Specific binding molecules

Abstract

To provide multi-domain specific binding molecules comprising VNARs, and a method for preparing the molecule, and also to provide specific binding domains that bind to Tumor Necrosis Factor alpha (TNFα).SOLUTION: Provided is a multi-domain specific binding molecule comprising two or more VNAR domains which bind to the same or different epitopes of one or more specific antigens. The multi-domain specific binding molecule further comprises a spacer sequence between the VNAR domains. The spacer sequence has independent functionality which is exhibited in the multi-domain specific binding molecule. The spacer sequence is an immunoglobulin Fc region, and two or more of the VNAR domains have a structure FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 in an N- to C-terminal direction respectively.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides multi-domain specific binding molecules comprising VNARs. The specific binding domain that binds to tumor necrosis factor α (TNFα) is involved in the formation of a dendritic cell membrane. Inns are also provided. [Background technology]

[0002] background In the search for specific, increasingly effective and diverse therapeutic weapons to combat disease A myriad of different modalities have been utilized, from traditional small molecules to increasingly larger biological Targeted drugs, e.g., single binding domains (10-15 kDa) to whole IgG (~150 kDa) A wide variety of single domains are currently being investigated as potential therapeutic agents. There are several distinct protein scaffolds, all with associated benefits and It comes with its drawbacks.

[0003] Such single domain scaffolds can be derived from an array of proteins from different species. A novel antigen receptor (IgNAR) was discovered in the serum of cartilaginous fish. It is a homodimeric protein of approximately 160 kDa (Greenberg AS, et al., Nature , 1995. 374(6518): p. 168-173, Dooley, H., et al, Mol. Immunol, 2003. 40(1): p. 25-33; Muller, MR, et al., mAbs, 2012. 4(6): pp. 673-685). Each molecule contains a single N It consists of a terminal variable domain (VNAR) and five constant domains (CNAR). IgNAR domains are members of the immunoglobulin superfamily. Binds to immunoglobulin and T cell receptor variable domains and to cell adhesion molecules It is a tightly folded domain with structural and some sequence similarity to classical Various immunoglobulin and T cell receptor N-terminal variable domains VNARs are called VNARs by their similarity to immunoglobulins. limited sequence homology, e.g., 25-30% similarity between VNAR and human light chain sequences (Dooley, H. and Flajnik, M.F., Eur. J. Immunol., 2005. 35(3): p. 936- 945).

[0004] Kovaleva M. et al. Expert Opin. Biol. Ther. 2014. 14(10): pp. 1527-1539 and Zie lonka S. et al. mAbs 2015. 7(1): p. 15-25 recently described the structural features and production of VNARs. provided herein are incorporated by reference.

[0005] VNARs do not appear to have evolved from a classical immunoglobulin antibody ancestor. The distinct structural features of the CDR2 loop are similar to those present in conventional immunoglobulin variable domains. Allows cleavage of equivalent sequences and association with light chain domains not normally present in the IgNAR structure lacking hydrophobic VH / VL interface residues that allow for the formation of CDRs N-terminally adjacent to CDRs 1 and 3 Canonical immunoglobulin superposition between cysteines in framework 1 and 3 regions canonical immunoglobulin superfamily bridge plus additional disulfide Any additional cysteine ​​residues in the CDR regions that are observed to form sulfide bridges It is present in several VNAR subtypes.

[0006] To date, there are three defined types of shark IgNAR, known as I, II, and III. These are under strong selective pressure and are therefore rarely replaced (Fig. 1). Classification based on the location of non-canonical cysteine ​​residues It has been done.

[0007] All three types contain the standard immunoglobulin with an invariant tryptophan at position 36. The classical immunoglobulin canonical sequences at positions 35 and 107 stabilize the conformation of the Calcisteine ​​(Kabat, EA et al. Sequences of proteins of immunologic l interest. 5th ed. 1991, Bethesda: US Dept. of Health and Human Services, PHS, CDR2 itself has not been defined, but TCR HV2 and The regions of sequence variation that compare more closely with HV1 and HV2 are in framework 2 and HV3, respectively. 3. Type I includes reproductive elements in Framework 2 and Framework 4. Cell line-encoded cysteine ​​residues and an even number of additional cysteines within CDR3 Type I isolated against lysozyme and in complex with lysozyme Crystal structure studies of IgNAR have allowed us to determine the contribution of these cysteine ​​residues. Both cysteines in frameworks 2 and 4 are located in the CDR3 loop and in the HV2 region. and disulfide in CDR3, which form a tightly packed structure that is held tightly against To date, type I IgNAR has been identified in nurse sharks. It has only been identified in the Elasmobranch family, and is different from all other Elasmobranchs, including members of the same order. Chi has only Type II or variations of this type.

[0008] Type II IgNARs are those that have cysteine ​​residues in CDR1 and CDR3. These are defined as the intercalated domains of the ribosomal nucleotides, which are bound by an intramolecular disulfide bond that holds these two domains in close proximity. Type I: The CDR3 forms a protruding CDR3 that guides the CDR3 into a binding pocket or groove (Figure 2). The sequences generally have longer CDR3s than type II, averaging 21 and 15 residues, respectively. This is because two or more cysteine ​​residues in the type I CDR3 are in their frame. Strong selective pressure to associate with Work 2 and 4 counterparts Studies on the accumulation of somatic mutations have shown that type I is more susceptible to type A mutations than type I. There are more mutations in the CDR1 of type II, but the HV2 region of type I This evidence supports the existence of these nucleotide sequences within the antigen-binding site. It correlates well with the determined location of the region.

[0009] A third IgNAR type, known as type III, has been identified in newborns. This member of the IgNAR family binds to the V gene through the D1 and D2 regions (CDR3). lack of diversity within CDR3 due to germline fusion of the two nucleotide sequences. All known clones, such as the 15 residue CD40, have little or no sequence diversity. It has an R3 length.

[0010] Another structural type of VNAR is called type (IIIb or IV) and has two canonical It contains only cysteine ​​residues. So far, this type has been mainly found in dogfish and sharks. It has been found in the dogfish shark (Liu, JL, et al. Mol. Immunol. 2007. 44(7 ): p. 1775-1783; Kovalenko OV, et al. J Biol Chem. 2013. 288(24): p. 17408-19) , and from a semi-synthetic V-NAR library derived from wobbegong sharks. Isolated (Streltsov, VA et al. (2004) Proc. Natl. Acad. Sci. USA 101(34): p. 12444-12449).

[0011] However, specific VNARs isolated from synthetic libraries formed from VNAR sequences can bind to other proteins with high affinity (Shao CY et al. Mol Immunol. 2 007. 44(4): p. 656-65; WO2014 / 173959), cartilaginous fish are antigens and antibodies. Immunization with the resulting responding IgNARs that bind to the antigen allows IgNARs to function as receptors in the adaptive immune system. (Dooley, H., et al., Mol. Immunol., 2003. 40(1): p. 25-33; WO2003 / 014161 IgNARs have been shown to have similar functions to immunoglobulin and T cell receptors. Mechanism for combinatorial joining of D and J sequences with V-like sequences It has been shown that the mechanism of action of mAbs is as follows (summarized in Zielonka S. et al. 2015. 7(1): p. 15-25). (It has been done).

[0012] The VNAR binding surface is distinct from the variable domains in other natural immunoglobulins and is The sequence is CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4. Multiple sequences connected by intervening framework sequences These are derived from four similar regions: CDR1, HV2, HV4 and CDR3 (Stanfield, R. L., et al, Science, 2004. 305(5691): p. 1770-1773; Streltsov, VA, et al, Prote in Sci., 2005. 14(11): p. 2901-2909; Stanfield, RL, et al., J Mol. Biol., 200 7. 367(2): pp. 358-372). Absence of a natural light chain partner and absence of CDR2 The combination of these makes VNARs the smallest naturally occurring binding domain in the vertebrate kingdom. This becomes:

[0013] IgNARs are found in the Camelidae family (camels, dromedaries and llamas, Hamers-Casterman, C. et al. Nature, 1993. 363, 446-448; Wesolowski, J., et al., Med Microbiol Immuno l, 2009. 198(3): pp. 157-74) ) share some incidental features. Unlike IgNAR, HCAb They are clearly derived from the immunoglobulin family and share significant sequence homology to standard immunoglobulins. Importantly, one key distinction between VNARs is that they are not classical immunoglobulin or Unlike HCAbs or HCAbs, the molecule has never had a partner light chain at any point in its evolution. Flajnik MF et al. PLoS Biol 2011. 9(8): e1001120 and Zie lonka S. et al. mAbs 2015. 7(1): p. 15-25, VNAR and camelid-derived immunoglobulins. Brin-derived V HH Similarities and differences between single binding domains and their different Commenting on evolutionary origins.

[0014] Binding derived from classical immunoglobulin light and heavy chains (VL and VH, respectively) The domains are linked together to form short peptide fragments of immunoglobulin VL and VH domains. Traunecker et al. (Traunecker A, et al. EMBO J.1991. 10, p 3655-36, Traunecker A, et al. Int J Cancer Suppl.7, 51-52; Neri D. J Mol Biol. 1995. 246(3): p. 367-73), scFv format (Bird et al., 1988; Huston et al. , 1988) or as diabodies (Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993. 90, 6444-6448; Holliger P. et al. Nat. Biotechnol. 15, 632-636. Another early example is Mack M, et al. Proc. Natl. Acad. Sci. USA 1995. 92, pp. 7021-702 5, Jost CR, et al. Mol. Immunol. 1996. 33, pp. 211-219) and bivalent or multivalent It has been shown that Tandabs can form bispecific conjugates. It contains two pairs of VL and VH domains linked to a tide chain (Kipriyanov SM et al., J. Mol. Biol. 293, 41-56, forming bispecific and bivalent molecules).

[0015] Furthermore, V HH can be linked together to form bivalent or multivalent bispecific conjugates It has been shown that (Els Conrath et al. J. Biol. Chem. 2001. 276(10) p.7346-7350). Similarly, variable domains from T cell receptors can be linked to immunoglobulin scFvs. , and bispecific formats (McCormack E. et al. Cancer Immunol Immunoth er. 2013. 62(4): p. 773-85). Single antibody variable domains (dA) derived from classical immunoglobulins Bs: Ward ES et al. Nature 1989, 341, pp. 544-546) can also dimerize. The overall concept of bispecific binding molecules and the current progress in their development has been recently reviewed, e.g. Kontermann R. mAbs 2012. 4(2): 185-197; Jost C. and Pluckthun A. Curr Opin S truct Biol. 2014. 27: p. 102-112; Spiess C. et al. Mol Immunol 2015. 67(2): 95-1 As outlined by 06.

[0016] In addition to bispecific molecules that recognize epitopes on separate molecules, The concept of linking two antibody binding domains that recognize adjacent epitopes (biparatopic (b iparatopic) has a long history (Neri D. J Mol Biol. 1995. 246(3): p. 367-73) (See Biparatopic V) HH The molecules are disclosed (e.g., Jahnichen S. et al. al Proc Natl Acad Sci US A. 2010. 107(47): p. 20565-70; Roovers RC et al Int J Cancer. 2011 129(8): p. 2013-24).

[0017] However, V HH Unlike VNARs, VNARs are unable to efficiently form dimeric fusion molecules. It has been suggested that this may not be the case (Simmons DP et al. Immunol Methods. 2006 3 15(1-2): p. 171-84). (Bispecific Antibodies Konterman RE Springer Publishing See also the 2011 commentary; Strohl WR and Strohl LM, Therapeutic Antibodies See also comments on pages 322 / 323 of dy Engineering, Woodhead Publishing 2012 ). Summary of the Invention

[0018] Summary of the Invention The present invention relates to the provision of multidomain-specific binding molecules comprising two or more VNAR domains. More particularly, the present invention relates to the provision of bivalent and multivalent VNARs. Recently, contrary to the general understanding in the art, it has been discovered that VNAR dimers and trimers are actually and demonstrated that bispecific fusions can be formed.

