed sequentially. nfrcezn / Lznz / q / γΐΛΐ

Claims

1. A modified lectin protein, wherein the modified lectin protein comprises an amino acid sequence selected from any of: i) SEQ ID NO. 1; or ii) an amino acid sequence having at least 60% homology with i), wherein the amino acid sequence of i) or ii) comprises at least one amino acid modification selected from one or more of the following (a) through (d): a) at least one amino acid modification at a carbohydrate-binding site of i) or ii); b) at least one amino acid modification at an N-terminal end of i) or ii); c) at least one amino acid modification at position 76; d) at least one amino acid modification at position 44 or 89, wherein the modified lectin protein does not consist of the amino acid sequence of any of SEQ ID NOS. 2 through 4.

2. The modified lectin protein as claimed in claim 1, wherein the modified lectin protein comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 97% or 99% homology with SEQ ID NO.

1.

3. The modified lectin protein as claimed in claim 1, wherein the modified lectin protein comprises at least one amino acid modification at a carbohydrate binding site of i) or i).

4. The modified lectin protein as claimed in claim 3, wherein the carbohydrate binding site is a primary and / or secondary carbohydrate binding site.

5. The modified lectin protein as claimed in claim 4, wherein the primary carbohydrate binding site comprises a position selected from one or more of: 27, 28, 47, 48, 70, 71, 72 and 105 in SEQ ID NO. 1 or a corresponding position in a sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it.

6. The modified lectin protein as claimed in claim 5, wherein the position of the amino acid modification is selected from one or more of: i) 27 and / or 28; ii) 47 and / or 48; ii) 70, 71 and / or 72; and / or iv) 105.

7. The modified lectin protein as claimed in claim 4, wherein the secondary carbohydrate binding site comprises a position selected from one or more of: 77, 78, 80, 101, nncrzn / i znz / zi / Yl· 112 and 114 in SEQ ID NO. 1 or a corresponding position in a sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it.

8. The modified lectin protein as claimed in claim 7, wherein the amino acid modification position is selected from one or more of: i) 77, 78 and / or 80; ii) 101; iii) 112 and / or 114.

9. The modified lectin protein as claimed in any of claims 1 to 8, wherein the amino acid modification is an amino acid substitution such that a substituent amino acid replaces an original amino acid.

10. The modified lectin protein as claimed in claim 6, wherein the amino acid substitution at the primary carbohydrate binding site is selected from one or more of: i) at position 27: a conservative, favorable, or unfavorable amino acid, wherein the conservative amino acid is nonpolar or acidic; the favorable amino acid is polar or basic, and the unfavorable amino acid is nonpolar; ii) at position 28: a conservative, favorable, neutral, or unfavorable amino acid, wherein the conservative amino acid is nonpolar; the favorable amino acid is polar, the neutral amino acid is acidic or basic, and the unfavorable amino acid is polar; iii) at position 47: an unfavorable amino acid, which is basic or nonpolar; iv) at position 48: an unfavorable amino acid, which is nonpolar; v) at position 70: an unfavorable amino acid, which is nonpolar; vi) at position 71: an unfavorable amino acid, which is nonpolar; vil) at position 72: an unfavorable amino acid, which is nonpolar;and / or vili) at position 105: a conservative, favorable, neutral or unfavorable amino acid, wherein the conservative amino acid is basic or nonpolar; the favorable one is polar, the neutral one is acidic, basic or polar and / or the unfavorable amino acid is polar, nonpolar or acidic.; 11. The modified lectin protein as claimed in claim 8, wherein the amino acid substitution at the secondary carbohydrate binding site is selected from one or more of: i) at position 77: an unfavorable amino acid that is nonpolar; ii) at position 78: an unfavorable amino acid that is nonpolar; iii) at position 80: an unfavorable amino acid that is nonpolar; iv) at position 101: a favorable, unfavorable, or neutral amino acid, wherein the favorable amino acid is polar or basic, the unfavorable amino acid is nonpolar, and the neutral amino acid is nonpolar or acidic; v) at position 112: an unfavorable amino acid that is nonpolar; vi) at position 114: an unfavorable amino acid that is polar.

