Stabilized DN-TNF mutein bioconjugates for selective soluble TNF neutralization

Abstract

A therapeutic composition is described including a selective soluble TNF-neutralizing bioconjugate for treating TNF-mediated inflammatory disorders. The bioconjugate includes a dominant-negative TNF (DN-TNF) mutein, which contains one or more amino acid substitutions in the TNF receptor interaction domain, trimer interface domain, or both, to reduce receptor binding while enhancing heterotrimer formation with wild-type TNF, thereby neutralizing soluble TNF. The DN-TNF mutein is covalently conjugated to a biocompatible stealth polymer via a hydrolyzed maleimide linker, preventing retro-Michael reaction and polymer dissociation. The composition is formulated as an injectable buffered aqueous solution for subcutaneous, intramuscular, intravitreal, or intravenous administration. Also disclosed are methods for stabilizing the bioconjugate through controlled hydrolysis of the maleimide linker and methods for treating TNF-mediated inflammatory disorders by administering the composition to a subject in need thereof.

Description

STABILIZED DN-TNF MUTEIN BIOCONJUGATES FOR SELECTIVE SOLUBLETNF NEUTRALIZATIONBACKGROUND

[0001] Technical Field

[0002] This invention relates to the field of biopharmaceuticals and protein therapeutics, specifically to tumor necrosis factor (TNF) inhibitors for the treatment of TNF- mediated inflammatory disorders. More particularly, the invention pertains to dominantnegative TNF (DN-TNF) muteins that selectively neutralize soluble TNF through heterotrimer formation while preventing receptor activation. The invention further encompasses bioconjugates of DN-TNF muteins covalently linked to biocompatible stealth polymers, stabilized through controlled hydrolysis of maleimide linkers, thereby improving therapeutic stability, pharmacokinetics, and resistance to polymer dissociation. The invention also relates to pharmaceutical compositions comprising stabilized DN-TNF bioconjugates, methods of manufacturing these compositions, and methods of treating TNF-associated diseases, including but not limited to rheumatoid arthritis, inflammatory bowel disease, psoriasis, neuroinflammation and neuroinflammatory disorders.

[0003] Background Art

[0004] Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays a central role in immune regulation, inflammation, and cell signaling. Dysregulated TNF activity contributes to various autoimmune and inflammatory diseases, including rheumatoid arthritis, Crohn’s disease, ulcerative colitis, psoriasis, ankylosing spondylitis, and are thought to be implicated in neuroinflammatory disorders, including Alzheimer’s disease and others. As a result, TNF inhibitors have been widely developed to counteract excessive TNF signaling.

[0005] Currently, anti-TNF monoclonal antibodies (e.g., infliximab, adalimumab, golimumab, certolizumab pegol) and TNF receptor fusion proteins (e.g., etanercept) are the most commonly used TNF inhibitors. These agents function by binding TNF and preventing its interaction with TNF receptors (TNFR1 and TNFR2). Importantly, conventional anti-TNF biologies neutralize both soluble TNF (sTNF) and transmembrane TNF (tmTNF). While this broad inhibition is effective in reducing inflammation, it also disrupts critical immune functions mediated by tmTNF, leading to immunosuppression.

[0006] In the central nervous system (CNS), one major concern associated with tmTNF inhibition is the risk of demyelination, as TNFR2 activation via tmTNF is crucial for myelin repair and neuroprotection. The blockade of tmTNF has been implicated in multiple sclerosis-like syndromes and progressive demyelinating disease in some patients receiving long-term anti-TNF therapy. Additionally, systemic immunosuppression increases susceptibility to opportunistic infections, including tuberculosis and fungal infections. Due to these risks, there is an unmet need for TNF inhibitors that selectively neutralize sTNF while sparing tmTNF function.

[0007] Dominant-negative TNF (DN-TNF) muteins represent an alternative therapeutic approach designed to selectively neutralize soluble TNF while preserving transmembrane TNF activity. DN-TNF muteins are engineered sTNF variants that preferentially form heterotrimers with wild-type sTNF, thereby rendering TNF inactive without direct receptor antagonism. Previous research, including Zalevsky et al., has demonstrated that specific mutations in TNF can disrupt receptor binding while maintaining trimerization, allowing for targeted neutralization of sTNF without blocking tmTNF. However, it was recently discovered that existing DN-TNF proteins face significant stability challenges, particularly when conjugated to pharmacokinetic-enhancing stealth polymers such as polyethylene glycol (PEG).

[0008] One major limitation of PEGylated DN-TNF conjugates is the instability of the maleimide-thiol linkage, which is prone to retro-Michael reaction under physiological conditions, leading to polymer dissociation and loss of therapeutic efficacy. Additionally, DN- TNF muteins must be carefully engineered to maximize their affinity for wild-type TNF while minimizing receptor binding, ensuring effective sTNF neutralization without off-target effects.SUMMARY

[0009] Technical Problem

[0010] Existing TNF inhibitors, including monoclonal antibodies and TNF receptor fusion proteins, effectively neutralize both soluble TNF (sTNF) and transmembrane TNF (tmTNF). However, the inhibition of tmTNF signaling is associated with adverse immunosuppressive effects, including increased risk of infections and demyelination-related neurotoxicity. To address these limitations, dominant-negative TNF (DN-TNF) muteins have been developed to selectively neutralize sTNF while preserving tmTNF function. DN-TNF muteins exert their effect by preferentially forming heterotrimers with wild-type TNF, thereby rendering TNF inactive without directly blocking tmTNF and TNFR1 / TNFR2 interactions. While DN-TNF muteins offer a promising alternative to conventional anti-TNF therapies, their clinical application is hindered by stability and pharmacologic challenges when conjugated to stealth polymers such as polyethylene glycol (PEG) or polysarcosine (PolySar).

[0011] A significant limitation of polymer-conjugated DN-TNF therapeutics, namely those conjugated with a maleimide linker, is the instability of the maleimide-thiol linkage, which can undergo retro-Michael reaction under physiological conditions, leading to polymer dissociation and loss of therapeutic efficacy. The instability of PEGylated or PolySar- conjugated DN-TNF muteins results in reduced circulation half-life, unpredictable or inconsistent pharmacokinetics, and loss of sTNF neutralization potency. Additionally, mutational modifications to DN-TNF must be carefully designed to both reduce receptor binding affinity and preserve or enhance heterotrimer formation with wild-type TNF, ensuring optimal dominant-negative function without destabilizing the sTNF trimer. There is therefore an unmet need for a stabilized DN-TNF therapeutic composition that: (i) selectively binds wildtype TNF to form inactive heterotrimers, (ii) neutralizes soluble TNF while sparing transmembrane TNF, (iii) prevents polymer dissociation, and (iv) demonstrates enhanced pharmacokinetic stability and immune evasion (reduced immunogenicity, prolonged circulation time, reduced recognition by proteases and antibodies, and decreased opsonization and phagocytosis) in an injectable formulation.

[0012] Solution to Problem

[0013] The present invention provides a stabilized dominant-negative TNF (DN-TNF) bioconjugate that effectively neutralizes soluble TNF (sTNF) while preserving transmembrane TNF (tmTNF) function, thereby mitigating the immunosuppressive and demyelination risks associated with conventional anti-TNF therapies. To achieve this, the invention introduces DN- TNF muteins containing amino acid substitutions in the TNF receptor interaction domain, the trimer interface domain, or both. These substitutions are designed to reduce TNF receptor binding affinity while enhancing heterotrimer formation with wild-type TNF, ensuring effective dominant-negative inhibition. Unlike traditional TNF inhibitors, this strategy selectively neutralizes soluble TNF without interfering with tmTNF-mediated physiological functions.

[0014] To overcome the stability limitations of polymer-conjugated DN-TNF muteins, the DN-TNF mutein is first covalently conjugated to a biocompatible stealth polymer via cysteine thiol engagement with a maleimide linker, followed by controlled hydrolysis of the maleimide linker to a maleamic acid derivative. This hydrolysis step prevents retro-Michael reaction, which can cause polymer dissociation and reduce therapeutic half-life. The invention ensures that at least 85%, 90%, or 95% of the maleimide linker is hydrolyzed to a maleamic acid derivative, leading to a highly stable DN-TNF bioconjugate with prolonged circulation time and consistent pharmacokinetics. Additionally, site-specific cysteine conjugation isutilized to optimize polymer attachment while minimizing protein aggregation and maintaining bioactivity.

[0015] The manufacturing process includes first conjugating the DN-TNF mutein to the stealth polymer via maleimide-thiol coupling, then subjecting the conjugate to a controlled hydrolysis step by adjusting the pH to between 8.0 and 9.5, followed by incubation at a controlled temperature (18°C to 25°C) for at least twelve hours to induce maleimide ring opening. The final step involves adjusting the pH to a physiologically compatible range (5.5 to 7.5) to yield a stable injectable formulation suitable for subcutaneous, intramuscular, intravitreal, or intravenous administration.

[0016] Advantageous Effects of Invention

[0017] The present invention provides a stabilized dominant-negative TNF (DN-TNF) bioconjugate with significant advantages over existing TNF inhibitors, including monoclonal antibodies, TNF receptor fusion proteins, and earlier DN-TNF constructs. By selectively neutralizing soluble TNF (sTNF) while preserving transmembrane TNF (tmTNF) function, the invention avoids the immunosuppressive and neurotoxic effects associated with non-selective TNF inhibition, which can lead to increased infection risk and demyelination disorders. This targeted approach enables effective sTNF blockade while maintaining physiological tmTNF signaling, reducing the likelihood of adverse neurological and immunological side effects.

[0018] A key innovation is improved stability and pharmacokinetic profile, achieved through site-specific conjugation of DN-TNF muteins to a stealth polymer via a hydrolyzed maleimide linker. Conventional maleimide-thiol linkages suffer from retro-Michael reaction, leading to polymer dissociation and loss of therapeutic efficacy. The claimed invention solves this instability problem by inducing controlled hydrolysis of the maleimide linker to a maleamic acid derivative, preventing polymer dissociation and ensuring a stable, long-acting sTNF-neutralizing bioconjugate. By achieving at least 85%, 90%, or 95% hydrolysis of the maleimide linker, the invention ensures improved stability, extended serum half-life, consistent drug exposure, and reduced dosing frequency, improving patient experience and therapeutic efficacy.