[0019] Recently, Muller MR et al. mAbs 2012. 4(6): p. 673-685; WO2013 / 167883) reported a Dual-specific domains containing VNARs with specificity for human serum albumin (HSA) The present invention discloses an unusual VNAR, which is a bivalent structure that binds to HSA in serum, thereby inhibiting viral replication. This allows for an extension of the biological half-life of the HSA-binding VNAR. Fusion of VNARs at both the N- and C-termini preserves the function of the HSA-binding domain More recently, in WO 2014 / 173975, it was demonstrated that B cells, activated monoclonal antibodies A cell surface antigen constitutively expressed on antigen-presenting cells (APCs) such as leukocytes and dendritic cells It can bind to ICOSL (CD275), a B7 family member, ICO VNARS, a ligand for S (CD278), has been disclosed (Yoshinaga. S., K., et al. I., Int. Immunol., 2000. 12(10): p. 1439-1447). Some can be linked to HSA-binding VNARS, and both domains retain functionality. It was shown that each of the different antigens (hICOSL, mICOSL, and HSA) A trimer that recognizes the α-terminal domain was prepared, and each domain was shown to retain its function.

[0020] However, bispecific or multispecific antigens that recognize the same or different epitopes on the same antigen may also be used. It has not previously been shown that atypical VNARs can be formed. , this form of bispecific molecule can be used in combination with the constituent monomers, or the monomeric form itself, or HSA. improved properties over bivalent molecules formed from monomers bound to VNARs that recognize show.

[0021] The present invention relates to multivalent or multispecific peptides that have the ability to be combined into multivalent or multispecific entities. Within the VNAR domain entity, specific VNAR domain arrangements are used where each domain retains its binding function. Regarding columns.

[0022] Thus, in a first aspect of the invention, the same or different epitopes of one or more specific antigens are Multi-domain specific binding molecules (multi-d) containing two or more VNAR domains that bind to the VNAR domain A domain-specific binding molecule (Bmc) is provided.

[0023] In certain preferred embodiments, the multidomain specific binding molecule of the first aspect of the invention The VNARs in each molecule bind to the same antigen on the specific antigen.

[0024] In a further preferred embodiment, the VNAR of the multidomain specific binding molecule These embodiments of the multi-domain specific The binding molecule may be a bi-paratopic molecule, as further described herein. It can be called a quantum molecule.

[0025] In one embodiment, the specific VNAR binding domain sequences are multivalent or multispecific entities. Within that multi-domain entity, each domain The binding domains retain binding function, and the binding domains recognize different epitopes on a single antigen. .

[0026] A preferred embodiment of the present invention is a method for detecting dendritic cells in which the binding specificities are directed to different epitopes on a single specific antigen. The resulting entities are improved compared to individual VNAR binding domains. Bispecific or bispecific antibodies containing two (or more) different VNAR domains exhibiting specific properties. or multispecific binding molecules. Examples of improved properties include those with This can result in increased agonist or antagonist effects.

[0027] Preferably, the VNAR domain of the multidomain specific binding molecule of the invention comprises a spacer More preferably, the spacer sequence is not present in the binding molecule. In one embodiment, the spacer sequence is a VNAR domain. or a functional fragment thereof. In a particular example, the spacer is human serum albumin or or ICOSL, or a functional fragment thereof that binds to serum albumin. In certain embodiments, the spacer sequence is selected from the group consisting of SEQ ID NOs: 67, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87 or 88 or a functional fragment thereof having at least 60% sequence identity thereto. In certain embodiments, the spacer sequence may be, but is not limited to, a human immunoglobulin F The improved properties may be, for example, the Fc portion of an immunoglobulin, such as the Fc region. By passively separating VNAR domains in space or The in vivo half-life of the target entity may be increased by binding to serum albumin or other specific molecules. The specific properties of the drug or the potential for a second therapeutic autoimmune target such as ICOSL may be important. by recognition of the Fc region of immunoglobulins (therapeutic auto-immune target) or In this case, the spacer is enhanced by introducing the ability to engage with cells of the immune system or complement. It may be partially or completely due to the characteristics.

[0028] Multidomains of the invention comprising two or more VNAR domains separated by a spacer sequence. An embodiment of the main specific binding molecule is herein referred to as Quad-X format (Quad-X format).

[0029] In other preferred embodiments, the multidomain-specific binding molecule comprises one or more non-VNAR domains. The one or more non-VNAR domains may further comprise a domain. Typically, and in a preferred embodiment, the non-VNA The R domain may be C-terminal or N-terminal to the VNAR domain.

[0030] Two or more VNAR domains and a non-VNAR domain C-terminal or N-terminal to the VNAR domain Embodiments of the multidomain-specific binding molecules of the invention comprising a VNAR domain are described herein. In literature, this can be called the Quad-Y format.

[0031] Exemplary non-VNAR domains include TNF R1 and immunoglobulin Fc. However, the present invention is not limited to these.

[0032] Specific antigens include cytokines, growth factors, enzymes, and cell surface-associated molecules. (cell surface associated molecule), cell-surface membrane component nt), intracellular molecules, extracellular matrix components, interstitial antigens, serum proteins, skeletal antigens (skel etal antigen, microbial antigen, or normally immune-privileged location The antigen may be from the group consisting of antigens of the same or similar origin (eged location).

[0033] A further aspect of the present invention is a specific combination of VNAR binding domains that recognize cytokines. It's a combination.

[0034] The present invention also provides a method for identifying and targeting human TNF-α, a nucleotide sequence that recognizes human TNF and is currently used to treat diseases. It has an epitope distinct from all other well-characterized anti-TNF antibodies and VHH binders. Specific domains that bind to the topes are provided.

[0035] Thus, in a second aspect, the present invention provides a method for the production of a polypeptide comprising the following CDRs and hypervariable regions (HV): a TNF-α-specific VNAR binding domain containing, or having at least 60% sequence identity with, and functional variants thereof, which are:

[0036] [ka]

[0037] In a particularly preferred embodiment, the TNF-α-specific VNAR binding domain is SEQ ID NO:2, 7 or 12 amino acid sequence, or a functional equivalent thereof with at least 60% sequence identity. Includes mutants.

[0038] In a preferred embodiment, the TNF-α-specific VNAR domain of the present invention is While retaining functional binding activity for a specific epitope of or similar techniques to reduce the likelihood of immunogenicity in vivo. The amino acid sequence is modified at position .

[0039] One embodiment of the present invention is directed to a method for the treatment of TNFα, which recognizes TNFα and, in the form outlined in the present invention, The resulting multi-domain binding molecules provide improved functional properties over the binding domains It is a specific combination of VNAR binding domains to TNFα. It is known that the IL-16 receptors can produce VNARs that are claimed to recognize the IL-16 receptor (Camacho-Villega) s T, et al MAbs. 2013. 5(1): P. 80-85; Bojalil R, et al BMC Immunol. 2013. 14:17 ; WO2011 / 056056; US20110129473; US20140044716). However, these VNARs are dimeric. Furthermore, the monomeric form is not linked to form a monospecific or bispecific form. These domains of the WT are 70 times larger than the monomeric anti-TNF VNAR domains described here. It is 200 times to 200 times less potent.

[0040] Thus, the TNF-α-specific VNAR binding domain of the second aspect of the invention comprises a first As one or both VNAR domains in the multidomain specific binding molecules of the embodiments Thus, in a preferred embodiment, one or more VNAR domains can be used. is an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 7 or 12, or The multi-domain of the first aspect, further comprising a functional variant thereof having 60% sequence identity. In another preferred embodiment, two or more VNAR domains are provided. an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 7 or 12, or at least The multi-domain peptide of the first aspect, having a functional variant thereof with 60% sequence identity. Heterologous binding molecules are provided.

[0041] Other preferred embodiments of the first aspect of the invention include the sequence of SEQ ID NO: 65 or 66 or an amino acid sequence thereof having at least 60% sequence identity. The multi-domain peptide of the first aspect comprises one or more VNAR domains having functional variants. In yet another embodiment of the first aspect, the binding molecule is SEQ ID NO: 65 or 66, or an amino acid sequence having at least 60% sequence identity thereto. The multidomain of the first aspect, comprising two or more VNAR domains having functional variants thereof There is a main specific binding molecule.

[0042] The one or more VNAR domains used in the first aspect of the invention may be For example, humanized, deimmunized, or modified while retaining functional binding activity for a specific epitope. or similar techniques to modify one or more amino acids to reduce their immunogenic potential in vivo. The modifications can be made at any sequence position.

[0043] The present invention also provides a method for producing a binding molecule according to any aspect or embodiment described herein. Further provided herein is an isolated nucleic acid comprising a polynucleotide sequence that encodes host cells containing the polynucleotide under conditions such that the host cells produce the binding molecule; and, if necessary, further isolating said binding molecule. A method for preparing a binding molecule according to the invention is provided, comprising:

[0044] According to a further aspect of the present invention, a specific antigen-binding molecule and / or a method for the treatment of a disease caused by a disease of the present invention Pharmaceutical compositions comprising the multidomain specific binding molecules of the invention are provided.

[0045] The pharmaceutical compositions of the present invention may be administered in the presence of any suitable and pharmaceutically acceptable carrier, diluent, adjuvant, The composition may include a solution or buffer solution. The composition may include an additional pharmaceutically active agent. Such carriers include saline, buffered saline, dextrose, liposomes, , water, glycerol, ethanol and combinations thereof. Such compositions may contain, but are not limited to, additional pharmaceutically active agents as indicated. The additional agent may include a therapeutic compound, e.g., an anti-inflammatory agent, a cytotoxic agent, a cytostatic agent, Such additional medications may be administered to patients in need thereof. The compounds may be administered in any suitable form, and such administration may be simultaneous, separate, or sequential. The above components may be prepared in the form of a kit, which may optionally include instructions. Cut.

[0046] Pharmaceutical compositions can be administered, for example, orally, topically, intravenously, intramuscularly, intranasally, or intradermally, among others. Administered in any effective and convenient manner effective to treat the patient's condition, including administration by any route. In therapy or prophylaxis, the active agent can be administered in an injectable composition. The compound may be administered to an individual as a liquid, for example, as a sterile, preferably isotonic, aqueous dispersion.

[0047] For administration to mammals, particularly humans, the daily dose of the active agent is 0. 0.01 mg / kg body weight or more, typically about 1 mg / kg, 2 mg / kg, 10 mg / kg In any case, the doctor should take into account the individual's age, Determine the actual dosage that is most appropriate for an individual, which will depend on factors including weight, sex, and response. The dosages given above are exemplary of the average case. Of course, higher dosages may be used. There may be instances where higher or lower doses are beneficial, and such instances are within the scope of the present invention. The present invention also provides a kit comprising a pharmaceutical composition as defined herein together with instructions for use. do.

[0048] According to a further aspect of the present invention there is provided a pharmaceutical composition according to the previous aspect for use in medicine. Such use involves the administration of a therapeutically effective amount of the pharmaceutical composition of the invention as defined above. The interaction between the target antigen of the binding domain of the invention and its ligand partner by The composition comprises at least one specific compound of the present invention. a specific antigen-binding molecule (VNAR domain) or a multi-domain specific binding molecule, or and / or humanized variants thereof.

[0049] According to this aspect of the invention, the target antigen of the binding domain of the invention and its ligand partner The present invention provides a composition for use in the manufacture of a medicament for the treatment of a disease associated with an interaction between will be done.

[0050] Such compositions may contain additional pharmaceutically active agents as indicated. The additional agent may be a therapeutic compound, for example, an anti-inflammatory agent, a cytotoxic agent, a cytostatic agent, or an antibiotic agent. Such additional agents may be in a form suitable for administration to a patient in need thereof. The components may be present in a single dose, and such administration may be simultaneous, separate, or sequential. can be prepared in the form of a kit which may optionally include instructions.

