Method for preparing secretory IgA and secretory IgM and their use for treating necrotizing enterocolitis - Patent Application 20070122999

Abstract

Methods for synthesizing and separating secretory IgA from a mixture of IgA monomers and IgA dimers are provided. The methods include covalently attaching affinity-tagged or epitope-tagged recombinant secretory component to the IgA dimers in the mixture, and then binding the affinity-tagged or epitope-tagged secretory IgA to a binding moiety immobilized on a solid-phase support resin to which the affinity tag or epitope tag is attached, and then eluting the affinity-tagged or epitope-tagged secretory IgA with a release buffer. Methods for synthesizing and separating secretory IgM from a mixture of IgM and other plasma proteins are also provided. Methods for suppressing or preventing symptoms of necrotizing enterocolitis in a subject are provided, comprising orally administering to the subject human polyclonal secretory IgA formed by conjugating human recombinant secretory component with dimeric and polymeric forms derived from pooled human plasma.

Description

[Technical Field]

[0001] [Government support] This invention was made with government support under Grant 1R44DK130749-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] [Related Applications] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 479,811, filed January 13, 2023; the contents of which are incorporated herein by reference.

[0003] [Technical field] The present invention relates generally to methods for preparing human semi-synthetic secretory IgA and secretory IgM, including recombinant human secretory component or molecular variations of human secretory component derived from recombinant human secretory component, and IgA and / or IgM, and specifically to methods that are scalable to allow commercial-scale production of pharmaceuticals containing them, including for the treatment of necrotizing enterocolitis. [Background technology]

[0004] Necrotizing enterocolitis is caused by intestinal inflammation in preterm infants. It can be superficial, affecting only the mucosal lining of the intestinal tract, or severe, involving the entire intestinal wall and presenting with perforation caused by inflammation (Zani and Pierro, 2019).

[0005] The incidence of necrotizing enterocolitis is approximately 5%–7% in preterm infants born before 33 weeks of gestation (Zani and Pierro, 2019) or weighing less than 1500 grams (Hackam and Caplan, 2018). Symptoms range from bradycardia to shock (Zani and Pierro, 2019). Maternal IgA in breast milk has been found to protect preterm infants from necrotizing enterocolitis (Gopalakrishna, 2019; Hand, NIH published report NIH / R01-DK120697-01A1, 2020).

[0006] Probiotics have been found to contribute to the prevention of this syndrome (Zani and Pierro, 2019). Medical management includes gut rest by cessation of nutritional intake for gut rest, and overall support such as maintaining adequate ventilation, tissue perfusion, and blood pressure, as well as antibiotics as needed (Zani and Pierro, 2019). Furthermore, maternal breast milk, which contains secretory IgA, has been found to be effective in preventing necrotizing enterocolitis (Gopalakrishna et al., 2019). Breast milk contains secretory IgA, which regulates the gut microbiota and promotes intestinal homeostasis ( Rogier , et al., 2014). New therapeutic perspectives include hypothermia and stem cell therapy (Zani and Pierro, 2019). Oral treatment with human immunoglobulins has also shown efficacy in the treatment of necrotizing enterocolitis (Eibl et al., 1988, Eibl et al., 1990, Wolf and Eibl, 1991). However, these authors did not use secretory immunoglobulins or envision their oral use.

[0007] However, the prior art has not evaluated orally administered human polyclonal semi-synthetic secretory IgA, including recombinant human secretory components and plasma-derived polyclonal human IgA, as a drug candidate for the treatment of necrotizing enterocolitis, even though its future use has been envisioned (EP 2636682). The prior art has not anticipated the relevant industrial production methods to ensure the high purity and high throughput disclosed herein.

[0008] Thus, there is a need for IgA and IgM therapeutics that are resistant to degradation in the digestive tract. There is also a need for human polyclonal secretory IgA therapeutics for the treatment of necrotizing enterocolitis. Furthermore, there is a need to provide such therapeutics in dosage forms that are highly suitable for treating infected subjects, including infants. Human plasma-derived IgA has been successfully combined with recombinant secretory components to generate biologically active secretory IgA (Longet et al., 2013) (Simon et al., 2014) (Chiari et al., 2021). Summary of the Invention

[0009] A method for suppressing symptoms of necrotizing enterocolitis in a subject suffering from the disease is provided, comprising orally administering a semisynthetic polyclonal human secretory IgA to the subject having or at risk for necrotizing enterocolitis. When administered in a therapeutic amount based on the subject's characteristics and the type of IgA, the symptoms of necrotizing enterocolitis are suppressed in the subject. The administered immunoglobulin is readily formed from a polyclonal source and recombinant human secretory components.