[0019] Furthermore, the invention incorporates precisely engineered amino acid substitutions in the DN-TNF mutein to enhance heterotrimer formation with wild-type TNF while minimizing receptor binding affinity. This unique property enables potent TNF neutralization through heterotrimer sequestration, rather than direct receptor blockade, further reducing the risk of off-target effects. The site-specific conjugation strategy also minimizes protein aggregation and immunogenicity, ensuring optimal bioactivity and manufacturability.

[0020] Additionally, the invention provides a well-defined and reproducible manufacturing process that includes precise control over pH and temperature during hydrolysis stabilization.

[0021] The final buffered aqueous injectable formulation (pH 5.5-7.5) is designed for subcutaneous, intramuscular, intravitreal, or intravenous administration, offering flexibility in clinical use and broad therapeutic applicability.

[0022] By integrating selective sTNF neutralization, enhanced stability, prolonged circulation time, and improved manufacturability, the present invention represents a major advancement in TNF inhibitor technology. The claimed therapeutic composition incorporating the stable DN-TNF bioconjugate is expected to offer superior clinical outcomes with fewer side effects and greater consistency, making it an ideal candidate for treating TNF-mediated inflammatory diseases and immunological diseases induced by chronic inflammation.BRIEF DESCRIPTION OF DRAWINGS

[0023] Now, turning to the drawings, the embodiments will be further described with reference to the figures, wherein:

[0024] FIG.1 shows a table illustrating a non-exhaustive list of amino acid substitutions forming various DN-TNF muteins that reduce TNF receptor binding while maintaining heterotrimer formation.

[0025] FIG.2 shows a table illustrating a non-exhaustive list of amino acid substitutions in the trimer interface domain to enhance DN-TNF binding to wild-type sTNF subunits forming neutralizing heterotrimers.

[0026] FIG.3 shows a table illustrating a non-exhaustive list of amino acid substitutions forming various DN-TNF muteins, taken along with amino acid substitutions C69V and C101 A, providing a single cysteine amino acid for site specific thiol conjugation with a stealth polymer, some of which substitutions are in the receptor binding domain effectively inducing an inherent steric hinderance preventing heterotrimer binding with TNFRs.

[0027] FIG.4 shows a schematic representation of DN-TNF conjugation with a maleimide-PEG according to various embodiments.

[0028] FIG.5 illustrates a stabilization mechanism for a DN-TNF maleimide-polymer bioconjugate through controlled maleimide hydrolysis, preventing retro-Michael reaction and thiol exchange.

[0029] FIG.6 shows a schematic representation of DN-TNF conjugation with a maleimide-PolySar according to various embodiments.

[0030] FIG.7 illustrates a stabilization mechanism for a DN-TNF maleimide-polymer bioconjugate through controlled maleimide hydrolysis, preventing retro-Michael reaction and thiol exchange.

[0031] FIG. 8 shows a summary of experiments related to a hydrolysis process development step for implementation after polymer conjugation with a DN-TNF mutein and prior to final pH adjustment and formulation as a drug product for injection.DETAILED DESCRIPTION OF EMBODIMENTS

[0032] The following detailed description provides exemplary embodiments of the present invention and is not intended to limit the invention to the particular embodiments described. It is to be understood that various modifications, substitutions, and equivalents will be apparent to those skilled in the art and are intended to be encompassed within the spirit and scope of the invention as defined by the appended claims. The embodiments described herein are provided for illustrative purposes and are not meant to be exhaustive. Features from one embodiment may be combined with features from another embodiment without departing from the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. While the invention is described in connection with certain specific embodiments, it should be understood that the invention is capable of further modifications without departing from the scope and spirit thereof. The present disclosure is also intended to encompass routine variations, substitutions, additions, and modifications that would be appreciated by a person of skill in the field based on the teachings herein.

[0033] Definitions

[0034] In the context of the present invention, a dominant-negative tumor necrosis factor (DN-TNF) mutein refers to a modified form of soluble TNF (sTNF) that is engineered to lack the ability to bind and activate TNF receptors (TNFR1 or TNFR2) while retaining its ability to form heterotrimers with wild-type sTNF. By sequestering wild-type sTNF into inactive heterotrimers, DN-TNF muteins selectively neutralize soluble TNF activity while sparing transmembrane TNF (tmTNF) function. The DN-TNF muteins of the present invention are based on the amino acid sequence of SEQ ID NO: 1 and are modified with one or more amino acid substitutions in either the TNF receptor interaction domain, the trimer interface domain, or both, to enhance their selective neutralization properties.

[0035] The TNF receptor interaction domain encompasses amino acid residues that mediate the binding of sTNF to TNF receptors TNFR1 and TNFR2. In SEQ ID NO: 1, the TNFreceptor interaction domain includes residues Q21, A22, E23, N30, R31, R32, A33, K65, G66, Q67, G68, S86, Y87, V91, A109, E110, D140, andA145. Substitutions at these residues disrupt TNF receptor binding, thereby preventing receptor activation and reducing downstream pro- inflammatory signaling while maintaining the ability to form heterotrimers with wild-type TNF.

[0036] The trimer interface domain refers to the amino acid residues that facilitate the formation of homotrimers and heterotrimers in sTNF. In SEQ ID NO: 1, the trimer interface domain includes residues V13, L57, 158, Y59, L93, 197, K98, Y115, G116, P117, 1118, Y119, L120, G121, G122, V123, G148, and 1154. Substitutions at these residues can enhance the affinity of DN-TNF muteins for wild-type TNF monomers, thereby promoting the formation of heterotrimers that sequester wild-type sTNF and neutralize its inflammatory effects. By optimizing mutations in this domain, the therapeutic potency of DN-TNF can be increased while maintaining its ability to form stable trimeric structures.

[0037] A biocompatible stealth polymer is a polymer that, when conjugated to a therapeutic protein, enhances pharmacokinetics by reducing renal clearance, proteolysis, and immune recognition. Herein, the biocompatible stealth polymer may include polyethylene glycol (PEG), polysarcosine (PolySar), hydroxyethyl starch (HES), poly(N-(2- hydroxypropyl)methacrylamide) (pHPMA), polyglutamic acid (PGA), poly(2-oxazoline) (POZ), poly(vinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), dextran, or combinations thereof. These polymers improve the circulating half-life of DN-TNF muteins and help prevent rapid enzymatic degradation or immune clearance. The present invention provides site-specific conjugation of such stealth polymers via a thiol-maleimide linkage to improve stability and retention of the bioconjugate in circulation. While examples include PEG and PolySar illustrations, it should be appreciated by one with skill in the art that other maleimide polymers can be similarly implemented.

[0038] A maleimide linker is a chemical moiety that contains a maleimide functional group, which reacts with thiol groups on cysteine residues in proteins to form a stable thioether bond. The DN-TNF muteins are conjugated to a biocompatible stealth polymer via a maleimide linker to improve pharmacokinetic properties. However, conventional maleimide-thiol conjugates suffer from instability due to the occurrence of the retro-Michael reaction, wherein the thioether bond undergoes cleavage, leading to the loss of the conjugated polymer and degradation of the therapeutic.

[0039] A hydrolyzed maleimide derivative is a maleimide moiety that has undergone controlled hydrolysis, converting the thiosuccinimide ring of the maleimide into a stablemaleamic acid derivative. Hydrolysis of the maleimide linker prevents the retro-Michael reaction and thiol exchange, thereby stabilizing the DN-TNF bioconjugate. In the present invention, at least 85%, 90%, or 95% of the maleimide linkers in the therapeutic composition are hydrolyzed through a controlled pH adjustment step, significantly reducing the risk of polymer dissociation and enhancing therapeutic stability.

[0040] The retro-Michael reaction is a chemical process in which a maleimide-thiol conjugate undergoes cleavage, resulting in the loss of the conjugated polymer. This reaction is undesirable in therapeutic bioconjugates because it leads to instability and reduced bioavailability. By hydrolyzing the maleimide linker under controlled conditions, the present invention eliminates this instability, ensuring that the DN-TNF bioconjugate remains intact for prolonged therapeutic efficacy.

[0041] The term steric hindrance refers to a spatial blocking effect caused by the conjugation of a biocompatible stealth polymer at a receptor binding site, which physically prevents TNF heterotrimer from interacting with TNF receptors TNFR1 and TNFR2. In certain embodiments of the present invention, the site-specific conjugation of a stealth polymer at a cysteine-modified receptor binding domain residue results in steric hindrance that further reduces TNF receptor binding. This provides an additional mechanism for selective inhibition of soluble TNF without interfering with transmembrane TNF function.

[0042] A selective soluble TNF neutralizing bioconjugate is a DN-TNF mutein that has been covalently conjugated to a biocompatible stealth polymer via a maleimide linker, wherein at least 85%, 90%, or 95% of the maleimide linkers are hydrolyzed to prevent polymer dissociation. The bioconjugate selectively neutralizes soluble TNF while preserving transmembrane TNF function, offering an advantage over traditional TNF inhibitors that indiscriminately block both soluble and transmembrane TNF activity.

[0043] An injectable formulation refers to a pharmaceutical composition suitable for administration via at least subcutaneous, intramuscular, intravitreal, or intravenous injection. The formulation typically consists of a stabilized DN-TNF bioconjugate in a buffered aqueous solution, with a pH adjusted between 5.5 and 7.5 to maintain protein stability. The formulation may also include stabilizers, surfactants, or excipients to optimize solubility, bioavailability, and storage conditions.

[0044] A pH-controlled hydrolysis step is a process in which the pH of a DN-TNF bioconjugate solution is adjusted to between 8.0 and 9.5 after polymer conjugation to induce hydrolysis of the maleimide linker. The solution is incubated at a temperature between 18°C and 25°C for a period sufficient to hydrolyze at least 85% of the maleimide linkers, therebypreventing retro-Michael reaction and polymer dissociation. Following hydrolysis, the pH is adjusted to a physiologically acceptable range of 5.5 to 7.5 for final formulation.

[0045] The term heterotrimer formation refers to the assembly of DN-TNF muteins with wild-type TNF monomers into inactive heterotrimers, thereby neutralizing soluble TNF activity. In the present invention, modifications in the trimer interface domain are designed to preserve or enhance heterotrimer formation, ensuring effective sequestration of wild-type TNF.