[0051] According to the present invention, the antigen-specific antigen-binding molecule of the present invention for use in medicine is c antigen binding molecule) or multidomain specific binding molecules are provided. Thus, this aspect of the invention provides a method for the manufacture of a medicament for the treatment of a disease in a patient in need thereof. In such an antigen-specific antigen-binding molecule or multidomain binding molecule of the present invention, The antigen-specific antigen-binding molecules of the present invention can also be used in the above-mentioned pharmaceutical compositions of the present invention. A fusion protein comprising a specific binding molecule or a multidomain binding molecule as defined above. Such a use can also be used to prepare antigen-specific antigen-binding proteins of the present invention. of a pharmaceutical composition as defined herein comprising a molecule or a multidomain binding molecule. Treatment of a disease in a patient in need of treatment comprising administering to the patient a therapeutically effective dose. The method includes:

[0052] As used herein, the term "treatment" refers to treatment of a human or non-human animal. The treatment of "non-human animals" in veterinary medicine includes any regimen that can provide benefit. livestock, including horses and companion animals (e.g., cats and dogs), and Covers the treatment of farm / agricultural animals including horses, goats, pigs, cattle and members of the equine family. The treatment may be a therapeutic treatment for any existing condition or disease, or may be prophylactic (preventive The treatment may be for a genetic or acquired disease. The treatment may be for: The treatment may be for an acute or chronic condition. The treatment may be for conditions associated with inflammation and / or cancer. The antigen-specific antigen-binding molecule or multidomain molecule of the present invention may be used for the treatment of a condition / disease. Specific binding molecules include, but are not limited to, those for osteoarthritis, scleroderma, kidney disease, rheumatoid arthritis, and the like. inflammatory bowel disease, multiple sclerosis, atherosclerosis, or any inflammatory disease It can be used to treat any disease.

[0053] In a further aspect, the present invention provides a therapeutic composition of the present invention that specifically binds to TNFα. A method of treating a condition mediated by TNFα is provided, comprising administering an effective amount of

[0054] In a further aspect, the present invention provides a composition of the present invention that specifically binds to ICOSL. EP 1 187 033 A1 2 5 10 15 20 25 30 35 40 45 50 55 Method for treating at least one condition mediated by ICOSL, comprising administering an effective amount of to provide.

[0055] A further aspect of the present invention is the use of specific combinations of VNAR binding domains that recognize cell surface molecules. In certain embodiments, the VNARs of the multidomain binding molecule are combinations of different classes. One non-limiting example included herein is wherein at least one VNAR domain binds to a target associated with an autoimmune disease, and The method of the first aspect of the invention, wherein another VNAR domain binds to a target associated with an inflammatory response. There are multi-domain specific binding molecules.

[0056] A particularly preferred multidomain-specific antigen-binding molecule of the present invention is a TNF-specific VNA. R and ICOSL-specific VNARs. Preferably, TNF-specific VNARs. is a VNAR according to the second aspect of the present invention.

[0057] In one embodiment of the present invention, ICOSL is recognized and the format outlined in the present invention is The resulting matrix provides improved functional properties over the individual binding domains in the set. There are specific combinations of VNAR binding domains to VNAR binding molecules. [Brief explanation of the drawings]