[0010] The present invention identifies an industrial method for producing polyclonal human secretory IgA (sIgA), which cannot otherwise be obtained in quantities suitable for pharmaceutical use. sIgA is readily administered in dimeric or polymeric forms, including recombinant human secretory component. The recombinant human secretory component may contain N-terminal modifications that facilitate efficient purification, necessary for pharmaceutical use. The secretory component may be modified to contain an affinity tag or epitope tag to form affinity- or epitope-tagged secretory IgA and / or secretory IgM, which are useful for capture by a solid-phase support resin. The resulting protein solution containing affinity- or epitope-tagged secretory IgA or affinity- or epitope-tagged secretory IgM can then be applied to the solid-phase support resin. The exposed affinity- or epitope-tagged secretory IgA or secretory IgM adheres to the resin, while unwanted components flow through and are removed. The desired product, secretory IgA or secretory IgM, is then eluted from the solid support resin using a releasing agent. [Brief explanation of the drawings]

[0011]

Figure 1

Figure 2

Figure 3

[0012] The present invention is useful for preparing secretory IgA or secretory IgM. In some embodiments, the IgA is derived from a mixture of monomeric and dimeric plasma IgA; in other embodiments, other plasma proteins are also included; and in still other embodiments, the preparation of secretory IgM is derived from a mixture of IgM and other plasma proteins. The present invention is superior to orally administered monomeric IgA or pentameric IgM because the presence of secretory components confers resistance to degradation and protects the IgA or IgM from digestion in the gastrointestinal tract (U.S. Patent No. 9,932,392). Without intending to be bound by any particular theory, it is believed that the improved efficacy of the present invention is achieved with secretory IgA or secretory IgM due to the tendency of monomeric IgA and pentameric IgM to be easily degraded in the gastrointestinal tract. The resulting administration requirements reduce the cost of treatments using secretory IgA or IgM. Although the invention will be further detailed primarily with respect to IgA, it will be understood that the methods and resulting medicaments are equally applicable to IgM and the resulting secretory IgM, whether the tag is retained or removed. The use of an N-terminal affinity-tagged secretory component further improves the efficiency of purification and therapy.

[0013] The present invention is also useful for treating or preventing necrotizing enterocolitis. Methods of treatment or prevention include treatment with dimeric or polymeric polyclonal secretory IgA. Polyclonal dimeric or polymeric IgA can be recovered from Cohn Fraction III precipitate or equivalent, which is the waste product of plasma fractionation (Simon, 2014). It can also be recovered from ion exchange plasma fractionation processes used to recover other plasma proteins (U.S. Patent Nos. 9,828,418 B2, 10,385,117 B2, and 9,828,418 B2) or from the strip solution of an anion exchange column used to recover IgG.

[0014] Where a range of values ​​is given, it should be understood that the range is intended to encompass not only the endpoints of the range, but also intermediate values ​​expressly included within the range and varied by the last significant digit of the range. As an example, the stated range of 1 to 4 is intended to include 1 to 2, 1 to 3, 2 to 4, 3 to 4, and 1 to 4.

[0015] As used herein, a "subject" is defined as a human.

[0016] As used herein, "dimeric and polymeric IgA" is defined as a construct comprising two or more IgA monomers linked by at least one J chain (joining chain).

[0017] Because the present invention uses immunoglobulins rather than metabolic or immunological inhibitors, it provides effective treatment or prevention without interfering with normal metabolism in the body as with other methods.

[0018] The preferred embodiment(s) described below are merely exemplary in nature and are in no way intended to limit the invention, its application, or uses.

[0019] Secretory IgA molecules are polyclonal and dimeric or polymeric; and are all known in the art, as supported by, for example, the references incorporated herein.

[0020] Because allogeneic immunoglobulins are naturally present in the gastrointestinal tract, their direct administration to the gastrointestinal tract has minimal or no side effects. The dimeric and polymeric IgA of the present invention are conjugated to recombinant human secretory component to mimic the subject's naturally secreted intestinal secretory IgA. Administration of semisynthetic secretory IgA compensates for the lack of secretory IgA naturally secreted in breast milk, which normally provides secretory IgA in newborns.

