Genetically modified cells into which a nucleic acid encoding a stefin a protein variants specifically binding to tnfr2 or a fusion protein comprising the same has been introduced, and uses thereof

Abstract

The present invention relates to a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 and / or a fusion protein including the same is introduced into a host cell, the conditioned cell culture medium thereof, the pharmaceutical preparations of the genetically modified cell, and uses of the genetically modified cell in the treatment of immune diseases, including inflammatory, autoimmunity and cancer.

Description

GENETICALLY MODIFIED CELLS INTO WHICH A NUCLEIC ACID ENCODING A STEFIN A PROTEIN VARIANTS SPECIFICALLY BINDING TO TNFR2 OR A FUSION PROTEIN COMPRISING THE SAME HAS BEEN INTRODUCED, AND USES THEREOF

[0001] The present invention relates to a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 and / or a fusion protein including the same is introduced into a host cell, and the use thereof.

[0002]

[0003] The immune system is a biological network composed of various cells and organs built to protect an organism from invasion from the outside. The immune system operates based on the specific functions of the organs and cells that form the same and the signaling and interaction between cells. The immune system maintains immune homeostasis by balancing between immune tolerance that regulates and modulates immunity and immune response that enhances immunity. Imbalance of immune tolerance and immune response may be caused by various causes, and this imbalance in immunity leads to the development of various diseases. For example, when the immune tolerance mechanism is strengthened, occurrence of cancer or invasion of external infectious agents is facilitated, thereby causing cancer, infectious diseases, and the like. Conversely, when the immune response mechanism is strengthened, inflammatory diseases such as autoimmune diseases and allergies may occur.

[0004] Recently, thorough research into immune synapses, which are signaling systems between immune cells constituting the immune system or between target cells and immune cells, is ongoing. Immune synapses are composed of various cytokines and signal transmitters secreted from cells, and various costimulatory molecules and receptors expressed on the cell surface. As various factors involved in maintaining homeostasis of the immune system have been reported, interest in immunotherapeutic agents for regulating immune responses by targeting such factors is increasing. Most immunotherapeutic agents developed to date are antibody drugs against cytokines or cell surface molecules, but the use thereof is very limited due to insufficient efficacy or side effects. Recently, in order to more effectively treat immune diseases, cell therapeutic agents using differentiated immune cells such as T cells, NK cells, etc. or stem cells capable of differentiating into various immune cells are being developed. Cell therapeutic agents based on differentiated immune cells such as CAR-T or CAR-NK are expensive due to the use of autologous cells and have limited targets, and mesenchymal stromal cells have a low therapeutic effect relative to the cost of treatment and have limitations in that it is difficult to explain a clear mechanism of action (Blood Cancer J. 11, 69 (2021); World J. Stem Cells. 2019;11(4):212-221). Maintaining control of the cell-mediated and humoral immune responses is an important dimension of healthy immune system activity. The abnormal regulation of T cell and B cell driven immune reactions has been associated with a wide array of human diseases, as the inappropriate increase of an immune response against various self and foreign antigens plays a causal role in such pathologies as autoimmune disorders, asthma, allergic reactions, graft versus host disease (GvHD), transplantation graft rejection, and a variety of other immunological disorders including cancer.

[0005] These diseases are mediated by T and B lymphocytes that exhibit reactivity against self-antigens and those derived from non-threatening sources, such as allergens or transplantation allografts. Regulatory T cells (Tregs) have evolved to inhibit the activity of immune cells that are cross reactive with "self" major histocompatability complex (MHC) proteins and other benign antigens. The most well understood populations of Tregs are CD4+, CD25+, FoxP3+ Tregs and CD17+ Tregs.

[0006] Tumor necrosis factor receptor (TNFR) subtypes 1 and 2 have been identified specifically on the Tregs surface. The activation of TNFR1 by soluble TNF-alpha, enhances the caspase signaling cascade leading to Treg apoptosis. On the other hand, activation of TNFR2 by mainly membrane bound TNF-alpha induces signaling through the mitogen activated protein kinase (MAPK) signaling pathway, which orchestrates the TRAF2 / 3 and NF-κB mediated transcription of genes that promote cell proliferation and escape from apoptosis.

[0007] Importance of TNFR2’s role in Treg proliferation and function was shown in various studies. TNFR2 deficient mice had reduced numbers of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 - / - Tregs were not able to control inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492). In humans, Tregs were shown to express a higher level of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_ Annunziato F, et al. PMID: 12163566) and TNFR2+ Tregs exhibited the most potent suppression of proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID: 20127680). Furthermore, TNFR2 has been shown to play a role in carcinogenesis and tumour growth by mediating TNF responses in immunosuppressive cells, allowing for immune escape and tumour development (Sheng Y., et al. doi: 10.3389 / fimmu.2018.01170).

[0008] Moreover, in autoimmune diseases affecting the central nervous system (CNS), specifically Multiple sclerosis (MS), pathogenic lymphocytes are triggered in the periphery to infiltrate the CNS and cause local inflammation and demyelination. Demyelination is the damage to the protective covering (myelin sheath) that surrounds nerve fibers in the brain. Protecting against demyelination or revert it are strategies explored to tackle MS. Interestingly, several studies have reported TNFR2 involvement in neuroprotection. For example, in a cuprizone induced mouse model of demyelination, it was shown that TNFR2 is critical for regeneration of oligodendrocyte the cells primarily responsible for maintenance and generation of the myelin sheath that surrounds axons, whereas TNF signaling via TNFR1 promoted nerve demyelination (2001_HA Arnett, et al. PMID: 11600888).

[0009] Due to its role in directing cell survival and growth, TNFR2 represents an attractive target for treating diseases such as autoimmune disorders, GvHD, allograft rejection, allergic reactions, asthma and cancer.

[0010] AFFIMER®, developed by Avacta Life Sciences Limited, is a small stable protein molecule engineered based on a Stefin A protein, which is an in-vivo protein. AFFIMER® includes two short peptide sequences having a random sequence and an N-terminal sequence, and is able to bind to a target material with high affinity and specificity in a manner similar to a monoclonal antibody. AFFIMER® shows remarkably improved binding affinity and specificity compared to the free peptide library, and has a very small size and high stability compared to antibodies, and is therefore receiving great attention as a next-generation alternative pharmaceutical platform to replace antibodies (U.S. Patent Nos. 9447170, 8853131, etc.).

[0011] Avacta Life Sciences Limited develop polypeptides that specifically bind to TNFR2 by engineering the natural Stefin A protein, resulting in the development of AFFIMER® polypeptides capable of binding to TNFR2 with excellent affinity and specificity.

[0012] Against this background, the present inventors have made great efforts to develop new types of cell therapeutic agents that exhibit superior immunomodulatory effects and therapeutic effects on immune-related diseases based on Stefin A protein variant technology and cell-based immunotherapeutic agents, and thus developed genetically modified cells that express the Stefin A protein variant specifically bind to TNFR2 by introducing a gene encoding the Stefin A protein variant specifically bind to TNFR2 into immune cells, stem cells, or somatic cells, and ascertained that the genetically modified cells may not only exhibit highly enhanced immunomodulatory activity compared to host cells, but also lower the immune rejection response due to allogeneic cell transplantation, and also that the genetically modified cells may exhibit excellent therapeutic effects on immune-related diseases such as inflammatory diseases and autoimmune diseases, or cancer thus culminating in the present invention.

[0013]

[0014] SUMMARY

[0015] It is an object of the present invention to provide a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 or a fusion protein including the same is introduced into a host cell.

[0016] It is another object of the present invention to provide a conditioned cell culture medium of the genetically modified cell.

[0017] It is still another object of the present invention to provide a cell therapeutic agent including the genetically modified cell or the conditioned cell culture medium thereof.

[0018] It is yet another object of the present invention to provide a use of the genetically modified cell or the conditioned cell culture medium thereof for the manufacture of a cell therapeutic agent.

[0019] It is still yet another object of the present invention to provide a use of the genetically modified cell or the conditioned cell culture medium thereof for the prevention or treatment of an immune disease or cancer.

[0020] It is yet another object of the present invention to provide use of the genetically modified cell or the conditioned cell culture medium thereof for immunomodulation.

[0021] It is yet another object of the present invention to provide use of the genetically modified cell or the conditioned cell culture medium thereof for culturing regulatory T cell.

[0022] It is yet another object of the present invention to provide use of the genetically modified cell or the conditioned cell culture medium thereof for manufacturing a composition or medium for culturing regulatory T cell.

[0023] It is a further object of the present invention to provide a composition for drug delivery including the genetically modified cell of the present invention or the conditioned cell culture medium thereof.

[0024] It is still a further object of the present invention to provide the use of the genetically modified cell of the present invention or the conditioned cell culture medium thereof for drug delivery.

[0025]

[0026] In order to accomplish the objects of the present invention, the present invention provides a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 and / or a fusion protein including the same is introduced into a host cell, or a culture thereof.

[0027] The present invention also provides a cell therapeutic agent including the genetically modified cell and / or the conditioned cell culture medium thereof.

[0028] The present invention also provides the use of the genetically modified cell and / or the conditioned cell culture medium thereof for the manufacture of a cell therapeutic agent.

[0029] The present invention also provides a pharmaceutical composition for preventing or treating an immune disease or cancer including the genetically modified cell and / or the conditioned cell culture medium thereof.

[0030] The present invention also provides the use of the genetically modified cell and / or the conditioned cell culture medium thereof for the prevention or treatment of an immune disease or cancer.

[0031] The present invention also provides the use of the genetically modified cell and / or the conditioned cell culture medium thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of an immune disease or cancer.

[0032] The present invention also provides a method of preventing or treating an immune disease or cancer including administering the genetically modified cell and / or the conditioned cell culture medium thereof to a subject.

[0033] The present invention also provides a composition for immunomodulation comprising the genetically modified cell and / or the conditioned cell culture medium thereof.

[0034] The present invention also provides use of the genetically modified cell and / or the conditioned cell culture medium thereof for immunomodulation.

[0035] The present invention also provides a method for immunomodulation comprising administering the genetically modified cell and / or the conditioned cell culture medium thereof to a subject.

[0036] The present invention also provides a composition or medium for culturing regulatory T cell comprising the genetically modified cell and / or the conditioned cell culture medium thereof.

[0037] The present invention also provides a method for culturing regulatory T cell comprising, culturing regulatory T cell in presence of the genetically modified cell.

[0038] The present invention also provides use of the genetically modified cell and / or the conditioned cell culture medium thereof for culturing regulatory T cell.

[0039] The present invention also provides use of the genetically modified cell and / or the conditioned cell culture medium thereof for manufacturing a composition or medium for culturing regulatory T cell.

[0040] The present invention also provides a composition for drug delivery including the genetically modified cell of the present invention or the conditioned cell culture medium thereof.

[0041] The present invention also provides the use of the genetically modified cell of the present invention or the conditioned cell culture medium thereof for drug delivery.

[0042]

[0043] Figure 1. Schematic of the selection strategy for the human TNFR2 selection campaign. Both libraries underwent solution and passive selections. Stringency was introduced by decreasing hTNFR2 concentration as the rounds progressed and increasing the number of wash steps between round 1 (5x PBS / 0.1% Tween 20, 2x PBS) and round 2(15x PBS / 0.1% Tween 20, 2x PBS). For the passive selections using Avacta’s proprietary Type 3 AFFIMER® phage library (US Pat. No. 9.932.575), the deselection and competition approaches were simultaneously explored. For the deselection approach, the Fc concentration in round n+1 was the concentration of hTNFR2 in round n. For the competition approach, the Fc concentration was 10x molar excess of the hTNFR2 concentration specified in the given round. For the passive selections using Avacta’s proprietary Type 1 AFFIMER® phage library (US Pat. No. 8,841,491), the deselection approach was exclusively performed and a round 4 was introduced since early-stage screening of the round 3 outputs indicated a high diversity and hit rate. For the solution selections, both libraries underwent the competition approach for reducing enrichment of Fc-binding phage-displayed AFFIMER® polypeptides. Streptavidin- and neutravidin-coated beads were alternated between rounds to reduce enrichment for streptavidin and / or neutravidin-binding phage-displayed peptides.

[0044] Figure 2. Stefin A protein variant binding to Expi293F™ cells overexpressing human TNFR2.

[0045] Figure 3. Titration experiments data compilation. % inhibition at 3 μM and IC50 for best blockers.

[0046] Figure 4. SEAP results in duplicate, ranking of 20 clones.

[0047] Figure 5. Cross-reactivity of clones of Stefin A protein variant binding to hTNFR2 with cynomolgus TNFR2, in triplicate.

[0048] Figure 6 and 7. Characterization of eMSCs(engineered Mesenchymal Stromal Cells) Expression of mesenchymal stromal cell specific markers by eMSCs CD29, CD44, CD73, CD90, and CD105(figure 6), but not cell markers CD45, CD14, CD19, HLA-DR, SSEA-3, TRA-1-60, and TRA-1-81(figure 7) (Data represent a representative experiment).

[0049] Figure 8. Analysis of Stefin A protein variant specifically binding to TNFR2 expressed on the cell surface. Cell lysates from eMSCs transduced with Stefin A protein variant were probed for surface expression by Western blotting and compared to signals from each Stefin A protein variant clone on the same gel. β-actin Western blot serves as loading control for the protein lysates from various eMSCs.

[0050] Figure 9. Stefin A protein variant quantification on cell surface. The number of Stefin A protein variant on the cell surface was quantified by QuantiBRITE PE fluorescence quantification kit for flow cytometric analysis. Antibody for Stefin A protein variant conjugated to phycoerythrin (PE) were used for the flow cytometric analysis.

[0051] Figure 10. A SEAP release of HEK-Blue TNFα reporter cell line triggered by 7 best Stefin A protein variants on the eMSCs surface. HEK-Blue TNFα cells seed 20,000 cells / well. eMSCs seed at 1 / 2 serial dilution from 40,960 cells / well. Data represents the mean of triplicate (Mean ± SD).

[0052]

[0053] DETAILED DESCRIPTION

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those typically understood by those skilled in the art to which the present invention belongs. In general, the nomenclature used herein is well known in the art and is typical.

[0055] The term, “Protein” or “Polypeptides” are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing at least one analog of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0056] For the most part, the amino acids and amino acids sequence used in the application are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups, and isomer thereof (e.g. D- or L- stereoisomers).

[0057] Amino acid residues further include analogs, derivatives and congeners of any specific amino acid referred to herein, as for instance, the subject AFFIMER® polypeptide (particularly if generated by chemical synthesis) can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy- phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid.

[0058] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. In the present invention, two nucleic acid or amino acid sequences can be “substantially identical”, which means that the two sequences are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, identity may exist over a length of at least about 10, 20, 40-60, 60-80, 80-100 or more residues. When calculating percent identity, any residues defined as ‘Xaa’ or ‘X’ in a reference sequence herein are included in the percentage identity calculation, i.e. any amino acid in this position in a comparison sequence matches the reference sequence.

[0059]

[0060] The protein or polypeptide described herein, for example, a Stefin A protein variant, a fusion protein, or a fusion protein configuration, may include not only the amino acid sequence described in regard thereto, but also a protein or polypeptide in which a portion of the amino acid sequence is substituted through conservative substitution.

[0061] As used herein, "conservative substitution" refers to a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause loss of biological or biochemical functions of the polypeptide.

[0062] A conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, proteins of the present invention do not cause functional loss, for example, a Stefin A protein variant that specifically binds to TNFR2 does not abrogate its binding to TNFR2 by its conservative substitution. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.

[0063] A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0064] TNFR2, along with TNFR1, is a Type 1 membrane bound receptors that binds TNFα. TNFR2, also known as p75, TNF Receptor Superfamily Member 1B or CD120b, is encoded in humans as TNFRSF1B which expresses a 48 kDa protein of 461 amino acids in length (UniProt P20333). In mice, the TNFR2 protein is 474 amino acids long and has a 50 kDa mass.It is understood that wherever embodiments are described herein with the language "comprising" otherwise analogous embodiments described in terms of "consisting of” and / or "consisting essentially of" are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of" otherwise analogous embodiments described in terms of "consisting of" are also provided.

[0065] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0066] The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0067] The phrase “at least one” may be used interchangeably with “one or more.” It should be understood that “a” is not limited to one but rather means “at least one.”

[0068]

[0069] Avacta has screened for Stefin A protein variants which can bind to TNFR2 with excellent affinity and specificity by using Affimer® platform technology, and has filed a patent application for a Stefin A protein that specifically binds to TNFR2.

[0070] Accordingly, the following inventions are claimed in the patent application filed by Avacta Life Sciences Limited for Stefin A protein variants that specifically bind to TNFR2, and are excluded from the scope of the present invention:

[0071] (i) a genetically engineered cell for use only in the manufacturing of a Stefin A protein variants that specifically binds to TNFR2 or a fusion protein comprising the same;

[0072] (ii) use only for manufacturing of a Stefin A protein variants that specifically binds to TNFR2 of the genetically engineered cells or a fusion protein or conjugate containing the same; and

[0073] (iii) A method for manufacturing a Stefin A protein variants that specifically binds to TNFR2 using the genetically engineered cells, or a fusion protein or conjugate containing the same.

[0074] As used herein, the term “manufacturing” means production to manufacture Stefin A protein variants that specifically binds to TNFR2 or a fusion protein containing the same as a final product.

[0075]

[0076] In the present invention, in order to develop cells having excellent immunomodulatory activity through genetic modification, a genetically modified cell that stably expresses Stefin A protein variants specifically binding to TNFR2 by introducing a gene encoding the Stefin A protein variants specifically binding to TNFR2 was produced.

[0077] In particular, in the examples of the present invention, it was confirmed that genetically engineered cells expressing Stefin A protein variants specifically binding to TNFR2 showed an excellent agonistic effect on TNFR2. The TNFR2-specific binding ability of the Stefin A protein variants of the present invention can be usefully used for culturing or activating Treg and immune system regulation (preferably suppression) by replacing TNF-alpha.

[0078] Accordingly, in one aspect, the present invention relates to a genetically modified cell in which a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 or a fusion protein including the same is introduced into a host cell.

[0079]

[0080] Stefin A Protein Variants Specifically Binding to TFNR2

[0081] As used herein, the term "Stefin A protein variant (or Stefin A protein variants)" a scaffold based on a Stefin A polypeptide, meaning that it has a sequence which is derived from a Stefin A polypeptide, for example, a mammalian Stefin A polypeptide, for example, a human Stefin A polypeptide. In the present invention, the “Stefin A protein Variant” may be used interchangeably in substantially the same sense as “Stefin A protein variants” or “AFFIMER® protein”. In the present invention, the “Stefin A protein Variant” may be used interchangeably in substantially the same sense as “Stefin A polypeptide variant” or “AFFIMER®”. Unless otherwise specified, the Stefin A protein variant refers to the Stefin A protein variant specifically binding to TNFR2 of the present invention.