[0046] A thiol conjugation site is a single cysteine substitution engineered in a DN- TNF mutein to allow site-specific thiol-maleimide conjugation with a stealth polymer. The cysteine substitution may be introduced at a surface-exposed residue, such as E23C, A38C, L43C, R44C, D45C, D140C, or within the receptor binding domain at Q21C, R31C, A145C, or E146C. These modifications enable controlled conjugation, improving the consistency and stability of the resulting bioconjugate.

[0047] DN-TNF Mutein Design and Engineering

[0048] Dominant-negative TNF (DN-TNF) muteins are engineered variants of soluble tumor necrosis factor (sTNF) that are designed to selectively neutralize soluble TNF while preserving transmembrane TNF (tmTNF) function. These muteins are derived from the wildtype sTNF sequence (SEQ ID NO: 1) and contain specific amino acid substitutions in one or both of two critical functional domains: (i) the TNF receptor interaction domain and (ii) the trimer interface domain. The DN-TNF mutein may include the amino acid sequence of SEQ ID NO: 1 (wild type sTNF) modified with 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions in the receptor interaction domain, the trimer interface domain, or a combination thereof. By introducing strategic substitutions in the receptor interaction domain, DN-TNF muteins lose the ability to bind TNF receptors (TNFR1 and TNFR2), thereby eliminating receptor-mediated inflammatory signaling. Simultaneously, substitutions in the trimer interface domain preserve or enhance affinity for wild-type TNF monomers, allowing DN-TNF muteins to efficiently incorporate into heterotrimers with endogenous sTNF. This mechanism enables functional sequestration of sTNF, rendering it incapable of receptor activation.

[0049] The TNF receptor interaction domain includes amino acid residues Q21, A22, E23, N30, R31, R32, A33, K65, G66, Q67, G68, S86, Y87, V91, A109, El 10, D140, andA145. Substitutions at these positions are designed to abolish receptor binding without impairing trimer formation. The trimer interface domain consists of V13, L57, 158, Y59, L93, 197, K98, Y115, G116, P117, 1118, Y119, L120, G121, G122, V123, G148, and 1154, and modifications at these positions preserve or increase the affinity of DN-TNF for wild-type sTNF, facilitating heterotrimerization. One or a combination of these modifications results in a DN-TNF muteinthat effectively neutralizes soluble TNF while preserving tmTNF signaling, thereby reducing the risk of adverse effects such as immunosuppression and demyelination.

[0050] FIG. 1 illustrates a table summarizing amino acid substitutions in DN-TNF designed to reduce TNF receptor binding while maintaining heterotrimer formation. The table identifies specific TNF residues that, when substituted, disrupt interactions with TNF receptors TNFR1 and TNFR2. The substitutions modify electrostatic charge, hydrophobic contacts, or hydrogen bonding interactions, thereby weakening receptor binding affinity. These modifications ensure that DN-TNF remains competent for heterotrimer formation with wildtype TNF, allowing for effective sequestration and neutralization of soluble TNF without triggering receptor activation.

[0051] FIG. 2 presents a table outlining amino acid substitutions in the trimer interface domain, which enhance the ability of DN-TNF to bind wild-type TNF subunits and form stable heterotrimers. The substitutions reinforce hydrophobic interactions, hydrogen bonding networks, and electrostatic complementarity, improving monomer-monomer affinity within the TNF trimer. The structural reinforcement imparted by these modifications increases trimer stability, ensuring that DN-TNF remains active and resistant to dissociation under physiological conditions.

[0052] Production of DN-TNF Muteins

[0053] The production of DN-TNF muteins follows standard recombinant protein expression and purification techniques. DN-TNF muteins can be expressed in bacterial, yeast, insect, or mammalian cell expression systems, with Escherichia coli (E. coli) and Chinese Hamster Ovary (CHO) cells being preferred due to high expression yield and proper protein folding.

[0054] The gene encoding the DN-TNF mutein is cloned into an expression vector using conventional molecular biology techniques, such as polymerase chain reaction (PCR), site-directed mutagenesis, and restriction enzyme cloning. The expression vector typically includes a strong promoter (e.g., CMV for mammalian cells or T7 for bacterial systems), a ribosome binding site (for prokaryotic expression), and a selection marker (e.g., antibiotic resistance genes for stable cell line selection).

[0055] In bacterial expression systems, the recombinant protein may be produced as inclusion bodies, requiring denaturation and refolding under controlled conditions to ensure correct trimer formation. Alternatively, expression in mammalian cells (e.g., CHO or HEK293) allows for proper post-translational modifications and native disulfide bond formation. Following expression, DN-TNF muteins are purified using affinity chromatography (e.g., His-tag or antibody-based capture), size-exclusion chromatography (SEC), and ion-exchange chromatography. The final purified protein can be assessed for trimer formation, receptor binding affinity, and stability using biophysical assays such as analytical ultracentrifugation, dynamic light scattering (DLS), and surface plasmon resonance (SPR).

[0056] Mechanism of Action of DN-TNF Muteins

[0057] The primary mechanism of action of DN-TNF muteins is heterotrimerization with wild-type TNF monomers, leading to functional inhibition of sTNF signaling. In contrast to traditional anti-TNF monoclonal antibodies and soluble TNF receptor decoys, which bind and neutralize TNF monomers or trimers, DN-TNF muteins act by sequestering sTNF into inactive heterotrimers that cannot bind TNF receptors.

[0058] Upon administration, DN-TNF muteins circulate in the bloodstream and encounter endogenous soluble TNF. Due to their affinity for trimer formation, DN-TNF muteins incorporate into wild-type TNF trimers, replacing one or more subunits of the TNF homotrimer. These mixed heterotrimers remain structurally stable but are unable to activate TNF receptors (TNFR1 and TNFR2) due to the receptor binding-disruptive mutations in the DN-TNF mutein subunits. This effectively prevents sTNF-mediated pro-inflammatory signaling while preserving the physiological functions of transmembrane TNF.

[0059] In some embodiments, an additional mechanism of TNF inhibition may involve steric hindrance induced by site-specific conjugation of a biocompatible stealth polymer (e.g., PEG or PolySar) to the DN-TNF mutein. By introducing a single cysteine substitution at a receptor binding domain residue, the present invention enables controlled thiol-maleimide conjugation of the stealth polymer to the DN-TNF mutein. The size and spatial orientation of the conjugated polymer physically block receptor binding, further preventing TNF receptor activation even if some heterotrimers contain wild-type sTNF subunits.

[0060] Advantages of DN-TNF Muteins Over Conventional TNF Inhibitors

[0061] The DN-TNF muteins disclosed herein offer several key advantages over conventional anti-TNF biologies, including monoclonal antibodies and soluble TNF receptors. Unlike monoclonal antibodies (e.g., infliximab, adalimumab), which neutralize both soluble TNF and transmembrane TNF, DN-TNF muteins selectively inhibit soluble TNF while preserving transmembrane TNF function. This reduces the risk of immunosuppression and prevents unwanted side effects such as demyelination and increased susceptibility to infections.

[0062] Additionally, DN-TNF muteins have a unique mechanism of action that does not rely on Fc-mediated clearance. Conventional monoclonal antibodies function by binding TNF and facilitating its clearance via Fc receptor interactions, whereas DN-TNF muteins actby incorporating into heterotrimers and neutralizing TNF within the extracellular space. This alternative mode of inhibition leads to longer duration of action and reduced immunogenicity.

[0063] The conjugation of DN-TNF muteins to biocompatible stealth polymers further enhances pharmacokinetic properties by reducing renal clearance and proteolysis. The present invention employs controlled hydrolysis of the maleimide linker to improve conjugate stability, preventing retro-Michael reaction and polymer dissociation, thus maintaining extended halflife in circulation.

[0064] Substitution of VIM for Improved Expression of DN-TNF Mutein

[0065] An amino acid substitution VIM may be imposed at the N-terminal position of the DN-TNF mutein to enhance recombinant protein expression and improve translation efficiency in various heterologous expression systems, including bacterial, yeast, insect, and mammalian cells. In many recombinant protein production platforms, the presence of a valine residue at the N-terminus can interfere with efficient translation initiation, particularly in systems that rely on strong ribosomal binding sequences. By substituting valine (V) with methionine (M) at position 1, the DN-TNF mutein benefits from optimized translation initiation, leading to increased protein yield and improved expression stability.

[0066] In bacterial expression systems such as Escherichia coli (E. coli), the initiation codon for protein synthesis is typically AUG (methionine), and translation efficiency can be compromised when non-methionine residues follow the start codon. The VIM substitution ensures that the recombinant DN-TNF mutein maintains a favorable N-terminal sequence that aligns with the natural methionyl-tRNA recognition mechanism, thereby promoting efficient ribosome loading and elongation. In mammalian and yeast expression systems, N-terminal methionine is often post-translationally cleaved, meaning the VIM substitution has no adverse impact on the final protein product but still enhances initial expression efficiency. This modification is particularly beneficial for large-scale biopharmaceutical manufacturing, where maximizing expression yield and consistency is essential for cost-effective production and commercial scalability.

[0067] Engineering DN-TNF Muteins for Site-Specific Conjugation

[0068] To enable precise and controlled conjugation of DN-TNF muteins to biocompatible stealth polymers, the present invention introduces specific amino acid modifications to eliminate endogenous cysteine residues and incorporate a single site-specific cysteine substitution at a strategically selected position. This approach ensures highly efficient and site-directed thiol-maleimide conjugation, minimizing heterogeneity and maximizing the stability of the conjugated biotherapeutic.

[0069] The wild-type soluble TNF (sTNF) monomer contains two endogenous cysteine residues at positions C69 and C101, which are known to form nonessential intramolecular disulfide bonds. These native cysteines can interfere with controlled polymer conjugation due to their inherent reactivity. To prevent nonspecific conjugation, oxidative instability, or disulfide scrambling, the present invention introduces C69V and C101A substitutions in DN- TNF muteins. The C69V substitution replaces cysteine with valine, a hydrophobic, non- reactive residue, while C101A replaces cysteine with alanine, a small, neutral amino acid. These mutations effectively eliminate all endogenous cysteine residues in DN-TNF, thereby creating a blank molecular background that allows for a single site-specific cysteine modification at a designated position.