[0021] Affinity or epitope tags useful in the present invention are any of the following peptide tags: Avi-tag, a peptide GLNDIFEAQKIEWHE (SEQ ID NO: 1) that allows biotinylation by the enzyme BirA, so that the protein can be isolated with streptavidin; calmodulin-tag, a peptide KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 2) that is bound by the protein calmodulin; FLAG-tag, a peptide DYKDDDDK (SEQ ID NO: 3) that is recognized by an antibody; hemagglutinin-tag, a peptide YPYDVPDYA (SEQ ID NO: 4) that is recognized by an antibody; His-tag, 5-10 histidines bound by a nickel or cobalt or other divalent cation chelate HHHHHH (SEQ ID NO: 5); Myc-tag, a short peptide EQKLISEEDL (SEQ ID NO: 6) that is recognized by an antibody; S-tag KETAAAKFERQHMDS (SEQ ID NO: 7); SBP-tag, a peptide that binds to streptavidin. MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 8); Softag 1, for mammalian expression SLAELLNAGLGGS (SEQ ID NO: 9); Softag 3, for prokaryotic expression TQDPSRVG (SEQ ID NO: 10); V5 tag, peptide recognized by antibody GKPIPNPLLGLDST (SEQ ID NO: 11); Xpress tag DLYDDDDK (SEQ ID NO: 12); biotin carboxyl carrier protein, protein domain recognized by streptavidin; glutathione S-transferase tag, protein that binds to immobilized glutathione; green fluorescent protein tag, protein that spontaneously fluoresces and can be bound by nanobodies; maltose binding protein tag, protein that binds to amylose agarose; Nus tag; Strep-tag, peptide that binds to streptavidin or a modified form of streptavidin called streptactin Strep-tag II: WSHPQFEK (SEQ ID NO: 13); thioredoxin tag; TC tag; or Ty tag.

[0022] Plasma IgA contains a mixture of monomers and dimers (Delacroix et al. 1981; Delacroix et al. 1983; Longet et al. 2013, Simon et al. 2014). In some embodiments of the present invention, plasma dimeric IgA from a naturally occurring monomer-dimer mixture is covalently linked in vitro to affinity peptide-tagged secretory component. In another embodiment of the present invention, natural secretory component is covalently linked to one or more amino acid residues through conventional synthetic techniques (Hermanson GT 1996). As an example, a histidine tag is used to understand that a single histidine residue or a polyhistidine, typically containing 2 to 20 histidine residues, is added to the N-terminus of the secretory component, whether produced recombinantly, synthetically, or by other techniques. Secretory IgA becomes histidine-tagged by bivalent binding of the histidine-tagged recombinant secretory component to the naturally occurring IgA dimer. A novel method of obtaining the purified secretory IgA thus formed is to remove the tagged secretory IgA by affinity binding of any of the above-mentioned tags to a binding moiety immobilized on a resin, as a further example, nickel. +2 This is accomplished by immobilizing the secretory component on a resin via its N-terminal histidine tag. Alternatively, other immobilized divalent metal ions, such as cobalt, zinc, copper, or iron, can also be used. Alternatively, in one embodiment of the invention, a FLAG peptide is used, and an antibody against the FLAG peptide is immobilized on a solid support resin. The FLAG tag is described in detail elsewhere, e.g., in U.S. Pat. No. 4,703,004. The resulting secretory IgA is useful, for example, for treating C. difficile-associated diseases, such as Clostridium difficile colitis, pseudomembranous colitis, and antibiotic-associated diarrhea, particularly for secretory immunoglobulin A (IgA) compositions administered in the form of pharmaceutical compositions. The above method is equally applicable to IgM to form purified secretory IgM.

[0023] In one embodiment, the present invention provides a method for human medical treatment involving oral administration of secretory IgA, which can be derived from multiple sources. One such source of IgA is pooled human plasma following the Cohn cold ethanol fractionation method to produce a fraction III precipitate, as performed by those skilled in the art of protein separation. The IgA by-product can be further purified by adsorption to jack bean lectin (jacalin) and / or to ion exchange media under neutral or slightly acidic conditions, as performed by those skilled in the art of protein purification (Kabir S, 1998; and U.S. Patent No. 9,828,418).

[0024] A more detailed description of an exemplary isolation procedure for the IgA component as a by-product from pooled human plasma or pooled hyperimmune human plasma follows. Ethanol fractionation of pooled human plasma is a well-known method for preparing immunoglobulin G. Pooled human plasma is first obtained from licensed plasmapheresis centers in the United States and tested for various pathogens, including HIV. The initial manufacturing step for most commercially available immunoglobulin G preparations involves a modified cold ethanol fractionation according to the Cohn method to produce Cohn Fraction II. In this fractionation method, many infectious viruses are removed from the pooled human plasma. After fractionation, Cohn Fraction II undergoes adsorption to an ion exchange medium. This step can selectively reduce the IgA concentration to less than 0.1%. Such a step is important for producing immunoglobulin G for intravenous infusion in humans because some individuals experience anaphylactoid reactions when treated with intravenous IgG containing IgA as an impurity.

[0025] Modified cold ethanol fractionation according to the Cohn method involves a series of fractionations using varying levels of ethanol, pH, and temperature to produce Fraction II, which is further processed to produce immunoglobulins as described above. In this fractionation method, pooled human plasma is first processed to produce cryoprecipitate and cryosupernatant. Alternatively, it is understood that the source plasma can be autologous or hyperimmune human plasma, pooled or from a single individual immunized against a specific disease.