[0082] In one embodiment of the present invention, a Stefin A protein variant that specifically binds to TNFR2 was developed. The “Stefin A protein variant that specifically binds to TNFR2” may be used interchangeably with “TNFR2 Stefin A protein variant” and “TNFR2 AFFIMER” in substantially the same meaning.

[0083] As used herein, the term “Stefin A protein” or (Stefin polypeptides)” encompass a subgroup of proteins in the cystatin superfamily, a family which encompasses proteins that contain multiple cystatin-like sequences. The Stefin subgroup of the cystatin family includes relatively small (around 100 amino acids) single domain proteins. They receive no known post-translational modification, and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A itself is a monomeric, single chain, single domain protein of 98 amino acids. The structure of Stefin A has been solved, facilitating the rational mutation of Stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed for exhaustive testing for residual biological activity of the engineered proteins.

[0084] As used herein, the term “Stefin A protein variant” refers to a small, highly stable protein that is an engineered variant of a Stefin polypeptide. AFFIMER® proteins display two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a similar manner to monoclonal antibodies. Stabilization of the two peptides by the Stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. Variations to other parts of the Stefin A polypeptide sequence can be carried out, with such variations improving the properties of these affinity reagents, such as increase stability, make them robust across a range of temperatures and pH and the like. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from Stefin A, sharing substantial identify with a Stefin A wild type sequence, such as human Stefin A. It will be apparent to a person skilled in the art that modifications may be made to the scaffold sequence without departing from the disclosure. In particular, an AFFIMER® polypeptide can have an amino acid sequences that is at least 25%, 35%, 45%, 55% or 60% identity to the corresponding sequences to human Stefin A, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identical, e.g., where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target (such as TNFR2), and e.g., which do not restore or generate biological functions such as those which are possessed by wild type Stefin A but which are abolished in mutational changes described herein. A target protein-specific binding platform using such a Stefin A protein variant is disclosed in detail in US Patent No. 9447170 and No. 8853131. In some embodiments, the Stefin A protein variant may be the fragment of the Stefin A protein variant can specifically bind to TNFR2 comprising a portion the Stefin A protein variant that binds human TNFR2.

[0085] In some embodiments, the Stefin A protein variant can specifically bind to TNFR2, a target protein, with high affinity and specificity through engineering of the Stefin A protein.

[0086] In some embodiments, the Stefin A protein variant may not bind to TNFR1.

[0087] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may exhibits an agonistic effect on TNFR2.

[0088] "Agonist" and "agonistic" refer to agents that are capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target or target pathway. "Agonist" is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.

[0089] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to TNFR2, thereby activating the TNFR2 sub-pathway.

[0090] As used herein, the term “TNFR2”, along with TNFR1, is a Type 1 membrane bound receptors that binds TNFα. TNFR2, also known as p75, TNF Receptor Superfamily Member 1B or CD120b. For examples, not limited thereto, TNFR2 is encoded in humans asTNFRSF1Bwhich expresses a 48 kDa protein of 461 amino acids in length (UniProt P20333). In mice, the TNFR2 protein is 474 amino acids long and has a 50 kDa mass. In some embodiments, the TNFR2 sequences from various organisms are well known, and a person skilled in the art can easily predict and modify the sequence therefrom.

[0091] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may comprise at least one of the solvent accessible loops from the wild-type Stefin A protein having the ability to bind TNFR2.

[0092] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to TNFR2 with Kd of 10-6M or less.

[0093] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 is derived from the wild-type human Stefin A polypeptide having a backbone sequence and in which one or both of loop 2 [designated (Xaa)n] and loop 4 [designated (Xaa)m] are replaced with alternative loop sequences (Xaa)n and (Xaa)m.In some embodiments, the Stefin A protein variant specifically binds to TNFR2 may comprise an amino acid sequence represented by Formula (I):

[0094] FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I)

[0095] wherein

[0096] FR1 comprises or is a sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1) or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 1, wherein X is any number of independently selected amino acids, more suitably three or fewer independently selected amino acids, or more suitably X is V, D or LA; and / or

[0097] FR2 comprises or is a sequence comprising the amino acid sequence of GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2) or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 2;

[0098] FR3 comprises or is a sequence comprising the amino acid sequence of EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3) or a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to the amino acid sequence of SEQ ID NO: 3; and

[0099] Xaa, individually for each occurrence, is an amino acid residue; and

[0100] n and m are each, independently, an integer from 3 to 20.

[0101] In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 1. In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 1; In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 2. In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 2; In some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 3. In some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 3.

[0102] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 comprises an amino acid sequence represented in the general Formula (II):

[0103] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4)

[0104] wherein

[0105] Xaa, individually for each occurrence, is an amino acid residue, and

[0106] n and m are each, independently, an integer from 3- 20.

[0107] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of:

[0108] MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5), wherein

[0109] Xaa, individually for each occurrence, is any number of an amino acid residue, more suitably three or fewer independently selected amino acids, and

[0110] n and m are each, independently, an integer from 3- 20.

[0111] In some embodiments, Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu, more preferably Gly, Ala, Arg or Lys, and more even more preferably Gly or Arg; Xaa2 is Val, Asp or ‘Leu-Ala’; Xaa3 is Gly, Ala, Val, Ser or Thr, more preferably Gly or Ser; Xaa4 is Arg, Lys, Asn, Gln, Ser, Thr, more preferably Arg, Lys, Asn or Gln, and even more preferably Lys or Asn; Xaa5 is Gly, Ala, Val, Ser or Thr, more preferably Gly or Ser; Xaa6 is Ala, Val, Ile, Leu, Gly or Pro, more preferably Ile, Leu or Pro, and even more preferably Leu or Pro; Xaa7 is Gly, Ala, Val, Asp or Glu, more preferably Ala, Val, Asp or Glu, and even more preferably Ala or Glu; and Xaa8 is Ala, Val, Ile, Leu, Arg or Lys, more preferably Ile, Leu or Arg, and even more preferably Leu or Arg.

[0112] In some embodiments, n is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12 or 7 to 9.

[0113] In some embodiments, m is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12 or 7 to 9.

[0114] In some embodiments, Xaa, independently for each occurrence, is an amino acid that can be added to a polypeptide by recombinant expression in a prokaryotic or eukaryotic cell, and even more preferably one of the 20 naturally occurring amino acids.

[0115] In some embodiments of the above sequences and formulas, (Xaa)n comprises or is an amino acid sequence selected from SEQ ID NOs: 6 to 102, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with a sequence selected from SEQ ID NOs: 6 to 102. In some embodiments, (Xaa)n is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 6 to 102.

[0116] In some embodiments of the above sequences and formulas, (Xaa)n comprises or is an amino acid sequence selected from SEQ ID NOs: 6, 11, 24 and 65, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with a sequence selected from SEQ ID NOs: 6 to 102. In some embodiments, (Xaa)n is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 6, 11, 24 and 65.

[0117] In some embodiments of the above sequences and formulas, (Xaa)m comprises or is an amino acid sequence selected from SEQ ID NOs: 103 to 199, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with a sequence selected from SEQ ID NOs: 103 to 199. In some embodiments, (Xaa)m is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 103 to 199.

[0118] In some embodiments of the above sequences and formulas, (Xaa)m comprises or is an amino acid sequence selected from SEQ ID NOs: 103, 108, 121 and 162, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with a sequence selected from SEQ ID NOs: 103 to 199. In some embodiments, (Xaa)m is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 103, 108, 121 and 162.

[0119] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 comprises or is an amino acid sequence selected from SEQ ID NOs: 200 to 296, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues. In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence having at least 70%, 75% 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 200 to 296, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues.

[0120] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 more preferably comprises or is an amino acid sequence selected from SEQ ID NOs: 200, 205, 218 and 259. In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence having at least 70%, 75% 80%, 85%, 90%, 95% or even 98% identity with a sequence selected from SEQ ID NOs: 200, 205, 218 and 259.

[0121]

[0122]

[0123]

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[0139] In some embodiments, the amino acid sequence 'AAAEQKLISEEDLAAHHHHHH' optionally excludes from SEQ ID NOs: 200 to 296.

[0140]

[0141] Nucleic acids encoding the Stefin A protein variants specifically binding to TNFR2

[0142] As used herein, “Nucleic acid” is a polynucleotide of any length and may comprise DNA, RNA or a combination of DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In the present invention, the nucleic acids is, for example but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2 '-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

[0143] As used herein, “nucleic acid encoding ~” is nucleic acid sequence encoding a specific protein or polypeptide. As used herein, “nucleic acid encoding ~” is nucleic acid sequence encoding a specific protein or polypeptide. In the art, when the sequence of a specific protein or polypeptide has been known, methods for designing or deriving a nucleic acid encoding the same are well known.

[0144] Therefore, the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 of the present invention can be easily understood from the description of the “Stefin A protein variant specifically binding to TNFR2”

[0145]

[0146] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 comprises an amino acid sequence that is encoded by a nucleic acid having a coding sequence at least 70%, 75% 80%, 85%, 90%, 95% or even 98% identical with a sequence selected from SEQ ID NOs: 297 to 393, optionally excluding the nucleotides encoding twenty one carboxy terminal amino acids. In some embodiments, the Stefin A protein variant specifically binding to TNFR2 comprises an amino acid sequence that is encoded by a nucleic acid that having a coding sequence that hybridizes to a sequence selected from SEQ ID NOs: 297 to 393, optionally excluding the nucleotides encoding the twenty one carboxy terminal amino acids under stringent conditions (such as in the presence of 6X sodium chloride / sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C.

[0147]

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[0167] Furthermore, minor modifications may also include small deletions or additions - beyond the loop 2 and loop 4 inserts described above - to the Stefin A or Stefin A derived sequences disclosed herein, such as addition or deletion of up to 10 amino acids relative to Stefin A or the Stefin A variant.

[0168] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to human TNFR2 as a monomer with a dissociation constant (KD) of about 1 uM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0169] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to human TNFR2 as a monomer with an off-rate constant (Koff), such as measured by BIACORE™ assay, of about 10-3s-1(e.g., unit of 1 / second) or slower; of about 10-4s-1or slower or even of about 10-5s-1or slower.

[0170] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to human TNFR2 as a monomer with an association constant (Kon), such as measured by BIACORE™ assay, of at least about 103M-1s-1or faster; at least about 104M-1s-1or faster; at least about 105M-1s-1or faster; or even at least about 106M-1s-1or faster.

[0171] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may bind to human TNFR2 as a monomer with an IC50 in a competitive binding assay with human TNFR2 of 1 uM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0172] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 has a melting temperature (Tm, e.g., temperature at which both the folded and unfolded states are equally populated) of 65°C or higher, and preferably at least 70°C, 75°C, 80°C or even 85°C or higher. Melting temperature is a particularly useful indicator of protein stability. The relative proportions of folded and unfolded proteins can be determined by many techniques known to the skilled person, including differential scanning calorimetry, UV difference spectroscopy, fluorescence, circular dichroism (CD), and NMR (Pace et al. (1997) "Measuring the conformational stability of a protein" in Protein structure: A practical approach 2: 299-321).

[0173] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may be expressed in the genetically modified cell, and may be secreted and / or anchored to the membrane and presented on the cell surface.

[0174] In some embodiments, the Stefin A protein variant specifically binds to TNFR2, preferably anchored to the membrane of the genetically modified cell or presented to the cell surface. In particular, when the Stefin A protein variant specifically binds to TNFR2 is presented on the membrane or surface of the genetically modified cell, the genetically modified cell may have agonistic effect on TNFR2.

[0175] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may be expressed in the genetically modified cell, and may be localized to a specific organ or location within the cell.

[0176] In some embodiments, when the Stefin A protein variant is anchored to a membrane or expressed on a cell surface, the Stefin A protein variant may be directly / indirectly linked (or fused) to a transmembrane domain, and may be expressed in the form of a fusion protein including a transmembrane domain. In the case of membrane-anchored or cell-surface-expressed Stefin A protein variant, the fusion protein comprising the transmembrane will be described in detail in the section on fusion proteins below.

[0177] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 is monomer.

[0178] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may form a multimer.

[0179] In some embodiments, the Stefin A protein variant specifically binding to TNFR2 may form a dimer, trimer, tetramer, pentamer or more multimer.

[0180] In some embodiments, the multimer may be formed by covalently or non-covalently linked by an interaction between amino acid residues of the Stefin A protein variant.

[0181] In some embodiments, the multimer may be a fusion protein formed through a fusion domain fused with a Stefin A protein variant.

[0182] In some embodiments, the multimer may be formed by in-line fusion of a Stefin A protein variant, and the in-line fusion protein in the form of such a multimer will be described in detail in the section on fusion proteins below.

[0183]

[0184] Fusions Proteins - General

[0185] In some embodiments, the fusion protein may further comprise an additional insertion, substitution and / or deletion that modulates biological activity of the Stefin A protein variant or that gives additional biological functions. For example, the additions, substitutions and / or deletions may modulate at least one property or activity of modified the Stefin A protein variant. For example, the additions, substitutions or deletions may modulate affinity for the the Stefin A protein variant, e.g., for binding to TNFR2 and activating TNFR2 pathway, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the Stefin A protein variant, modulate cleavage by proteases, modulate dose, modulate release or bioavailability, facilitate purification, decrease deamidation, improve shelf-life, or improve or alter a particular route of administration. For examples, the fusion protein may further comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity-based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection, purification or other traits of the polypeptide, but is not limited thereto.

[0186] In some embodiments, the fusion protein may be one in which an additional peptide sequence (a fusion domain) is fused to one end and / or the other end of the Stefin A protein variant.

[0187] As used herein, the term “fusion domain” refers to an additional domain or moiety that may be incorporated directly or indirectly by being fused to the Stefin A protein variant specifically binding to TNFR2 of the present invention.

[0188] In some embodiments, the fusion protein may comprise at least one fusion domain.

[0189] In some embodiments, the fusion domain, for example, may be fused to confer expression properties such as secretion from the cell, or anchoring to the cell surface(e.g. cell membrane), or intracellular localization; to serve as substrate or other recognition sequences for post-translational modifications; to create multimeric structures aggregating through protein-protein interactions; to alter (often to extend) serum half-life; or to alter tissue localization or tissue exclusion and other ADME properties; to add other functional proteins or peptides.

[0190] For example, some fusion domains are particularly useful for isolation and / or purification of the fusion proteins, such as by affinity chromatography. Well known examples of such fusion domains that facilitate expression or purification include, merely to illustrate, affinity tags such as polyhistidine (e.g., a His6 tag), Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, c-Myc tag, thioredoxin, protein A and protein G.

[0191] In order for the Stefin A protein variant or the fusion protein to be secreted, it will generally contain a signal sequence that directs the transport of the protein to the lumen of the endoplasmic reticulum and ultimately to be secreted (or retained on the cell surface if a transmembrane domain or other cell surface retention signal). Signal sequences (also referred to as signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Many signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence.

[0192] In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

[0193] In some embodiments, the signal peptide is a native signal peptide from a human protein. In other embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding native secreted human protein, and can include at least one (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitution, insertions and / or deletions.

[0194] In some embodiments, the signal peptide is a signal peptide or mutant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g. HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently secrete a protein from a cell. Exemplary signal peptides include but are not limited to.

[0195] Table 3 below lists examples of signal peptides that can be used for secretion of Stefin A protein variants or fusion proteins thereof of the present invention, but is not limited thereto.

[0196]

[0197] In some embodiments, the fusion protein may further comprise at least one linker separating the Stefin A protein variant and or the fusion domains. In some embodiments, the “Linker” may inserted between a first polypeptide (e.g. the Stefin A protein variant specifically binding to TNFR2) and a second polypeptide (e.g. other Stefin A protein variant or the fusion domain).

[0198] In some embodiments, one or more Stefin A protein variants and one or more fusion domains can be directly linked without a linker.

[0199] Many natural linkers exhibited α-helical structures. The α-helical structure was rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff α-helical linkers can act as rigid spacers between protein domains. George et al. (2002) “An analysis of protein domain linkers: their classification and role in protein folding” Protein Eng. 15(11):871-9. In general, rigid linkers exhibit relatively stiff structures by adopting α-helical structures or by containing multiple Pro residues. Under many circumstances, they separate the functional domains more efficiently than the flexible linkers. The length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains. As a result, rigid linkers are chosen when the spatial separation of the domains is critical to preserve the stability or bioactivity of the fusion proteins. In this regard, alpha helix-forming linkers with the sequence of (EAAAK)n (SEQ ID NO: 421) have been applied to the construction of many recombinant fusion proteins. Another type of rigid linkers has a Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.

[0200] Merely to illustrate, exemplary linkers include GRA, poly(Gly), poly(Ala) and those provided in Table 4.

[0201]

[0202] In some embodiments, besides the basic role in linking the fusion domains together, the linker may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.

[0203] In some embodiments, the linkers, preferably, should not adversely affect the expression, secretion, or activity of each domain. In some embodiments, the linkers, preferably, should not be antigenic and should not elicit an immune response.

[0204] In some embodiments, the fusion protein may include a fusion domain selected from the group consisting of an antigen-binding protein (domain), a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell-penetrating domain, but is not limited thereto. In some embodiments, the fusion protein may further include a therapeutic peptide or protein.

[0205] In some embodiments, the “therapeutic peptide or protein” refers to any peptide or protein having a preventive or therapeutic effect on a specific disease. In some embodiments, the therapeutic peptide or protein may be a Stefin A protein variant (which may be the same as or different from the Stefin A protein variant specifically binding to TNFR2 of the present invention). It includes, without limitation, peptides and proteins reported to have preventive or therapeutic effects on a specific disease in the art.

[0206] In some embodiments, the fusion protein may include a binding domain.

[0207] In some embodiments, when the fusion protein includes a binding domain, it may have multispecificity capable of binding to at least one target molecule in addition to TNFR2.

[0208] In some embodiments, the binding domain may be selected from the group consisting of, for example, a Stefin A protein variant (which may be the same as or different from the Stefin A protein variant specifically binding to TNFR2 of the present invention), an antibody or fragment thereof, an antibody-like material, an antigen-binding peptide, a ligand-binding site of a receptor (e.g. a receptor trap polypeptide), a receptor-binding ligand (e.g. a cytokine or a growth factor), an engineered T-cell receptor, and an enzyme or a catalytic fragment thereof, but is not limited thereto.

[0209] In some embodiments, examples of the binding domain may include, but are not limited to, adnectins / monobodies, affilins, affibodies, affitins, anticalin, atrimers, avimers, bicyclic peptides, C7 peptide, centyrin, carbohydrate-binding module (CBM), cys-knots, darpin, el-tandem, fynomers, knottin, Kunitz domains, O bodies, pronectin, scFv, Sac7d, Sso7d, Tn3, and the like.

[0210] In some embodiments, the fusion domain fused to the Stefin A protein variant may be the same or different Stefin A protein variant specifically binding to TNFR2, and / or a Stefin A protein variant specifically binding to another target.