[0070] With the elimination of C69 and C101, a new single cysteine residue is introduced at a strategically selected site that is either surface-exposed or located within the TNF receptor interaction domain. The site-specific cysteine substitution allows for precise thiol-maleimide conjugation of DN-TNF to a biocompatible stealth polymer such as polyethylene glycol (PEG) or polysarcosine (PolySar). Preferred substitution sites for cysteine incorporation include E23C, A38C, L43C, R44C, D45C, D140C, Q21C, R31C, A145C, or E146C. These sites were selected based on their solvent accessibility, minimal impact on trimerization, and optionally an ability to induce steric hindrance upon polymer conjugation.

[0071] In embodiments where the cysteine substitution is introduced within the TNF receptor interaction domain, such as Q21C, R31C, A145C, or E146C, conjugation of the stealth polymer physically blocks receptor binding through steric hindrance, further ensuring that DN- TNF cannot activate TNFR1 or TNFR2. This feature enhances the neutralization selectivity of DN-TNF muteins, making them functionally superior to conventional anti-TNF agents that indiscriminately block both soluble and transmembrane TNF activity.

[0072] To facilitate efficient conjugation, the single cysteine-substituted DN-TNF muteins are reacted with maleimide-functionalized stealth polymers under mild reducing conditions, ensuring that the polymer selectively reacts with the engineered thiol group. Following conjugation, the maleimide linker undergoes controlled hydrolysis, stabilizing the conjugate and preventing retro-Michael reaction and polymer dissociation. The resulting DN- TNF bioconjugate maintains high affinity for wild-type sTNF, efficiently sequesters soluble TNF into inactive heterotrimers, and retains prolonged therapeutic activity in vivo.

[0073] FIG. 3 details site-specific cysteine substitutions engineered for thiol conjugation with stealth polymers. The table identifies TNF residues that can be mutated to cysteine (Cys, C) to provide a reactive thiol group, facilitating site-directed conjugation withmaleimide-functionalized polyethylene glycol (PEG) or polysarcosine (PolySar). Certain substitutions are located within the TNF receptor binding domain, meaning that conjugation at these sites not only attaches a polymer but also induces steric hindrance, further preventing receptor interaction. Other substitutions occur at surface-exposed residues, allowing conjugation without disrupting heterotrimerization or protein stability.

[0074] Stealth Polymer Conjugation & Stabilization viaMaleimide Hydrolysis

[0075] The DN-TNF muteins of the present invention are conjugated to a biocompatible stealth polymer via site-specific thiol-maleimide chemistry, ensuring prolonged circulation time and enhanced therapeutic stability. To achieve precise polymer attachment, a single cysteine substitution is introduced at a strategically selected surface-exposed residue or within the TNF receptor interaction domain following the removal of wild-type cysteines (C69V / C101A). This allows for controlled thiol conjugation to a maleimide-functionalized stealth polymer, which may include polyethylene glycol (PEG), polysarcosine (PolySar), hydroxyethyl starch (EES), poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), polyglutamic acid (PGA), poly(2-oxazoline) (POZ), poly(vinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), dextran, or combinations thereof. The stealth polymer protects the bioconjugate from enzymatic degradation, immune recognition, and rapid renal clearance, thereby increasing half-life and bioavailability.

[0076] The conjugation reaction is carried out under mild reducing conditions, ensuring selective reaction of the engineered cysteine residue with the maleimide-functionalized polymer. This results in a stable thioether bond, effectively linking the DN-TNF mutein to the stealth polymer. However, maleimide-thiol conjugates are inherently unstable due to their susceptibility to retro-Michael reaction and thiol exchange, which can lead to polymer dissociation and reduced therapeutic efficacy. The present invention overcomes this limitation by intentionally hydrolyzing the maleimide linker under controlled conditions, yielding a hydrolyzed maleimide derivative that is resistant to further thiol exchange.

[0077] Following polymer conjugation, the pH of the bioconjugate solution is adjusted to between 8.0 and 9.5, and the solution is incubated at a temperature of 18°C to 25°C for a period sufficient to hydrolyze at least 85% of the maleimide linkers to their corresponding maleamic acid derivatives, generally 12 hours to 48 hours. The hydrolysis process prevents retro-Michael reaction, ensuring that the stealth polymer remains stably attached to the DN- TNF mutein throughout storage and its therapeutic duration. Unlike conventional approaches that focus on preventing maleimide hydrolysis, the present invention takes the counterintuitive approach of inducing hydrolysis in a controlled manner, transforming a liability into a stability-enhancing feature. The hydrolyzed bioconjugate is then adjusted to a physiologically acceptable pH of 5.5-7.5, more preferably 6.0-7.0, formulated in a buffered aqueous solution suitable for injection, and stabilized for storage.

[0078] FIG. 5 illustrates the stabilization mechanism of DN-TNF bioconjugates via controlled maleimide hydrolysis. The schematic highlights how non-hydrolyzed maleimide linkers are susceptible to retro-Michael reaction and thiol exchange, leading to polymer dissociation and potential loss of stability. The controlled hydrolysis process converts maleimide into a stable ring-opened maleamic acid derivative, preventing undesired reactivity and ensuring that the conjugated polymer remains permanently attached to DN-TNF.

[0079] FIG. 7 provides another schematic representation of DN-TNF stabilization via maleimide hydrolysis, reinforcing the mechanism shown in FIG. 5. It emphasizes the importance of buffer conditions, pH, and incubation time in achieving controlled hydrolysis, preventing retro-Michael reaction and polymer dissociation.

[0080] The resulting DN-TNF bioconjugate exhibits superior storage and in vivo stability, extended half-life, and improved pharmacokinetics compared to non-hydrolyzed maleimide conjugates. Additionally, in embodiments where the stealth polymer is conjugated at a cysteine-substituted residue within the receptor binding domain, the polymer introduces steric hindrance, further reducing the potential for TNF receptor activation. The combination of selective sTNF neutralization, site-specific conjugation, polymer stabilization, and / or steric blockade enables a highly optimized TNF inhibitor with enhanced therapeutic properties.

[0081] Molecular Weight Selection for Biocompatible Stealth Polymers in TNF Bioconjusates

[0082] The molecular weight (MW) of biocompatible stealth polymers, such as polyethylene glycol (PEG) and polysarcosine (PolySar), plays a critical role in modulating pharmacokinetics, stability, and clearance of protein bioconjugates. Selection of an appropriate MW range is essential to maximize circulation half-life while minimizing rapid renal clearance, immune recognition, and potential toxicological concerns. Based on known pharmacokinetic principles and prior studies on PEG and polysarcosine-protein conjugates, the optimal MW range for PEG is between 5 kDa and 40 kDa, while for polysarcosine, it is between 3 kDa and 30 kDa.

[0083] For polyethylene glycol (PEG), molecules smaller than 5 kDa are subject to rapid renal clearance, as they fall below the glomerular filtration threshold (-30-50 kDa for proteins, but smaller for flexible polymers like PEG). Conversely, PEG sizes above 40 kDa can exhibit reduced clearance, leading to potential tissue accumulation and PEG-associatedvacuolation, a phenomenon observed in long-term studies of PEGylated drugs. Within the 5- 40 kDa range, PEG provides an optimal balance between enhanced solubility, immune evasion, and extended half-life, as evidenced by the FDA-approved PEGylated biologies such as Pegasys (PEGylated interferon, ~40 kDa), Pegloticase (PEGylated uricase, -10-40 kDa), and Pegfilgrastim (PEGylated G-CSF, -20 kDa). These clinical examples support the rationale that PEG in this MW range achieves a prolonged circulation time without compromising safety or efficacy.

[0084] For polysarcosine (PolySar), the optimal MW range is slightly lower (3-30 kDa) due to its superior biodegradability and clearance properties compared to PEG. PolySar molecules as small as 3 kDa have been shown to provide effective steric shielding and stealth properties, reducing opsonization and macrophage uptake. Unlike PEG, higher MW polysarcosine chains (>30 kDa) do not accumulate in tissues but may slow clearance excessively, limiting dosing flexibility. Studies indicate that 10-30 kDa poly sarcosine achieves optimal plasma half-life extension while preserving renal clearance, making it a strong alternative to PEG for therapeutic conjugates. The emerging field of PolySar-based protein conjugation ("PolySarylation") is gaining interest due to its lower immunogenicity and superior biocompatibility compared to PEG, making it an attractive stealth polymer for TNF bioconjugates.

[0085] Moreover, our data suggests a polymer MW between 5 kDa and 10 kDa conjugated to the -17 kDa DN-TNF mutein results in a bioconjugate that can effectively cross the blood brain barrier, which is desirable for treating TNF mediated disorders of the CNS or involving neuroinflammation, whereas a polymer MW between 15 kDa and 40 kDa conjugated to the -17 kDa DN-TNF mutein results in a bioconjugate that cannot effectively cross the blood brain barrier, which may be desirable for indications involving peripheral inflammation.

[0086] Given these considerations, limiting the PEG MW range to 5-40 kDa and the PolySar MW range to 3-30 kDa ensures optimal pharmacokinetics while preventing potential issues such as rapid clearance (low MW) or unwanted tissue accumulation (high MW). This selection provides a well-balanced stealth effect, maintaining therapeutic efficacy while minimizing toxicity risks.

[0087] Formulation o f the DN-TNF Bioconjusate

[0088] The therapeutic composition comprising stabilized DN-TNF bioconjugate is formulated in a buffered aqueous solution with a pH range of 5.5 to 7.5, ensuring protein stability, solubility, and compatibility for injection. The selected pH range is critical for maintaining bioconjugate integrity while preventing aggregation, degradation, or loss oftherapeutic activity. Following the controlled hydrolysis of the maleimide linker at pH 8.0-9.5, the solution is adjusted to the physiologically relevant range of 5.5-7.5, and more preferably 6.0 - 7.0, to optimize long-term storage stability and in vivo tolerability. This formulation strategy ensures that the DN-TNF bioconjugate remains stable and bioactive upon administration.