[0026] In another embodiment, the IgA component is prepared from plasma by ion exchange chromatography.

[0027] In yet another embodiment, the IgA component is prepared by hybridoma technology to provide antigen-specific dimeric IgA. Hybridoma technology was first described in Kohler and Milstein, Nature 1975; 256:495-497, with more recent developments summarized in Berzofsky et al., Fundamental Immunology, Third Edition, 1993, pp 455-62.

[0028] Regardless of source, cryosupernatant undergoes a first ethanol fractionation to yield Supernatant I. Supernatant I undergoes a second ethanol fractionation to yield Fractions II+III. Fractions II+III undergo a third ethanol fractionation step to yield Supernatant III and Fraction III precipitate.

[0029] The IgA-enriched Fraction III precipitate is typically discarded as an unwanted by-product. According to the present invention, IgA is obtained from this unwanted Fraction III precipitate via ion exchange adsorption or affinity chromatography purification, followed by further treatment by incubation with immobilized hydrolytic enzymes to inactivate viruses and vasoactive substances. Such treatment has been demonstrated to remove many tested viruses, including HIV, Sindbis virus, and vaccinia virus. Other antiviral treatments known to those skilled in the art may also be used, consisting of solvent / detergent treatment, nanofiltration, and / or heat inactivation. Typically, three antiviral steps are performed. After incubation to remove viruses, the concentration of the active ingredient is adjusted with sterile saline or buffer solution to achieve a fixed amount of active ingredient per milliliter of reconstituted product. Finally, the solution containing the fixed amount of reconstituted product is sterilized by filtration before use.

[0030] Ethanol fractionation according to the Cohn method is well known in the art and is described in Cohn et al., J. Am. Chem. Soc. 1946; 68:459-475, more particularly in Oncley et al., J. Am. Chem. Soc. 1949; 71:541-550, pp. 576-602, and most particularly in the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 3, second edition (1963), pp. 576-602. Alternatively, ion exchange chromatography can be used to isolate dimeric and polymeric IgA by-products during the production of intravenous immunoglobulin. Dimeric and polymeric IgA account for 4% to 22% of plasma IgA (Delacroix et al. 1981; Delacroix et al. 1983). The resulting dimeric IgA-J chain is purified for pharmaceutical use. In certain embodiments, the compositions of the invention contain, in addition to the IgA component, recombinant human secretory component. Human secretory component can be produced by recombinant techniques such as those described in Crottet et al., 1999.

[0031] The dimeric and polymeric IgA present in the monomer-polymer mixture of plasma IgA is further conjugated to secretory component, which may be recombinantly produced human secretory component containing an N-terminal histidine tag or another of the tags described above; or human secretory component subsequently covalently linked to a peptide tag, such as a histidine or polyhistine oligopeptide. In a specific inventive embodiment, the dimeric and polymeric IgA of the present invention are conjugated to recombinant human secretory component to mimic the naturally secreted intestinal secretory IgA present in the subject. Administration of semisynthetic secretory IgA compensates for the lack of secretory IgA naturally secreted in breast milk, which normally provides secretory IgA. Human secretory component is illustrated as a space-filling model in Figure 2, showing two lobes and prominently sterically exposed N-terminus (amino terminus) extending therefrom. According to some invention embodiments, a polyhistidine tag is added extending from the N-terminus, which is directed away from the majority of the newly formed secretory immunoglobulin into the ambient solution and is accessible for metal affinity resin chromatography. This steric exposure is readily exploited for efficient purification compared to a similar C-terminal tag on the secretory component, which is located between the secretory component and its bound IgA dimer and is not exposed to the ambient solution.

[0032] In one embodiment of the invention, binding of IgA to secretory component is achieved by disulfide bond formation under mildly oxidizing conditions (Jones RML, Schweikart F., Frutiger S., Jaton JC., Hughes GJ. Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component. Biochimica et Biophysica Acta 1998; 1429:265-274). Dimeric and polymeric IgA containing both J chain and secretory component is repurified from the mixture by immobilized metal ion affinity chromatography, e.g., ion exchange and size exclusion chromatography, and / or ultrafiltration, as described in Lullau et al., 1996; Corthesy, 1997; and Crottet et al., 1999; and performed by those skilled in the art of protein purification.

[0033] Purified dimeric and polymeric secretory IgA containing recombinant human secretory component may be stabilized, for example, by the addition of human serum albumin to a final concentration of 5%. The presence of human secretory component in the compositions of the invention provides physiologically effective doses of immunoglobulin A, whereas compositions lacking secretory component do not. Furthermore, the present invention identifies an industrial method for producing polyclonal human secretory IgA composed of recombinant human secretory component and IgA dimers and higher polymers derived from natural human plasma, which are otherwise not obtainable in sufficient quantities for commercial medical use.