[0211] In some embodiments, when the fusion domain is a Stefin A protein variant specifically binding to TNFR2, the two or more Stefin A protein variants included in the fusion protein of the present invention may bind to the same or different sites of TNFR2. In some embodiments, the fusion protein may bind to two sites (biparatopic) or two or more sites (multiparatopic) of TNFR2.

[0212] In some embodiments, the fusion domain may be an immune checkpoint protein, an immune costimulatory receptor, a receptor (or receptor agonist), a cytokine, a growth factor, or a tumor-associated antigen, but is not limited thereto.

[0213] In some embodiments, the cytokine is used as a generic term for secretory proteins that play an important role in signaling between cells. Examples of the cytokines may include, but are not limited to, chemokines, interferons, lymphokines, interleukins, tumor necrosis factors, and the like.

[0214] In some embodiments, the growth factor refers to a naturally-occurring material or a variant thereof that may stimulate cell proliferation, wound healing, and / or cell differentiation. Examples of the growth factors may include, but are not limited to, GH, EGF, VEGF, FGF, bFGF, HGF, BMPs, M-CSF, G-CSF, GM-CSF, EPO, GDNF, IGF, KGF, BDNF, NGF, PDGF, TPO, TGF, and the like.

[0215] In some embodiments, the enzyme is used as a generic term for proteins that catalyze a biological reaction. Examples of the enzyme may include, but are not limited to, α-chymotrypsin, lysozyme, urate oxidase, acetylcholinesterase, Thermomyces lanuginosus lipase, glucose oxidase, superoxie dismutase, caspase, β-glucosidase, Trametes versicolor laccase, alcohol oxidase, Cas9, Cas12, Cas13, Cas14, zinc-finger nuclease, TALLEN, dimethyl sulfoxide, uricase, agalsidase beta, agalsidase alfa, imiglucerase, taliglucerase alfa, velaglucerase alfa, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, alglucosidase alpha, and the like.

[0216] In some embodiments, the cell-penetrating peptide is a short peptide that promotes cellular uptake and absorption of various molecules. In some embodiments, when the fusion protein includes a cell-penetrating peptide, the Stefin A protein variant may be absorbed into the cell. Examples of the cell-penetrating peptide may include, but are not limited thereto, Tat, penetratin, transporant, Pept1, Pept 2, pVEC, DPV3, DPV6, R8, R9, MPG, MAP, Bip4, C105Y, melittin, and the like.

[0217] In some embodiments, a nucleic acid encoding the fusion protein is introduced into a genetically modified cell. the genetically modified cell may be may express a fusion protein and secrete extracellularly, and / or preferably may be anchored on a cell membrane and presented on the cell surface.

[0218] In some embodiments, the fusion protein is expressed in a genetically modified cell, and may be localized to a specific organ or location within the cell.

[0219] In some embodiments, the fusion protein is a secretory fusion protein and / or a membrane-anchored fusion protein.

[0220] In some embodiments, when the fusion protein is fixed to a cell membrane or is expressed on a cell surface, the fusion protein may further include a transmembrane domain. As used herein, the term “transmembrane domain” refers to a protein domain that spans the width of a cell membrane. In some embodiments, the transmembrane domain preferably has an alpha-helical structure, but is not limited thereto.

[0221] In some embodiments, the transmembrane domain may be a transmembrane domain derived from, for example, CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulin (e.g. IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc. or a variant thereof, but is not limited thereto.

[0222] In some embodiments, the fusion protein may further include a hinge domain in addition to the transmembrane domain. As used herein, the term “hinge domain” refers to a series of amino acid sequences that exist between the extracellular domain and the transmembrane domain of a membrane-anchoring protein. In some embodiments, the hinge domain may be located between the Stefin A protein variant specifically binding to TNFR2 and the transmembrane domain.

[0223] In some embodiments, the hinge domain may be a hinge domain derived from, for example, CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulin (e.g. IgG1, IgG4, IgD, etc.), PDGFR, PTGFRN, etc., or a variant thereof, but is not limited thereto.

[0224] In some embodiments, the fusion protein may further include a coiled coil domain. As used herein, the term “coiled coil domain” refers to a structural motif of a protein in which two to seven alpha helices are wound like a rope strand. Preferably, the coiled coil domain is configured such that two or three alpha helices are wound.

[0225] In some embodiments, the coiled coil domain may be a coiled coil domain derived from a leucine zipper, foldon, cardiac phospholamban, a water-soluble analogue of a membrane phospholamban, COMP (cartilage oligomeric matrix protein), thrombospondin 3, thrombospondin 4, or VASP (vasodilator-stimulated phosphoprotein), or a variant thereof, but is not limited thereto.

[0226] In some embodiments, the coiled coil domain may be located between the Stefin A protein variant specifically binding to TNFR2 and the transmembrane domain.

[0227] In some embodiments, the fusion protein may further include a virus-derived peptide or protein. In some embodiments, examples of the virus-derived peptide or protein may include, but are not limited to, syncytin-1, syncytin-2, VSVG (vesicular stomatitis virus glycoprotein), F and G proteins of Nipah virus, F and H proteins of measles virus, F and H proteins of Tupaia paramyxovirus, F and G proteins, F and H proteins, or F and HN proteins of paramyxovirus, F and G proteins of Hendra virus, F and G proteins of Henipavirus, F and H proteins of Morbillivirus, F and HN proteins of respirovirus, F and HN proteins of Sendai virus, F and HN proteins of rubulavirus, F and HN proteins of avulavirus, variants thereof, and combinations thereof.

[0228] In some embodiments, the fusion protein may further include an immunomodulatory domain or an intracellular signaling domain.

[0229] In some embodiments, the immunomodulatory domain or intracellular signaling domain is a domain located in the cytoplasmic direction of a membrane-anchoring protein, and indicates a site that activates or inhibits an immune response when a target antigen is bound to the extracellular domain.

[0230] In some embodiments, the immunomodulatory domain or intracellular signaling domain may be an immunomodulatory domain derived from CD3, CD28, CD40L, ICOS, OX40, 4-1BB, TNFR2, etc., but is not limited thereto.

[0231] In some embodiments, the fusion protein may be a chimeric antigen receptor (CAR). In some embodiments, when the fusion protein is a chimeric antigen receptor, the Stefin A protein variant specifically binding to TNFR2 may function as an extracellular binding domain.

[0232] In some embodiments, when the fusion protein is a chimeric antigen receptor, it may further include the above-described transmembrane domain, hinge domain, and intracellular signaling domain, but the present invention is not limited thereto. The fusion protein may be easily designed and prepared by changing the extracellular antigen-binding domain of various chimeric antigen receptors known in the art or analogues thereof to the Stefin A protein variant specifically binding to TNFR2 of the present invention.

[0233] In some embodiments, the fusion protein may further include a localization domain. In some embodiments, when the fusion protein is expressed intracellularly, it is preferable to further include a localization domain. As used herein, the term “localization domain” refers to a peptide or protein sequence that functions to localize a protein to a specific organ within a cell or a specific location within a cell. In some embodiments, the localization domain may be an organ-specific localization domain or an intracellular protein localization domain.

[0234] In some embodiments, the localization domain may be a nucleus-specific localization domain derived from VACM-1 / CUL5, CXCR4, VP1, 53BP1, ING4, IER5, ERK5, Hrp1, UL79, EWS, PTHrP, Pho4, and rpL23a, a mitochondria-specific localization domain derived from ATP synthase F1b, cytochrome c oxidase polypeptide VIII, SOD2, citrate synthase, Tu translation elongation factor, etc., or a peroxisome localization domain derived from PTS1, PTS2, etc., but is not limited thereto.

[0235] In some embodiments, the fusion protein may further include a half-life extension domain. In some embodiments, the half-life extension domain is a domain or moiety that is fused to extend the half-life of the Stefin A protein variant of the present invention, and examples of the half-life extension domain may include, but are not limited to, an Fc domain, an Fc-binding protein or peptide, albumin (e.g. HSA), an albumin-binding protein or peptide, transferrin, transferrin-binding protein or peptide, etc.

[0236] Still other modifications that can be made to the Stefin A protein variant or the fusion protein sequence or to a flanking polypeptide moiety provided as part of a fusion protein is at least one sequence that is a site for post-translational modification by an enzyme. These can include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.

[0237]

[0238] Multispecific Fusion Proteins

[0239] In some embodiments, the fusion protein is a multispecific polypeptide including, for example, a first Stefin A protein variant specifically binding to TNFR2 and at least one additional binding domain. The additional binding domain may be a polypeptide sequence selected from amongst, to illustrate, a second the Stefin A protein variant (which may be the same or different than the first the Stefin A protein variant), an antibody or fragment thereof or other antigen binding polypeptide, a ligand binding portion of a receptor (such as a receptor trap polypeptide), a receptor-binding ligand (such as a cytokine, growth factor or the like), engineered T-cell receptor, an enzyme or catalytic fragment thereof.

[0240] In some embodiments, the fusion protein includes at least one additional the Stefin A protein variant sequence that is also directed to TNFR2. The additional the Stefin A protein variant(s) specifically binding to TNFR2 may be the same or different (or a mixture thereof) as the first Stefin A protein variant specifically binding to TNFR2 in order to create a multispecific fusion protein. The fusion protein can bind the same or overlapping sites on TNFR2 or can bind two different sites such that the fusion protein can simultaneously bind two sites on the same TNFR2 protein (biparatopic) or more than two sites (multiparatopic).

[0241] In some embodiments, the fusion protein includes at least one antigen binding site from an antibody. The resulting fusion protein can be a single chain including both the Stefin A protein variant specifically binding to TNFR2 and the antigen binding site (such as in the case of an scFv) or can be a multimeric protein complex such as in antibody assembled with heavy and / or light chains to which the sequence of the TNFR2 antibody has also been fused.

[0242] In some embodiments, with respect to a multispecific fusion protein comprising a full-length immunoglobulin, the fusion of the AFFIMER® polypeptide sequence to the antibody will preserve the Fc function of the Fc region of the immunoglobulin. For example, the fusion protein may be capable of binding, via its Fc portion, to the Fc receptor of Fc receptor-positive cells. In some further embodiments, the fusion protein may activate the Fc receptor-positive cell by binding to the Fc receptor-positive cell, thereby initiating or increasing the expression of cytokines and / or co-stimulatory antigens. Furthermore, the AFFIMER® agent may transfer at least a second activation signal required for physiological activation of the T cell to the T cell via the co-stimulatory antigens and / or cytokines.

[0243] In some embodiments, resulted from the binding of its Fc portion to other cells that express Fc receptors present on the surface of effector cells from the immune system, such as immune cells, hepatocytes, and endothelial cells, the AFFIMER® agent may possess antibody-dependent cellular cytotoxicity (ADCC) function, a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigen has been bound by an antibody, and therefore, trigger tumor cell death via ADCC. In some further embodiments, the AFFIMER® agent is capable of demonstrating ADCC function.

[0244] As described above, apart from the Fc-mediated cytotoxicity, the Fc portion may contribute to maintaining the serum levels of the fusion protein, critical for its stability and persistence in the body. For example, when the Fc portion binds to Fc receptors on endothelial cells and on phagocytes, the fusion protein may become internalized and recycled back to the blood stream, enhancing its half-life within the body.

[0245] Exemplary targets of the additional Stefin A protein variant include but are not limited to, another immune checkpoint protein, and immune co-stimulatory receptor (particularly if the additional Stefin A protein variant (s) can agonize the co-stimulatory receptor), a receptor, a cytokine, a growth factor, or a tumor-associated antigen, merely to illustrate. In some embodiments, the immunoglobulin portion may be a monoclonal antibody against at least one autoimmune target. In some embodiments, the Stefin A protein variant specifically binding to TNFR2 is part of a fusion protein that includes one or more binding domains that bind to a protein upregulated in autoimmune conditions.

[0246] See William BA et al. J. Clin. Med. 2019; 8(8): 1261 for a review of the Stefin A protein variant and the fusion protein formats encompassed by the present disclosure.

[0247] In some embodiments, the multispecific fusion protein may further comprise a half-life extension moiety, such as any of those described herein. For example, the fusion protein may comprise at least one the Stefin A protein variant specifically binding to TNFR2 linked through a peptide linker to a binding domain specific for at least one immune cell (e.g., T cell and / or NK cell) binding domain (e.g., CD3ε chain or CD16) further linked to a half-life extension moiety, such as a fragment crystallizable (Fc) domain (e.g., an FcγR null-binding Fc), human serum albumin (HSA), or an Stefin A protein variant specifically binding to HSA. In some embodiments, the half-life extension moiety is a fragment crystallizable (Fc) domain. In some embodiments, the half-life extension moiety is a human serum albumin (HSA). In some embodiments, the half-life extension moiety is an the Stefin A protein variant specifically binding to HSA

[0248] Engineering PK and ADME Properties

[0249] In some embodiment, the fusion protein may not have a half-life and / or PK profile that is optimal for the route of administration, such as parenteral therapeutic dosing. A “half-life” is the amount of time it takes for a substance, such as a fusion protein or Stefin A proein variant of the present invention, to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”). To address this shortcoming, there are a variety of general strategies for prolongation of half-life that have been used in the case of other protein therapeutics, including the incorporation of half-life extending moieties as part of the fusion protein.

[0250] The term “half-life extending moiety” refers to a pharmaceutically acceptable moiety, domain, or molecule covalently linked (chemically conjugated or fused) to an Stefin A protein variant to form an fusion protein described herein, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing modification of the Stefin A protein variant, increases half-life, and / or improves or alters other pharmacokinetic or biophysical properties including but not limited to increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and / or target selectivity of the modified Stefin A protein variant, increasing manufacturability, and / or reducing immunogenicity of the modified AFFIMER® polypeptide, compared to a comparator such as an unconjugated form of the modified AFFIMER® polypeptide. The term “half-life extending moiety” includes non-proteinaceous, half-life extending moieties, such as a water soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), a lipid, a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group; and proteinaceous half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) adnectins), Fc domain, and unstructured polypeptide, such as XTEN and PAS polypeptide (e.g. conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser), and a fragment of any of the foregoing.

[0251] Half-life extending moieties that can be used in the production of the fusion proteins of the present disclosure are well known in the art and can be used by one skilled in the art to prepare fusion proteins using them without limitation.

[0252] In some embodiments, the half-life extending moiety extends the half-life of the resulting fusion protein circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety (such as relative to the AFFIMER® polypeptide alone). In some embodiments, half-life is extended by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold., 5.0-fold, or 6.0-fold. In some embodiments, half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety.

[0253] As means for further exemplification, half-life extending moieties that can be used in the generation of fusion protein of the disclosure include:

[0254] * Genetic fusion of the pharmacologically AFFIMER® sequence to a naturally long-half-life protein or protein domain (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion. See, for example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs. 3:1-2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 4:1015-28; Huang et al. (2009) “Receptor-Fc fusion therapeutics, traps, and Mimetibody technology” Curr Opin Biotechnol. 2009; 20:692-9; Keefe et al. (2013) “Transferrin fusion protein therapies: acetylcholine receptor-transferrin fusion protein as a model. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; p. 345-56; Weimer et al. (2013) “Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 297-323; Walker et al. (2013) “Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 325-43.

[0255] * Genetic fusion of the pharmacologically AFFIMER® sequence to an inert polypeptide, e.g., XTEN (also known as recombinant PEG or ‘‘rPEG’’), a homoamino acid polymer (HAP; HAPylation), a proline-alanine-serine polymer (PAS; PASylation), or an elastin-like peptide (ELP; ELPylation). See, for example, Schellenberger et al. (2009) “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009; 27:1186-90; Schlapschy et al. Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007; 20:273-84; Schlapschy (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 26:489-501. Floss et al. (2012) “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnol. 28:37-45. Floss et al. “ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p. 372-98.

[0256] * Increasing the hydrodynamic radius by chemical conjugation of the pharmacologically active peptide or protein to repeat chemical moieties, e.g., to PEG (PEGylation) or hyaluronic acid. See, for example, Caliceti et al. (2003) “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates” Adv Drug Delivery Rev. 55:1261-77; Jevsevar et al. (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113-28; Kontermann (2009) “Strategies to extend plasma half-lives of recombinant antibodies” BioDrugs. 23:93-109; Kang et al. (2009) “Emerging PEGylated drugs” Expert Opin Emerg Drugs. 14:363-80; and Mero et al. (2013) “Conjugation of hyaluronan to proteins” Carb Polymers. 92:2163-70.

[0257] * Significantly increasing the negative charge of fusing the pharmacologically active peptide or protein by polysialylation; or, alternatively, (b) fusing a negatively charged, highly sialylated peptide (e.g., carboxy-terminal peptide [CTP; of chorionic gonadotropin (CG) b-chain]), known to extend the half-life of natural proteins such as human CG b-subunit, to the biological drug candidate. See, for example, Gregoriadis et al. (2005) “Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids” Int J Pharm. 2005; 300:125-30; Duijkers et al. “Single dose pharmacokinetics and effects on follicular growth and serum hormones of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed females” (2002) Hum Reprod. 17:1987-93; and Fares et al. “Design of a longacting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit” (1992) Proc Natl Acad Sci USA. 89:4304-8. 35; and Fares “Half-life extension through O-glycosylation.

[0258] * Binding non-covalently, via attachment of a peptide or protein-binding domain to the bioactive protein, to normally long-half-life proteins such as HSA, human IgG, transferrin or fibronectin. See, for example, Andersen et al. (2011) “Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain” J Biol Chem. 286:5234-41; O’Connor-Semmes et al. (2014) “GSK2374697, a novel albumin-binding domain antibody (albudAb), extends systemic exposure of extendin-4: first study in humans-PK / PD and safety” Clin Pharmacol Ther. 2014; 96:704-12. Sockolosky et al. (2014) “Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice” PLoS One. 2014; 9:e102566.

[0259] Classical genetic fusions to long-lived serum proteins offer an alternative method of half-life extension distinct from chemical conjugation to PEG or lipids. Two major proteins have traditionally been used as fusion partners: antibody Fc domains and human serum albumin (HSA). Fc fusions involve the fusion of peptides, proteins or receptor exodomains to the Fc portion of an antibody. Both Fc and albumin fusions achieve extended half-lives not only by increasing the size of the peptide drug, but both also take advantage of the body’s natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in the endosome. Fusions based on these proteins can have half-lives in the range of 3-16 days, much longer than typical PEGylated or lipidated peptides. Fusion to antibody Fc domains can improve the solubility and stability of the peptide or protein drug. An example of a peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein exploited by the fatty acylated peptides is the other popular fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. A major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA leading to presentation of a fused peptide as a dimer or a monomer depending on the choice of fusion partner. The dimeric nature of an AFFIMER®-Fc fusion can produce an avidity effect if the AFFIMER® targets are spaced closely enough together or are themselves dimers. This may be desirable or not depending on the target.