[0058] This application makes reference to a number of drawings, including: [Figure 1] Figure 1 shows anti-hTNF-alpha IgNAR titration of immunized nurse shark plasma using an anti-nurse shark IgNAR hybridoma antibody. ELISA titration of sera from immunized animals, pre-immunization, and post-bleed 5 (bleed 5) serum, combined with ELISA measurement of anti-hTNF-alpha IgNAR titer in immunized nurse sharks. Detection was performed using the GA8 monoclonal anti-nurse shark IgNAR antibody, with an anti-mouse IgG-HRP-conjugated antibody used as the secondary antibody. [Figure 2]Figure 2 shows the neutralization of hTNF-α-induced cytotoxicity in L929 cells. This assay measured the ability of anti-TNF domains (D1 and C4) and a control anti-human serum albumin domain (BA11) to neutralize the activity of hTNF-α in a cell bioassay. Both the D1 and C4 domains showed similar levels of concentration-dependent neutralization (see Table 2 for calculated values). The BA11 control does not recognize hTNF, and no neutralization was observed even at the highest concentration. hTNF-α plus actinomycin-D served as a control, demonstrating classical cytotoxicity in the absence of a neutralizing domain. [Figure 3] Figure 3 shows an in vitro rhTNFα neutralization assay in the L929 fibrosarcoma cell line. A. Neutralization of 1×LD80 [0.3 ng / ml rhTNFα], n=3 replicates per experiment, ±SEM; B. Neutralization of 10×LD80 [3 ng / ml rhTNFα], n=2 replicates per experiment, ±SD. TNF30-Fc is a fusion of an anti-rhTNFα VHH nanobody isolated from immunized camelids fused to IgG Fc (Coppieters et al., Arthritis and Rheumatism, 2006, 54 (6): 1856-1866; Riechmann et al., J. Immunol. Methods, 1999. 231: 25-38). Alb8-Fc is a VHH domain that recognizes HSA fused to IgG Fc. 2V is a negative control that does not recognize a known target. 2V-Fc is a fusion of 2V with IgG Fc. Only hTNF-specific binders (D1, C4, and TNF-30) were able to neutralize the activity of free hTNF in a concentration-dependent manner. Neutralizing potency was enhanced by conversion from a monomeric to a bivalent Fc format. The combination of D1-Fc and C4-Fc resulted in a neutralizing potency superior to that of D1-Fc or C4-Fc alone (see Table 2 for all calculated neutralization values). Both controls, Alb-8 and 2V, were unable to neutralize even in this bivalent Fc format. [Figure 4]FIG. 4 is a diagram of bivalent and bispecific construct formats. [Figure 5] Figure 5 shows ELISA binding of dimeric VNARs and a TNF-30 VHH control (TNF30-TNF30). All VNAR domains tested, D1, C4, and B4, were paired with themselves (e.g., D1-D1, C4-C4, etc.) or with each other (e.g., D1-C4, D1-B4), in both possible orientations (e.g., C4-D1, B4-D1). ELISA ranking ranked the D1-D1 dimer pair as the best (lowest concentration of VNAR required to reach a saturating signal), and B4-C4 and B4-B4 as the worst performers in this ELISA format. Numerous VNAR pairs performed better than the VHH dimer control. [Figure 6] Figure 6 shows the L929 assay for measuring TNF neutralization by VNAR dimer pairs. The neutralizing potency of the anti-TNFα VNAR dimer pairs D1-D1, D1-C4, D1-B4, and a positive control anti-TNF VHH dimer (TNF30-TNF30) was assessed using appropriate bioassays. The domain pair showing the most potent neutralizing activity in this assay format was the VNAR pair D1-C4 (see Table 2 for calculated neutralization values). hTNFα + actinomycin-D treated cells, which do not contain a neutralizing domain, provided a suitable classical, non-inhibitory cytotoxicity control. [Figure 7]Figure 7 shows neutralization of hTNFα-induced cytotoxicity in L929 cells using trimeric anti-hTNFα VNAR. Lead anti-hTNFα VNAR dimers (D1-D1 and D1-C4) were reformatted into multivalent trimeric constructs by incorporating an anti-HSA humanized VNAR (soloMER™ BA11) into the center of both dimeric constructs, resulting in D1-BA11-D1 and D1-BA11-C4, respectively. The ability of these multivalent trimeric constructs, as well as Humira (Adalimumab) and TNF30-BA11-TNF30, to neutralize hTNFα was evaluated in a classical L929 assay. The D1-BA11-C4 construct exhibited neutralization potency comparable to AdalImumab and showed significantly improved potency over the VHH trimer constructs and the anti-hTNFα dimer (D1-C4 and D1-D1) VNARs (see Table 2 for ND50 calculations). [Figure 8]Figure 8 shows Caco2 epithelial permeability in polarized Caco2 cells. Caco-2 cells were treated and incubated with 10 ng / mL TNFα, LPS, and IFNγ + / - anti-TNFα protein for 18 hours. Five microliters of 10 mg / mL FITC-dextran [3000-5000 kDa] was added to the apical chamber, and transport across the membrane to the basolateral chamber was measured 24 hours later. Treatment with VNAR / VHH monomers and VNAR control proteins was performed at 50 nM concentrations, while treatment with VNAR dimers (2C and 2D) and Adalimumab was performed at 25 nM. BA11 and 2V are non-TNFα-binding VNAR controls, and B4 is a non-neutralizing TNF-binding VNAR. The ability of anti-hTNFα VNAR constructs (monomers D1, C4, B4; dimers D1-D1, D1-C4; trimers D1-BA11-D1, D1-BA11-C4), VHH constructs TNF30, TNF30-TNF30, TNF30-BA11-TNF30, and Adalimumab to prevent intestinal barrier dysfunction in cytokine-treated Caco-2 cells was evaluated using this classical assay. VNAR domains D1-C4 and D1-BA11-C4 showed efficacy comparable to Adalimumab. Negative controls BA11 and 2V failed to prevent intestinal barrier dysfunction. [Figure 9]Figure 9 shows epithelial resistance in polarized Caco2 cells. Differentiated Caco-2 cells were treated and incubated with 10 ng / mL TNFα and IFNγ + / - anti-TNFα for 24 hours. The effect of cytokine treatment on transepithelial resistance was measured using a volt-ohm meter. Resistance was normalized to the surface area under treatment (ohms cm2). Treatment with VNAR / VHH monomers and VNAR control proteins was performed at 50 nM concentration, while treatment with VNAR dimers (2C and 2D) and Adalimumab was performed at 25 nM. BA11 and 2V are non-TNFα-binding VNAR controls, and B4 is a non-neutralizing TNF-binding VNAR. [n=1 ± SD with ≥8 replicates per treatment, one-way ANOVA using GraphPad Prism 5, and Dunnett's post-hoc test]. The efficacy of anti-hTNFα VNAR domains in restoring epithelial resistance in cytokine-treated Caco-2 cells was investigated in comparison with that of VHH TNF30 and clinically available Adalimumab at equimolar dose ranges. Anti-hTNFα dimeric and trimeric VNAR domains demonstrated significant ability to restore epithelial resistance in a manner comparable to that observed with Adalimumab. Negative controls BA11 and 2V did not restore epithelial resistance. [Figure 10] Figure 10 shows the format of the ICOSL VNAR-Fc fusion. [Figure 11] Figure 11 shows ICOSL ELISA binding data. Binding ELISA of different anti-ICOSL Quad-X™ constructs to both human and mouse ICOS ligand. [Figure 12] Figure 12 shows a multivalent and multispecific VNAR format of the invention incorporating a TNF R1 domain, an ICOSL VNAR and human IgG Fc. [Figure 13]Figure 13 shows efficacy data for multivalent and multispecific VNARs incorporating the TNF R1 domain, ICOSL. The VNAR and human IgG Fc provide further improved functional properties. The VNAR-TNFR1 Fc bifunctional constructs demonstrate specific and potent potency in cell-based neutralization assays. Format 1: anti-TNFα scFv; Format 2: anti-mICOSL VNAR (CC3); Format 3: anti-hICOSL scFv; Format 4: anti-hICOSL VNAR (2D4). [Figure 14] Figure 14 shows the hTNF-α binding profile between the VNAR T43 horn shark clone and VNAR Nurse shark D1 and C4. Binding ELISA of horn shark VNAR T43 and VNAR D1 and C4 to 1 μg / ml hTNFα coated wells. The binding profile of the T43 clone could not be determined at the experimental concentrations used. [Figure 15] Figure 15 shows the hTNF-α neutralization potency in L929 cells of the VNAR T43 horn shark clone and VNAR Nurse shark D1 and C4. Comparison of the neutralization potency of anti-hTNFα VNAR monomers D1 and C4 compared to the horn shark T43 VNAR at an equimolar dose range. The T43 domain does not show any dose-dependent neutralizing effect and has a similar profile to unprotected cells treated with hTNFα and actinomycin-D (see Table 2). [Figure 16]Figure 16 shows the binding profile of a successfully humanized anti-hTNF-α D1 (also known as D1 soloMER™). Binding profiles of multiple progressively improved framework humanized versions of the VNAR D1 domain. D1-v1, D1-v2, D1-v3, and D1-v4 represent various degrees of humanization, and VNAR D1(wt) is the parent VNAR D1 domain. Substitution of nurse shark framework amino acid residues with those of DPK-9 did not disrupt the ability of the humanized D1 versions to recognize hTNFα with human germline kappa. [Figure 17] Figure 17 shows the neutralization potency of D1 SoloMER™ in L929 cells. The ability to neutralize hTNFα-mediated cytotoxicity in L929 cells was evaluated with humanized VNAR D1 variants. SoloMER D1-v2 retained its neutralizing potency against hTNFα-induced cytotoxicity. [Figure 18] Figure 18 shows a multivalent and multispecific VNAR format of the invention incorporating human IgG Fc. [Figure 19] Figure 19 shows the hTNF-α binding profiles of multivalent / multispecific VNAR-Fc constructs. The binding profiles of biparatopic / bispecific D1-Fc-C4 (Quad-X™) versus biparatopic VNAR Fc constructs D1-Fc and C4-Fc are shown. The anti-hTNF-α VNAR Quad-X™ D1-Fc-C4 retains binding to hTNFα with a binding profile comparable to, and slightly improved over, D1-Fc and C4-Fc. [Figure 20]Figure 20 shows the evaluation of the hTNF-α neutralizing activity of multivalent / multispecific VNAR-Fc constructs in L929. The neutralizing potency of VNAR Quad-X™ D1-Fc-C4 versus Humira (Adalimumab) is evaluated in an hTNFα-mediated cytotoxicity assay using L929 cells. VNAR Quad-X™ D1-Fc-C4 retained neutralizing potency and showed superior neutralizing activity compared to Humira (see Table 2 for ND50 values). [Figure 21] Figure 21 shows a multivalent bi-paratopic VNAR format of the invention incorporating an anti-mouse TNF-α VNAR; and an anti-HSA soloMER™ BA11 or ICOSL VNAR domain, A5 or mouse IgG2a Fc. [Figure 22]Figure 22A shows the mouse TNF-α binding profile of bi-paratopic anti-mouse TNF-α VNAR constructs. The VNAR anti-mouse TNFα S17 domain is reformatted as a multivalent / multispecific trimer incorporating either the anti-ICOS ligand VNAR A5 or the anti-HSA humanized VNAR, soloMER™ BA11. Both constructs retained recognition for mouse TNF-α. Figure 22B shows the HSA binding profile of bi-paratopic anti-mouse TNF-α VNAR constructs. The VNAR anti-mouse TNFα S17 domain is reformatted as a multivalent / multispecific trimer incorporating either the anti-ICOS ligand VNAR A5 or the anti-HSA humanized VNAR, soloMER™ BA11. S17-BA11-S17 retained binding to HSA, while the negative control, S17-A5-S17, did not recognize HSA. Figure 22C shows the mouse ICOS ligand binding profile of biparatopic dimeric / trimeric anti-ICOSL VNAR constructs. The binding profiles of the reformatted S17-A5-S17 and A5-A5 homodimers to mouse ICOS ligand showed that the reformatted trimeric construct incorporating the central anti-mouse ICOS ligand VNAR A5 as S17-A5-S17 retained binding to mouse ICOS ligand. [Figure 23] Figures 23A and 23B show the mouse TNF-α neutralization in L929 profiles of bi-paratopic and IgG2a Fc-fused anti-mouse TNF-α VNAR S17 constructs, respectively. The neutralization potency of anti-mouse TNFα constructs (S17-A5-S17, S17-BA11-S17, S17-Fc) was evaluated in the mouse TNFα-mediated cytotoxicity L929 assay. Both the trimeric S17 and bi-paratopic S17-Fc constructs demonstrated neutralizing activity against mouse TNFα-mediated cytotoxicity in L929 cells. S17-A5-S17 demonstrated the highest potency of the three constructs. BA11 was the negative control in the assay, and hTNFα plus actinomycin D demonstrated the classical cytotoxic effect observed in the absence of anti-mouse TNFα inhibitors / neutralizers. Cells alone represent healthy, untreated cells. [Figure 24] Figure 24 shows a CHO-based huICOS / recombinant mouse ICOS Ligand-Fc (ICOSL-Fc) Neutralization (Blocking) Assay—ELISA based. In this blocking assay, the multivalent VNAR constructs demonstrated significant ability to block the interaction of mouse ICOSL-Fc with its cognate binding partner, ICOS, on CHO cells. This leads to reduced / compromised detection of the Fc portion of mouse ICOSL-Fc using an anti-human Fc-HRP antibody in a cell-based ELISA format. The A5-A5 dimer was the most potent blocker, followed by S17-A5-S17, while the S17 monomer served as a negative control in this assay. [Figure 25] Figure 25 shows the differences in binding cross-reactivity of anti-hTNF-α VNARs with VHH TNF30 and Humira®. This figure shows the binding cross-reactivity profile of VNAR D1-C4 compared to VHH TNF30 and Humira. VNAR D1-C4 binds only human, dog, and cynomolgus TNFα; VHH TNF30, which includes binding to human, dog, and cynomolgus TNFα, binds weakly to porcine TNFα and human TNFβ. Humira binds to human, dog, cynomolgus, and mouse TNFα. See also Tables 3A and 3B for detailed binding and neutralization profiles of these anti-TNFα domains. [Figure 26]Figure 26 shows a BIAcore™ T200 epitope binning analysis of an anti-hTNF-α VNAR tetramer versus a VHH TNF30 dimer. The epitope binning data indicates that VNAR D1-C4 recognizes and interacts with a different epitope on the hTNFα molecule than that recognized by the VHH TNF30 domain. The assay involves reaching epitope saturation available in the first binding domain (in this case, VNAR D1-C4 using a saturating concentration determined as 100 times its KD value) and then the second binding domain (TNF30). [Figure 27] FIG. 27 shows functional binding to hTNF-α by Quad-X™ and Quad-Y™ constructs in an ELISA format. [Figure 28] Figure 28 shows the evaluation of the hTNF-α neutralizing activity of multivalent / multispecific VNAR-Fc constructs in L929. The neutralizing potency of VNAR Quad-X™ D1-Fc-C4, Quad-Y™ D1-C4-Fc, and C4-D1-F versus Humira (Adalimumab) is evaluated in an L929 cell-based assay of hTNFα-mediated cytotoxicity. The VNAR Quad-Y™ construct retains neutralizing activity, and the D1-C4-Fc construct exhibits neutralizing activity equivalent to Quad-X™ in the presence of 0.3 ng / ml or 3 ng / ml TNFα (see Table 2 for ND50). [Figure 29] Figure 29 shows the effect of D1-Fc-C4 (Quad-X™) and Humira® on weight gain in experimental Tg197 mice. From week 3 until the end of the study (10 weeks of age), the mean body weights for all groups treated twice weekly were as follows: G1-Vehicle = 19.3 ± 1.4 g, G4-Humira® 10 mg / kg = 24.4 ± 1.5 g, G2-D1-Fc-C4 3 mg / kg = 24.1 ± 1.5 g, G5-D1-Fc-C4 10 mg / kg = 24.1 ± 1.7 g, and G3-D1-Fc-D4 30 mg / kg = 23.4 ± 1.4 g. Control mice at week 3 had a mean body weight of 9.8 ± 0.2 g. Error bars indicate the standard error of the mean. [Figure 30] Figure 30 shows the effects of D1-Fc-C4 (Quad-X™) and Humira™ on in vivo arthritis scores in experimental Tg197 mice. Through the end of the study (10 weeks of age), mean in vivo disease severity scores for all groups treated twice weekly from week 3 were as follows: G1-Vehicle = 1.36 ± 0.07, G4-Humira® 10 mg / kg = 0.25 ± 0.05, G2-D1-Fc-C4 3 mg / kg = 0.17 ± 0.04, G5-D1-Fc-C4 10 mg / kg = 0.17 ± 0.04, and G3-D1-Fc-D4 30 mg / kg = 0.17 ± 0.04. Control mice at week 3 had an in vivo arthritis score = 0.13 ± 0.05. Error bars indicate standard error of the mean. [Figure 31] Figure 31 shows the effects of D1-Fc-C4 (Quad-X™) and Humira® on arthritis histopathology scores in experimental Tg197 mice. From week 3 through the end of the study (10 weeks of age), the mean arthritis histopathology scores for all groups treated twice weekly were as follows: G1-Vehicle = 2.94 ± 0.12, G4-Humira® 10 mg / kg = 0.42 ± 0.07, G2-D1-Fc-C4 3 mg / kg = 0.41 ± 0.03, G5-D1-Fc-C4 10 mg / kg = 0.50 ± 0.05, and G3-D1-Fc-D4 30 mg / kg = 0.42 ± 0.07. Control mice at week 3 had a histopathology score = 1.22 ± 0.10. Error bars indicate the standard error of the mean. [Figure 32]FIG. 32 is a comparison of the effects of D1-Fc-C4 (Quad-X™) and Humira® on in vivo arthritis scores versus ankle histopathology scores in experimental Tg197 mice. Through the end of the study (10 weeks of age), mean disease severity scores for all groups treated twice weekly from week 3 onwards were as follows: G1-Vehicle = 2.94 ± 0.12 (HS) and 1.36 ± 0.07 (AS), G4-Humira® 10 mg / kg = 0.42 ± 0.07 (HS) and 0.25 ± 0.05 (AS), G2-D1-Fc-C4 3 mg / kg = 0.41 ± 0.03 (HS) and 0.17 ± 0.04 (AS), G5-D1-Fc-C4 10 mg / kg = 0.50 ± 0.05 (HS) and 0.17 ± 0.04 (AS), and G3-D1-Fc-D4 30 mg / kg = 0.42 ± 0.07 (HS) and 0.17 ± 0.04 (AS). Error bars indicate the standard error of the mean. [Figure 33] Figure 33 is an efficacy evaluation of 0.5, 1, and 3 mg / kg D1-Fc-C4 (Quad-X™) and 30 mg / kg D1-BA11-C4 versus 1 mg / kg and 3 mg / kg Humira® in ameliorating arthritis pathology in the Tg197 model of arthritis. [Figure 34] Figure 34 shows the effects of 0.5, 1, and 3 mg / kg D1-Fc-C4 (Quad-X™) and 30 mg / kg D1-BA11-C4 versus 1 mg / kg and 3 mg / kg Humira® on mean group weight in the Tg197 model of arthritis. [Figure 35] Figure 35 shows the effect of different Humira® dosing regimens on in vivo arthritis and histopathology scores, which was performed in a separate experiment using the same test method as described for Figures 29-32. [Figure 36]Figure 36 shows that 12 rats were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce experimental autoimmune uveitis (EAU). Four animals each were treated with 20 mg / kg (rodent protein-specific) anti-TNFα VNAR-Fc by intraperitoneal injection on days 8, 10, and 12; four animals were treated intraperitoneally with dexamethasone on the same days, and four animals were similarly treated with vehicle. Optical coherence tomography (OCT) of both the anterior and posterior segments of the rat eyes was performed on days 0, 7, 10, 12, 13, and 14. To minimize any scientific bias in the results, OCT images were scored for total inflammation by an experimentally blinded observer using a validated scoring system. The experiment also included a vehicle control and a positive control using a standard dose of dexamethasone steroid. [Figure 37] Figures 37A and 37B show evaluation of the hTNF-α neutralizing activity of soloMER VNAR dimer constructs in L929 cells. [Figure 38] Figure 38 shows an evaluation of the hTNF-α neutralizing activity of S17-Fc vs S17-Fc-S17 (Quad-X™) constructs in L929 cells. The Fc used in the S17 construct is derived from murine IgG2a. [Figure 39] Figures 39A, 39B and 39C show the cross-reactive binding profiles of S17-Quad-X™ and D1-C4 Quad-X™ to human and mouse TNF-α. DETAILED DESCRIPTION OF THE INVENTION

[0059] Various nucleotide and amino acid sequences are provided herein as follows:

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[0081] Detailed Description of the Invention definition Amino acids are referred to herein as either the one-letter code or the three-letter code, or It is expressed as both.

[0082] The term "affinity purification" refers to the process by which a molecule is bound or purified to a partner moiety. or a combination that allows the substance to remain attracted and be separated from impurities. The specific attraction of a molecule to a chemical or binding partner to form a complex Or refers to the purification of molecules based on binding.