[0034] It has been previously found that recombinant proteins can be isolated from cell supernatants by producing proteins tagged with a histidine affinity tag or other affinity tags mentioned above. The recombinant protein-containing cell supernatant is passed over a nickel-bound solid support resin. The histidine tag or other tag is retained by attachment to nickel or other suitable tag-specific binding moieties, while unwanted proteins are washed away. In the present invention, the tagged secreted immunoglobulin protein is subsequently recovered by elution with an imidazole buffer, if there is an amide-metal bond between the target protein and the resin (Block H et al. 2009).

[0035] The mixture of histidine-tagged secretory IgA and remaining plasma proteins is buffer-exchanged into a binding buffer containing a low concentration of imidazole (≦40 mM). Alternative release agents that function to exchange histidine-tagged secretory IgA or secretory IgM illustratively include: (1) 10 mM ethylenediaminetetraacetic acid (EDTA) and (2) an elution buffer with a pH of 5.5 or less. Typical imidazole concentrations in the binding buffer range from 0.1 to 40 millimolar (mM). It is understood that the pH of the initial binding buffer can vary to some extent, but is readily determined through routine testing for a given buffer chemistry and concentration. Chromatographic media functional herein are selected to be stable in the presence of the binding buffer and capable of separating histidine-tagged secretory IgA. Examples of these metal-bound solid support resins include nickel, cobalt, and zinc immobilized on cross-linked beaded agarose (SEPHAROSE®). In a preferred embodiment, the affinity medium is washed in a wash buffer containing 0-40 mM imidazole to remove unbound monomeric IgA and other nonspecifically bound residual proteins. The bound histidine-tagged secretory IgA is recovered using an elution buffer with a higher imidazole concentration (e.g., 100-1000 mM). Sequential elution results in strict separation of the monomeric form from the dimeric IgA bound to the histidine-tagged secretory component. It is understood that the method of the present invention is scalable to produce sufficient quantities to treat multiple subjects. It is understood that similar selective binding pairs can be achieved between other tagged secretory component-containing immunoglobulin proteins of the invention and prior art resins for each of the above-mentioned tags.

[0036] As a specific example, the binding and washing buffer is 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, adjusted to pH 8. A mixture of IgA monomers and secretory IgA is dissolved in this buffer. The elution buffer is identical to the binding buffer, except that the imidazole concentration is higher, e.g., 100-1000 mM.

[0037] The remaining histidine-tagged secretory IgA is then eluted from the bivalent immobilized metal resin using an elution buffer according to conventional techniques and conditions, including an exemplary basic pH of, for example, 8-10 (see Figure 1).

[0038] In some embodiments, purified secretory IgA comprising histidine-tagged secretory component is stabilized by the addition of human serum albumin, for example, to a final concentration of 5% of the total weight.

[0039] In another embodiment, the tag is removed from the recovered secretory IgA, allowing the original secretory IgA to be used as a pharmaceutical. For histidine-tagged secretory IgA, procedures for tag removal are known in the art (Kopera E et al 2012).

[0040] In summary, the method of the present invention involves adding amino-terminally tagged secretory component, either recombinantly or post-expression tagged, to a mixture of plasma-derived IgA monomers and dimers, where the tagged secretory component binds to the IgA dimers to form secretory IgA, and then recovering the newly formed secretory IgA by adhesion to immobilized divalent metal ions or other solid phase moieties from which it is subsequently eluted.

[0041] In an embodiment of the invention, the affinity tag is located at the amino terminus (N-terminus) of the secretory component molecule. This tag location allows steric access to a nickel affinity column after the secretory component binds to the IgA dimer to form secretory IgA, as shown in Figure 2, facilitating recovery of the entire secretory IgA molecule using the affinity tag. In yet another embodiment, the mature N-terminal affinity tag, such as a histidine tag, contains a capping amino acid, dipeptide, or oligopeptide of 3 to 9 amino acids. This cap is optionally a residue from cleavage of the signaling sequence.