[0260] Fc Fusions

[0261] the fusion protein may include an immunoglobulin Fc domain (an Fc domain) or a fragment or variant thereof, for example, a functional Fc region. In some embodiments, the Fc region is a FcγR null-binding Fc region. In some embodiments, the fusion protein may comprise at least one Stefin A protein variant specifically binding to TNFR2 covalently linked through a peptide backbone (directly or indirectly) to an Fc region of an immunoglobulin. In some embodiments, the fusion protein may comprise the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and the Stefin A protein variant specifically binding to TNFR2 as part of the same polypeptide. An immunoglobulin Fc region may also be linked indirectly to at least one Stefin A protein variant specifically binding to TNFR2. Various linkers are known in the art for use in the fusion proteins of the invention. In some embodiments, the fusion protein comprising Fc domain may be used as dimer, and may be used as a homodimer or a heterodimer.

[0262] In some embodiments, various Fc domain sequences that can be used for Fc domain fusion and functionality thereof are well known in the art, and a person skilled in the art can selects an appropriate Fc domain according to the purpose and fuses it to the Stefin A protein variant of the present invention.

[0263] In some embodiments, the Stefin A protein variant may be part of a fusion protein with an immunoglobulin Fc domain ("Fc domain"), or a fragment or variant thereof, such as a functional Fc region. In some embodiments, the Fc region is a FcγR null-binding Fc region. In this context, an Fc fusion (“Fc-fusion”), such as a Fc fusion protein, is a polypeptide comprising at least one Stefin A protein variant specifically binding to TNFR2 sequence covalently linked through a peptide backbone (directly or indirectly) to an Fc region of an immunoglobulin. An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and a Stefin A protein variant specifically binding to TNFR2 sequence as part of the same polypeptide. An immunoglobulin Fc region may also be linked indirectly to at least one Stefin A protein variant specifically binding to TNFR2. Various linkers are known in the art and can optionally be used to link an Fc to a polypeptide including a Stefin A protein variant specifically binding to TNFR2 sequence to generate an Fc-fusion. In some embodiments, Fc-fusions can be dimerized to form Fc-fusion homodimers, or using non-identical Fc domains, to form Fc-fusion heterodimers.

[0264] In some embodiments, an Fc-fusion homodimer comprises a dimer of a fusion protein that comprises a Stefin A protein variant specifically binding to TNFR2 linked to an Fc domain linked to Stefin A protein variant specifically binding to TNFR2 (TNFR2 AFFIMER® polypeptide-Fc domain-TNFR2 AFFIMER® polypeptide).

[0265] There are several reasons for choosing the Fc region of human antibodies for use in generating fusion protein. The principle rationale is to produce a stable protein, large enough to demonstrate a similar pharmacokinetic profile compared with those of antibodies, and to take advantage of the properties imparted by the Fc region; this includes the salvage neonatal FcRn receptor pathway involving FcRn-mediated recycling of the fusion protein to the cell surface post endocytosis, avoiding lysosomal degradation and resulting in release back into the bloodstream, thus contributing to an extended serum half-life. Another obvious advantage is the Fc domain’s binding to Protein A, which can simplify downstream processing during production of the fusion protein and permit generation of highly pure preparation of the fusion protein.

[0266] In general, an Fc domain will include the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domain may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region in isolation, or this region in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein.

[0267] In some embodiments, the Fc As used herein, a “functional Fc region” refers to an Fc domain or fragment thereof which retains the ability to bind FcRn. A functional Fc region binds to FcRn but does not possess effector function. The ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0268] In an exemplary embodiment, the Fc domain is derived from an IgG1 subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. An exemplary sequence of a human IgG1 immunoglobulin Fc domain which can be used is:

[0269]

[0270] In some embodiments, the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule. An exemplary hinge region comprises the core hinge residues spanning positions 1-16 (e.g., DKTHTCPPCPAPELLG ((SEQ ID NO: 536)) of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In some embodiments, the AFFIMER®-containing fusion protein may adopt a multimeric structure (e.g., dimer) owing, in part, to the cysteine residues at positions 6 and 9 within the hinge region of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In other embodiments, the hinge region as used herein, may further include residues derived from the CH1 and CH2 regions that flank the core hinge sequence of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above. In yet other embodiments, the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 443) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 444).

[0271] In some embodiments, the hinge sequence may include at least one substitution that confer desirable pharmacokinetic, biophysical, and / or biological properties. Some exemplary hinge sequences include:

[0272] EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO: 445);

[0273] EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446);

[0274] EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 447);

[0275] EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448);

[0276] DKTHTCPPCPAPELLGGPS (SEQ ID NO: 449); and

[0277] DKTHTCPPCPAPELLGGSS (SEQ ID NO: 450).

[0278] In some embodiments, the residue P at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to ablate Fc effector function; this replacement is exemplified in hinges having the sequences

[0279] EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 452), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 453).

[0280] In another embodiment, the residues DK at positions 1-2 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with GS to remove a potential clip site; this replacement is exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448). In another embodiment, the C at the position 103 of the heavy chain constant region of human IgG1 (e.g., domains CH1-CH3), may be replaced with S to prevent improper cysteine bond formation in the absence of a light chain; this replacement is exemplified by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 452).

[0281] In some embodiments, the Fc is a mammalian Fc such as a human Fc, including Fc domains derived from IgG1, IgG2, IgG3 or IgG4. The Fc region may possess at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fc region and / or with an Fc region of a parent polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity with a native Fc region and / or with an Fc region of a parent polypeptide.

[0282] In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 454-467 or an Fc sequence from the examples provided by SEQ ID NOs: 454-467. It should be understood that the C-terminal lysine of an Fc domain is an optional component of a fusion protein comprising an Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 454-467, except that the C-terminal lysine thereof is omitted. In some embodiments, the Fc domain comprises the amino acid sequence selected from SEQ ID NOs: 454-467. In some embodiments, the Fc domain comprises the amino acid sequence selected from SEQ ID NOs: 454-467 except the C-terminal lysine thereof is omitted.

[0283]

[0284]

[0285]

[0286] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins.

[0287] In some embodiments, the fusion protein includes an Fc domain sequence for which the resulting fusion protein has no (or reduced) ADCC and / or complement activation or effector functionality. For example, the Fc domain may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. One example comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering).

[0288] In other embodiments, the fusion protein includes an Fc domain sequence for which the resulting fusion protein will retain some or all Fc functionality for example will be capable of one or both of ADCC and CDC activity, as for example if the fusion protein comprises the Fc domain from human IgG1 or IgG3. Levels of effector function can be varied according to known techniques, for example by mutations in the CH2 domain, for example wherein the IgG1 CH2 domain has at least one mutation at positions selected from 239 and 332 and 330, for example the mutations are selected from S239D and I332E and A330L such that the antibody has enhanced effector function, and / or for example altering the glycosylation profile of the antigen-binding protein of the disclosure such that there is a reduction in fucosylation of the Fc region.

[0289] Albumin Fusions

[0290] In some embodiments, the fusion protein is a fusion protein comprising, in addition to at least one Stefin A protein variant, an albumin sequence or an albumin fragment. In other embodiments, the AFFIMER® agent is conjugated to the albumin sequence or an albumin fragment through chemical linkage other than incorporation into the polypeptide sequence including the Stefin A protein variant. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment. Albumin serum proteins comparable to HSA are found in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats. Of the non-human species, bovine serum albumin (BSA) is the most structurally similar to HSA. See, e.g., Kosa et al., (2007) J Pharm Sci. 96(11):3117-24. The present disclosure contemplates the use of albumin from non-human species, including, but not limited to, albumin sequence derived from cyno serum albumin or bovine serum albumin.

[0291] Mature HSA, a 585 amino acid polypeptide (approx. 67 kDa) having a serum half-life of about 20 days, is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands. The protein has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges. In some embodiments, the fusion protein can be an albumin fusion protein including at least one Stefin A protein variant sequence and the sequence for mature human serum albumin (SEQ ID NO: 468) or a variant or fragment thereof which maintains the PK and / or biodistribution properties of mature albumin to the extent desired in the fusion protein.

[0292] The albumin sequence can be set off from the Stefin A protein variant sequence or other flanking sequences in the fusion protein by use of linker sequences as described above.

[0293] While unless otherwise indicated, reference herein to “albumin” or to “mature albumin” is meant to refer to HSA. However, it is noted that full-length HSA has a signal peptide of 18 amino acids (MKWVTFISLLFLFSSAYS (SEQ ID NO: 483) followed by a pro-domain of 6 amino acids (RGVFRR) (SEQ ID NO: 469); these 24 amino acid residue peptide may be referred to as the pre-pro domain. The AFFIMER®-HSA fusion proteins can be expressed and secreted using the HSA pre-pro-domain in the recombinant proteins coding sequence. Alternatively, the AFFIMER®-HSA fusion can be expressed and secreted through inclusion of other secretion signal sequences, such as described above.

[0294] In alternative embodiments, rather than provided as part of a fusion protein with the Stefin A protein variant, the serum albumin polypeptide can be covalently coupled to the Stefin A protein variant containing polypeptide through a bond other than a backbone amide bond, such as cross-linked through chemical conjugation between amino acid sidechains on each of the albumin polypeptide and the Stefin A protein variant-containing polypeptide.

[0295] In some embodiments, a chemical modification method that can be applied in the generation of the subject Stefin A protein variant or fusion protein to increase protein half-life is lipidation, which involves the covalent binding of fatty acids to peptide side chains. Originally conceived of and developed as a method for extending the half-life of insulin, lipidation shares the same basic mechanism of half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration. However, the lipid moiety is itself relatively small and the effect is mediated indirectly through the non-covalent binding of the lipid moiety to circulating albumin. One consequence of lipidation is that it reduces the water-solubility of the peptide but engineering of the linker between the peptide and the fatty acid can modulate this, for example by the use of glutamate or mini PEGs within the linker. Linker engineering and variation of the lipid moiety can affect self-aggregation which can contribute to increased half-life by slowing down biodistribution, independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res. 29(8):2104-14.

[0296] Other examples of albumin binding moieties for use in the generation of certain fusion protein include albumin-binding (PKE2) adnectins (See WO2011140086 “Serum Albumin Binding Molecules”, WO2015143199 “Serum albumin-binding Fibronectin Type III Domains” and WO2017053617 “Fast-off rate serum albumin binding fibronectin type iii domains”), the albumin binding domain 3 (ABD3) of protein G of Streptococcus strain G148, and the albumin binding domain antibody GSK2374697 (“AlbudAb”) or albumin binding nanobody portion of ATN-103 (Ozoralizumab).

[0297] AFFIMER® XT

[0298] In some embodiments, the molecule that binds a serum protein such as HSA comprises a Stefin A protein variant specifically binding to HSA. Examples of such Stefin A protein variant specifically binding to HSA can be found in WO2022 / 023540. The Stefin A protein variant specifically binding to HSA provided herein, in some embodiments, is linked to another molecule and extend the half-life of that molecule (e.g., a therapeutic polypeptide). These Stefin A protein variant specifically binding to HSA have been shown in in vivo pharmacokinetic (PK) studies to extend, in a controlled manner, the serum half-life of any other Stefin A protein variant therapeutic to which it is conjugated in a single genetic fusion, for example, that can be made in E. Coli. AFFIMER® XT™ polypeptides can also be used to extend the half-life of other peptide or protein therapeutics, such as the Stefin A protein variant specifically binding to TNFR2 of the present invention.

[0299] In some embodiments, a Stefin A protein variant specifically binding to HSA extends the serum half-life of the TNFR2 AFFIMER® polypeptide in vivo. For example, a Stefin A protein variant specifically binding to HSA may extend the half-life of the TNFR2 AFFIMER® polypeptide by at least 2-fold, relative to the half-life of the molecule not linked to a Stefin A protein variant specifically binding to HSA. In some embodiments, a Stefin A protein variant specifically binding to HSA extends the half-life of the Stefin A protein variant specifically binding to TNFR2 by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, relative to the half-life of the Stefin A protein variant specifically binding to TNFR2 not linked to an Stefin A protein variant specifically binding to HSA. In some embodiments, an Stefin A protein variant specifically binding to HSA extends the half-life of the Stefin A protein variant specifically binding to TNFR2 by 2-fold to 5-fold, 2-fold to 10-fold, 3-fold to 5-fold, 3-fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, relative to the half-life of the Stefin A protein variant specifically binding to TNFR2 not linked to an Stefin A protein variant specifically binding to HSA. In some embodiments, a Stefin A protein variant specifically binding to HSA extends the half-life of the Stefin A protein variant specifically binding to TNFR2 by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, for example, at least 1 week after in vivo administration, relative to the half-life of the molecule not linked to a Stefin A protein variant specifically binding to HSA.

[0300] A Stefin A protein variant specifically binding to HSA comprises an AFFIMER® polypeptide in which at least one of the solvent accessible loops is from the wild-type Stefin A protein having amino acid sequences to enable an AFFIMER® polypeptide to bind HSA, selectively, and in some embodiments, with a Kd of 10-6M or less.

[0301] In some embodiments, the Stefin A protein variant specifically binding to HSA is derived from the wild-type human Stefin A protein having a backbone sequence and in which one or both of loop 2 (designated (Xaa)n) and loop 4 (designated (Xaa)m) are replaced with alternative loop sequences (Xaa)n and (Xaa)m, to have the general Formula (I):

[0302] FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I),

[0303] wherein FR1 is an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 470); FR2 is an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2); FR3 is an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); Xaa, individually for each occurrence, is an amino acid; and n is an integer from 3 to 20, and m is an integer from 3 to 20. Additional variations of this general structure can be found in WO2022 / 023540.

[0304] In all embodiments, each amino acid of (Xaa)n may be the same or selected from any amino acid. The same applies for (Xaa)m.

[0305] In some embodiments, the fusion protein further comprises a Stefin A protein variant specifically binding to HSA comprising an amino acid sequence selected from any one of SEQ ID NOs: 471-477 (Table 6). In some embodiments, the Stefin A protein variant specifically binding to TNFR2 has an extended serum half-life and comprises an amino acid sequence that is at least 70%, 75% 80%, 85%, 90%, 95% or even 98% identical with a sequence selected from SEQ ID NOs: 471-477 (Table 6). Additional Stefin A protein variant specifically binding to HSA sequences for use with the present invention can be found in WO2022 / 023540.

[0306]

[0307] Host cell and genetically modified cell

[0308] In some embodiments, a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 described above and / or the fusion protein including the same is introduced into a host cell.

[0309] As used herein, the term “host cell” refers to a cell before introduction, for introducing a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 of the present invention and / or the fusion protein including the same.

[0310] As used herein, the term “genetically modified cell” refers to a cell expressing the Stefin A protein variant and / or the fusion protein by introducing a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 of the present invention and / or the fusion protein including the same.

[0311] In some embodiments, the genetically modified cell may include additional genetic modification in addition to the introduction of a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 of the present invention and / or the fusion protein including the same.

[0312] In some embodiments, the genetically modified cell may be characterized in that a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced, in order to produce the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same.

[0313] In some embodiments, when the genetically modified cell is constructed for the production of the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same, all types of cells such as eukaryotic cells, prokaryotic cells, etc. may be used, and examples of the host cell may include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc., yeast cells, fungal cells such as Pichia pastoris, etc., insect cells such as Drosophila, Spodoptera Sf9 cells, etc., animal cells such as CHO, COS, NSO, 293, and bow melanoma cells, and plant cells.

[0314] In an embodiment of the present invention, a genetically modified mesenchymal stromal cell that expresses the Stefin A protein variant or the fusion protein by introducing a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 of the present invention or the fusion protein including the same into a mesenchymal stromal cell was produced.

[0315] In some embodiments, the host cell may be selected from the group consisting of a stem cell, an immune cell, and a somatic cell.

[0316] In some embodiments, the host cell is a cell derived from nature, for example, an animal, preferably a mammal, more preferably a human, or a cell engineered through cell engineering or genetic engineering.

[0317] As used herein, the term “stem cell” refers to a cell capable of differentiating into various types of cells constituting a biological tissue, and collectively refers to undifferentiated cells in the pre-differentiation stage which may be obtained from each tissue of embryo, fetus, and adult body. Stem cells are differentiated into specific cells by differentiation stimuli (environment), and unlike cells in which differentiation is completed and cell division is stopped, stem cells are capable of self-renewal by cell division to thus enable proliferation (expansion), and may be differentiated into other cells by different environments or different differentiation stimuli, meaning they have plasticity in differentiation.

[0318] In some embodiments, examples of the stem cells may include, but are not limited to, pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on the differentiation potential thereof.

[0319] In some embodiments, the pluripotent stem cells are stem cells capable of differentiating into three germ layers constituting a living body, and examples thereof may include, but are not limited to, embryonic stem cells, induced pluripotent stem cells (iPS), and the like.

[0320] In some embodiments, the multipotent stem cells are cells having the potential to differentiate progenitor cells into cells belonging to a certain family. Examples of the multipotent stem cells may include, but are not limited to, hematopoietic stem cells, mesenchymal stromal cells, neural stem cells, and the like.

[0321] In some embodiments, the stem cells may be mesenchymal stromal cells. As used herein, the term “mesenchymal stromal cell” refers to a cell capable of differentiating into osteoblasts, adipocytes, chondrocytes, and the like, which may be differentiated from mesoderm among the three germ layers of embryonic tissue. The mesenchymal stromal cells may be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular bone, subpatellar fat pad, etc. The mesenchymal stromal cells are known to 1) inhibit the activity and proliferation of T lymphocytes and B lymphocytes, 2) inhibit the activity of natural killer cells (NK cells), and 3) enable allotransplantation and xenotransplantation by virtue of immunomodulatory activity of regulating the functions of dendritic cells and macrophages.

[0322] The Stefin A protein variant specifically binding to TNFR2 of the present invention exhibits a TNFR2 agonistic effect, and thus an immunosuppressive effect such as inhibition of T-cell and / or B-cell activity. Therefore, in an embodiment of the present invention, by introducing a nucleic acid encoding the Stefin A protein variant into a mesenchymal stromal cell (MSC) as the host cell, novel immunomodulatory activity was acquired through the TNFR2 agonistic effect by the Stefin A protein variant while maintaining the immunomodulatory effect of the mesenchymal stromal cell (e.g. T-cell activation inhibitory effect), confirming that immunity was very effectively inhibited and also that a vastly superior therapeutic effect on immune-related diseases such as GVHD or autoimmune diseases was exhibited.

[0323] In some embodiments, the mesenchymal stromal cell may be derived from a pluripotent stem cell. In some embodiments, the mesenchymal stromal cell enables long-term subculture. The method of producing the mesenchymal stromal cell from the pluripotent stem cell and the long-term subculture method are well known in the art, and for example, Korean Patent Application Publication No. 10-2021-0072734, Korean Patent No. 10-1135636, etc. disclose a method of maintaining undifferentiation potency and marker expression characteristics even after tens of passages.

[0324] In some embodiments, the mesenchymal stromal cell may express at least one selected from among CD29, CD44, CD73, CD90, and CD105. In some embodiments, the mesenchymal stromal cell may not express at least one cell surface marker selected from among c-kit, CD11b, CD19, CD14, CD34, CD45, CD14, CD79, HLA-DR, TRA-1-60, and TRA-1-81.