[0083] The term "complementarity determining region" or CDR (i.e., CDR1 and CDR3) refers to the amino acid residues in the VNAR domain whose presence is required for antigen binding. Each complementary amino acid sequence typically has CDR regions identified as CDR1 and CDR3. The sex-determining region is composed of amino acid residues in the "complementarity-determining region" and / or the "hypervariable loop" (H In some cases, the complementarity determining region may comprise amino acid residues of CDR regions and The amino acids in both the hypervariable loops may be included. According to this nomenclature, there is no CDR2 region.

[0084] "Framework regions" (FW) are VNAR residues other than the CDR residues. R are typically identified as FW1, FW2, FW3a, FW3b, and FW4 VNAR domains typically have five framework regions. From -terminus to C-terminus, FW1-CDR1-FW2-HV2-FW3a-HV4-FW It has the structure 3b-CDR3-FW4.

[0085] "Cell," "cell line," and "cell culture" are used interchangeably (unless the context indicates otherwise). are used interchangeably and such designations include all progeny of the cell or cell line. Thus, for example, terms such as "transformant" and "transformed cell" include those derived from the primary subject cell. This includes cultures derived from the above, regardless of cell type and number of transfers. The offspring may not be exactly identical in DNA content due to deliberate or inadvertent mutations. It is understood that the screening is not limited to the original transformed cells. Mutant progeny that retain the same function or biological activity are included.

[0086] "Control sequences," when referring to expression, refer to the sequences to which they are operably linked in a particular host organism. The term "control sequences" refers to the DNA sequences required for expression of a coding sequence that is suitable for prokaryotes. It contains a promoter, an operator sequence if necessary, a ribosome binding site, etc. Eukaryotic cells contain regulatory elements such as promoters, polyadenylation signals, and enhancers. Use the control array.

[0087] The term "coat protein" refers to a protein, at least a portion of which is present on the surface of a virus particle. From a functional standpoint, coat proteins are proteins that are present in the host cell. During the viral assembly process, they associate with virus particles and then infect other host cells. It is a protein that remains associated with the assembled virus.

[0088] The "detection limit" of a chemical entity in a particular assay is determined by the background The minimum concentration of the entity that can be detected above the round level. In a page ELISA, the "detection limit" for a particular phage displaying a particular antigen-binding fragment is A specific phage is compared to the signal produced by a control phage that does not display an antigen-binding fragment. is the phage concentration that produces an ELISA signal above 0.05.

[0089] "Fusion proteins" and "fusion polypeptides" refer to proteins in which two moieties are covalently linked together. wherein each of the portions has different properties. The properties may be biological properties, such as in vitro or in vivo activity. The above properties may also be simple chemical or functional properties such as binding to a target antigen or catalysis of a reaction. The two moieties may be linked by a single peptide bond or by a physical property. They may also be directly linked via a peptide linker comprising one or more amino acid residues. , the two portions and the linker will be in reading frame with each other. Preferably, the two portions of the polypeptide are derived from heterologous or different polypeptides. .

[0090] The term "fusion protein" as used herein means, in general terms, a protein that is a fusion protein formed by hydrogen bonding. or by chemical means, such as salt bridges, or by peptide bonds via protein synthesis. It refers to one or more proteins bound together by a bond or both.

[0091] "Heterologous DNA" is any DNA that is introduced into a host cell. A variety of sources including cDNA, synthetic DNA, and fusions or combinations thereof. The DNA may be derived from the same cell or cell type as the host or recipient cell. DNA from a different cell type, e.g., DNA from a homologous or heterologous source. A. The DNA may optionally contain a marker or selection gene (e.g., an antibiotic The gene may contain a gene for resistance to microbial growth factors, such as a microbial resistance gene, a temperature resistance gene, etc.

[0092] A "highly diverse position" is a position that is known and / or naturally occurring. A comparison of the amino acid sequences of antibodies or antigen-binding fragments present at a certain position reveals that The term "amino acid positions" refers to amino acid positions located in the variable regions of the light and heavy chains that have different amino acids. The highly diverse positions are typically in the CDR regions.

[0093] "Identity" refers to the degree of identity between two or more polypeptide sequences or or describes the relationship between two or more polynucleotide sequences. A polypeptide sequence or polynucleotide sequence determined by a match between strings of sequences such as It refers to the degree of sequence relatedness (homology) between two polypeptide sequences. Although there are many methods for determining identity between two polynucleotide sequences, Commonly used methods to determine identity are codified in computer programs. A preferred computer program for determining identity between two sequences is For example, the GCG program package (Devereux, et al., Nucleic Acids Res, 1984, 12 , 387 BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. (1990) 215, 403 ), but are not limited to:

[0094] Preferably, the amino acid sequence of the protein is determined according to the Human Genome Mapping Project (HGMP) The BLAST computer program provided by Atschul et al., J. Mol. Biol. 1990 215, 403-410) using the default parameters of the amino acid sequence disclosed herein. The amino acid sequence has at least 60% identity at the amino acid level.

[0095] More preferably, the protein sequences are nucleic acid sequences relative to the amino acid sequences shown herein. Acid or amino acid levels of at least 65%, 66%, 67%, 68%, 69%, 70% %, 75%, 80%, 85%, 90%, and even more preferably 95% (even more preferably may have at least 96%, 97%, 98% or 99% identity.

[0096] Proteins can also be identified using the BLAST computer program provided by HGMP. The sequences disclosed herein and at least 60 %, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, or 99% identity to the sequence.

[0097] A "library" refers to a plurality of VNAR or VNAR fragment sequences or sequences thereof. The origin of the library refers to nucleic acids that encode the nucleic acid. is a non-natural source of natural frameworks with properties designed to be combined V can be of natural or synthetic origin, or isolated from RNA extracted from immunized animals. It may be derived from natural sources, as exemplified by the NAR domain.

[0098] "Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. For the purpose of joining two fragments, the ends of the fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, to make it suitable for ligation, it is first typically produced after endonuclease digestion. It may be necessary to convert the resulting staggered ends to blunt ends. To make the ends blunt, the DNA is cleaved in the presence of four deoxyribonucleotide triphosphates. About 10 units of DNA polymerase I or the Klenow fragment of T4 DNA polymerase The DNA is then treated with HCl in an appropriate buffer at 15°C for at least 15 minutes. by phenol-chloroform extraction and ethanol precipitation or by silica purification The DNA fragments to be ligated together are placed in approximately equimolar amounts in a solution. Also, add ATP, ligase buffer, and ligase (e.g., 0.5 μg / µg of DNA). Approximately 10 units of T4 DNA ligase will be included. To do this, the vector is first linearized by digestion with the appropriate restriction endonuclease. The linearized fragments were then treated with bacterial alkaline phosphatase or calf intestinal phosphatase. This prevents self-ligation during the ligation process.

[0099] A "mutation" is a deletion of a nucleotide relative to a reference nucleotide sequence, such as a wild-type sequence. deletion, insertion, or substitution.

[0100] "Natural" or "naturally occurring" VNARs are, for example, those obtained ex vivo. from non-synthetic sources, such as from tissue sources, or from animals of the subclass Elasmobranchii These VNARs include natural or induced VNARs identified in the serum of VNARs can also be generated by any type of immune response. Naturally occurring VNARs are amino acid sequences. The present invention includes the nucleic acid sequences and the nucleotide sequences that constitute or encode these antibodies. As used in the specification, naturally occurring VNARs are distinct from "synthetic VNARs" and refer to synthetic VNARs. represents, for example, the substitution of one or more amino acids at a particular position by a different amino acid. refers to a VNAR sequence that has been altered from the source or template sequence by substitution, deletion, or addition of a sequence The different amino acids provide an antibody sequence that differs from the original antibody sequence.

[0101] The term "nucleic acid construct" generally refers to a nucleic acid construct that is a DNA, cDNA, or cloned Any length of RNA may be used, such as mRNA obtained by chemical synthesis or produced by chemical synthesis. DNA refers to nucleic acids of a certain length. DNA can be single-stranded or double-stranded. Single-stranded DNA is It may be the sense strand, or the non-coding or antisense strand. For purposes of use, the nucleic acid construct is preferably in a form that can be expressed in the subject to be treated. is.

[0102] "Operably linked," when referring to nucleic acids, means that the nucleic acid is linked to another It means a functional relationship with a nucleic acid sequence, e.g., a presequence. Alternatively, secretory leader DNA may be a promoter involved in the secretion of polypeptides. When expressed as a protein, it is operably linked to the DNA of the polypeptide; A promoter or enhancer is engineered into a coding sequence if it affects the transcription of the sequence. or the ribosome binding site is positioned to facilitate translation. In this case, it is operably linked to a coding sequence. "(linked)" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, means that it is in the same reading frame as the enhancer. They do not necessarily have to be contiguous; joining is accomplished by ligation at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adaptor or The linker is used according to conventional practice.

[0103] "Phage display" refers to the process by which mutant polypeptides are displayed on phage (e.g., as a fusion protein to at least a portion of a coat protein on the surface of a phage particle Phage display technology allows the detection of targets with high affinity. Random proteins that can be rapidly and efficiently selected for antigen-binding sequences Large libraries of protein variants can be prepared. Library display identifies millions of polypeptides with specific binding properties. Multivalent phage display can be used to screen for novel phage vectors. The coat proteins of the pill, pVIII, pVI, pVII, or pIX small random peptides and small proteins via fusion to genes encoding has been used to display

[0104] A "phagemid" is a vector containing a bacterial origin of replication, e.g., ColEI, and a bacteriophage. A phagemid is a plasmid vector that contains a copy of the intergenic region of a gene. Any known bacteriophage, such as bacteriophages and lambdoid bacteriophages The plasmids can also be used in phages, which are generally used for selection for antibiotic resistance. The DNA segments cloned into these vectors will contain markers. It can be propagated as a plasmid and contains all the genes necessary for the production of phage particles. When cells containing these vectors are provided, the mode of replication of the plasmids is rolling sequence. This changes to rolling circle replication, which produces a single-stranded copy of the plasmid DNA. Phagemids are used to generate and package phage particles. Phagemids can contain infectious or non-infectious phage. The term refers to the ability of a heterologous polypeptide to be displayed on the surface of a phage particle. The phage polypeptide is linked to a heterologous polypeptide gene as a gene fusion, as described above. The present invention also encompasses phagemids containing a nucleotide protein gene or a fragment thereof. An example of a spray vector is pWRIL-1.

[0105] The term "phage vector" refers to a bacteriophage vector that contains a heterologous gene and is capable of replication. A phage vector refers to the double-stranded replicative form of a phage. The phage preferably has an origin of replication that allows the formation of M13, fl, filamentous bacteriophages, such as fd, Pf3 phage or its derivatives, or lambda , 21, phi80, phi81 or their derivatives.

[0106] The term "protein" generally refers to a group of multiple molecules joined together by peptide bonds. It means an amino acid residue. It is a peptide, oligopeptide, oligomer, or poly Peptides are used interchangeably and have the same meaning as glycoproteins and their derivatives. The term "protein" includes fragments, analogs, variants and derivatives of proteins. It is understood that the term "antibody" encompasses fragments, analogs, variants, or derivatives of the reference protein. Retains essentially the same biological activity or function. Examples of Protein Analogs and Derivatives These include peptide nucleic acids, and DARPins (designed ankyrin repeat proteins) (Designed Ankyrin Repeat Proteins). The "polypeptide" of the present invention is defined herein as It is a defined TNFα-specific antigen-binding molecule.

[0107] A protein fragment, analog, variant, or derivative is a derivative of the original protein sequence from which it is derived. Depending on the length of the row, at least 25, preferably at least 30 or 40, or 5 It can be 0 or 100 or less, or 60 to 120 amino acids in length. A length of 0 to 110 amino acids may be convenient in some cases.