[0042] The amino acid sequence of native secretory component is shown (SEQ ID NO: 15) and is shown in Figure 3 along with additional N-terminal sequence. It is understood that the FLAG sequence detailed above with respect to the N-terminal tag also functions herein. In some inventive embodiments, a spacer is provided intermediate the polyhistidine sequence and the N-terminus of the native secretory component. It is understood that the spacer is highly variable and functions to expose the polyhistidine from the folded IgA-binding secretory component. When present, the spacer is 2 to 20 residues in length. An exemplary polyhistidine-spacer-secretory component is shown (SEQ ID NO: 16). In yet other embodiments, an endopeptidase recognition sequence is provided as part of the spacer and adjacent to the native secretory component. The endopeptidase recognition sequence provides a cleavage site for optional removal of the affinity tag and optional spacer from the fusion protein to yield the native IgA-binding secretory component or to provide a site of attachment for additional purification steps. An exemplary endopeptidase recognition sequence that functions herein is the tobacco etch virus (TEV) NIa (nuclear-inclusion-a) endopeptidase recognition sequence. TEV protease is known as a highly sequence-specific cysteine ​​protease. Other endopeptidase recognition sequences that function herein include, by way of example, those of trypsin, factor Xa, elastase, chymotrypsin, enterokinase recognition sequence (A. Hillar), and collagenase.

[0043] In yet other invention embodiments, the recombinant IgA secretory component comprises a signal sequence. The signal sequence functions to facilitate expression from transfected cells. For expression of the recombinant IgA secretory component in non-lytic insect cells, signal sequences functional herein include sequences derived from honeybee melittin or the major envelope protein from nuclear polyhedrosis viruses, such as AcNPV or OpNPV (Brown et al., 2011). For expression and secretion from mammalian cell culture, the signal sequence can be rat PAM (ZH Jiang et al.) or human cyclooxygenase 2 (Venkatesan et al., 2021). An exemplary honeybee melittin-polyhistidine-spacer-IgA secretory component is shown (SEQ ID NO: 18). In some invention embodiments, cleavage of the signal sequence is partial, leaving a cap adjacent to the affinity tag. The alanine cap is shown in Figure 3 and SEQ ID NO: 17.

[0044] Without intending to be bound by any particular theory, the signal sequence is cleaved while the protein is in the lumen of the endoplasmic reticulum of the expressing cell. The mature protein in Figure 3 begins with an N-terminal alanine, which is the portion of the signal sequence that remains after cleavage. This is directly followed by an affinity tag, e.g., a His tag. There is a spacer, followed by the Tobacco Etch Virus (TEV) NIa endopeptidase recognition sequence. TEV protease is known as a highly sequence-specific cysteine ​​protease.

[0045] The present invention further encompasses variants and equivalents that are substantially homologous to secretory component and still retain the ability to selectively bind to polymeric IgA, IgM, or both. These can include, for example, conservative substitution mutations, i.e., replacing one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of one amino acid for another amino acid within the same general class, such as the substitution of one acidic amino acid for another, one basic amino acid for another, or one neutral amino acid for another.

[0046] The secretory component of the present invention can be a recombinant or synthetic secretory component that retains IgA or IgM binding properties. It is recognized in the art that portions of the amino acid sequences of the invention can be altered without significantly affecting the structure or function of the protein (Patent Application 2004 / 002294). Accordingly, the present invention further encompasses variations of secretory component that exhibit substantial activity; such variants include deletions, insertions, inversions, repeats, and type substitutions. Secretory component variants that function herein illustratively include amino acid substitutions relative to SEQ ID NO: 16. Other sequence variants that function herein are detailed in Stadtmueller et al. It is understood that other mutations at different amino acid positions will function similarly. It is further understood that mutation of a conserved amino acid at any particular position to glycine or alanine is preferred. It is further understood that mutation to any neutral, charged, hydrophobic, hydrophilic, synthetic, non-natural, non-human, or other amino acid will function similarly.

[0047] The structure (primary, secondary, or tertiary) of the secretory component proteins encompassed by the compounds of the present invention may be modified or altered, which may or may not result in molecules with properties similar to the exemplary polypeptides disclosed herein. It is understood that changes in conserved amino acid bases are likely to affect the activity of the resulting protein. However, it is further understood that changes in amino acids that function in receptor interaction, resistance or promotion of proteolysis, intracellular or extracellular transport, secretion, protein-protein interactions, post-translational modifications such as glycosylation, phosphorylation, sulfation, etc., may enhance or decrease the activity of the compounds of the present invention, while retaining some ability to alter or maintain biological activity. It is known that substitution of certain amino acids in a sequence with other amino acids does not result in a significant decrease in activity.

[0048] In making such changes, the hydropathic index of the amino acid is considered. According to the present invention, an amino acid may be substituted for another amino acid having a similar hydropathic index and still result in a polypeptide with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0049] Without intending to be limited to a particular theory, it is believed that the relative hydropathic properties of amino acids determine the secondary structure of a resulting polypeptide, which in turn determines the interaction of that polypeptide with other molecules. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index while still obtaining a functionally equivalent polypeptide. In such changes, substitution with amino acids having a hydropathic index within ±0.2 is preferred, with those within ±0.1 being particularly preferred, and those within ±0.05 being even more preferred.