[0325] In some embodiments, the mesenchymal stromal cell may express at least one cell surface marker selected from among CD29, CD44, CD73, and CD105.

[0326] In some embodiments, the mesenchymal stromal cell is capable of maintaining at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of expression of the cell surface marker after at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 16 passages, at least 17 passages, at least 18 passages, at least 19 passages, or at least 20 passages.

[0327] In some embodiments, the passage may be based on an increase in the number of cells by a certain fold or more, and for example, proliferation of the number of cells at least 2-fold, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, or 8-fold or more as compared to the start of the previous passage may be determined to be 1 passage, but the present invention is not limited thereto.

[0328] In some embodiments, the mesenchymal stromal cell may not express at least one cell surface marker selected from among CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81.

[0329] In some embodiments, the host cell may be an immune cell.

[0330] As used herein, the term “immune cell” refers to all types of cells constituting the immune system. A cell therapeutic agent using the immunomodulatory activity of immune cells is used for the treatment of various diseases such as cancer and autoimmune diseases, but due to the non-specific effects thereof, immune cell therapeutic agents engineered to enable target-specific immune regulation, such as chimeric antigen receptors, are of great interest.

[0331] The Stefin A protein variant specifically binding to TNFR2 of the present invention targets TNFR2 involved in immune regulation, and in particular, since it has an agonistic ability to TNFR2, when a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced into immune cells, a vastly superior immunomodulatory effect may be exhibited.

[0332] In some embodiments, the immune cells may be selected from the group consisting of T cells, B cells, natural killer (NK) cells, nkT cells, and dendritic cells, but are not limited thereto.

[0333] In some embodiments, the immune cells are those isolated from the human body, blood, or peripheral blood mononuclear cells (PBMCs), or those differentiated from stem cells (preferably pluripotent stem cells), but are not limited thereto.

[0334] In some embodiments, when a nucleic acid encoding a fusion protein (e.g. a chimeric antigen receptor) including the Stefin A protein variant specifically binding to TNFR2 as an extracellular binding domain is introduced into the immune cells, the resulting cells may be used as a cell therapeutic agent, such as CAR-T or CAR-NK.

[0335] In some embodiments, the host cell may be a somatic cell.

[0336] As used herein, the term “somatic cell” refers to any type of cells except gametes, constituting the body of an animal, preferably a human. Examples of cell therapeutic agents using somatic cells are well known in the art. For example, epidermal cells such as keratinocytes, fibroblasts, and mucous membranes may be used for the treatment of skin burns, scars, cosmetic purposes, etc., and chondrocytes, adipocytes, islet cells, and skeletal myoblasts may be used for the treatment of diseases such as degenerative arthritis, subcutaneous fat obliteration, etc., but the present invention is not limited thereto.

[0337] When a gene encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced into a somatic cell as the host cell of the present invention, an immunomodulatory effect through inhibition of TNFR2 activity may be exhibited.

[0338] In some embodiments, the genetically modified cell enables secretion of the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same, expression thereof on the cell membrane, and / or localization thereof to a specific site in the cell.

[0339] In some embodiments, most preferably, the genetically modified cell expresses the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein and presents it on the cell surface (or membrane).

[0340] In vivo, the ligand of the TNF receptor, the TNF molecule, acts in a trimer. A trimeric TNF molecule interacts with three molecules of either TNFR1 or TNFR2. Importantly, membrane TNF activates both TNF receptors, while only TNFR1 responds strongly to sTNF(soluble TNF). Triggering of TNFR2-associated signaling pathways requires secondary clustering of initially formed multimeric TNF-TNFR2 complexes. This occurs spontaneously for membrane TNF-induced TNF-TNFR2 complexes but not sTNF-TNFR2 complexes.

[0341] When the genetically modified cell of the present invention expresses and presents the Stefin A protein variant specifically binding to TNFR2 on the cell surface, clustering of ligand-TNFR2 complexes and activation of the TNFR2 pathway mimicked. As a result, the genetically modified cell presenting the Stefin A protein variant specifically binding to TNFR2 on the cell surface has a strong agonistic effect on TNFR2 and can induce activation of the TNFR2 pathway.

[0342]

[0343] Method of introducing nucleic acid into host cell

[0344] As used herein, the term “introduction” refers to allowing the host cell to receive a foreign gene (nucleic acid) that the host cell does not have.

[0345] In some embodiments, a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 or a fusion protein including the same may be introduced into a host cell using a vector including the same.

[0346] As used herein, a “vector” is a means for expressing a target gene in a host cell, and examples of the vector may include, but are not limited to, viral vectors such as adenoviral vector, retroviral vector, adeno-associated viral vector, and vectors derived from viruses such as vaccinia virus (Puhlmann M. et al., Human Gene Therapy, 10:649-657 (1999); Ridgeway, 467-492 (1988); Baichwal and Sugden, In: Kucherlapati R., ed. Gene transfer. New York: Plenum Press, 117-148 (1986) and Coupar et al., Gene, 68:1-10(1988)), lentivirus (Wang G. et al., J. Clin. Invest., 104(11):R55-62(1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA, 92:1411-1415 (1995)), poxvirus (GCE, NJL, Krupa M., Esteban M., Curr. Gene Ther. 8(2):97-120 (2008)), reovirus, measles virus, Semliki Forest virus, and poliovirus, and non-viral vectors such as plasmid vectors (Sambrook et al., 1989) and mini circles (Yew et al. 2000 Mol. Ther. 1(3), 255-62).

[0347] The vector may typically include at least one component selected from among a signal sequence, an origin of replication, at least one antibiotic resistance marker gene, an enhancer element, a promoter, and a transcription termination sequence, but the present invention is not limited thereto. The nucleic acid encoding the Stefin A protein variant of the present invention or the fusion protein including the same may be operably linked with a promoter and a transcription termination sequence.

[0348] As used herein, the term “operably linked” means a functional linkage between a nucleic acid expression control sequence (e.g. a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and a different nucleic acid sequence, whereby the control sequence serves to control the transcription and / or translation of the different nucleic acid sequence.

[0349] When a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g. a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosome-binding site for initiation of translation, and a transcription / translation termination sequence are generally included. In addition, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g. a metallothionine promoter, β-actin promoter, human hemoglobin promoter or human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g. an adenovirus late promoter, vaccinia virus 7.5k promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), or promoter of Rous sarcoma virus (RSV)) may be used, and a polyadenylation sequence is generally used as a transcription termination sequence

[0350] In some embodiments, the promoter may be a eukaryotic promoter, is preferably selected from among a cytomegalovirus (CMV) promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and a UbC promoter, and is most preferably selected from among a CMV promoter, an EF1α promoter, and a CBh promoter, but is not limited thereto.

[0351] In some embodiments, the promoter may further include an enhancer sequence, but is not limited thereto.

[0352] In some cases, the vector may be fused with another sequence in order to facilitate purification of the antibody expressed therefrom. Examples of the sequence that is fused therewith include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexa-histidine; Qiagen, USA)).

[0353] The vector may include, as a selective marker, an antibiotic resistance gene that is commonly used in the art, for example, a gene conferring resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, and tetracycline, but is not limited thereto.

[0354] In some embodiments, a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 or the fusion protein including the same may be incorporated and introduced into the gene of the host cell.

[0355] In some embodiments, the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 or the fusion protein including the same may be constructed through chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides may be designed based on the amino acid sequence of the desired polypeptide and by selecting codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. A polynucleotide sequence encoding the isolated polypeptide of interest may be synthesized using standard methods. For example, a reverse-translated gene may be constructed using a complete amino acid sequence. In addition, a DNA oligomer containing a nucleotide sequence encoding a particular isolated polypeptide may be synthesized. For example, several small oligonucleotides encoding portions of a desired polypeptide may be synthesized and then ligated. Individual oligonucleotides generally contain 5’ or 3’ overhangs for complementary assembly.

[0356] In some embodiments, when the nucleic acid sequence encoding the Stefin A protein variant specifically binding to TNFR2 or the fusion protein including the same is obtained, a vector including the same may be produced through recombinant DNA technology using a technique well known in the art. An expression vector containing a sequence encoding the Stefin A protein variant of the present invention or the fusion protein including the same and appropriate transcriptional and translational control signals may be constructed using methods well known to those skilled in the art. Examples of such methods may include in-vitro recombinant DNA techniques, synthesis techniques, and in-vivo genetic recombination (e.g. Sambrook et al., 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN Molecular Biology, John Wiley & Sons, NY).

[0357] In some embodiments, the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 or the fusion protein including the same or the non-viral expression vector including the same may be delivered to the host cell using typical techniques (e.g. electroporation, liposome transfection, and calcium phosphate precipitation).

[0358] The vector may be introduced into the host cell through a method such as transduction or transfection. As used herein, the term “transduction” refers to introduction of DNA into a host such that the DNA becomes replicable either as an extrachromosomal factor or through chromosomal integration. As used herein, the term “transfection” means that an expression vector is accommodated by the host cell, regardless of whether or not any coding sequence is actually expressed. In order to introduce the vector, a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells, for example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection may be used, but the present invention is not limited thereto.

[0359] It is to be understood that not all vectors and expression control sequences function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art will be able to make an appropriate selection from among various vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, a vector may be selected in consideration of the host. This is because the vector has to be able to replicate in the host. Also, the number of copies of a vector, ability to control the number of copies, and expression of another protein encoded by the vector, for example, an antibiotic marker, have to be taken into consideration. In selecting the expression control sequence, various factors have to be considered. For example, the relative strength of the sequences, controllability thereof, compatibility with the DNA sequences of the present invention, etc., should be taken into account, particularly with regard to possible secondary structures. The single-celled host should be selected in consideration of factors such as the selected vector, the toxicity and secretory properties of the product encoded by the DNA sequence of the invention, the ability to correctly fold the protein, culture and fermentation requirements, ease of purification of the product encoded by the DNA sequence of the present invention from the host, and the like. Within the scope of these parameters, those skilled in the art may select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture. Examples of a screening method of cloning cDNA by expression cloning may include a binding method, a panning method, a film emulsion method, etc.

[0360] In an embodiment of the present invention, a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 or the fusion protein including the same was introduced into a host cell using a lentivirus.

[0361] In particular, as in an embodiment of the present invention, a transduction enhancer may be used when introducing a gene using a lentivirus.

[0362] In some embodiments, the transduction enhancer may be selected from among, for example, polybrene, protamine sulfate, and LentiBOOST from Sirion, and is most preferably polybrene, but is not limited thereto.

[0363] In some embodiments, the host cell that is attached or not attached may be infected with both a transduction enhancer and a lentivirus, and infection before cell attachment is preferable, but the present invention is not limited thereto.

[0364] Another aspect of the present invention related to a genetically modified cell expressing the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein comprising the same.

[0365]

[0366] Method of producing genetically modified cell and culture of genetically modified cell

[0367] Still another aspect of the present invention pertains to a method of producing a genetically modified cell into which a nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced, including:

[0368] (a) introducing a nucleic acid encoding a Stefin A protein variant specifically binding to TNFR2 and / or a fusion protein including the same into a host cell; and

[0369] (b) selecting and obtaining the host cell into which the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced.

[0370] In some embodiments, step (a) may be performed through various means known in the art.

[0371] In some embodiments, step (a) may be performed using a lentivirus containing the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same.

[0372] In some embodiments, the lentivirus may be introduced with a vector including the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same.

[0373] In some embodiments, the vector may further include at least one selected from among a signal sequence, an origin of replication, at least one antibiotic resistance marker gene, an enhancer element, a promoter, and a transcription termination sequence.

[0374] In some embodiments, the promoter may be a eukaryotic promoter, is preferably selected from among a CMV promoter, a PGK promoter, an EF1α promoter, an EFS promoter, a CBh promoter, an MSCV promoter, an SFFV promoter, and a UbC promoter, and is most preferably selected from among a CMV promoter, an EF1α promoter, and a CBh promoter, but is not limited thereto.

[0375] In some embodiments, the promoter may further include an enhancer sequence, but is not limited thereto.

[0376] In some embodiments, the enhancer is a short DNA region of about 50 to 1500 bp in length that may bind to a transcriptional regulatory protein. The enhancer may be located in the transcription start site or upstream or downstream of the promoter. Enhancers for various promoters are well known in the art, and may be selected and applied without limitation by those skilled in the art.

[0377] In some embodiments, step (a) may include transducing the host cell by infecting the host cell with the lentivirus.

[0378] In some embodiments, transducing the host cell by infecting the host cell with the lentivirus may be performed by adding a transduction enhancer.

[0379] In some embodiments, the transduction enhancer is preferably a cationic polymer, making it easy to incorporate a negatively charged nucleic acid or gene into the host cell.

[0380] In some embodiments, the transduction enhancer may be a cationic polymer. For example, the transduction enhancer may be selected from among polybrene, protamine sulfate, and LentiBOOST from Sirion, and is most preferably polybrene, but is not limited thereto.

[0381] In some embodiments, transducing the host cell by infecting the host cell with the lentivirus may be performed by treating the host cell with the lentivirus after attaching the host cell, or by treating and infecting the host cell with the lentivirus before attaching the host cell.

[0382] In some embodiments, a method of infecting the host cell with the lentivirus during the process of attaching the host cell by treating the host cell with the lentivirus before attaching the host cell is also known as reverse transduction.

[0383] In some embodiments, in the transduction of the host cell by infecting the host cell with the lentivirus, it is preferable to infect the host cell through treatment with the lentivirus before attaching the host cell, but the present invention is not limited thereto.

[0384] In some embodiments, step (b) may be characterized in that the host cell into which the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same is introduced is selected using an antibiotic and a resistance gene thereto.

[0385] The method of selecting the transduced cell into which the gene is introduced using a vector including an antibiotic and a resistance gene thereto is well known in the art.

[0386] In some embodiments, step (b) may be characterized in that the genetically modified cell into which the nucleic acid is introduced is selected through treatment with an aminoglycoside-based antibiotic.

[0387] In some embodiments, examples of the antibiotic may include, but are not limited to, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, puromycin, blasticidin, hygromycin, geneticin, neomycin, tetracycline, and the like.

[0388] In some embodiments, the genetically modified cell may further include a neomycin resistance gene introduced thereto, in addition to the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same.

[0389] In some embodiments, treatment with the antibiotic for 3 to 7 days at a concentration of 250 to 500 μg / mL, for 5 days at a concentration of about 125 μg / mL, or for 7 days at a concentration of about 62.5 μg / mL is possible, but the present invention is not limited thereto.

[0390] Yet another aspect of the present invention pertains to a culture fluid of the genetically modified cell. In some embodiments, the conditioned cell culture medium may be prepared by culturing the genetically modified cell using suitable conditions and media depending on the type of host cell.

[0391] In some embodiments, the genetically modified cell is capable of expressing and secreting the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same. As such, the conditioned cell culture medium of the genetically modified cell may include not only the genetically modified cell, but also the Stefin A protein variant specifically binding to TNFR2 and / or the fusion protein including the same, which are secreted thereby.

[0392] In some embodiments, when the host cell is a mesenchymal stromal cell, a long-term subculture method thereof is well known in the art, and, for example, Korean Patent Application Publication No. 10-2021-0072734, Korean Patent No. 10- No. 1135636, and the like disclose a method of maintaining undifferentiation potency and marker expression characteristics even after tens of passages.

[0393]

[0394] USE

[0395] The present invention excludes use only for the manufacture of a Stefin A protein variant that specifically binds to TNFR2 of the genetically engineered cells and / or a fusion protein containing the same.

[0396] Uses of the genetically engineered cells of the present invention include, without limitation, all uses except for the manufacture of Stefin A protein variants and / or fusion proteins containing the same. Preferred examples include, but are not limited to, medical, pharmaceutical, and clinical uses.

[0397]

[0398] Use - Cell therapeutic agent or pharmaceutical composition

[0399] In one example of the present invention, the nucleic acid encoding the Stefin A protein variant specifically binding to TNFR2 was introduced into PSC-derived mesenchymal stromal cells to prepare genetically modified cells. In order for the Stefin A protein variant to be presented on the cell surface, the Stefin A protein variant specifically binding to TNFR2 was fused with the human PDGFR (platelet-derived growth factor receptor) transmembrane domain. The prepared genetically modified cells showed a high level of Stefin A protein variant surface presentation while maintaining the characteristics of MSC.

[0400] In another example of the present invention, the genetically modified cells were co-cultured with HEK-Blue TNF-alpha cells to evaluate the TNFR2 agonistic function. As a result, it was confirmed that the Stefin A protein variant presented on the surface of the genetically modified cell of the present invention binds to TNFR2 of HEK-Blue TNF-alpha cells and exhibits an excellent agonistic effect.

[0401] Various prior studies in this field report that activation of the TNFR2 pathway is very important for Treg proliferation and activation. TNFR2 deficient mice had reduced numbers of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 - / - Tregs were not able to control inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492). In humans, Tregs were shown to express a higher level of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_ Annunziato F, et al. PMID: 12163566) and TNFR2+ Tregs exhibited the most potent suppression of proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID: 20127680).

[0402] Therefore, it can be obviously predicted that the genetically modified cells of the present invention will be able to exhibit Treg proliferation and activation, further reducing the immune response or suppressing the immune system from the agonistic effect on TNFR2 of the genetically modified cells of the present invention.

[0403] In conclusion, the genetically modified cells of the present invention can be used for preventing and / or treating various immune diseases caused by an abnormal homeostasis of immunity, preferably an excessive or abnormal response of the immune system. More specifically, it can be used for the prevention and / or treatment of various diseases that have been reported to be treatable using Treg proliferation and activation as a mechanism.

[0404] In another aspect, the present invention relates to a cell therapeutic agent including the genetically modified cell and / or the conditioned cell culture medium thereof.

[0405] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for the manufacture of a cell therapeutic agent.

[0406] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating an immune disease or cancer including the genetically modified cell and / or the conditioned cell culture medium thereof.

[0407] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for the prevention or treatment of an immune disease or cancer.

[0408] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of an immune disease or cancer.

[0409] In another aspect, the present invention relates to a method of preventing or treating an immune diseases or cancer including administering the genetically modified cell and / or the conditioned cell culture medium thereof to a subject.

[0410] Immune diseases for which preventive and / or therapeutic effects can be expected using the genetically modified cells of the present invention include, for example, Inflammatory disease, autoimmune disease or cancer, but is not limited thereto.

[0411] As used herein, the term “immune disease” refers to a disease that may be directly caused by an abnormality in the immune system, and may be selected from the group consisting of dermatitis, allergies, rhinitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, tendonitis, type 1 diabetes, scleroderma, neurodegenerative disease, type 2 diabetes, silicosis, atherosclerosis, vitiligo, conjunctivitis, and autoimmune disease, but is not limited thereto.

[0412] As used herein, the term “autoimmune disease” refers to a disease that occurs when immune cells in an organism recognize the organism's own tissues or cells, rather than an external invading antigen, as an antigen and attack the same. The autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic scleroderma, atopic dermatitis, psoriasis, asthma, Guillain-Barre syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, temporal arteritis, childhood diabetes, alopecia areata, blisters, aphthous stomatitis, Crohn's disease, and Behcet's disease, but is not limited thereto.