[0089] The buffered formulation may include phosphate, citrate, histidine, acetate, or other physiologically acceptable buffering agents to maintain the desired pH range. In addition to the buffer system, the formulation may also contain osmotic stabilizers, surfactants, and excipients such as sodium chloride, trehalose, sucrose, polysorbates, or amino acids to enhance protein solubility, prevent aggregation, and protect against freeze-thaw degradation. The final formulation is designed for subcutaneous, intramuscular, intravitreal, or intravenous administration, allowing for flexible therapeutic applications while ensuring maximum bioavailability and efficacy.

[0090] Therefore, according to the embodiments herein, a therapeutic composition comprises a selective soluble TNF neutralizing bioconjugate in an injectable formulation, the selective soluble TNF neutralizing bioconjugate comprising: (i) a dominant negative TNF (DN- TNF) mutein comprising the amino acid sequence of SEQ ID NO: 1 modified with 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions in a TNF receptor interaction domain, a trimer interface domain, or a combination thereof, and (ii) a linker-polymer moiety covalently conjugated to the DN-TNF mutein via a thioether bond, the linker-polymer moiety comprising a maleimide linker and a biocompatible stealth polymer, wherein at least 85% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative, thereby preventing retro-Michael reaction and polymer dissociation.

[0091] In various embodiments, the hydrolyzed maleimide derivative is maleamic acid.

[0092] In various embodiments, the biocompatible stealth polymer of the linker- polymer moiety comprises polyethylene glycol (PEG) or polysarcosine (PolySar).

[0093] In various embodiments, the biocompatible stealth polymer comprises a molecular weight between: 5 kDa and 40 kDa for PEG, or 3 kDa and 30 kDa for PolySar.

[0094] In various embodiments, the biocompatible stealth polymer of the linker- polymer moiety is selected from the group consisting of: polyethylene glycol (PEG), polysarcosine (PolySar), hydroxyethyl starch (HES), poly(N-(2- hydroxypropyl)methacrylamide) (pHPMA), polyglutamic acid (PGA), poly(2-oxazoline) (POZ), polyvinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), dextran, or a combination thereof.

[0095] In various embodiments, at least 90% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative.

[0096] In various embodiments, at least 95% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative.

[0097] In various embodiments, wherein with respect to SEQ ID NO: 1 said amino acid substitutions in the TNF receptor interaction domain comprise substitutions at residues selected from the group consisting of: Q21, A22, E23, N30, R31, R32, A33, K65, G66, Q67, G68, S86, Y87, V91, A109, El 10, D140, A145, thereby reducing or eliminating TNF receptor binding. In various embodiments, the amino acid substitutions in the TNF receptor interaction domain are selected from the group consisting of: Q21A, Q21C, Q21E, Q21G, Q21R, Q21S, A22D, A22E, E23A, E23Q, N30A, N30D, N30E, R31C, R31D, R31E, R31I, R31S, R32D, R32E, R32S, A33D, A33E, K65D, K65E, K65I, K65M, K65N, K65Q, K65S, K65T, K65V, K65W, G66D, G66E, G66K, G66Q, Q67D, Q67E, Q67K, Q67R, Q67S, Q67V, Q67W, Q67Y, G68D, G68E, G68K, S86D, S86E, S86Q, S86R, Y87A, Y87H, Y87Q, Y87R, Y87S, V91D, V91E, A109D, A109E, E110A, E110Q, D140A, D140K, D140R, A145C, A145D, A145E, A145F, A145G, A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, and A145Y.

[0098] In various embodiments, wherein with respect to SEQ ID NO: 1 said amino acid substitutions in the trimer interface domain comprise amino acid substitutions at residues selected from the group consisting of: V13, L57, 158, Y59, L93, 197, K98, Y115, G116, P117, 1118, Y119, L120, G121, G122, V123, G148, 1154. In various embodiments, the amino acid substitutions in the trimer interface domain are selected from the group consisting of: V13I, V13L, V13F, V13M, L57I, L57V, L57F, L57W, I58L, I58M, I58V, I58F, Y59H, Y59R, Y59Q, Y59K, L93I, L93F, L93M, L93W, I97F, I97L, I97M, I97T, I97V, K98R, K98H, K98Q, K98E, Y115H, Y115R, Y115Q, Y115E, G116S, G116T, G116Q, P117A, P117S, P117N, I118L, I118M, I118V, I118F, Y119F, Y119W, Y119H, L120I, L120V, L120F, L120M, G121S, G121T, G121N, G121Q, G122S, G122T, G122N, G122Q, V123I, V123L, V123F, V123M, G148S, G148T, G148N, G148Q, I154L, 1154V, I154M, and I154F, wherein said amino acid substitutions increases the affinity of the DN-TNF mutein for wild-type TNF monomers, thereby promoting heterotrimer formation and neutralizing soluble TNF..

[0099] In various embodiments, the DN-TNF mutein further comprises the amino acid substitutions C69V / C101 A relative to SEQ ID NO: 1.

[0100] In various embodiments, the DN-TNF mutein comprises a single cysteine substitution at a surface-exposed residue selected from the group consisting of: E23C, A38C, L43C, R44C, D45C, D140C, or a single cysteine substitution at a position within the receptorbinding domain selected from the group consisting of Q21C, R31C, A145C, and E146C, whereby the DN-TNF mutein is adapted for site specific cysteine thiol conjugation with the stealth polymer.

[0101] In various embodiments, the DN-TNF mutant protein further comprises amino acid substitution VIM relative to SEQ ID NO: 1.

[0102] In various embodiments, the DN-TNF mutein further comprising up to twelve amino acid deletions at the N-terminal end thereby forming a truncated DN-TNF mutein.

[0103] In various embodiments, the injectable formulation is a buffered aqueous solution with a pH between 5.5 and 7.5.

[0104] In various embodiments, the therapeutic composition is formulated for administration via subcutaneous, intramuscular, intravitreal, or intravenous injection.

[0105] In another aspect, a method of treating a TNF-mediated inflammatory disorder in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the therapeutic composition as described herein to the subject via subcutaneous, intramuscular, intravitreal, or intravenous injection, wherein the composition comprises a stabilized DN-TNF mutein conjugated to a biocompatible stealth polymer, and wherein at least 85%, 90%, or 95% of the selective soluble TNF neutralizing bioconjugate comprises a maleamic acid derivative linking the DN-TNF mutein to the stealth polymer, thereby preventing biocompatible stealth polymer dissociation and prolonging therapeutic activity.

[0106] In another aspect, a method for preparing a therapeutic composition comprising a stabilized selective soluble TNF-neutralizing bioconjugate, the method comprising: conjugating a dominant-negative TNF (DN-TNF) mutein comprising the amino acid sequence of SEQ ID NO: 1 modified with 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions in a TNF receptor interaction domain, a trimer interface domain, or a combination thereof, to a biocompatible stealth polymer via a maleimide linker to form a bioconjugate; adjusting the pH of the bioconjugate solution to between 8.0 and 9.5 to induce hydrolysis of the maleimide linker; incubating the solution at a temperature between 18°C and 25°C for a period sufficient to hydrolyze at least 85% of the maleimide linkers to a maleamic acid derivative, thereby preventing retro-Michael reaction and polymer dissociation; and adjusting the pH of the solution to between 5.5 and 7.5 to obtain a stable injectable formulation.

[0107] In another aspect, a method for stabilizing a selective soluble TNF-neutralizing bioconjugate, the method comprising: conjugating a DN-TNF mutein to a maleimide- functionalized biocompatible stealth polymer via a thioether bond; subjecting the conjugated bioconjugate to a controlled hydrolysis step at a pH of 8.0 to 9.5 and a temperature of 18°C to25°C for at least 12 hours; allowing at least 85%, 90%, or 95% of the maleimide linkers to be hydrolyzed to their corresponding maleamic acid derivatives, thereby preventing polymer dissociation and degradation; formulating the stabilized bioconjugate in an aqueous injectable solution with a pH of 5.5 to 7.5.Examples

[0108] Example 1 - Production of a DN-TNF Mutein with VIM, C69V, C101A, R31C, A145R, and Y87H Substitutions

[0109] A DN-TNF mutein was designed incorporating the following amino acid substitutions relative to SEQ ID NO: 1 : VIM, C69V, C101A, R31C, A145R, and Y87H. This DN-TNF mutein is sometimes referred to as “DN-550” and is further represented by SEQ ID NO:2. The VIM substitution was introduced to enhance translation efficiency in heterologous expression systems, while C69V and C101A were engineered to eliminate native cysteines and prevent disulfide scrambling, ensuring site-specific conjugation at R31C. The R31C substitution provides a single cysteine residue for thiol-maleimide conjugation to a stealth polymer, and A145R and Y87H further reduce receptor binding while maintaining heterotrimerization capability with wild-type TNF.

[0110] To produce this DN-TNF mutein, a synthetic gene encoding the modified TNF sequence was synthesized and codon-optimized for expression in Escherichia coli (E. coli). The gene was cloned into a pET expression vector under the control of a T7 promoter, allowing for inducible high-yield expression in E. coli. The construct included an N-terminal methionine start codon, which aligned with the VIM substitution, and an affinity purification tag (e.g., His- tag or Strep-tag) to facilitate downstream purification. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, and successful transformants were selected on LB agar plates containing ampicillin (100 pg / mL).

[0111] For protein expression, an overnight starter culture was used to inoculate a 1 L culture of LB medium supplemented with ampicillin. The culture was grown at 37°C with shaking at 250 rpm until the optical density at 600 nm (OD600) reached 0.6-0.8, at which point expression was induced by adding 0.5 mM isopropyl Q-D- 1 -thiogalactopyranoside (IPTG). The culture was then incubated at 30°C for 4 hours to promote soluble protein expression. After induction, the cells were harvested by centrifugation at 6,000 * g for 10 minutes at 4°C, and the resulting cell pellet was stored at -80°C until further processing.

[0112] To extract the DN-TNF mutein, the cell pellet was resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole and disruptedusing sonication on ice (10 cycles of 10-second pulses with 30-second rest intervals). The lysate was clarified by centrifugation at 15,000 x g for 30 minutes at 4°C, and the supernatant containing the soluble DN-TNF mutein was loaded onto a nickel-affinity chromatography column (Ni-NTA). The column was washed with buffer containing 50 mM imidazole to remove nonspecifically bound proteins, and the DN-TNF mutein was eluted using a buffer containing 250 mM imidazole.