[0050] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account some of the above characteristics are well known to those of skill in the art and include the following (original residue: exemplary substitution): (Ala:Gly,Ser), (Arg:Lys), (Asn:Gln,His), (Asp:Glu,Cys,Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn,Gln), (Ile:Leu,Val), (Leu:Ile,Val), (Lys:Arg), (Met:Leu,Tyr), (Ser:Thr), (Thr:Ser), (Trp:Tyr), (Tyr:Trp,Phe), and (Val:Ile,Leu).

[0051] Secretory components and analogs can be further modified to contain additional chemical moieties not normally part of the protein. These derivatized moieties can improve the solubility, biological half-life, absorption, or binding affinity of the protein. These moieties can also reduce or eliminate any undesirable side effects of the protein. A review of these moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0052] It will be appreciated that recombinant secretory component modified as detailed above for IgA is applicable to the purification of secretory IgM, through either N- or C-terminal modification.

[0053] Plasma IgM can be recovered from by-products of intravenous immunoglobulin production. One example of such a by-product is Cohn Fraction III precipitate. IgM is most easily solubilized from Cohn Fraction III precipitate with 20 mM sodium acetate. Other plasma proteins, along with IgM, are similarly solubilized. Plasma IgM in this protein mixture is covalently bound in vitro to recombinant histidine-tagged secretory component to form secretory IgM in the protein mixture. The secretory IgM becomes tagged by bivalent binding of the tagged secretory component to the IgM. A novel method for obtaining purified secretory IgM thus formed involves the use of an immobilized nickel catalyst that is part of a resin column. +2 or other divalent metal ions, or other suitable binding moieties within the art. The resulting semi-synthetic secretory IgM is useful, for example, for the treatment of Clostridium difficile-associated diseases, such as Clostridium difficile colitis, pseudomembranous colitis, necrotizing enterocolitis, and antibiotic-associated diarrhea, particularly for secretory immunoglobulin M compositions administered in the form of pharmaceutical compositions.

[0054] In another embodiment, the tag is removed from the recovered secretory IgM, allowing the original secretory IgM to be used as a pharmaceutical. For histidine-tagged secretory IgM, procedures for tag removal are known in the art (Kopera E et al. 2012). In another embodiment, the spacer is also removed, resulting in the original secretory IgM, regardless of whether part of the spacer functions as an endopeptidase recognition sequence.

[0055] Thus, the methods of the present invention provide a method in which a peptide-tagged secretory component is added to a mixture of plasma-derived IgM and other plasma proteins, where the peptide-tagged secretory component binds to the IgM to form secretory IgM, whereby the newly formed secretory IgM is recovered by adhesion to binding moieties on a solid support resin to which the peptide is bound, and then eluted therefrom using an elution buffer.

[0056] In yet other embodiments, IgA is combined with pasteurized human breast milk or with human breast milk prepared in a bioreactor (Deng M, 2022).

[0057] Secretory IgA antibodies can be administered alone or in combination with various pharmaceutical adjuvants.

[0058] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonicity agents, for example, sugars, sodium chloride, etc. Prolonged retention of IgA in the intestinal lumen can be achieved by the use of absorption delaying agents, for example, aluminum monostearate and gelatin.

[0059] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), (b) binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), (c) humectants (e.g., glycerol), (d) disintegrants (e.g., agar, calcium carbonate, (e.g., sodium, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate), (e) solution retarders (e.g., paraffin), (f) absorption enhancers (e.g., quaternary ammonium compounds), (g) wetting agents (e.g., cetyl alcohol and glycerol monostearate), (h) adsorbents (e.g., kaolin and bentonite), and (i) lubricants (e.g., calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof). In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0060] Solid compositions of a similar type may also be employed as fillers in soft and hard capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0061] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art; these are detailed, for example, in U.S. Pat. Nos. 4,017,647; 4,385,078; 4,518,433; and 4,556,552.

[0062] Such solid preparations may contain opacifying agents, and may be of such composition that they release the active ingredient(s) in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.

[0063] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. The liquid dosage form consists of secretory IgA dissolved in 250 mM glycine solution in saline. In addition to the active ingredient, the liquid dosage form may contain the following: an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, specifically cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures of these substances.

[0064] Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0065] Suspensions may contain, in addition to the active ingredient, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium or other metal hydroxides, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0066] Because the effectiveness of IgA antibodies depends on their reaching the small intestine, preferred tablets or capsules are enteric coated. Alternatively, the active IgA antibodies themselves may be microencapsulated prior to formulation. Preparation of IgA antibody microcapsules and enteric-coated tablets or capsules can be achieved by conventional methods as detailed above.