[0108] A fragment, derivative, variant, or analog of a protein is one in which (i) one or more amino acid residues are conserved; Conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such The substituted amino acid residue may or may not be one encoded by the genetic code. or (ii) one or more amino acid residues containing a substituent group; or (iii) Additional amino acids may be added to the mature polypeptide, such as a leader or auxiliary sequence used in purifying the polypeptide. Such fragments, derivatives, variants and and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0109] "Oligonucleotides" may be synthesized by known methods (e.g., phosphotriesters, phosphatase inhibitors, etc.) using solid phase techniques. Short chains, single strands chemically synthesized by sulfite or phosphoramidite chemistry A further method is to clone the entire gene. Used when the nucleic acid sequence is known or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, the target amino acid sequence can be determined by PCR. In some cases, potential sequences are generated using known and preferred coding residues for each amino acid residue. The oligonucleotides can be separated by polyacrylamide gel or molecular weight to determine the nucleic acid sequence. It can be purified on molecular sizing columns or by precipitation. When DNA is separated from non-nucleic acid impurities (which can be polar, non-polar, ionic, etc.), In this case, the DNA is "purified."

[0110] As used herein, a "source" or "template" VNAR is: The antigen-binding sequence serves as a template sequence upon which diversification according to the criteria described herein is performed. The term "antigen-binding sequence" refers to a VNAR or VNAR antigen-binding fragment that functions in a manner consistent with the VNAR antigen-binding sequence. The antigen-binding sequence generally comprises: Within a VNAR, it is preferred to include at least one CDR, preferably including a framework region. It is preferable that:

[0111] "Transcriptional regulatory elements" include the following components: enhancer elements, promoters, The gene may include one or more of a promoter sequence, a repressor gene, and a transcription termination sequence. cormorant.

[0112] "Transformation" refers to the process by which a cell takes up DNA and becomes a "transformant." The DNA uptake may be permanent or transient. phenotypes associated with (e.g., conferred by proteins encoded by DNA) cells that have taken up and maintained the DNA, as evidenced by the expression of antibiotic resistance do.

[0113] Fusion proteins (polypeptides) or heterologous polypeptides (heterologous to the phage) Any "variant" of a starting or reference polypeptide (e.g., a source VNAR or its CDRs) A "variant" or "mutant" is (1) a variant of a starting or reference polypeptide; (2) have an amino acid sequence that differs from that of the original amino acid sequence, whether naturally occurring or artificially induced; A polypeptide derived from a starting or reference polypeptide by any of the following: Such variants include, for example, deletions of residues within the amino acid sequence of the polypeptide of interest, and and / or insertions and / or substitutions. For example, (source VNAR or antigen-binding fragment) variant amino acid (with respect to the amino acid found at the corresponding position in the fragment) An oligonucleotide containing a nonrandom codon set that encodes a sequence having Fusion polypeptides of the invention produced using oligonucleotides may be derived from a source VNAR or is a variant polypeptide with respect to the antigen-binding fragment. CDRs (variant CDRs) are CDRs derived from the starting or reference polypeptide sequence (source VNAR or antigen As used herein, a mutated CDR refers to a CDR that contains a mutated sequence relative to the CDR of a target polypeptide (such as that of a target polypeptide-binding fragment). The amino acids are those of the starting or reference polypeptide sequence (source VNAR or antigen-binding fragment). Deletions, insertions, and substitutions refer to amino acids that differ from the amino acids at the corresponding positions in a gene. Any combination of may be used, provided that the final construct has the desired functional characteristics. Amino acid changes can be made to arrive at the final variant or mutant construct. It also alters post-translational processes of polypeptides, such as changing the number or location of glycosylation sites. The alteration may be in the coat protein or the CDRs of the source VNAR. of the "wild-type" or "reference" sequence or "wild-type" or "reference" protein / polypeptide The sequence of the peptide is a reference to the sequence of the variant polypeptide derived by the introduction of a mutation. Generally, the "wild-type" sequence of a given protein may be the sequence that is most common in nature. It is an array of

[0114] Similarly, a "wild-type" gene sequence is the sequence of the gene most commonly found in nature. A mutation is a mutation that occurs when a "wild-type" gene (a gene that has been modified by natural processes or artificial means) is altered. The product of such a process is is a "variant" or "mutant" of the original "wild-type" protein or gene.

[0115] General methods for DNA manipulation, transfection methods and culture methods are known in the art. In this regard, Molecular Cloning: A Laboratory Manual (Fourth Edition) (edition) Cold Spring Harbor Publishing.

[0116] Isolation of VNARs VNAR domains were constructed using tissue from targeted immunized sharks. It can be obtained from a prey library (Dooley, H., et al. Mol Immunol, 2003. 40( 1): p. 25-33; Nuttall, SD, et al, Proteins, 2004. 55(1): p. 187-97; and Dooley , H., et al., Proc Natl Acad Sci USA, 2006. 103(6): p. 1846-51), WO 200 No. 3,014,161 (incorporated by reference) describes immunizing sharks to extract binding domains. Useful methods for obtaining them are described.

[0117] VNAR binding domains can also be obtained from synthetic libraries containing VNAR sequences. WO 2014 / 173959 (incorporated by reference) describes a method for preparing a VNAR library. Useful methods for developing and obtaining binding domains are described.

[0118] Additionally, a synthetic diversity library targeting CDR3 was used to identify VNARs. It has been shown that structure-based binding domains can be obtained (Nuttall, SD, et al. l. Mol Immunol, 2001. 38(4): p. 313-26; Nuttall, SD, et al. Eur J Biochem, 200 3. 270(17): p. 3543-54; Shao, CY, et al. Mol Immunol, 2007. 44(4): p. 656-65 a nd Liu, JL, et al. BMC Biotechnol, 2007. 7: p. 78; WO2005 / 118629.

[0119] The VNARS of the present invention are designed to reduce potential immunogenicity when administered to humans. can be further adapted (humanized).

[0120] Humanization of antibody variable domains is a process whereby the humanized forms are compatible with human subjects. and targeting a therapeutically useful target so as to avoid undesirable immunological reactions when administered to a subject. For antibodies produced in species other than humans, techniques well known in the art for modifying the antibodies have been developed. Related methods for humanization are discussed in Almagro JC and William Strohl W. Antibody Eng. ineering: Humanization, Affinity Maturation, and Selection Techniques in Therape utic Monoclonal Antibodies: Bench to Clinic. Edited by An J. 2009 John Wiley & Sons, Inc. and Strohl WR and Strohl LM, Therapeutic Antibody Engineerin g, Woodhead Publishing 2012.

[0121] IgNARs have a different origin compared to immunoglobulins, and are derived from immunoglobulin variable domains. Although there is very little sequence homology between the immunoglobulin variable domain and IgNAR compared to There are some structural similarities between the variable domains, and as a result, similar processes occur in the VN This can be applied to the AR domain. See, for example, WO 2013 / 167883 (incorporated by reference). A description of VNAR humanization is provided in Kovalenko OV, et al. J Biol See also Chem. 2013. 288(24): p. 17408-19.

[0122] Protein expression A nucleic acid encoding an antigen-specific antigen-binding molecule or a multidomain-specific binding molecule of the present invention The nucleic acid sequence can be present in a nucleic acid construct. Such a nucleic acid construct can be a vector, e.g., an expression vector. The vector may be in the form of a chromosomal, episomal, or virally derived vector, among others. Vectors, e.g., bacterial plasmids, bacteriophages, transposons, yeast epitopes chromosomes, insertion elements, yeast chromosomal elements, viruses, e.g., baculovirus, S Papovaviruses such as V40, vaccinia viruses, adenoviruses, fowlpox viruses vectors derived from rabies, pseudorabies virus and retroviruses, as well as e.g. Plasmid and bacteriophage genetic elements, such as cosmids and phagemids and vectors derived from combinations thereof, such as vectors derived from the vector of interest. suitable for maintaining, propagating or expressing nucleic acids to express a polypeptide in a host. Any suitable vector may be used for expression in this regard.

[0123] The nucleic acid construct suitably comprises a promoter or other regulatory sequence that controls expression of the nucleic acid. The promoter and other regulatory sequences that control the expression of the nucleic acid have been identified and and are well known in the art. Those skilled in the art will appreciate that the entire promoter or other regulatory sequence may be utilized. You may know that only the minimum essential regulatory elements are required. It is necessary, and indeed such elements may be present in chimeric sequences or other promoters. Of course, essential requirements may be organizational and / or time-specific. The promoter may be any suitable known promoter (e.g., , human cytomegalovirus (CMV) promoter, CMV immediate early promoter, H SV thymidine kinase, early and late SV40 promoters, or retroviral L a promoter of a TR (e.g., a Rous sarcoma virus (RSV) promoter), and a metallothionine promoter (e.g., mouse metallothionine-I promoter) The promoter is compromised for the minimum (TA TA element, and if necessary, does not contain enhancer elements), e.g., CMV It may contain a minimal sequence of a promoter. Preferably, the promoter is adjacent to the nucleic acid sequence. do.

[0124] As described herein, the nucleic acid construct may be in the form of a vector. often require the selection of cells transfected (or transformed) with them. allowing for the selection of cells containing vectors that incorporate heterologous DNA, It contains one or more expression markers. Appropriate start and stop signals will generally be present. cormorant.

[0125] The vector may be any suitable expression vector, such as pET. Additional regulatory sequences, if desired, such as selectable markers (e.g., antibiotic resistance, fluorescence, etc.) , transcriptional regulatory sequences and promoters (including initiation and termination sequences).

[0126] The promoter drives the expression of the protein encoded by the nucleic acid sequence of the present invention. A suitable promoter for activating the gene (e.g., CMV promoter, human phosphoglycerate kinase The promoter may be a human PGK promoter.

[0127] Such vectors can be present in host cells for expression of the nucleic acid constructs of the present invention. Representative examples of suitable host cells include those that allow encapsulation of nucleic acids into viral vectors. Virus packaging cells; Streptococci , Staphylococci, E. coli, Streptomyces and Ba Bacterial cells, such as Saccharomyces cerevisiae; yeast cells, such as Saccharomyces cerevisiae; single cells, such as myces cerevisiae and Aspergillus cells cells; such as Drosophila S2 cells and Spodoptera Sf9 cells, Insect cells, CHO, COS, C127, 3T3, PHK.293, and Bowes animal cells, such as human leukemia cells and other suitable human cells; and plant cells, e.g. Arabidopsis thaliana. Suitably, the host cell is CH The cells are eukaryotic cells, such as 0 cells or HEK293 cells.

[0128] The expression vector was introduced into the host cells using calcium phosphate transfection, DEA E-dextran-mediated transfection, microinjection, cationic lipid Cationic - lipid-mediated transfection, electroporation ration, transduction, scrape loading, ballistic introduction This may be achieved by stic introduction, infection, or other methods. mbrook et al, Molecular Cloning, a Laboratory Manual, Second Edition, Cold Sprin Harbor Laboratory Press, Cold Spring Harbor, NY (1989), among many other standard It is described in the laboratory manual.

[0129] The mature protein can be expressed in mammalian cells (CHO cells, yeast) under the control of an appropriate promoter. The vector can be expressed in a host cell, such as a mammalian, bacterial, or other cell. The RNA derived from the nucleic acid construct of the third aspect of the invention is used to produce such proteins. Suitable cloning and transcription factors for use with prokaryotic and eukaryotic hosts may also be used. The expression vectors are described in Sambrook et al., Molecular Cloning, a Laboratory Manual, Second Edition. Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). It will be described.

[0130] The present invention also relates to the polynucleotides and / or vectors of the invention described herein. According to the present invention, a host cell containing any one of the antigen-specific antigen binding vectors of the present invention is provided. A method for producing a composite or multidomain specific binding molecule as defined herein, comprising: and expressing a nucleic acid sequence encoding said molecule in a suitable host cell such that Provided.

[0131] Proteins can be purified by ammonium sulfate or ethanol precipitation, acid extraction, anionic or cationic precipitation, or by precipitation with ammonium sulfate or ethanol. Ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography Chromatography, affinity chromatography, hydroxyapatite chromatography standard methods including chromatographic, lectin and / or heparin chromatography Thus, they can be recovered and purified from recombinant cell culture. For example, nucleic acid constructs in the form of recombinant vectors can be prepared using methods such as those described in Sambrook et al., Molecular Cloning, a aboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spri such as by column chromatography means as described in [Illegible] Harbor, NY (1989). , can be purified by techniques known in the art.