[0113] To remove the affinity tag and further purify the DN-TNF mutein, the eluate was incubated with a protease (e.g., TEV or thrombin) for tag cleavage, followed by passage through a size-exclusion chromatography (SEC) column equilibrated in phosphate-buffered saline (PBS, pH 7.4). The SEC step ensured removal of protein aggregates and yielded a highly pure DN-TNF mutein in trimeric form. The final protein concentration was determined using a bicinchoninic acid (BCA) assay, and purity was confirmed by SDS-PAGE and analytical ultracentrifugati on .

[0114] To prepare the DN-TNF bioconjugate, the purified mutein was reacted with maleimide-functionalized polyethylene glycol (PEG-MAL, 10 kDa) under mild reducing conditions to allow selective conjugation at R31C. The reaction mixture was incubated at pH 6.0-6.5 at 20±2°C for 1 hour, ensuring efficient thiol-maleimide conjugation.

[0115] FIG. 4 provides a schematic representation of DN-TNF conjugation with maleimide-PEG. The diagram shows the DN-TNF protein linked via a maleimide-thiol reaction to a polymer moiety. The methoxy-maleimide-PEG linker is depicted as covalently attached to the DN-TNF mutein, forming a DN-TNF-PEG bioconjugate. This conjugation process improves pharmacokinetics, reduces renal clearance, and enhances biocompatibility, ensuring prolonged therapeutic activity.

[0116] In an additional embodiment, an amount of the DN-TNF mutein containing the VIM, C69V, C101A, R31C, A145R, and Y87H substitutions was conjugated to polysarcosine (PolySar), an alternative biocompatible stealth polymer selected for its low immunogenicity, non-fouling properties, and enhanced pharmacokinetic profile. Following recombinant expression and purification, the DN-TNF mutein was selectively conjugated to maleimide- functionalized PolySar (PolySar-MAL, 15 kDa) via the engineered cysteine at position R31C, using a thiol-maleimide conjugation reaction under mild reducing conditions (pH 6.1, 25°C, 1 hour).

[0117] FIG. 6 depicts DN-TNF conjugation with mal eimide-Poly Sar, demonstrating an alternative stealth polymer to PEG. The process follows the same thiol-maleimide conjugation chemistry used for PEGylation, but instead employs polysarcosine, which offers lowimmunogenicity and non-fouling properties. PolySar conjugation enhances the pharmacokinetic profile of DN-TNF while maintaining trimer integrity and TNF neutralization function.

[0118] Example 2 - Production of a DN-TNF Mutein with VIM, C69V, C101A, R31C, A145R, and 197 T Substitutions

[0119] In a further example, a DN-TNF mutein was engineered to incorporate the following amino acid substitutions: VIM, C69V, C101A, R31C, A145R, and I97T. This DN- TNF mutein is sometimes referred to as “DN-346” and is further represented by SEQ ID NO:3. As in the prior example, the VIM substitution was introduced to enhance translation efficiency in heterologous expression systems, particularly in E. coli, by optimizing initiation codon recognition and ribosomal loading. The C69V and C101 A substitutions were made to eliminate native cysteine residues, thereby preventing unwanted disulfide bond formation and enabling site-specific conjugation at R31C, a substitution that provides a single reactive cysteine for thiol-maleimide conjugation with a stealth polymer. The A145R substitution disrupts TNF receptor binding, further reducing the potential for TNF-mediated signaling, while the newly introduced I97T mutation in the trimer interface domain was designed to enhance the affinity of DN-TNF for wild-type TNF monomers, further improving heterotrimer formation and TNF neutralization efficiency.

[0120] As in the previous example, the modified TNF gene was synthesized with codon optimization for expression in E. coli, cloned into a pET expression vector under the control of a T7 promoter, and transformed into BL21(DE3) competent E. coli cells. The transformation was confirmed by colony PCR and DNA sequencing, ensuring that the desired mutations were correctly incorporated. Positive clones were selected, and an overnight starter culture was used to inoculate a 1 L LB culture supplemented with ampicillin. The culture was grown at 37°C to an OD600 of 0.6-0.8, and IPTG induction (0.5 mM) was used to initiate DN-TNF mutein expression. The culture was then maintained at 30°C for 4 hours to promote soluble protein expression.

[0121] The bacterial cells were harvested, lysed using sonication in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and clarified by centrifugation at 15,000 x g. The DN-TNF mutein was purified using nickel-affinity chromatography, washed with 50 mM imidazole, and eluted with 250 mM imidazole-containing buffer. Tag cleavage was performed with TEV protease, and the protein was further purified using size-exclusion chromatography (SEC) in phosphate-buffered saline (PBS, pH 7.4) to ensure the recovery of properly folded DN-TNF trimers.

[0122] For polymer conjugation, the purified DN-TNF mutein was reacted with maleimide-functionalized polyethylene glycol (PEG-MAL, 10 kDa) at pH 6.1, allowing selective attachment to the engineered cysteine at position R31C.

[0123] In an additional embodiment, an amount of the DN-TNF mutein containing the VIM, C69V, C101A, R31C, A145R, and I97T substitutions was conjugated to polysarcosine (PolySar), an alternative biocompatible stealth polymer selected for its low immunogenicity, non-fouling properties, and enhanced pharmacokinetic profile. Following recombinant expression and purification, the DN-TNF mutein was selectively conjugated to maleimide- functionalized PolySar (PolySar-MAL, 15 kDa) via the engineered cysteine at position R31C, using a thiol-maleimide conjugation reaction under mild reducing conditions (pH 6.1, 25°C, 1 hour).

[0124] By incorporating I97T in the trimer interface domain, this DN-TNF mutein exhibited enhanced affinity for wild-type TNF monomers, leading to more efficient heterotrimer formation and improved TNF neutralization. The introduction of A145R in the receptor binding domain further ensured that the bioconjugate could not activate TNF receptors, reinforcing the selective inhibition of soluble TNF while preserving transmembrane TNF function.

[0125] A luciferase TNF potency assay confirmed potency of all DN-TNF bioconjugates made in these examples.

[0126] Example 3 - First Attempt at Bioconjugate Hydrolysis Failed

[0127] With protein drug product manufactured, each of the DN-550-PEG, DN-550- PolySar, DN-346-PEG, and DN-346-PolySar bioconjugates were intentionally subjected to a forced hydrolysis process.

[0128] Experiments were conducted to evaluate the effect of controlled mal eimide hydrolysis on the stability and activity of the DN-TNF bioconjugates using hydrolysis conditions adapted from a previously reported method for stabilizing antibody-drug conjugates (ADCs) (Tumey LN et al., 2014). The goal was to stabilize the DN-TNF bioconjugates by hydrolyzing the succinimide ring of the maleimide linker, thereby preventing retro-Michael reaction and polymer dissociation. The study sought to determine whether ring-opening hydrolysis could be applied to DN-TNF in a similar manner to ADCs, yielding a stable therapeutic with improved pharmacokinetic properties.

[0129] To this end, each of the DN-346 and DN-550 muteins were expressed and purified as previously described. The purified protein was conjugated to maleimide- functionalized polyethylene glycol (PEG-MAL, 10 kDa) via the engineered R31C residue,allowing site-specific thiol-maleimide conjugation. The resulting PEGylated DN-TNF conjugate was then subjected to a hydrolysis process based on parameters disclosed in the referenced ADC study, where the succinimide-thioether ring was opened under controlled conditions. Similarly, an amount of both DN-346 and DN-550 muteins was conjugated with PolySar in accordance with the disclosures herein.

[0130] In accordance with the published protocol, for each sample, the conjugate was buffer-exchanged into 50 mM borate buffer at pH 9.2 and incubated at 37°C for 24 hours to induce hydrolysis of the maleimide linker. The reaction was characterized by mass spectrometry (MS) and high-performance liquid chromatography (HPLC) to assess the extent of hydrolysis. At the conclusion of the reaction, the solution was adjusted to physiological pH (6.5-7.0), and the conjugates were subjected to stability testing and functional evaluation.

[0131] Despite successful hydrolysis of the maleimide linker, the DN-TNF conjugates exhibited significant loss of structural integrity and biological activity. Reverse-phase HPLC (RP-HPLC) and size-exclusion chromatography (SEC) revealed that a substantial portion of the DN-TNF proteins had aggregated or degraded following the hydrolysis step.

[0132] Importantly, functional TNF neutralization assays revealed that the hydrolyzed DN-TNF conjugates had lost ability to form heterotrimers with wild-type TNF and no longer effectively inhibited TNF receptor activation in a cell-based TNF potency bioassay.

[0133] The findings were unexpected, as the same hydrolysis conditions had been successfully applied in the prior study to stabilize ADC conjugates. However, the results indicate that DN-TNF is highly sensitive to prolonged exposure to elevated pH and temperature, possibly leading to conformational instability and functional inactivation under these conditions. The inability of DN-TNF to retain bioactivity following hydrolysis suggests that the structural constraints of TNF trimerization impose unique stability requirements, which differ from those of monoclonal antibodies or ADCs.

[0134] Example 4 - Another Failed Attempt at Forced Hydrolysis

[0135] Next, a study was conducted to evaluate an alternative approach for stabilizing DN-TNF bioconjugates using hydrolysis conditions adapted from a previously reported process (Shinmi D et al., 2016). The process had been used to induce succinimide ring hydrolysis under alkaline conditions to enhance the stability of antibody-drug conjugates (ADCs). The goal was to determine whether similar hydrolysis conditions could be applied to DN-TNF bioconjugates to achieve a stabilized formulation resistant to retro-Michael reaction and thiol exchange.

[0136] Four DN-TNF mutein bioconjugates were prepared for evaluation. Each mutein contained the substitutions VIM, C69V, C101A, R31C, and A145R, while also including either Y87H (DN-550) or I97T (DN-346). Each DN-TNF mutein was conjugated to either maleimide- functionalized polyethylene glycol (PEG, 10 kDa) or maleimide-functionalized poly sarcosine (PolySar, 15 kDa), generating the following four conjugates: DN-550-PEG, DN-550-PolySar, DN-346-PEG, and DN-346-PolySar. The DN-TNF muteins were expressed, purified, and site- specifically conjugated via thiol-mal eimide chemistry at the R31C residue, ensuring controlled conjugation with either PEG-MAL or PolySar-MAL.