[0067] It is understood that the therapeutic amount of sIgA depends on its form, with larger doses required for forms that are susceptible to degradation in the gastrointestinal tract. Typically, the amount of sIgA used is about 0.005 mg to 50 g per day, preferably 1 mg to 40 g per day. Generally, secretory IgA is effective as a sole therapeutic agent when administered to a patient at about 10 g per day. Forms of IgA that are easily degraded in the gastrointestinal tract are typically effective at doses at least 80% greater than the secretory form. For example, about 5 g of secretory IgA can be administered to a subject in a single dose once daily or in divided doses 3 to 4 times daily. Preferably, multiple doses are administered with meals that may contain food allergens. It is understood that a physician can easily adjust the dose of IgA to be administered based on the subject's response to treatment. Many factors are taken into consideration when adjusting the dose. Doses of secretory IgA contemplated by the present invention that are considered therapeutically effective range from about 5 mg to 5 g, although it should be understood that the dose can be readily adjusted to provide an appropriate amount of IgA antibody.

[0068] The present invention is distinguished from the prior art in that its dimeric and polymeric IgA components are derived from pooled healthy human plasma. The present invention is further distinguished in that the dimeric and polymeric IgA components are conjugated to an amino-terminal histidine affinity-tagged recombinant human secretory component, which is required for normal activity of secretory IgA in the intestine. Importantly, the present invention identifies an industrial method for producing polyclonal semisynthetic human secretory IgA that cannot otherwise be obtained in quantities suitable for widespread medical use.

[0069] The present invention is further described with reference to the following detailed examples, and exemplary process procedures are described below. These examples are not meant to limit the scope of the invention set forth in the foregoing description. Variations within the spirit of the invention will be apparent to those skilled in the art. [Example]

[0070] [Example 1] Dimeric IgA is obtained by affinity purification from pooled healthy human plasma and conjugated with recombinant amino-terminal histidine affinity-tagged human secretory component produced according to Figure 3 to form secretory IgA. Secretory IgA is stabilized by the addition of human serum albumin to a final concentration of 5%. The final solution is adjusted to a therapeutic dose of 100 mg of secretory IgA per day. Secretory IgA is administered once daily to infants suffering from necrotizing enterocolitis. One week after initiating treatment, patients with necrotizing enterocolitis show a reduction in their physiological abnormalities.

[0071] [Example 2] The method of Example 1 is repeated with enteric-coated secretory IgA administered at a higher dose of 1 g per day to achieve similar results.

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[0173] The patent applications and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains and are hereby incorporated by reference to the same extent as if each individual application or publication was specifically and individually incorporated by reference.

[0174] The above description illustrates specific embodiments of the present invention, but is not meant to limit its practice. The following claims, including all equivalents thereof, are intended to define the scope of the present invention.

Claims

1. 1. A recombinant secretory component comprising: a secretory component having an affinity tag attached to its amino terminus, Recombinant secretory components.

2. 2. A recombinant secretory component according to claim 1, comprising: the affinity tag is a histidine sequence of 2 to 20 residues; Recombinant secretory components.

3. 2. A recombinant secretory component according to claim 1, comprising: further comprising a spacer peptide sequence intermediate the amino terminus and the affinity tag. Recombinant secretory components.

4. 14. A recombinant secretory component according to claim 13, comprising: the spacer comprises an endopeptidase recognition sequence; Recombinant secretory components.

5. A recombinant secretory component according to any one of claims 1 to 4, further comprising the entire or residues of a signal sequence attached to the end of the affinity tag; Recombinant secretory components.

6. 2. A recombinant secretory component according to claim 1, comprising: having any of SEQ ID NOs: 14, 15, or 16; Recombinant secretory components.

7. 1. A method for suppressing symptoms of or preventing necrotizing enterocolitis in a subject suffering therefrom, comprising: The method comprises: orally administering to said subject suffering from necrotizing enterocolitis purified polymeric secretory IgA comprising recombinant human secretory component and human plasma-derived IgA dimers and higher polymers; and allowing a sufficient time for the secretory IgA to suppress the symptoms of necrotizing enterocolitis in the subject; Including, method.

8. 8. The method according to claim 7, further comprising the step of microencapsulating said compound prior to said administration. method.

9. 8. The method according to claim 7, The secretory IgA is stabilized by the addition of human serum albumin prior to or in conjunction with the administration. method.

10. The method according to any one of claims 7 to 9, The secretory IgA is stabilized by delivery with an antacid. method.

11. The method according to any one of claims 7 to 9, The secretory IgA is dissolved in an aqueous glycine solution. method.

12. The method according to any one of claims 7 to 9, The secretory IgA is produced by an industrial method. method.

13. The method according to any one of claims 7 to 9, the subject is a human infant. method.

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