[0132] Thus, this aspect of the invention provides recombinant production of the fusion protein by expression in a host cell. production, peptide bond linkage, hydrogen or salt bonds or chemical crosslinking procedures The present invention also extends to methods for preparing the fusion proteins of the present invention, including purification of the expressed fusion protein by steps. In some embodiments of this aspect of the invention, the fusion protein can be a peptide. Using certain hydrogen or salt bonds or polymerisation, e.g. dimerisation or trimerisation It can be prepared.

[0133] The antigen-specific antigen-binding molecule or multidomain-specific binding molecule may be any of the molecules described herein. prior to use, which may aid in purification and / or isolation during the process for the production of molecules such as For example, the C-terminus of the molecule may contain additional N- or C-terminal sequences that are cleaved into Ala)3(His)6.

[0134] Some, for example, 5-10, or 1-5, or 1-3, 2, 1, or 0 The amino acid sequence of a protein in which amino acid residues are substituted, deleted, or added in any combination. Variants, analogs, derivatives and fragments having the same sequence are also included in the present invention. Among these are silent substitutions, additions, and deletions that do not alter the properties and activity of the protein. It is particularly preferred that the properties of the protein of the invention are preserved in the mutant form compared to the original form. Conservative substitutions that are not specific to the antigens of the present invention are also particularly preferred in this regard. There are fusion proteins that contain heterologous antigen-binding molecules.

[0135] As noted above, exemplary variants of the present invention include those in which one or more amino acids are replaced with one or more other amino acids. Those skilled in the art will recognize that various amino acids have similar properties. One or more such amino acids in a substance often contribute to the desired properties of that substance. may be substituted by one or more other such amino acids without interfering with or eliminating the activity of Such substitutions are called "non-conservative" amino acid substitutions. These can be called id substitutions.

[0136] Thus, the amino acids glycine, alanine, valine, leucine and isoleucine , can often be substituted for each other (amino acids with aliphatic side chains). These possible substitutions Among these, glycine and alanine are used to replace each other (they are relatively short side chains), valine, leucine, and isoleucine are used to replace each other are preferred (because they have larger aliphatic side chains that are hydrophobic). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine, and thiamin Tryptophan (an amino acid with an aromatic side chain); lysine, arginine and histidine ( amino acids with basic side chains; aspartic acid and glutamic acid (with acidic side chains) amino acids); asparagine and glutamine (amino acids with amide side chains); and These include cysteine ​​and methionine (amino acids with sulfur-containing side chains). Substitutions of the amino acid sequence are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

[0137] Amino acid deletions or insertions may also be made to the amino acid sequence of the fusion protein. Thus, for example, it may have no or little substantial effect on the activity of the polypeptide. Amino acids may be deleted that do not eliminate such activity. This is advantageous because it allows the total length and molecular weight of the polypeptide to be reduced while maintaining the This can reduce the amount of polypeptide needed for a particular purpose. For example, the dosage level may be reduced.

[0138] Amino acid insertions into the sequences of the above fusion proteins may also be made. To alter the properties of the substance of the invention (e.g., as described above for fusion proteins), Such analyses can be performed to aid in identification, purification, or expression.

[0139] Amino acid changes to the sequence of the fusion protein of the invention can be made using any suitable technique, e.g. For example, this can be done by using site-directed mutagenesis.

[0140] Amino acid substitutions or insertions included within the scope of the present invention may be naturally occurring or non-naturally occurring. It should be understood that this can be done using amino acids that do not exist in the body. Whether or not an acid is used, it is preferred that only L-amino acids are present.

[0141] The protein of the present invention may further comprise N-terminal and / or C-terminal amino acid sequences. Such sequences may be provided for a variety of reasons (e.g., glycosylation).

[0142] The fusion protein is a protein in which the antigen-specific antigen-binding molecule of the present invention is incorporated into the fusion protein by structural elements. In other embodiments, the peptide or protein may be fused to a heterologous peptide or protein sequence that provides a The fusion protein is a molecule having biological activity in which the antigen-specific antigen-binding molecule of the present invention is a molecule having biological activity. The molecule may be fused to a peptide or protein sequence, or to another biological The compound may be an environmentally active molecule.

[0143] For example, an antigen-specific antigen-binding molecule may comprise a polyamino acid sequence, e.g., a sequence of multiple histidine residues. a group or a plurality of lysine residues (suitably 2, 3, 4, 5, or 6 residues), or It may be fused to a heterologous peptide sequence, which may be a globulin domain (e.g., an Fc domain). do.

[0144] Reference to heterologous peptide sequences includes sequences derived from other mammalian species (e.g., mouse and human). sequences, as well as heterologous peptide sequences derived from other VNAR domains.

[0145] The fusion protein is a fusion protein in which the antigen-specific antigen-binding molecule of the present invention is fused to a molecule having biological activity. When the biologically active moiety is an enzyme, immunoglobulin, cytokine or It may be a biologically active peptide or protein, such as a fragment thereof. , biologically active molecules include antibiotics, anticancer drugs, NSAIDs, steroids, painkillers, toxins or other pharmaceutically active agents. Anti-cancer agents include cytotoxic or cytostatic Sexual drugs may be included.

[0146] In some embodiments, the fusion protein comprises an antigen-specific antigen-binding molecule of the present invention. fused to an immunoglobulin variable or constant region, or another antigen-specific antigen-binding molecule of the present invention In other words, the antigen-specific antigen-binding molecules of the present invention can be of variable length, e.g., dimeric fusions of monomers, trimers, tetramers, or higher multimers (i.e., pentamer, hexamer, heptamer, etc.) In certain embodiments, this may be a monomer, octamer, nonamer, or decamer (or larger). It can be represented as a multimer of VNAR subunits.

[0147] In the fusion protein of the present invention, the antigen-specific antigen-binding molecule is a The element may be directly fused or linked via a linker moiety. The linker may be a peptide, peptide nucleic acid, or polyamide linker. The carcinomas are (Gly)4, (Gly)5, (Gly)4Ser, and (Gly)4(Ser). (Gly)4, or combinations or multimers thereof (e.g., dimers, trimers, a plurality of amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, It may contain 8, 9, 10, 15, 20 or 25 amino acids. For example, a suitable linker is Alternative linkers include (Ala)(HIs) or The BLAST computer program provided by HGMP Use the default parameters of the ram and set it to at least 50%, 60%, 70%, 80% Also included are sequences with 90%, 95% or 99% identity.

[0148] The vectors constructed as described in accordance with the present invention may be used for amplification and / or expression. Vectors are introduced into host cells for transfection by electroporation, calcium phosphate precipitation, or other methods. The vector can be introduced into the host cell using standard transformation methods such as In the case of an infectious particle such as a vector, the vector itself provides entry into the host cell. 1. Establishing a host cell containing a replicable expression vector encoding the gene fusion by standard procedures Transfection and production of phage particles allows the fusion protein to be incorporated into the phage particles. Phage particles are provided that are displayed on the surface of the

[0149] Replicable expression vectors are introduced into host cells using a variety of methods. Alternatively, the vector can be introduced into cells using the following: Incubate at about 37°C for about 6-48 hours, if necessary. Grow the cells in standard culture broth for 1 hour (or until OD600 = 0.6-0.8). After growing the cells in PBS, the broth is centrifuged and the supernatant is removed (e.g., decanted). The initial purification is preferably carried out in a buffer solution (e.g., 1.0 mM HEPES pH 7.4 The cell pellet was resuspended in PBS, followed by recentrifugation and removal of the supernatant. Resuspend the collected cell pellet in diluted glycerol (e.g., 5–20% v / v) and resuspend again. Centrifuge to form a cell pellet and remove the supernatant. The final cell concentration is determined by the cell pellet concentration. obtained by resuspending in water or diluted glycerol to the desired concentration do.

[0150] By using a higher DNA concentration (approximately 10-fold) during electroporation, It increases the transformation efficiency and the amount of DNA that is transformed into the host cells. Using a lower cell concentration increases the efficiency (approximately 10-fold). The higher the amount, the greater the diversity and the greater the number of unique members of the combinatorial library. A larger library representing specific members is generated. Typically, the bacteria are selected by growing them on antibiotic-containing medium.

[0151] Pharmaceutical Compositions and Uses According to the present invention, the antigen-specific antigen-binding molecule or multidomain-specific binding molecule of the present invention A pharmaceutical composition comprising the antigen-specific antigen-binding molecule is provided. Includes fusion proteins.

[0152] The pharmaceutical composition also includes an antigen-specific antigen-binding molecule of the present invention fused to a therapeutic protein, or fragments thereof. Therapeutic proteins may include hormones, growth factors (e.g., T GFβ, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (N GF), colony-stimulating factor (CSF), hepatocyte growth factor, insulin-like growth factor, placenta formation factor length factors); differentiation factors; blood clotting factors (e.g., factor VIIa, factor VIII, factor IX) factor, von Willebrand Factor or Protein C); or Other proteins from the blood clotting cascade (e.g., antithrombin); cytokines , for example, interleukins (e.g., IL1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 9, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL -26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 also or IL-33), or interferons (e.g., IFN-α, IFN-β, and IFN-γ), tumor necrosis factor (TGF), IFN-γ-inducing factor (IGIF), bone morphogenesis Proteins (BMPs, e.g., BMP-1, BMP-2, BMP-4, BMP-5, BMP P-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP- 12, BMP-13); interleukin receptor antagonists (e.g., IL-1 ra, IL-RII); chemokines (e.g., MIP (macrophage inflammatory protein) ), such as MIP1α and MIP1β; MCP (monocyte chemotactic protein), such as MCP1, 2, or 3; RANTES (regulated by expression and secretion of activated, normal T cells) induced upon activation by normal T-cells (expressed and secreted); trophic factors; sites kine inhibitors; cytokine receptors; enzymes, e.g., free radical scavenging enzymes (free-radical scavenging enzyme), e.g., superoxide dismutase or catabolite enzymes or prodrug-converting enzymes (e.g., angiotensin-converting enzymes, deaminases) , dehydrogenases, reductases, kinases and phosphatases); peptidomimetics (peptide mimetic); protease inhibitor; tissue inhibitor of metalloproteinases tRNA (TIMP, e.g., TIMP1, TIMP2, TIMP3, or TIMP4) or may be a serpin (an inhibitor of serine proteases).

[0153] In other embodiments of the invention, the therapeutic protein in the fusion protein is an antibody, or ab, Fc, F(ab')2 (including chemically linked F(ab')2 chains), Fab', scFv (including its multimeric forms, i.e., di-scFv, or tri-scFv) ), sdAb, or BiTE (bispecific T cell engager), Antibody fragments may also include variable domains and fragments thereof, as well as other Contains VNAR-type fragments (IgNAR molecules).

[0154] Pharmaceutical compositions may contain multiple antigen-specific antigen-binding molecules of the invention, e.g., dimers, trimers, or or higher multimers, i.e., 2-, 3-, 4-, 5-, 6-, 7-, or 8-mers, are therapeutic proteins. It can be constructed by fusion with a protein.

[0155] The fusion of the antigen-specific antigen-binding molecules of the present invention to therapeutic proteins can be carried out by any The cleavage may be performed at any convenient site, including N-terminal, C-terminal and / or N- / C-terminal fusion. In one embodiment of the present invention, the fusion of the antigen-specific antigen-binding molecule of the present invention may be , directed to both the N- and C-termini of the therapeutic protein.

[0156] The pharmaceutical compositions of the present invention may be prepared in the presence of suitable pharmaceutically acceptable carriers, diluents, adjuvants or The composition may include a buffer solution. The composition may include an additional pharmaceutically active agent. Such carriers include saline, buffered saline, dextrose, liposomes, water, and These may include, but are not limited to, glycerol, ethanol, and combinations thereof. do not have.

[0157] Such compositions may contain additional pharmaceutically active agents as indicated. The agent may be a therapeutic compound, such as an anti-inflammatory, cytotoxic, cytostatic or antibiotic agent. Such additional agents may be in a form suitable for administration to a patient in need thereof. The components may be present in a variety of dosage forms, and such administration may be simultaneous, separate, or sequential. It may also be prepared in the form of a kit which may optionally include instructions.

[0158] Pharmaceutical compositions can be administered, for example, orally, topically, intravenously, intramuscularly, intranasally, or intradermally, among others. Administered in any effective and convenient manner effective to treat the patient's condition, including administration by any route In therapy or prophylaxis, the active agent can be administered in an injectable composition. The compositions may be administered to an individual as a liquid, for example, as a sterile, preferably isotonic, aqueous dispersion.