[0413] As used herein, the term “inflammatory disease” is a generic term for a disease accompanied by inflammation as a main lesion, particularly one selected from the group consisting of edema, allergies, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, severe myasthenia gravis, and multiple sclerosis, but is not limited thereto.

[0414] An another example, the disease include, systemic lupus erythermatosis (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn’s disease and colitis / ulcerative colitis), Graft versus Host Disease (GvHD) (relating to stem cell transplants) also called allograft rejection, transplant / Solid Organ Transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, Myasthenia gravis, Grave's disease, glomerulosclerosis and / or cancer, but is not limited thereto.

[0415] As used herein, the term “prevention” refers to any action that inhibits or delays the onset of an immune diseases or cancer by administering the pharmaceutical composition provided in the present invention to a subject who is expected to develop an immune diseases or cancer.

[0416] As used herein, the term “treatment” refers to any action that clinically intervenes to alter the natural process of a subject or cell to be treated, and may be performed during the course of or to prevent a clinical pathology. The desired therapeutic effect includes prevention of occurrence or recurrence of disease, alleviation of symptoms, inhibition of all direct or indirect pathological consequences of disease, prevention of metastasis, reduction of disease progression rate, alleviation or temporary alleviation of disease state, and prognosis improvement. For the purpose of the present invention, the treatment may be interpreted as including all actions of ameliorating the symptoms of an autoimmune disease by administering the pharmaceutical composition of the present invention to a patient suffering from an autoimmune disease including psoriasis, but the present invention is not particularly limited thereto.

[0417] Systemic Lupus Erythermatosis

[0418] Systemic lupus erythematosus (SLE), commonly referred to simply as lupus, is a chronic autoimmune disease that can cause swelling (inflammation) and pain throughout the body. There are several different types of lupus. Systemic lupus erythematosus is the most common. Other types of lupus include:

[0419] Cutaneous lupus erythematosus: This type of lupus affects the skin - cutaneous is a term meaning skin. Individuals with cutaneous lupus erythematosus may experience skin issues like a sensitivity to the sun and rashes. Hair loss can also be a symptom of this condition.

[0420] Drug-induced lupus: These cases of lupus are caused by certain medications. People with drug-induced lupus may have many of the same symptoms of systemic lupus erythematosus, but it is usually temporary.

[0421] Neonatal lupus: A rare type of lupus, neonatal lupus is a condition found in infants at birth. Children born with neonatal lupus have antibodies that were passed to them from their mother - who either had lupus at the time of the pregnancy or may have the condition later in life. Not every baby born to a mother with lupus will have the disease.

[0422] Therapies that may be used in combination with the genetically modified cell provided herein include, for example: Steroids (corticosteroids, including prednisone); Hydroxychloroquine (Plaquenil®); Azathioprine (Imuran®); Methotrexate (Rheumatrex®); Cyclophosphamide (Cytoxan®) and mycophenolate mofetil (CellCept®); Belimumab (Benlysta®); and / or Rituximab (Rituxan®).

[0423] Lupus Nephritis

[0424] Lupus nephritis is a frequent complication in people who have systemic lupus erythematosus - more commonly known as lupus. Lupus nephritis occurs when lupus autoantibodies affect structures in your kidneys that filter out waste. This causes kidney inflammation and may lead to blood in the urine, protein in the urine, high blood pressure, impaired kidney function or even kidney failure. As many as half of adults with systemic lupus develop lupus nephritis. Systemic lupus causes immune system proteins to damage the kidneys, harming their ability to filter out waste.

[0425] Rheumatoid Arthritis

[0426] Rheumatoid arthritis is a type of chronic (ongoing) arthritis that occurs in joints on both sides of the body, such as hands, wrists and knees. The short-term goals of rheumatoid arthritis medications are to reduce joint pain and swelling and / or to improve joint function. The long-term goal is to slow or stop the disease process, particularly joint damage.

[0427] Arthritis is a general term that describes inflammation in joints. Rheumatoid arthritis is a type of chronic (ongoing) arthritis (resulting in pain and swelling) that occurs generally in joints symmetrically (on both sides of the body, such as hands, wrists and knees). This involvement of several joints helps distinguish rheumatoid arthritis from other types of arthritis.

[0428] In addition to affecting the joints, rheumatoid arthritis may occasionally affect the skin, eyes, lungs, heart, blood, nerves or kidneys.

[0429] Therapies that may be used in combination with the genetically modified cell provided herein to treat rheumatoid arthritis can include, for example:

[0430] Drugs that decrease pain and inflammation. These products include non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen (MOTRIN®), naproxen (ALEVE®), and other similar products. Another type of drug - the COX-2 inhibitor - also falls into this drug category, providing relief of the signs and symptoms of rheumatoid arthritis. Celecoxib (CELEBREX®), one COX-2 inhibitor, is available and used in the United States. The COX- 2 inhibitors were designed to have fewer bleeding side effects on the stomach.

[0431] Disease-modifying antirheumatic drugs (DMARDs). Unlike other NSAIDs, DMARDs can actually slow the disease process by modifying the immune system. Older DMARDs include methotrexate (TREXALL®), gold salts, penicillamine (CUPRIMINE®), hydroxychloroquine (PLAQUENIL®), sulfasalazine (AZULFIDINE®), cyclosporine (SANDIMMUNE®), cyclophosphamide (CYTOXAN®) and leflunomide (ARAVA®). Currently, methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are the most commonly used. (Cyclosporine, cyclophosphamide, gold salts, and penicillamine are not typically used anymore.)

[0432] Biologics. Beyond these more "traditional" DMARDs, newer medications have been approved. Currently, there are seven different classes of medications and, in some cases, there are different kinds in each class. (Some of them, such as the class anti-TNFs, have been used since 2000.) Collectively, these DMARDs are known by another name - biologic agents (or biologic response agents). Compared with the traditional DMARDs, these products target the molecules that cause inflammation in rheumatoid arthritis. Inflammatory cells in the joints are involved in the development of rheumatoid arthritis itself. The biologic agents cut down the inflammatory process that ultimately causes the joint damage seen in rheumatoid arthritis. The older DMARDs work one step further out than the biologics; they work by modifying the body's own immune response to the inflammation. By attacking the cells at a more specific level of the inflammation itself, biologics are considered to be more effective and more specifically targeted. The biologic agents include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), anakinra (KINARET®), abatacept (ORENCIA®), rituximab (RITUXAN®), certolizumab pegol (CIMZIA®), golimumab (SYMPONI®), tocilizumab (ACTEMRA®) and tofacitinib (XELJANJ®). Some of the biologics are used in combination with the traditional DMARDs, especially with methotrexate.

[0433] Multiple Sclerosis

[0434] Multiple sclerosis (MS) is an autoimmune disease. With these conditions, the immune system mistakenly attacks healthy cells. In people with MS, the immune system attacks cells in the myelin, the protective sheath that surrounds nerves in the brain and spinal cord. Damage to the myelin sheath interrupts nerve signals from the brain to other parts of the body. The damage can lead to symptoms affecting the brain, spinal cord and eyes.

[0435] There are four types of multiple sclerosis:

[0436] Clinically isolated syndrome (CIS): When someone has a first episode of MS symptoms, healthcare providers often categorize it as CIS. Not everyone who has CIS goes on to develop multiple sclerosis.

[0437] Relapsing-remitting MS (RRMS): This is the most common form of multiple sclerosis. People with RRMS have flare-ups -- also called relapse or exacerbation -- of new or worsening symptoms. Periods of remission follow (when symptoms stabilize or go away).

[0438] Primary progressive MS (PPMS): People diagnosed with PPMS have symptoms that slowly and gradually worsen without any periods of relapse or remission.

[0439] Secondary progressive MS (SPMS): In many cases, people originally diagnosed with RRMS eventually progress to SPMS. With secondary-progressive multiple sclerosis, one continues to accumulate nerve damage. Symptoms progressively worsen. While one may still experience some relapses or flares (when symptoms increase), one no longer has periods of remission afterward (when symptoms stabilize or go away).

[0440] Therapies that may be used in combination with the genetically modified cell provided herein include, for example:

[0441] Disease-modifying therapies (DMTs): Several medications have FDA approval for long-term MS treatment. These drugs help reduce relapses (also called flare-ups or attacks). They slow down the disease’s progression. And they can prevent new lesions from forming on the brain and spinal cord.

[0442] Relapse management medications: If there is a severe attack, a neurologist may recommend a high dose of corticosteroids. The medication can quickly reduce inflammation. They slow damage to the myelin sheath surrounding nerve cells.

[0443] Physical rehabilitation: Multiple sclerosis can affect physical function. Staying physically fit and strong will help maintain mobility.

[0444] Mental health counseling: Coping with a chronic condition can be emotionally challenging. MS can sometimes affect mood and memory. Working with a neuropsychologist or getting other emotional support is an essential part of managing the disease.

[0445] Inflammatory Bowel Disease

[0446] Inflammatory bowel disease (IBD) is a group of disorders that cause chronic inflammation (pain and swelling) in the intestines.

[0447] Crohn’s disease and ulcerative colitis are the main types of IBD. Types include:

[0448] Crohn’s disease causes pain and swelling in the digestive tract. It can affect any part from the mouth to the anus. It most commonly affects the small intestine and upper part of the large intestine.

[0449] Ulcerative colitis causes swelling and sores (ulcers) in the large intestine (colon and rectum).

[0450] Microscopic colitis causes intestinal inflammation that’s only detectable with a microscope.

[0451] Therapies that may be used in combination with the genetically modified cell provided herein include, for example: aminosalicylates (an anti-inflammatory medicine like sulfasalazine, mesalamine or balsalazide) minimize irritation to the intestines; antibiotics treat infections and abscesses; biologics interrupt signals from the immune system that cause inflammation; corticosteroids, such as prednisone, keep the immune system in check and manage flares; immunomodulators calm an overactive immune system; antidiarrheal medication; nonsteroidal anti-inflammatory drugs (NSAIDs); vitamins and supplements like probiotics.

[0452] Graft versus Host Disease

[0453] Graft versus host disease (GvHD) is a condition that might occur after an allogeneic transplant. In GvHD, the donated bone marrow or peripheral blood stem cells view the recipient’s body as foreign, and the donated cells / bone marrow attack the body.

[0454] There are two forms of GvHD: Acute graft versus host disease (aGvHD); and Chronic graft versus host disease (cGvHD).

[0455] Psoriasis

[0456] Psoriasis is a chronic skin disorder, which means a skin condition that doesn’t go away. People with psoriasis have thick, pink or red patches of skin covered with white or silvery scales. The thick, scaly patches are called plaques. Psoriasis usually starts in early adulthood, though it can begin later in life. In addition to red, scaly patches, symptoms of psoriasis include: itchiness, cracked, dry skin, scaly scalp, skin pain, nails that are pitted, cracked or crumbly, and joint pain.

[0457] Therapies that may be used in combination with the genetically modified cell provided herein include, for example: Steroid creams, Moisturizers for dry skin, anthralin (a medication to slow skin cell production), Medicated lotions, shampoos and bath solutions to improve scalp psoriasis, Vitamin D3 ointment, Vitamin A or retinoid creams, light therapy, PUVA (a treatment combines a medication called psoralen with exposure to a special form of UV light), methotrexate, retinoids, cyclosporine, and / or immune therapies.

[0458] Sjogren's Syndrome

[0459] Sjogren's syndrome is a lifelong autoimmune disorder that reduces the amount of moisture produced by glands in the eyes and mouth. It is named for Henrik Sjogren, a Swedish eye doctor who first described the condition. While dry mouth and dry eyes are the primary symptoms, most people who have these problems don't have Sjogren's syndrome. Dry mouth is also called xerostomia.

[0460] There are two forms of Sjogren's syndrome: primary Sjogren's syndrome, which develops on its own, not because of any other health condition, and secondary Sjogren’s syndrome, which develops in addition to other autoimmune diseases like rheumatoid arthritis, lupus and psoriatic arthritis.

[0461] Therapies that may be used in combination with the genetically modified cell provided herein include, for example, treatments for dry eyes (e.g., artificial tears, prescription eye drops, punctal plugs, surgery, autologous serum drops), treatments for dry mouth (e.g., saliva producers), treatments for joint or organ problems (e.g., pain relievers, anti-rheumatics, immunosuppressants, steroids, antifungals, and treatments for vaginal dryness.

[0462] Myasthenia gravis

[0463] Myasthenia gravis (MG) is an autoimmune disease, meaning the body’s immune system mistakenly attacks its own parts. MG affects the communication between nerves and muscles (the neuromuscular junction).

[0464] People with MG lose the ability to control muscles voluntarily. They experience muscle weakness and fatigue of various severity. They may not be able to move muscles in the eyes, face, neck and limbs. MG is a lifelong neuromuscular disease.

[0465] MG affects about 20 out of every 100,000 people. Experts estimate that 36,000 to 60,000 Americans have this neuromuscular disease. The actual number of people affected may be higher, as some people with mild cases may not know they have the disease. MG mostly affects women aged 20 to 40 and men aged 50 to 80. About one in 10 cases of MG occur in teenagers (juvenile MG). The illness can affect people of all ages but is rare in children.

[0466] Autoimmune MG is the most common form of this neuromuscular disease. Autoimmune MG may be:

[0467] Ocular: The muscles that move the eyes and eyelids weaken. The eyelids may droop, or you may not be able to keep your eyes open. Some people have double vision. Eye weakness is often the first sign of MG. Nearly half of people with ocular MG evolve into the generalized form within two years of the first symptom.

[0468] Generalized: Muscle weakness affects the eye and other body parts such as the face, neck, arms, legs and throat. It may be difficult to speak or swallow, lift the arms over the head, stand up from a seated position, walk long distances and climb stairs.

[0469] Therapies that may be used in combination with the genetically modified cell provided herein include, for example, Medications, Monoclonal antibodies, IV immunoglobulin (IVIG), Plasma exchange (plasmapheresis), and / or Surgery.

[0470] Cancer

[0471] The binding of TNF to TNFR2 has been shown promote activation in tumour cells and tumour-associated cell types, as well as being affecting the immune response, such as tumour infiltrating lymphocytes, to create an environment in which the tumour can progress. Indeed, TNFR2 expression in tumours is associated with disease progression and poor clinical outcomes (Sheng Y, et al.). Therefore, the genetically modified cell of the present invention may be useful in the treatment of cancer, either as monotherapy or in conjunction with other cancer treatments such as immunotherapies, e.g. CAR-T therapies, checkpoint inhibitors, and conventional cancer treatments.

[0472] The cancer includes, for example, benign, premalignant, and malignant tumors, or proliferative disease, a precancerous condition, but is not limited thereto.

[0473] In some embodiments, the cancer is a hematologic cancer, such as chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia, but is not limited thereto.

[0474] In some embodiments, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

[0475] Pharmaceutical preparations Formulations are prepared for storage and use by combining the genetically modified cell of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and / or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

[0476] In some embodiments, an AFFIMER® agent described herein is lyophilized and / or stored in a lyophilized form. In some embodiments, a formulation comprising the genetically modified cell described herein is lyophilized.

[0477] Suitable pharmaceutically acceptable vehicles include but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.).

[0478] The pharmaceutical compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

[0479] The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0480] The genetically modified cell described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.

[0481] In some embodiments, pharmaceutical formulations include the genetically modified cell of the present disclosure complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0482] In some embodiments, sustained-release preparations comprising the genetically modified cell described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an AFFIMER® agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0483] In some embodiments, in addition to administering the genetically modified cell described herein, the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and / or subsequently to, administration of the genetically modified cell. Pharmaceutical compositions comprising the genetically modified cell and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

[0484] Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the genetically modified cell.

[0485] In some embodiments of the methods described herein, the combination of the genetically modified cell described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the genetically modified cell. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and / or side effects of the genetically modified cell. In some embodiments, the combination therapy results in a decrease in the toxicity and / or side effects of the additional therapeutic agent(s).

[0486] Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.

[0487] It will be appreciated that the combination of the genetically modified cell described herein and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the genetically modified cell will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the genetically modified cell and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given the genetically modified cell while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In some embodiments, the genetically modified cell will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, the genetically modified cell will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, the genetically modified cell will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, the genetically modified cell will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (e.g., substantially simultaneously).

[0488]

[0489] Use - a composition or method for immunomodulation

[0490] As described above, it is well known that activation of the TNFR2 pathway can lead to suppression of the immune system. The stefin A protein variant and / or the fusion protein of the genetically modified cell binds to and activate the TNFR2 pathway thereby exhibiting the effect of proliferation and activation of Treg cells, and consequently, the regulation of immunity, that is, the effect of suppressing immune system or immune response.

[0491] Therefore, in another aspect, the present invention relates to a composition for immunomodulation comprising the genetically modified cell and / or the conditioned cell culture medium thereof.

[0492] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for immunomodulation.

[0493] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for immunomodulation.

[0494] In another aspect, the present invention relates to a method for immunomodulation comprising administering the genetically modified cell and / or the conditioned cell culture medium thereof to a subject.

[0495] As used herein, the term “immunomodulation” means relieving immune imbalance in the blood and maintaining immune homeostasis. Maintaining immune homeostasis refers to a condition in which immune tolerance which is a mechanism for inhibiting immunity, and immunity which promotes immune response, are balanced, and the maintenance of such a condition is an essential factor in the treatment of immune diseases, especially autoimmune diseases.

[0496] The term “immunosuppression” or “suppression of immune system” as used herein refers to various substances used to reduce or block the ability of a host to produce antibodies (humoral immune response) or the ability to elicit a cellular immune response to the action of an antigen.

[0497] In some embodiments, the genetically modified cell and / or the conditioned cell culture medium may exhibit a hyper-immunosuppressive effect. The hyper-immunosuppressive effect is an effect of suppressing hyper-immunity by inhibiting lymphocyte overexpression caused by a nonspecific stimulator in splenocytes.

[0498] The compositions according to the present invention may be administered in combination with immune-related proteins, in particular autoimmune- or allergy-related proteins. Specific examples of the proteins include autoantigens involved in autoimmune diseases. For example, the proteins may include autoantigens involved in rheumatoid arthritis, such as heat-shock proteins (HSPs), citrullinated filaggrin, glucose-6-phosphate isomerase, p205, collagen, and the like; autoantigens involved in Type I diabetes, such as insulin, Zinc transporter 8 protein (ZnT8), Pancreatic and duodenal homeobox 1 (PDX1), Chromogranin A (CHGA), and Islet amyloid polypeptide (IAPP); and autoantigens involved in myasthenia gravis, such as an acetylcholine receptor. In addition to all types of autoantigens known in autoimmune diseases, the proteins may include various allergens known to cause food allergies, such as peanuts, milk, eggs, tree nuts, beans, crustaceans such as shrimp and the like, fish-derived substances, and the like.