12. The modified lectin protein as claimed in claim 1, wherein the modified lectin protein comprises at least one amino acid modification at the N-terminal end of i) or ii), wherein the N-terminal end comprises a position selected from: 1 and / or 2 in SEQ ID NO. 1 or a corresponding position in the sequence having at least 60%, 70%, 80%, 90%, 95%, 97% or 99% homology with it.

13. The modified lectin protein as claimed in claim 12, wherein the amino acid modification is an amino acid substitution at position 1 and wherein a substituent amino acid is not threonine or valine.

14. The modified lectin protein as claimed in claim 13, wherein the substituent amino acid is selected from: alanine, glycine, proline, or serine.

15. The modified lectin protein as claimed in claim 12, wherein the amino acid modification is an amino acid substitution at position 2 and wherein a substituent amino acid is tryptophan.

16. The modified lectin protein as claimed in claim 12, wherein the cleavage of an initiator methionine is increased or decreased compared to a control.

17. The modified lectin protein as claimed in claim 1, wherein the amino acid modification at position 76 is an amino acid substitution by a nonpolar amino acid.

18. The modified lectin protein as claimed in claim 17, wherein the nonpolar amino acid is selected from: glycine, valine, or leucine.

19. The modified lectin protein as claimed in claim 1, wherein the amino acid modification at position 44 or 89 is an amino acid substitution by a nonpolar amino acid.

20. The modified lectin protein as claimed in claim 19, wherein the nonpolar amino acid is selected from: leucine, isoleucine, or valine.

21. The modified lectin protein as claimed in any of claims 1 to 20, wherein the modified lectin protein is soluble, partially soluble, or insoluble and / or has cytotoxicity. nfrcezn / Lznz / q / γΐΛΐ 22. The modified lectin protein as claimed in claim 21, wherein the modified lectin protein has a cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of a control.

23. The modified lectin protein as claimed in claim 21, wherein the modified lectin protein has a percentage of cytotoxicity that is less than 10% of a control or has no cytotoxicity.

24. The modified lectin protein as claimed in claim 21, wherein the modified lectin protein has a percentage of cytotoxicity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher compared to that of a control.

25. The modified lectin protein as claimed in any of claims 1 to 24, wherein the modified lectin protein has a length equal to or less than 500, 400, 300, 250, 200 or 150 amino acids.

26. A pharmaceutical composition comprising a modified lectin protein as claimed in any of claims 1 to 25 and a pharmaceutically acceptable diluent or excipient and optionally an additional therapeutic ingredient.

27. A method of treating cancer in a patient comprising administering the modified lectin protein as claimed in any of claims 1 to 25 or the pharmaceutical composition according to claim 26 to a patient.

28. A modified lectin protein as claimed in any of claims 1 to 25 or a pharmaceutical composition according to claim 26 for use in medicine.

29. The modified lectin protein or pharmaceutical composition as claimed in claim 28 for use in the treatment of cancer.

30. The modified lectin protein as claimed in any of claims 1 to 26 when used in the detection of a cancer cell, cancer diagnosis and / or cancer therapy.

31. A nucleic acid molecule comprising a nucleotide sequence encoding a modified lectin protein as claimed in any of claims 1 to 26.

32. A recombinant vector comprising an insert of a nucleic acid molecule as claimed in claim 31. nfrcezn / Lznz / q / Yl· 33. The recombinant vector as claimed in claim 32, comprising operatively linked in a 5' to 3' direction: a promoter functional in a host cell; a nucleotide sequence according to claim 31 encoding a modified lectin protein; and a termination signal.

34. The recombinant vector as claimed in claim 32 or 33, wherein the recombinant vector is capable of replicating, transcribing, translating and / or expressing itself in a unicellular organism.

35. A transformed host cell comprising the nucleic acid molecule according to claim 31 or the recombinant vector as claimed in any of claims 32 to 34.

36. The transformed host cell as claimed in claim 35, wherein the host cell is an Escherichia coh bacterium or a yeast cell.

37. A method for producing a recombinant lectin protein from Sclerotium rolfsii comprising: i) culturing a host cell containing the recombinant vector as claimed in any of claims 32 to 34 encoding a recombinant lectin protein; ii) expressing the recombinant lectin protein; iii) isolating a crude recombinant lectin protein from the culture.