[0137] Following conjugation, the four bioconjugates were subjected to hydrolysis conditions based on the prior study, which involved incubation in 50 mM bis-tris propane buffer at pH 9.5 for 6 hours at room temperature using an anion exchange column-based process. The reaction conditions were chosen based on prior reports demonstrating successful hydrolysis of succinimide rings in ADCs under these conditions, leading to improved plasma stability and reduced linker exchange reactions. The DN-TNF bioconjugates were processed under identical conditions and subsequently buffer-exchanged into phosphate-buffered saline (PBS) at pH 7.4 for final formulation and analysis.

[0138] Despite achieving successful hydrolysis of the maleimide linkers, all four DN- TNF bioconjugates exhibited significant protein degradation and loss of functional activity following the hydrolysis step. Analytical size-exclusion chromatography (SEC) revealed that a substantial portion of each bioconjugate had aggregated or fragmented, indicating protein unfolding or structural instability under these conditions. Reverse-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the presence of high-molecular- weight aggregates and lower-molecular-weight degradation products, suggesting that the elevated pH and prolonged incubation time resulted in irreversible protein modifications.

[0139] Most notably, TNF neutralization assays using a TNFR1 activation bioassay confirmed that the hydrolyzed DN-TNF bioconjugates no longer effectively neutralized TNF, suggesting that their functional integrity had been compromised.

[0140] The results of this study were unexpected, as the same pH 9.5 hydrolysis conditions had been previously reported to stabilize ADC conjugates by preventing maleimide- thiol exchange. However, in the case of DN-TNF bioconjugates, these conditions led to protein denaturation rather than stabilization. This outcome further suggests that unlike ADCs, DN- TNF muteins are highly sensitive to prolonged exposure to mild alkaline conditions, which may induce unfolding, aggregation, or non-native crosslinking reactions. The inability of DN-TNF to retain structural and functional integrity after hydrolysis under these conditions further highlights the unique stability requirements of TNF -based biotherapeutics.

[0141] This experiment underscores the importance of optimizing hydrolysis conditions specifically tailored for DN-TNF bioconjugates, rather than assuming that conditions effective for ADCs or other bioconjugates will translate to TNF -based proteins. The surprising instability of DN-TNF bioconjugates under these hydrolysis conditions demonstrates that not all biotherapeutics tolerate the same linker stabilization processes, reinforcing the need for a different approach to achieve maleimide hydrolysis while preserving TNF trimerization and bioactivity.

[0142] Example 5 - Hydrolysis of DN-TNF Bioconjugates Using Ultrasonication and Resulting Loss of Activity

[0143] Another study was conducted, this time to evaluate whether ultrasonicationbased hydrolysis, as described in a prior report (Huang W et al., 2019), could be used to stabilize DN-TNF bioconjugates by inducing maleimide ring hydrolysis while preserving protein function. The prior study demonstrated that applying solvodynamic shear forces through ultrasonication could accelerate maleimide ring-opening hydrolysis, yielding a conjugate resistant to retro-Michael reaction and thiol exchange. This study sought to determine whether the same hydrolysis approach could be applied to DN-TNF bioconjugates without compromising protein integrity.

[0144] Four DN-TNF mutein bioconjugates were tested: DN-550-PEG, DN-550- PolySar, DN-346-PEG, and DN-346-PolySar. Each DN-TNF mutein contained VIM, C69V, C101A, R31C, and A145R, with DN-550 additionally containing Y87H and DN-346 containing I97T. The muteins were site-specifically conjugated at R31C to either maleimide- functionalized PEG (10 kDa) or PolySar (15 kDa). The bioconjugates were subjected to pulsed ultrasonication at 20 kHz in PBS (pH 7.4) for 30 minutes while maintaining the temperature at 6°C to 9°C to prevent heat-induced degradation.

[0145] Post-treatment analysis by mass spectrometry and reverse-phase HPLC (RP- HPLC) confirmed that maleimide hydrolysis had occurred, forming a maleamic acid derivative. However, significant protein aggregation and fragmentation were observed using sizeexclusion chromatography (SEC) and SDS-PAGE, indicating that the ultrasonication process destabilized the DN-TNF trimeric structure.

[0146] Despite successful hydrolysis, TNF neutralization assays revealed a substantial loss of DN-TNF activity, with treated bioconjugates showing significantly reduced ability to inhibit TNF receptor activation compared to non-hydrolyzed controls. While some residualactivity remained, the loss was substantial, suggesting that ultrasonication-induced mechanical stress negatively impacted DN-TNF function.

[0147] The results were unexpected, as the same ultrasonication-based hydrolysis method had been successfully used in prior studies to stabilize antibody-polymer conjugates. However, in the case of DN-TNF bioconjugates, these conditions appear to have led to partial denaturation and significant loss of function, likely due to mechanically induced unfolding of the TNF trimer. This study underscores the fragility of DN-TNF under ultrasonication, highlighting the need for a tailored hydrolysis strategy that stabilizes the conjugate without disrupting TNF trimerization and bioactivity.

[0148] While these experiments were unsuccessful, it should be recognized that ultrasonication might be useful for hydrolysis given different experimental conditions.

[0149] Example 6 - Successful Maleimide Hydrolysis ofDNTNF Bioconjugates

[0150] Yet another experiment was conducted to determine the optimal pH conditions for controlled maleimide hydrolysis in DN-TNF bioconjugates, with the goal of stabilizing the conjugates by preventing retro-Michael reaction and polymer dissociation. The study investigated how different, and more gentle, temperature and pH conditions influenced the efficiency of maleimide ring-opening hydrolysis while monitoring the structural and functional integrity of the DN-TNF protein.

[0151] In the experiment, DN-550-PEG was subjected to a range of pH conditions over a fixed incubation period and temperature, as described in FIG. 8. The hydrolysis process was carried out in buffered aqueous solutions, with pH values ranging from mildly acidic to strongly basic conditions. The samples were incubated under controlled conditions at room temperature (21 °C) for 24 hours, a time period chosen to allow sufficient hydrolysis while minimizing potential protein degradation. After incubation, the reaction mixtures were neutralized and buffer-exchanged into phosphate-buffered saline (PBS, pH 7.4) for further analysis.

[0152] Mass spectrometry (MS) and high-performance liquid chromatography (HPLC) confirmed that hydrolysis was successfully achieved at various pH values, but the extent of hydrolysis varied depending on the pH. The data revealed that pH 8.5 was the optimal condition, resulting in efficient maleimide hydrolysis while preserving protein stability. At this pH, the conversion of the succinimide ring into its more stable maleamic acid derivative was maximized, preventing polymer dissociation without causing significant protein aggregation or loss of activity.

[0153] Functional evaluation via TNF neutralization assays confirmed that bioactivity was preserved, as the hydrolyzed DN-550-PEG maintained its ability to inhibit TNF receptor activation at levels comparable to non-hydrolyzed controls.

[0154] At pH values lower than 8.0, hydrolysis was incomplete, suggesting that acidic conditions were not sufficient to promote efficient maleimide ring-opening. Conversely, at pH values above 9.0, protein degradation and aggregation increased, likely due to the instability of DN-TNF under prolonged alkaline conditions. This finding was consistent with previous studies indicating that TNF -based proteins are highly sensitive to elevated pH, leading to structural unfolding and loss of function. However, unlike other studies, it was surprisingly discovered that controlled temperature and pH can be effective in hydrolyzing DN-TNF bioconjugates without losing TNF inhibiting potency, albeit there is a very narrow window in the useful conditions where the process can be successful.

[0155] FIG. 8 summarizes the hydrolysis pH optimization results detailing the experimental conditions used to evaluate maleimide linker stabilization in DN-TNF bioconjugates. The results demonstrate optimal hydrolysis conditions at pH 8.5 for 24 hours at 21 °C, ensuring that maleimide ring opening occurs efficiently without inducing DN-TNF degradation. The findings confirm that proper pH and temperature control are essential for maintaining DN-TNF integrity while stabilizing the polymer conjugate.

[0156] Although FIG. 8 specifically presents data for DN-550-PEG, similar experiments were performed for DN-550-PolySar, DN-346-PEG, and DN-346-PolySar, and the results were comparable. In each case, the optimal hydrolysis condition was found at pH 8.5 for 24 hours at 21°C, leading to efficient hydrolysis while maintaining conjugate stability and bioactivity. These findings confirm that controlled maleimide hydrolysis at pH 8.5 is a reproducible and effective stabilization method for DN-TNF bioconjugates, ensuring long-term conjugate integrity and therapeutic functionality. The range of hydrolyzed species was confirmed to always be greater than 85%, with three of the four DN-TNF bioconjugates exhibiting greater than 95% hydrolysis to a maleamic acid derivative. The process can be adjusted and monitored to ensure sufficient hydrolysis depending on the DN-TNF mutein, formulation, and other specifics for each therapeutic product.INDUSTRIAL APPLICABILITY