[0499]

[0500] Use - a composition or method for culturing regulatory T cell

[0501] As described above, Importance of TNFR2’s role in Treg proliferation and function was shown in various studies. TNFR2 deficient mice had reduced numbers of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 - / - Tregs were not able to control inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492). In humans, Tregs were shown to express a higher level of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_ Annunziato F, et al. PMID: 12163566) and TNFR2+ Tregs exhibited the most potent suppression of proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID: 20127680).

[0502] Therefore, in another aspect, the present invention relates to a composition or medium for culturing regulatory T cell comprising the genetically modified cell and / or the conditioned cell culture medium thereof.

[0503] In another aspect, the present invention relates to a method for culturing regulatory T cell comprising, culturing regulatory T cell in presence of the genetically modified cell and / or the conditioned cell culture medium thereof.

[0504] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for culturing regulatory T cell.

[0505] In another aspect, the present invention relates to a use of the genetically modified cell and / or the conditioned cell culture medium thereof for manufacturing a composition or medium for culturing regulatory T cell.

[0506] In some embodiments, the composition or medium for culturing T cell further comprises any one selected from the group consisting of energy source, amino acids, sugars, inorganic salts, and vitamins. fetal bovine serum (FBS), hydroxyethyl piperazine ethane sulfonic acid (HEPES), proteins, carbohydrates, mercaptoethanol, and growth factors.

[0507] In some embodiments, the composition or medium may further comprises - components required by cells for cell growth and survival in vitro, or comprises components that help cell growth and survival. Specifically, the components may be vitamins, essential or non-essential amino acids, and trace elements.

[0508] As used herein, the term "regulatory T cell (Treg or Treg cell)" comprises natural regulatory T cells (nTreg) or induced regulatory T cells (iTreg). The regulatory T cells maintain immune homeostasis and block an autoimmune response, and the like by inhibiting an immune response. In some embodiments, the regulatory T cells may be genetically engineered regulatory T cells. Examples of the genetically engineered regulatory T cells include, but are not limited to, CAR-Treg and TCR-Treg.

[0509] In some embodiments, the regulatory T cells are preferably CD4+Foxp3+ regulatory T cells, but are not limited thereto.

[0510] In some embodiments, the regulatory T cells express the TNFR2, preferably the TNFR2 is presented on surface (or membrane) of the regulatory T cells. In some embodiments, the regulatory T cells are isolated regulatory T cells.

[0511] In some embodiments, the regulatory T cells are cultured in presence of the genetically modified cell and / or the conditioned cell culture medium thereof.

[0512] In some embodiments, culturing the regulatory T cells in presence of the genetically modified cell and / or the conditioned cell culture medium thereof may be performed in vitro or ex vivo. In some embodiments, culturing the regulatory T cells may be performed co-culturing the regulatory T cells and the genetically modified cells. In the context of the present invention, the genetically modified cell and / or the conditioned cell culture medium thereof binds to the TNFR2 presented on the Treg cell surface and activates a TNFR2 pathway.

[0513] In some embodiments, the induced regulatory T cells(iTregs) may be obtained by co-culture with tolerogenic antigen-presenting cells with CD4+ T cells, but are not limited thereto.

[0514] In some embodiments, the use, composition, medium, or method for culturing Treg cells of the present invention may induce proliferation and / or activation of regulatory T cells.

[0515]

[0516] Use - Composition for drug delivery

[0517] Even yet a further aspect of the present invention pertains to a composition for drug delivery including the genetically modified cell described above.

[0518] In some embodiments, the genetically modified cell may include at least one drug that is supported thereby or attached to the surface thereof.

[0519] In some embodiments, the drug may be additionally loaded with at least one selected from the group consisting of a gene, a virus, and a small molecule compound.

[0520] In some embodiments, the drug may have immunomodulatory activity, preferably an immunosuppressive effect, but is not limited thereto.

[0521] The genetically modified cell of the present invention is capable of expressing the stefin A protein variant specifically binding to TNFR2, thereby activating the TNFR2-targeted attachment and activity of immune cells.

[0522] Also, in some embodiments, the genetically modified cell may be a mesenchymal stromal cell. When the host cell of the present invention is a mesenchymal stromal cell, the mesenchymal stromal cell has a homing function to biologically search for a damaged or infected site in the body, and thus has very high targeting ability, making it possible to effectively and accurately deliver the drug to a desired site of the body.

[0523] In some embodiments, the drug may be supported inside the mesenchymal stromal cell, attached to the surface thereof, or supported inside the cell and attached to the cell surface, but the present invention may not be limited thereto. The drug may be directly supported inside the mesenchymal stromal cells and / or attached to the surface thereof, but the present invention is not limited thereto. The drug may be loaded to a nanostructure, supported inside the mesenchymal stromal cell in a state of being attached to a specific molecule, and / or attached to the cell surface, but the present invention is not limited thereto. In some embodiments, the nanostructure may include inorganic nanoparticles, polymer nanoparticles, proteins, or liposomes. Examples of the inorganic nanoparticles may include, but are not limited to, iron oxide nanoparticles, quantum dot nanoparticles, metal oxide nanoparticles, and the like. The nanostructure may include, but is not limited to, a porous nanostructure. For example, the drug may be supported by the pores in the porous nanostructure or loaded to the mesenchymal stromal cell in a form of being attached to the surface of the nanostructure, but the present invention is not limited thereto. The polymer nanoparticles are nanoparticles frequently used for drug delivery, and are mainly made of polymers and fats.

[0524] In some embodiments, the genetically modified cell may be characterized in that the drug is released at a target site. In some embodiments, the drug carried in the genetically modified cell may be released. In some embodiments, the drug attached to the surface of the genetically modified cell may be detached and released from the surface depending on the target environment. In some embodiments, when the drug is supported by the nanostructure, the drug may be released by temperature-specific or pH-specific structural change of the nanostructure.

[0525]

[0526]

[0527] EXAMPLES

[0528] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that the following examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.

[0529]

[0530] Example 1. Selection of Stefin A protein variant specifically binding to TNFR2 from Phage Display Library

[0531] Identification of candidate clones

[0532] Stefin A protein variant specifically binding to TNFR2 of the present disclosure were identified by selection from a library of Stefin A protein variant with two random loop sequences, each loop having a length of about 9 amino acids displayed in a constant Stefin A protein variant framework backbone based on the amino acid sequence of Stefin A. Such selection procedures have been described (see, e.g., Tiede et al. Protein Eng Des Sel. 2014. 27(5): 145-155 and Hughes et al. Sci Signal. 2018. 10(505): eaaj2005). According to such procedures, suspensions of phage expressing Stefin A protein variant were incubated with human (or mouse in later experiments) TNFR2 as required, the sequences of which are shown in Table 7.

[0533]

[0534]

[0535]

[0536]

[0537] In some case the TNFR2 was biotinylated and captured on alternating streptavidin and neutravidin beads, alternatively the TNFR2 was passively absorbed to a surface. Unbound phage particles were then washed away and, following washing, bound phages were eluted. Elution of bound phage was accomplished by exposure to trypsin. Eluted phage particles were then used to infectEscherichia coli(E. coli), and infected bacteria were incubated under conditions suitable for replication of the bacteriophage. Following release of bacteriophage particles from these infected bacteria, the cycle of allowing phage particles to bind to the target antigen, eluting bound phage particles, propagating eluted phage particles in bacteria, and isolating released phage particles from infected bacteria was repeated to enrich the bacteriophage population for phage particles displaying proteins that bind the target antigen. Specific conditions were modified in these cycles, such as increasing the number of wash steps, reducing the amount of available antigen, or adding a blocking reagent, to select for phage particles displaying proteins that bind more tightly or specifically to the target antigen.

[0538] Following multiple rounds of phage display library selection and amplification, proteins expressed by phages were expressed and screened by enzyme-linked immunosorbent assay (ELISA). Briefly, Stefin A protein variant were overexpressed from phagemid vectors, the bacterial cells were lysed, and lysates were used as substrates in ELISAs. In these ELISAs, human, murine and cynomolgus TNFR2 was immobilized on a plate, lysates were added, and the amount of Stefin A protein variant specifically binding to TNFR2 in each plate was measured using a detector antibody specific to the Myc tag expressed on the candidate Stefin A protein variant. The phagemid vectors encoding Stefin A protein variant with the best human TNFR2-binding activity were sequenced to identify DNA sequences of candidate clones for further development. TNFR1 polypeptides were also used to distinguish specificity. The loop 2 and loop 4 amino acid sequences of each of these candidate clones are shown in Table 1.

[0539]

[0540] EXAMPLE 2: Stefin A protein variant production in mammalian cells as soluble protein and screening

[0541] Cloning into vector of interest

[0542] To assess the ability of the selected Stefin A protein variant to bind TNFR2 when expressed at the surface of a mammalian cell, transiently transfected cell pools were generated for 30 ‘fast tracked’ clones based on being the most repeated sequences during passive selection. These clones were used for initial characterisation and assay optimisation. Stefin A protein variant specifically binding to TNFR2 sequences and TNFR2 targets were cloned from the phagemid of Example 1 into the mammalian expression vector pD609kanR (ATUM custom vector) for expression in Expi293F™ cells (ThermoFisher). The average concentration obtained from expression was 2.8 mg / mL, with 19 clones selected based on purity and assessed by ELISA for degradation.

[0543] These 19 clones were screened on Expi293F™ hTNFR2 expressing cells by flow cytometry at 1 μM and 0.1 μM while being compared to the SQT-GLY and 3T0-GLY negative controls which contain glycines only in Loop 2 and Loop 4 and thus are not specific binders. The signal detected on transfected cells was subtracted by the signal obtained on the untransfected Expi293F™ cells. Of these, 16 clones demonstrate binding to hTNFR2- Expi293F™ cells at 1 μM >10% after binding to parental cell line was subtracted. Clones 15, 22 and 23 did not show such binding (Figure 2).

[0544] The hTNFR2 mammalian purified Stefin A protein variants were screened in the HEK Blue™ TNFα SEAP reporter assay (Invivogen) according to manufacturer’s to determine if they could trigger the NFkB pathway and the SEAP production via binding to TNFR2 when they are clustered by an anti-his antibody coated on the plate. In this experiment, 5 of the fast tracked clones (01, 09, 12, 21 and 26) showed the ability to trigger the release of SEAP, with clone 12 showing the most consistent agonism.

[0545] A further 90 Stefin A protein variants were cloned from phagemid and similarly characterised. 55 clones were taken forward and binding confirmed by Mirrorball analysis. Further characterisation was done by competition ELSA to test their ability to block the interaction between hTNFR2-hFc-his-avi and recombinant TNF-alpha. A series of experiments were carried out to determine the EC80 of hTNFR2-hFc-his-Avi when binding to coated hTNF alpha (593 pM). Stefin A protein variant were tested at three different concentrations (10, 1 and 0.1 μM) when possible, in presence of hTNFR2 antigen at its determined EC80. Detection of hTNFR2-hFc-his-Avi bound was done by anti-hFc-HRP antibody. Among the 75 clones tested, 32 clones, that showed a % inhibition greater than 65% at 10 μM, were selected to be characterized further to determine their IC50 (Figure 3). clones 06, 21, 26, 27, 44, 108 and 115 showed full inhibition of the interaction between TNFα and hTNFR2 at the tested concentrations.

[0546] At this point, these 55 clones as well as the 14 clones from the fast track group (13 agonist, one negative, clone 06) were cloned into the pDisplay vector (Thermo Fisher). That vector contains a PDGFR protein transmembrane domain in C-term to allow Affiner expression at the surface of the expressing cells and not as soluble proteins. Additiona tags such as HA in N-term are also encoded by this vector allowing an easy verification for Affimer expression. Once cloned these Affimer were transiently transfected into Expi293F™ cells for SEAP analysis as previously described. Taking all the data together, 43 clones were selected for additional characterisation.

[0547] EXAMPLE 3: Characterisation of 43 selected clones

[0548] Biacore assay

[0549] The selected 43 clones were tested in a Biacore kinetic analysis as soluble proteins. Briefly Multicycle kinetics were performed using a CM5 chip on which the dimeric antigen hTNFR2-hFc-his-avi was immobilized at 500. Stefin A protein variant were titrated down in HBS-EP+ buffer from 1μM in 1:2 dilution series. Association time and Dissociation used were 200 sec and 400 sec respectively at a 20 μl / min flow rate with a regeneration with 10 mM Glycine pH 1.5 for 30 sec at 30 μl / min. Twelve clones showed very low response and no KD value could be estimated for them. Clone 09 was fitted using the 1:1 binding model however, the reported KD value was too high (>E-05) to be trusted with the range of concentration used. For 16 clones, the reached signal was adequate and they could be fitted successfully using the 1:1 Binding model, the curve and their fit are compiled in. Two clones (21 and 69) that are dimeric were fitted using both 1:1 binding model and steady state in order to estimate there KD, while in 12 clones, low Rmax made it difficult to fit them using the 1:1 binding model and instead the Steady State model was used. In that case, the obtained KD value was not reported, an order of magnitude was instead. Results are shown in Figure 4.

[0550] Binding ELISA crossreactivity

[0551] Stefin A protein variant clones’ specificity for hTNFR2 was assessed by ELISA when the clones binding to TNFR2 was compared to their binding to TNFR1. None of the clones showed binding to TNFR1 as a recombinant antigen. hTNFR1 was also expressed in Expi293F™ cells, and no binding was observed to this cellularly expressed hTNFR1, confirming no crossreactivity between the Stefin A protein variant binding to hTNFR2 binders with hTNFR1.

[0552] In addition to their binding to human TNFR2, the clones were tested as well for binding to the mouse TNFR2 to verify if any of them was cross reactive. There were very little chances for this to happen as the homology between the two proteins is ~68% for the extra cellular domain, which was confirmed as no clone demonstrated binding to mTNFR2 expressing Expi293F™ cells.

[0553] HEK293 - HEK Blue assay characterization

[0554] The 43 lead clones were transiently transfected in HEK 293 cells, along with 4 controls, SQT-Gly(SEQ ID NO: 484), 3t0-Gly (SEQ ID NO: 485), Empty vector, and clone 06 which has shown to be negative. Approximately 29h post transfection the cells viability was determined using a cell counter and the expression of the Stefin A protein variant was determined by flow cytometry using anti-HA antibody. 48 hours post transfection the Stefin A protein variant expressing HEK293 cells were co-cultured with HEK Blue reporter cells at 4 different cell densities, 40000, 20000, 10000 and 5000 with a fixed number of HEK Blue cells of 50000 cells / well. In parallel, the positive control MR2-1 was also incubated with HEK Blue cells for 24h at a range of concentrations. After 24h, the release of SEAP in the supernatant was quantified using Quanti-Blue and measurement of Absorbance 640nm. Staining of the AFFIMER®s with monoclonal anti-HA antibody showed expression levels >90% in both experiments. In the co-culture reporter assay, the effect of the Stefin A protein variant specifically binding to TNFR2 displayed on HEK293 on the HEK-Blue cells was visible, as well as the MR2-1 clone effect, allowing the selection of 16 clones which were consistently ranked within the best 20 clones in both experiments. These results are compiled in Figure 4, with the following clones identified: 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.

[0555] Cell binding to Human and Cynomolgus TNFR2

[0556] The 16 best agonists were characterized in soluble format for its binding to human TNFR2 overexpressing Expi293F™ and cynomolgus TNFR2 over-expressing Expi293F™. The experiment was repeated 3 times and 2 different batches of transiently transfected cells were used. All clones showed consistently specific binding to hTNFR2-Expi293F™ cells. Clones 26 and 69 showed the best EC50s on both human and cynomolgus TNFR2 expressing cells. 13 out of 16 tested clones show cross reactivity with cynomolgus TNFR2. The 3 non-crossreactive clones were clones 001, 007 and 009. Without necessarily being the best binders, clones DAW02-013, DAW02-044 and DAW02-112 are the clones with the least difference between the binding to human TNFR2 and cynomolgus TNFR2 over-expressing cells when all 3 experiments are compiled (Figure 5).

[0557] EXAMPLE 4. In-line fusion homodimer constructs

[0558] Clones 001, 009 and 012 were chosen to be formatted as in-line fusion (ILF) homodimers, to investigate if that format would be an advantage to trigger agonism compared to the monomeric form of the clones, as soluble protein and when displayed on cells. Two constructs were used for the generation of the ILF homodimers with a flexible linker, one codon optimised for mammalian expression and one not codon optimised. Both constructs were cloned into mammalian secretion vector (pD609_KanR) to purify recombinant protein and assess their expression levels, protein quality, binding kinetics to hTNFR2-hFc-his-avi by Biacore and agonistic activity in reporter assay with HEK reporter cells. In addition, both constructs were cloned into pDisplay vector for expression on Expi293F™ cell surface and assessment of their expression levels, binding to hTNFR2-hFc-his-avi and agonistic activity in co culture reporter assay with HEK reporter cells. Codon optimisation did not improve yields or purity of the soluble ILF homodimers, which were confirmed by HPLC.

[0559] When analysed by Biacore, clones 001 and 009 showed increased affinity in their soluble ILF format as compared to the soluble monomer format. Clone 12 showed no response. No difference was observed between the codon optimisation constructs. In SEAP assays only Clone 12 showed improved profile as a soluble ILF format, however when displayed on cells, in SEAP assays as well as flow cytometry presentation assays, no difference could be determined between the ILF and monomer formats.

[0560]

[0561] Summary of Stefin A protein variant binding to hTNFR2 selection and characterisation.

[0562] More than 2500 monoclonal phage were screened by phage ELISA with a hit rate greater than 60% with good diversity (20-40%). In the screening in ELISA as cell crude extract, more than 400 clones showed binding to the hTNFR2 antigen. At this stage, it was decided to introduce another step in the selection screening campaign to reduce the number of clones to cloned into the mammalian expression vector. In parallel, it was also decided to fast-track 30 clones from the passive selection directly to expression as soluble AFFIMER® proteins to facilitate assay development and screening cascade building for the rest of the clones. 20 of these clones passed the protein production quality criteria of more than 80% purity by SEC-HPLC and were used to set up various assays as well as being characterised. 5 out of 20 clones demonstrated agonist activity in a preliminary agonist assay where monomeric soluble Stefin A protein variant were clustered with an anti-his antibody via their histidine tag and incubated with the HEK reporter cell line.

[0563] Alongside assay development with fast-tracked clones, the phage screening campaign was finalised and a total of 364 Stefin A protein variant specifically binding to human TNFR2 were purified from the phagemid and tested for binding to a TNFR2 over-expressing cell line. Following on from this assay, 90 Stefin A protein variant specifically binding to human TNFR2 were re-formatted into the mammalian expression vector. 55 of these clones passed the protein production quality criteria of more than 80% purity by SEC-HPLC and were carried forward for the rest of the assays.

[0564] The total number of clones (fast-tracked and non-fast tracked) was narrowed down from 75 to 43 following a series of experiment that looked at Stefin A protein variant soluble proteins binding to hTNFR2 overexpressing cells, agonist assay with HEK reporter cells and preliminary data from agonist assay using HEK reporter cells in co-culture with Stefin A protein variants expressed at the surface of Expi293F™ cells. These 43 best clones were characterised as soluble proteins for their binding to human TNFR2 by Biacore where 16 out of 43 had their KD successfully calculated using a 1:1 binding model. None of the clones showed binding to human TNFR1 by ELISA or mouse TNFR2 by cell binding. As Stefin A protein variant displayed at the surface of HEK293, these 43 clones were tested for their ability to trigger TNFR2 agonism in a HEK reporter cell line when in co-culture. A large majority of the tested clones demonstrated a dose dependent effect on the HEK reporter cells (80%). 16 Stefin A protein variants were selected as best agonists and taken through additional characterisation to assess cynomolgus TNFR2 binding and human TNFR1 binding on cells. The selected 16 Stefin A protein variant specifically binding to hTNFR2 polypeptides are clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.

[0565] The use of in-line fusion homodimers as soluble proteins vs monomers showed advantages for binding to the recombinant TNFR2 by Biacore and possible advantage in the anti-his clustering agonism assay with HEK reporter cells. However, no advantage was demonstrated compared to the monomers when the in-line fusion Stefin A protein variants were displayed on cells to trigger the reporter cell line agonism pathway via TNFR2. The reason for the in-line fusion homodimers not performing better than the monomers in this last assay could have been that the in-line fusion format was not well displayed on the cell surface and only one of the two Stefin A protein variants was available for binding or that the limitation of the reporter assay had been reached and it was not possible to discriminate between the two formats.

[0566] EXAMPLE 5. Stefin A protein variant specifically binding to Mouse TNFR2

[0567] Phage selection and screening campaign

[0568] Murine TNFR2 antigens having the sequence as shown in Table 7 but in different formats were tested for suitability for use in assays to select for mTNFR2 AFFIMER® polypeptides. Of the three formats (mFc, hFc and His tagged), mTNFR2-mFc was chosen for biotinylation and use in the Stefin A protein variant selections due to its better performance in the assays.

[0569] Selections

[0570] Passive and solution selections were performed on the two Stefin A protein variant phage libraries as described for the Stefin A protein variant specifically binding to hTNFR2 selection. Enrichment was observed when the mTNFR2 concentration was maintained at 10 nM at round 3. More than 2000 phage-displayed Stefin A protein variants were screened by monoclonal phage ELISA from round 2 and round 3 outputs. Of these clones, over 900 demonstrated mTNFR2 binding, with no detectable non-specific binding to mouse Fc, plastic, streptavidin or neutravidin. These were defined as “hits” based on the criteria of absorbance 450nm-630nm above 0.5 on mTNFR2-coated plates and below 0.05 on plates coated with control antigens (i.e. mouse Fc, plastic, neutravidin). Sequencing of phage ELISA hits identified 281 unique clone sequences, of which 109 demonstrated mTNFR2 binding after ELISA on the cell crude extracts. Further analysis of binding using mTNFR2-Expi293F™ cells narrowed this field to 58 clones, identified as clones 122-179 in Table 2.

[0571] Soluble protein characterisation

[0572] As described for the Stefin A protein variant specifically binding to hTNFR2 characterisation above, the identified Stefin A protein variant specifically binding to mTNFR2 were cloned from phagemid to mammalian vectors for expression in Expi293F™ cells. The expressed proteins were then assessed by SDS-PAGE and HPLC for purity and Biacore for affinity, as well as cell-based binding of mTNFR2 in mTNFR2-Expi293F™ cells. Specificity for mTNFR2 was tested using ELISA and cell-based assays using mTNFR1 and mTNFR2 as the targets, confirming the identified clones’ specificity. To complete the soluble protein characterisation, the Stefin A protein variants were tested for their ability to block mTNFR2-mFC binding to mTNFα in ELISA, with four clones (157, 160, 171 and 172) showing clear competitive ability at the range tested.

[0573] T cell stimulation assays

[0574] A T cell stimulation assay was used as a screening tool since activated T cells express high levels of TNFR2, when stimulated with HM102 (TNFR2 agonist) the CD25 expression increases.

[0575] The Stefin A protein variant specifically binding to mTNFR2 were first expressed on the cell surface of HEK293s. Then, these cells were co-cultured for 48 hours with BALB / c splenocytes and finally the CD25 expression increase was evaluated on CD4 and CD8 positive T cells. Expression of the Stefin A protein variant on the HEK293 cell surface was assessed by flow cytometry staining of the HA tag present at the beginning of the open reading frame prior to the Stefin A protein variant and cell anchoring protein. In order to assess the effect of the Stefin A protein variant-HEK on the CD4+ and CD8+ cells, after 48h incubation the cells were stained with the following panel of markers, Live / Dead stain, CD90.2, CD4, CD8 and CD25.

[0576] A dose response was seen in CD25 expression in CD4+ and CD8+ T cells in response to HEK293 cells expressing mTNFR2 AFFIMER® proteins, similar to the response observed for HM102 TNFR2 agonist. The response obtained in CD8+ T cells is stronger than in CD4+ T cells. However, when focusing on the two highest cell numbers used, a similar ranking of the clones was obtained between the CD8+ and CD4+ T cell subsets.

[0577] In two rounds of this assay experiment the six top-ranked Stefin A protein variant were consistent 174, 160, 175, 125, 157 and 179.

[0578]

[0579] Summary of Stefin A protein variant specifically binding to mTNFR2 selection and characterisation.

[0580] More than 2000 monoclonal phage were screened by phage ELISA with a moderate 35- 60% hit rate, with a good diversity (25-35%). In the screening in ELISA as cell crude extract, 109 clones showed binding to mTNFR2 antigen. Using the same screening cascade than for the human programme, it was decided to purify the identified binders from the phagemid and to test them for binding to a TNFR2 over-expressing cell line. Following on from this assay, 60 Stefin A protein variant specifically binding to mouse TNFR2 were reformatted into the mammalian expression vector, produced and tested for their biophysical properties as soluble proteins. 22 Stefin A protein variant passed the protein production quality criteria of purity by SEC-HPLC greater than 80%. The 22 clones were characterised in various assays as soluble proteins, then were expressed at the surface of HEK293 for a co-culture assay with splenocytes.

[0581] Although only 10 out of the 22 Stefin A protein variant showed triple digit nanomolar binding affinities (KD) when tested on Biacore, it was considered that even a Stefin A protein variant with a higher KD value could be a good agonist once displayed on the cell surface. Additionally, the performance of the clones as soluble proteins was also dependent on whether they were a natural dimer or trimer. For these reasons, Biacore data were considered in the context of mTNFR2 agonism activity and were not used to discriminate clones. When tested on cells expressing mouse TNFR2, 20 out of 22 clones demonstrated specific binding with a wide range of EC50 values. As for Biacore data, cell binding results were only considered in the context of mouse TNFR2 agonism activity and were not used to discriminate clones. None of the clones bound to TNFR1 either as recombinant proteins or expressed on a cell’s surface. 5 out of 22 clones demonstrated full inhibition of the interaction between TNF-alpha and TNFR2 in a competition ELISA.

[0582] The 22 mouse clones were also expressed at the surface of HEK293 cells for a co-culture assay with splenocytes. The main read out for the assay was the CD25 expression increase on CD4+ T cells and CD8+ T cells. A large majority of the tested clones demonstrated a dose dependent effect on the CD4+ T cells and CD8+ T cells (over 70%). Clones were ranked looking at the CD25 expression on both CD4+ and CD8+ T cells. Based on the results of this key assay, six Stefin A protein variant specifically binding to mTNFR2 were identified: 174, 160, 175, 125, 157 and 179.

[0583]

[0584] Examples 1-5 were performed by Avacta Life Sciences Limited.

[0585]

[0586] EXAMPLE 6: Characteristics of Stefin A protein variant specifically binding to TNFR2 expressed on the cell surface

[0587] Stable genetic modification of PSC-derived MSCs (Pluripotent Stem Cell-derived Mesenchymal Stromal Cells) using lentiviral vectors

[0588] Among the selected Stefin A protein variant specifically binding to human TNFR2 clones, the top 7 clone genes with high activity were cloned into a plasmid vector so that they could be expressed by the CBh promoter, and lentiviral vectors were produced respectively. Lentiviral vectors were constructed using a vector including a nucleic acids encoding Stefin A protein variant specifically binding to TNFR2 fused transmembrane domain (derived from PDGFR, SEQ ID NO: 486, Biotechnology and Bioengineering, 73(4), 313-323) and a neomycin resistance gene (Gene Volume 85, Issue 2, 28 December 1989, Pages 421-426). The frozen Naive PSC-derived MSC was thawed, mixed with 1 MOI of lentiviral vectors and 2-8 μg / mL of polybrene, and then inoculated into a cell culture flask. After culture at 37°C and 5% CO2 for 16 to 20 hours, the culture medium containing the lentivirus was thoroughly removed and then replaced with a fresh culture medium, followed by culture at 37°C and 5% CO2 for 48 hours. Thereafter, the cells were harvested and inoculated again at a cell density of 0.4 - 1.0 x 104 cells / cm2, followed by culture at 37°C and 5% CO2 for 18 to 24 hours. The culture medium was replaced with a culture medium containing 100~250 μg / mL of G418, followed by culture for 5 days, and the culture medium was replaced with a culture medium containing G418 every 2 days. When cell confluency reached 90% or more, the cells were harvested and then frozen.

[0589]

[0590] PSC-derived MSCs retain their stemness after lentiviral transduction with the nucleic acids encoding Stefin A protein variant specifically binding to TNFR2

[0591] Flow cytometric analysis of eMSCs, 5 passages after their transduction with the lentivirus encoding Stefin A protein variant specifically binding to TNFR2, indicate their stemness, which was comparable to Naive PSC-derived MSCs. The frozen cells at each passage were thawed and analyzed for purity and immune markers through flow cytometry. Expression of mesenchymal stromal cell surface markers CD29, CD44, CD73, and CD105, and expression of cell surface markers for hematopoietic stem cell-specific marker CD45, embryonic stem cell-specific markers SSEA-3, TRA-1-60, and TRA-1-81, and immune marker HLA-DR were comparatively analyzed with Naive MSCs. As shown in Figure 6 and 7, it was confirmed that the expression of the mesenchymal stromal cell surface markers CD29, CD44, CD73, and CD105 was maintained at 95%, regardless of gene introduction. Also, expression of CD45, SSEA-3, TRA-1-60, TRA-1-81, and HLA-DR was maintained at less than 1%, indicating that the characteristics of mesenchymal stromal cells were maintained well.

[0592]

[0593] Assessment of Stefin A protein variant specifically binding to TNFR2 expression

[0594] The human PDGFR (platelet-derived growth factor receptor) transmembrane domain was fused to the C-terminus of Stefin A protein variant specifically binding to TNFR2 as an extracellularly facing plasma membrane protein. Western blot and flow cytometry analyses were carried out to confirm functional expression of the recombinant fusion proteins PDGFR and Stefin A protein variant specifically binding to TNFR2 in PSC-derived MSCs.

[0595] eMSCs transduced with lentiviral vectors were harvested and protein lysates prepared after five passages. In Western analysis, all cells transduced with PDGFR transmembrane domain fused Stefin A protein variant specifically binding to TNFR2 demonstrated high levels expression of Stefin A protein variant specifically binding to TNFR2 (Figure 8).

[0596] The percentage of cells that express Stefin A protein variant are determined by flow cytometry using phycoerythrin (PE)-labelled antibodies to the AFFIMER®, and the mean absolute number of membrane-anchored AFFIMER® proteins per cell using QuantiBRITE PE calibration beads (BD Biosciences, San Jose, CA, USA). To estimate the absolute number of membrane-anchored proteins of AFFIMER®, we used QuantiBRITE PE beads. Tubes of QuantiBRITE PE beads contain a lyophilized pellet of beads that have been conjugated with four concentrations of PE. Each type of beads has a known number of PE molecules per bead. Using this kit, we plotted a calibration curve and established a formula that enabled MFI values to be converted into the number of AFFIMER® molecules on the cell surface. When Naive PSC-derived MSCs (non-genetically modified MSCs) were compared, the highest levels of AFFIMER® proteins were found in eMSC transduced with TNFR2 AFFIMER® proteins fused hPDGFR, respectively (Table 9 and Figure 10).

[0597]

[0598]

[0599] eMSC - HEK-Blue TNFα cell co-culture for TNFR2 agonistic function assessment

[0600] The 7 lead clones were stably transduced in PSC-derived MSCs, along with 2 controls, SQT-Gly and 3t0-Gly which has shown to be negative. The Stefin A protein variant expressing eMSCs were co-cultured with HEK Blue reporter cells at 10 different cell densities from 40,960 to 80 cell / well with a fixed number of HEK Blue cells of 20,000 cells / well. After 24h, the release of SEAP in the supernatant was quantified using Quanti-Blue and measurement of Absorbance 640nm. Among the seven types of AFFIMER® transduced eMSCs, AFX002-C013, AFX002-C016, and AFX002-C017 cells did not show TNFR2 agonist activity, and AFX002-C010 and AFX002-C011 cells used as negative control also did not show agonist activity. HEK-Blue TNF-α cell activation was detected in four candidate cell lines: AFX002-C012, AFX002-C014, AFX002-C015, and AFX002-C018 (Figure 10e. The soluble form Stefin A protein variants (AFFIMER®) specifically binding to human TNFR2 failed to induce activation of HEK-Blue TNFα cells. However, Stefin A protein variant genes transduced PSC-derived MSCs induce activation of TNFR2. Reportedly, triggering of TNFR2-associated signaling pathways requires secondary clustering of initially formed trimeric TNF-TNFR2 complexes (Front. Immunol. 10:2040, 2019). Our results support the fact that the expression of AFFIMER® on the cell membrane induces clustering between AFFIMER® and TNFR2, which can transduce signals.

[0601]

[0602] Although specific embodiments of the present invention have been described illustratively, those skilled in the art will appreciate that the present invention may be embodied in other specific forms without changing the technical spirit or essential features thereof. Thus, the embodiments described above should be understood to be non-limiting and illustrative in every way.

[0603]

[0604] Attached in Electronic File.

Claims

1.A genetically modified cell in which a nucleic acid encoding a stefin A protein variant specifically binding to TNFR2 or a fusion protein comprising the stefin A protein variant is introduced into a host cell.2.The genetically modified cell according to claim 1, wherein the stefin A protein variant exhibits a Kd value of 1x10-6M or less for TNFR2.3.The engineered polypeptide of claim 1 comprising an amino acid sequence having at least 80%, at least 90%, at least 95, or at least 98% identity to an amino acid sequence ofMIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV-Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4),wherein Xaa, individually for each occurrence, is any number of independently selected amino acids;Xaa2 is any number of independently selected amino acids; and n and m are each, independently, an integer from 3 to 20.4.The genetically modified cell according to claim 1, wherein the stefin A protein variant comprises an amino acid sequence having at least 80%, at least 90%, at least 95, or at least 98% identity to an amino acid sequence ofMIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5),wherein Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.5.The genetically modified cell according to claim 3 or 4, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 102.6.The genetically modified cell according to claim 3 or 4, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 to 199.7.The genetically modified cell according to claim 1, wherein the stefin A protein variant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 200 to 296.8.The genetically modified cell according to claim 7, wherein the amino acid selected from the group consisting of SEQ ID NOs: 200 to 296 excludes one or more of the twenty one carboxy terminal residues.9.The genetically modified cell according to claim 1, wherein the fusion protein further comprises at least one selected from the group consisting of a signal peptide, a Fc domain, a binding domain, a cytokine, a half-life extension domain, a growth factor, an enzyme, and a cell-penetrating domain.10.The genetically modified cell according to claim 1, wherein the fusion protein further comprises at least one selected from the group consisting of a transmembrane domain, a hinge domain, a coiled coil domain, a virus-derived domain, an intracellular signaling domain, and a localization domain.11.The genetically modified cell according to claim 1, wherein the fusion protein further comprises a therapeutic peptide or protein.12.The genetically modified cell according to claim 10, wherein the stefin A protein variant or the fusion protein comprising the stefin A protein variant further comprises a transmembrane domain.13.The genetically modified cell according to claim 12, wherein the transmembrane domain is derived from group consisted of CD3, CD4, CD5, CD8, CD28, CD99, immunoglobulin (e.g. IgG1, IgG4, IgD, etc.), PDGFR, and PTGFRN.14.The genetically modified cell according to claim 1, wherein the host cell is selected from the group consisting of a stem cell, an immune cell, and a somatic cell.15.The genetically modified cell according to claim 11, wherein the stem cell is selected from the group consisting of a pluripotent stem cell, a multipotent stem cell, and a unipotent stem cell.16.The genetically modified cell according to claim 1, wherein the host cell is a mesenchymal stromal cell.17.The genetically modified cell according to claim 16, wherein the mesenchymal stromal cell is differentiated from a human pluripotent stem cell.18.The genetically modified cell according to claim 16, wherein the mesenchymal stromal cell expresses at least one cell surface marker selected from among CD29, CD44, CD73, and CD105.19.The genetically modified cell according to claim 18, wherein at least 90% of expression of the cell surface marker is maintained in the mesenchymal stromal cell after at least 15 passages.20.The genetically modified cell according to claim 16, wherein the mesenchymal stromal cell does not express at least one cell surface marker selected from among CD34, CD45, HLA-DR, TRA-1-60, and TRA-1-81.21.The genetically modified cell according to claim 1, wherein the stefin A protein variant or the fusion protein comprising the stefin A protein variant is expressed on a cell surface.22.A conditioned cell culture medium of the genetically modified cell according to claim 1.23.A cell therapeutic agent for preventing or treating an immune diseases or cancer comprising the genetically modified cell according to claim 1.24.A pharmaceutical composition for preventing or treating an immune diseases or cancer comprising the genetically modified cell according to claim 1.25.The pharmaceutical composition according to claim 24, wherein the immune diseases is selected from the group consisting of lupus (SLE), lupus nephritis (e.g. drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g. Crohn’s disease and colitis / ulcerative colitis), graft-versus-host disease (GVHD) or allograft rejection, transplantation / solid organ transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, oophoritis, allergic rhinitis, asthma, Sjogren’s syndrome, atopic eczema, myasthenia gravis, Graves’ disease, and glomerulosclerosis.26.The pharmaceutical composition according to claim 24, wherein the cancer is selected from the group consisting of hematologic cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.27.A composition for immunomodulation comprising the genetically modified cell according to claim 1 or the conditioned cell culture medium according to claim 22.28.The method according to claim 27, wherein the immunomodulation is immunosuppression.29.A composition or medium for culturing regulatory T cell comprising the genetically modified cell according to claim 1 or the conditioned cell culture medium according to claim 22.30.A method for culturing regulatory T cell comprising, culturing a regulatory T cell in presence of the genetically modified cell according to claim 1.31.The method according to claim 30, wherein the regulatory T cell presents TNFR2 on the cell surface.

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