[0157] The claimed invention is industrially applicable in the biopharmaceutical sector, particularly in the development, manufacturing, and commercialization of biologic therapies for TNF-mediated inflammatory diseases and immunological diseases with a chronicinflammatory component. The claimed therapeutic compositions including a dominantnegative TNF (DN-TNF) bioconjugate can be manufactured at scale using established recombinant protein production techniques, followed by site-specific conjugation to a stealth polymer and controlled hydrolysis stabilization, ensuring reproducibility. Provided is a clinically viable TNF inhibitor that can be formulated as a buffered aqueous injectable solution, enabling broad therapeutic application across subcutaneous, intramuscular, intravitreal, and intravenous routes of administration. The stabilized bioconjugate offers improved stability, making it highly applicable in chronic inflammatory disease management. The ability to neutralize soluble TNF while sparing transmembrane TNF function offers a differentiated therapeutic profile compared to conventional anti-TNF biologies, thereby expanding market potential in autoimmune and neuroinflammatory indications. Given the increasing demand for next-generation TNF inhibitors with improved safety and pharmacokinetics, the present invention has strong commercial and clinical applicability in the global biopharmaceutical industry.SEQUENCE LISTING FREE TEXTSEO ID NO: 1Wild type human TNF (wtTNF)VRSSSRTPSD KPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLVVPSEGLYLIYS QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSPCQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFAESGQVYFGIIALFor the following non-exhaustive list of sequence identifications corresponding to example DN-TNF muteins, bold and underlined type indicates an amino acid substitution relative to SEQ ID NO:!.SEQ ID NO:2DN-550MRSSSRTPSD KPVAHVVANP QAEGQLQWLN CRANALLANG VELRDNQLVV PSEGLYLIYS QVLFKGQGVP STHVLLTHTI SRIAVSHQTK VNLLSAIKSP AQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFRESGQVYFGIIALSEP ID NO: 3DN-346MRSSSRTPSD KPVAHVVANP QAEGQLQWLN CRANALLANG VELRDNQLVV PSEGLYLIYS QVLFKGQGVP STHVLLTHTI SRIAVSYQTK VNLLSATKSP AQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFRESGQVYFGIIALCITATION LIST1. Steed PM, Tansey MG, Zalevsky J, et al. Inactivation of TNF signaling by rationally designed dominant-negative TNF variants. Science. 2003;301(5641): 1895-1898. doi: 10.1126 / science.10812972. Zalevsky J, Secher T, Ezhevsky SA, et al. Dominant-negative inhibitors of soluble TNF attenuate experimental arthritis without suppressing innate immunity to infection. J Immunol. 2007;179(3):1872-1883. doi:10.4049 / jimmunol.179.3.18723. Tumey LN, Charati M, He T, et al. Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy. Bioconjug Chem.2014;25(10): 1871-1880. doi: 10.1021 / bc500357n4. Shinmi D, Taguchi E, Iwano J, et al. One-Step Conjugation Method for Site-Specific Antibody-Drug Conjugates through Reactive Cysteine-Engineered Antibodies. Bioconjug Chem. 2016;27(5): 1324-1331. doi: 10.1021 / acs.bioconjchem.6b001335. Huang W, Wu X, Gao X, et al. Maleimide-thiol adducts stabilized through stretching. Nat Chem. 2019; 11(4):310-319. doi: 10.1038 / s41557-018-0209-26. U.S. Patent No. 8,658,682 B27. U.S. Patent No. 9,017,668 B28. U.S. Patent No. 9,937,173 B29. U.S. Patent No. 10,111,834 B210. U.S. Patent No. 11,365,229 B2

Claims

CLAIMSWhat is claimed is:

1. A therapeutic composition comprising a selective soluble TNF neutralizing bioconjugate in an injectable formulation, the selective soluble TNF neutralizing bioconjugate comprising: a dominant negative TNF (DN-TNF) mutein comprising the amino acid sequence of SEQ ID NO:1 modified with 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions in a TNF receptor interaction domain, a trimer interface domain, or a combination thereof, and a linker-polymer moiety covalently conjugated to the DN-TNF mutein via a thioether bond, the linker-polymer moiety comprising a maleimide linker and a biocompatible stealth polymer, wherein at least 85% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative, thereby preventing retro-Michael reaction and polymer dissociation.

2. The therapeutic composition of claim 1, wherein the hydrolyzed maleimide derivative comprises maleamic acid.

3. The therapeutic composition of claim 1, wherein said biocompatible stealth polymer of the linker-polymer moiety comprises polyethylene glycol (PEG) or polysarcosine (PolySar).

4. The therapeutic composition of claim 3, wherein the biocompatible stealth polymer comprises a molecular weight between: 5 kDa and 40 kDa for PEG, or 3 kDa and 30 kDa for PolySar.

5. The therapeutic composition of claim 1, wherein said biocompatible stealth polymer of the linker-polymer moiety is selected from the group consisting of: polyethylene glycol (PEG), polysarcosine (PolySar), hydroxyethyl starch (HES), poly(N-(2- hydroxypropyl)methacrylamide) (pHPMA), polyglutamic acid (PGA), poly(2-oxazoline) (POZ), poly(vinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), dextran, or a combination thereof.

6. The therapeutic composition of claim 1, wherein at least 90% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative.

7. The therapeutic composition of claim 1, wherein at least 95% of the selective soluble TNF neutralizing bioconjugate comprises a hydrolyzed maleimide derivative.

8. The therapeutic composition of claim 1, wherein with respect to SEQ ID NO: 1 said amino acid substitutions in the TNF receptor interaction domain comprise substitutions at residues selected from the group consisting of: Q21, A22, E23, N30, R31, R32, A33, K65, G66, Q67, G68, S86, Y87, V91, A109, El 10, D140, A145, thereby reducing or eliminating TNF receptor binding.

9. The therapeutic composition of claim 8, wherein said amino acid substitutions in the TNF receptor interaction domain are selected from the group consisting of: Q21 A, Q21C, Q21E, Q21G, Q21R, Q21S, A22D, A22E, E23A, E23Q, N30A, N30D, N30E, R31C, R31D, R31E, R31I, R31S, R32D, R32E, R32S, A33D, A33E, K65D, K65E, K65I, K65M, K65N, K65Q, K65S, K65T, K65V, K65W, G66D, G66E, G66K, G66Q, Q67D, Q67E, Q67K, Q67R, Q67S, Q67V, Q67W, Q67Y, G68D, G68E, G68K, S86D, S86E, S86Q, S86R, Y87A, Y87H, Y87Q, Y87R, Y87S, V91D, V91E, A109D, A109E, E110A, E110Q, D140A, D140K, D140R, A145C, A145D, A145E, A145F, A145G, A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, and A145Y.

10. The therapeutic composition of claim 1, wherein with respect to SEQ ID NO: 1 said amino acid substitutions in the trimer interface domain comprise amino acid substitutions at residues selected from the group consisting of: V13, L57, 158, Y59, L93, 197, K98, Y115, G116, P117, 1118, Y119, L120, G121, G122, V123, G148, 1154.

11. The therapeutic composition of claim 10, wherein said amino acid substitutions in the trimer interface domain are selected from the group consisting of: V13I, V13L, V13F, V13M, L57I, L57V, L57F, L57W, I58L, I58M, I58V, I58F, Y59H, Y59R, Y59Q, Y59K, L93I, L93F, L93M, L93W, I97F, I97L, I97M, I97T, I97V, K98R, K98H, K98Q, K98E, Y115H, Y115R, Y115Q, Y115E, G116S, G116T, G116Q, P117A, P117S, P117N, I118L, I118M, I118V, I118F, Y119F, Y119W, Y119H, L120I, L120V, L120F, L120M, G121S, G121T, G121N, G121Q, G122S, G122T, G122N, G122Q, V123I, V123L, V123F, V123M, G148S, G148T, G148N, G148Q, I154L, Il 54V, I154M, and I154F, wherein said amino acid substitutions increases the affinity of the DN-TNF mutein for wild-type TNF monomers, thereby promoting heterotrimer formation and neutralizing soluble TNF..

12. The therapeutic composition of claim 1, wherein the DN-TNF mutein further comprises the amino acid substitutions C69V / C101 A relative to SEQ ID NO: 1.

13. The therapeutic composition of claim 12, wherein the DN-TNF mutein comprises a single cysteine substitution at a surface-exposed residue selected from the group consisting of: E23C, A38C, L43C, R44C, D45C, D140C, or a single cysteine substitution at a position within the receptor binding domain selected from the group consisting of Q21C, R31C, A145C, and E146C, whereby the DN-TNF mutein is adapted for site specific cysteine thiol conjugation with the stealth polymer.

14. The therapeutic composition of claim 1, wherein the DN-TNF mutant protein further comprises amino acid substitution VIM relative to SEQ ID NO: 1.

15. The therapeutic composition of claim 1, the DN-TNF mutein further comprising up to twelve amino acid deletions at the N-terminal end thereby forming a truncated DN-TNF mutein.

16. The therapeutic composition of claim 1, wherein the injectable formulation is a buffered aqueous solution with a pH between 5.5 and 7.5.

17. The therapeutic composition of claim 1, wherein the therapeutic composition is formulated for administration via subcutaneous, intramuscular, intravitreal, or intravenous injection.

18. A method of treating a TNF-mediated inflammatory disorder in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the therapeutic composition of claim 1 to the subject via subcutaneous, intramuscular, intravitreal, or intravenous injection, wherein the composition comprises a stabilized DN- TNF mutein conjugated to a biocompatible stealth polymer, and wherein at least 85%, 90%, or 95% of the selective soluble TNF neutralizing bioconjugate comprises a maleamic acid derivative linking the DN-TNF mutein to the stealth polymer, thereby preventing biocompatible stealth polymer dissociation and prolonging therapeutic activity.

19. A method for preparing a therapeutic composition comprising a stabilized selective soluble TNF-neutralizing bioconjugate, the method comprising: conjugating a dominantnegative TNF (DN-TNF) mutein comprising the amino acid sequence of SEQ ID NO: 1 modified with 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions in a TNF receptor interaction domain, a trimer interface domain, or a combination thereof, to a biocompatible stealth polymer via a maleimide linker to form a bioconjugate; adjusting the pH of the bioconjugatesolution to between 8.0 and 9.5 to induce hydrolysis of the maleimide linker; incubating the solution at a temperature between 18°C and 25°C for a period sufficient to hydrolyze at least 85% of the maleimide linkers to a maleamic acid derivative, thereby preventing retro- Michael reaction and polymer dissociation; and adjusting the pH of the solution to between 5.5 and 7.5 to obtain a stable injectable formulation.

20. A method for stabilizing a selective soluble TNF-neutralizing bioconjugate, the method comprising: conjugating a DN-TNF mutein to a maleimide-functionalized biocompatible stealth polymer via a thioether bond; subjecting the conjugated bioconjugate to a controlled hydrolysis step at a pH of 8.0 to 9.5 and a temperature of 18°C to 25°C for at least 12 hours; allowing at least 85%, 90%, or 95% of the maleimide linkers to be hydrolyzed to their corresponding maleamic acid derivatives, thereby preventing polymer dissociation and degradation; formulating the stabilized bioconjugate in an aqueous injectable solution with a pH of 5.5 to 7.5.

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