[0159] For administration to mammals, particularly humans, the daily dose of active agent is 0.01 mg / kg body weight or more, typically about 1 mg / kg or more, 2 mg / kg or more, or 4 mg / kg In any event, it is up to the physician to determine the actual dosage that will be most appropriate for an individual. However, this amount will depend on factors including the age, weight, sex and response of the individual. The dosages given are illustrative of the average case. Of course, higher or lower dosages may be effective. There may be cases where this is beneficial, and such cases are within the scope of the present invention.

[0160] According to the present invention, an antigen-specific antigen-binding molecule or multimeric antibody of the present invention for use in medicine is provided. Domain-specific binding molecules are provided. Thus, this aspect of the invention is directed to those in need thereof. an antigen-specific antigen-binding molecule of the invention in the manufacture of a medicament for the treatment of a disease in a patient suffering from the disease; Such uses of the antigen-specific antibodies of the present invention extend to the use of a multi-domain or multi-domain specific binding molecule. The original binding molecule may also be a specific binding molecule as defined above in connection with the pharmaceutical compositions of the present invention. The method can be used to prepare fusion proteins containing the polypeptide.

[0161] Such uses also include methods of treating a disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the compound of the present invention. The antigen-specific antigen-binding molecules or multidomain-specific binding molecules defined herein include and administering to a patient a therapeutically effective dosage of such a pharmaceutical composition. Includes.

[0162] As used herein, the term "treatment" refers to any treatment that benefits a human or non-human animal. The treatment of "non-human animals" in veterinary medicine includes any regimen that can provide benefit. livestock and companion animals (e.g., cats and dogs), including horses, and sheep; It extends to the treatment of farm / agricultural animals, including goats, pigs, cattle and members of the equine family. Whether for therapeutic treatment of a current condition or disorder, or for prevention The treatment may be for a genetic or acquired disease. The treatment may be for an acute or chronic condition. The treatment may be for inflammation and / or cancer. The antigen-specific antigen-binding molecule or multidrug-resistant molecule of the present invention may be a symptom / disorder associated with the antigen. The main specific binding molecules are osteoarthritis, scleroderma, kidney disease, rheumatoid arthritis, and inflammatory bowel disease. , multiple sclerosis, atherosclerosis, or any inflammatory disease The compounds may be used to treat a wide variety of disorders.

[0163] The antigen-specific antigen-binding molecules or multidomain-specific binding molecules of the present invention can also be administered to patients. The antigen-specific antigen-binding molecules or multimeric antigen-binding molecules can be used to investigate the nature of disease states in the The thiazide domain-specific binding molecules are useful for imaging diagnostics such as X-ray, gamma-ray, or PET scanning. To prepare an image of a diseased area in a subject's body using imaging techniques. Thus, the present invention provides a method for detecting antigen-specific antigens that are suitably detectably labeled. The binding molecule or multidomain-specific binding molecule is administered to the subject, followed by administration of the binding molecule or multidomain-specific binding molecule to the subject's body. and a method for imaging a site of disease in a subject, the method comprising: scanning the Alternatively, the molecule can be administered to a subject, and the subject can then undergo treatment after administration of the molecule. A test result may be obtained by analyzing a sample from the specimen. The method includes administering an antigen-specific antigen-binding molecule or a multidomain-specific binding molecule of the present invention. The present invention may include a method for diagnosing a disease or medical condition in a subject, including the diagnosis of a disease or medical condition in a subject. Multidomain-specific binding molecules are particularly useful for binding multiple antigens targeting different epitopes on the same antigen. This can be particularly useful in terms of diagnostic sensitivity when VNARs are used.

[0164] Measurement of binding Detection and measurement of VNAR binding to targets can be achieved using methods such as ELISA and surface plasmon resonance. This can be measured in a number of ways well known in the art.

[0165] Functional activity The VNARs of the present invention are capable of binding to molecules such as cytokines and mediating the biological effects of those molecules. Neutralization, binding to receptors that prevent ligand binding, or causing biological effects after binding It can work in many ways, including rubbing.

[0166] Methods for measuring the functional activity of a binding domain are known in the art.

[0167] The present invention is further illustrated by the following non-limiting examples.

[0168] Example 1: Isolation of specific antigen-binding VNARs A. TNF-binding VNAR Immunization and selection Nurse sharks [Ginglymostoma cirratum], 1% (w / v) tricaine methanesulfonate [MS- After achieving the desired level of anesthesia, the animals were placed in a container containing artificial seawater containing

[222] . The sharks were removed for immunization or bleeding. Complete Freund's adjuvant 250 μg of hTNFα emulsified in CFA was injected into the side of the shark using a 20-gauge needle. Soluble antigen in phosphate buffered saline (PBS) [ The samples were 0.45 μM sterile filtered and booster doses were administered intravenously via the tail vein at 4-week intervals. Fluid samples were collected from the tail vein in 200 μl of porcine heparin [1000 U / ml in PBS]. The blood samples were collected in a 30 ml syringe containing 10 ml of PBS. The blood samples were centrifuged at 2000 rpm for 10 minutes. Blood cells were separated from the plasma. The plasma supernatant fraction was placed in a sterile tube containing RNA stabilizing buffer. The mixture was carefully removed and stored at -80°C.

[0169] Detection of hTNFα-specific IgNAR in shark serum ELISA plates were coated with 1 μg / ml rhTNFα and incubated at 37°C for 1 hour. After incubation, the plate was blocked in 4% (w / v) MPBS at 37°C for 1 hour. Shark serum (pre-bleed, bleeds 4 and 5) was diluted 1:2 as specified The plate was diluted 1:2 in PBS and incubated at 37°C for 1 hour. 100 μl / well of purified anti-Nurse shark IgNAR monoclonal antibody at a dilution of 0.00. The binding signal was measured with 0.1% (v / v) of the monoclonal antibody [GA8]. Anti-mouse IgG-H at a dilution of 1:2000 in Tween-20 PBS (PBST) The plates were incubated at room temperature for 1 hour. After the procedure, the cells were washed three times with PBST and then incubated with anti-mouse IgG-horseradish peroxidase (HRP) ) After incubation with conjugated antibody [Sigma], the cells were washed three more times with PBS. SureBlue TMB Microwell Peroxidase Substrate Peroxidase Substrate [Thermo Scientific] was added. The plate was developed with 1M H2SO4, and the reaction was stopped with 1M H2SO4. The absorbance was measured at a wavelength of 450 nm using a chromatograph.

[0170] Sera obtained after each boost were used to perform binding ELISA. The rhTNFα-specific IgNAR responses after each immunization boost were then measured. The mouse monoclonal antibody was diluted as hybridoma tissue culture supernatant in PBS. The anti-nurse shark IgNAR antibody, GA8, was used as the detection antibody (Haines et al., 2005; Muller, et al. 2012). The results show that, as shown in breeds 4 and 5, immunization After placement, a convincing trend of increased IgNAR over time was observed, and the prebleed samples The background response seen in the samples indicated that there was no significant rhTNFα response prior to immunization. This suggests the absence of a specific IgNAR response [Figure 1].

[0171] Total RNA isolation from PBLs and PCR amplification Peripheral blood lymphocytes (PBLs) were sorted into the breed with the best IgNAR response (Breed 5). Total RNA was prepared from the plasma of the collected PBLs. III First strand synthesis supermix [Invitrogen] was used as a template for cDNA synthesis. The framework-specific primers, NARF4Fo, r1[5'-ATA ATC AAG CTT GCG GCC GC A TTC AC A GTC ACG ACA GTG CCA CCT C-3' (SEQ ID NO: 74) and and NARF4For2 [5'-ATA ATC AAG CTT GCG GCC G C A TTC ACA GTC ACG GCA GTG CCA TCT C-3'] (SEQ ID NO: 75) was used to obtain cDNA (Dooley, H., et al., Mol. Immunol., 2003). 40(1): pp. 25-33). After cDNA synthesis, a consensus framework one-specific primer was inserted. The primers were NARF1Rev[5'-AT A ATA AGG AAT TCC ATG G CT CGA GTG GAC CA A ACA CCG-3' (SEQ ID NO: 76) was introduced and subjected to a three-step polymerase chain reaction ( IgNAR V-region DNA was amplified using a PCR amplification protocol. The 00 base pair PCR product was electrophoresed on a 1.5% agarose gel and the NAR V region was excised. The purified DNA was purified using the QIAquick purification kit (QIAGEN). I and NotI [New England Biolabs] with primers coding for the restriction sites [underlined]. Digested and repurified.

[0172] Library Construction The phagemid vector pHEN2 was digested with the restriction enzymes NcoI and NotI and PCR The product was purified [QIAquick PCR purification] and ligated to a similarly prepared PCR product. Materials were prepared by transfection with electroporation-competent E. coli TG1 cells (Electroporation-competent The transformed cells were incubated in 2% glucose solution. The plates were plated on TYE agar plates containing 100 μg / ml ampicillin and 100 μg / ml ethanol. The plates were grown overnight at 37°C. The library size was calculated and colonies were scraped from the plates. The library stock was then removed and aliquots were stored at -80°C.

[0173] Phage display selection OD 600 A single aliquot of the library stock, equivalent to a 0.1 saturation, was diluted in 2% glucose. 2xTY growth medium containing 100 μg / ml ampicillin, At 37 °C, mid-logarithmic phase [OD 600 After growing the cells until the β-saturation reached 0.4–0.6, M13K The plasmids were infected with 07 helper phage (New England Biolabs). Expression of the library was confirmed by ×TY medium, 0.2% glucose, 100 μg / ml ampicillin and 50 μg / ml The incubation was carried out in kanamycin at 30°C overnight. The phage was then incubated with polyethylene glycol (PEG). The library was precipitated from the culture supernatant with 1000 kJ / ml and used for bio-panning. Dynabeads® M-280 streptavidin beads [Dynabeads, Invi Panning was performed against biotinylated rhTNFα captured on [progen]. The gel and Dynabeads® M-280 streptavidin beads were In each well, with rotation at room temperature, add blocking solution [3% (w / v) milk in PBS, 1 % (w / v) BSA] for 1 hour. M] was added to the blocked beads and incubated for 1 hour with rotation at room temperature. In a separate tube, the library phage were incubated with pre-blocked streptavidin. The mixture was incubated with Dynabeads for 1 hour at room temperature with rotation. Unbound phages were collected using a magnetic rack (Dynabeads magnetic rack), and the collected phages were The phage is referred to here as the non-selected phage. Phages were deselected by incubation with beads. Biotin-rhTNFα-modified beads were incubated with unselected phages for 1 hour at room temperature with rotation. The beads were washed with 5x PBST and 5x PBS, and then incubated with 100 mM Elute with 400 μl of triethylamine (TEA) for 8 min and 1 M Tris-HCl The medium was neutralized by adding 200 μl of E. coli TG1 (pH 7.5). Cells [10 ml] were infected with 400 μl of eluted phage for 30 minutes at 37°C. TYE agar plate containing 2% glucose (w / v) and 100 μg / ml ampicillin The cells were grown overnight at 37°C on a 100% PBS-free plate. Three additional rounds of selection were performed to identify the cells with biotin-rhTN By reducing the concentration of Fα to 200 nM, stringency was increased in rounds 3 and 4. Increased frequency.

[0174] Clone screening and selection Enrichment of antigen-binding monoclonal phages by blocking with 4% [w / v] milk-PBS Evaluation was performed using ELISA plates coated with 1 μg / ml rhTNFα. Binding was detected using anti-M13-HRP-conjugated monoclonal antibody (GE Healthcare). In addition, the selectivity and affinity of the antibody to streptavidin- and HSA-coated ELISA plates were confirmed. The monoclonal phages were analyzed for their activity and specificity.

[0175] The library was subjected to four iterative panning rounds against rhTNFα. The starting concentration was kept constant in rounds 1 and 2, but the concentration was adjusted to favor high affinity binders. The number of positive monoclonal phage binders was halved in the next round of panning to prevent further degradation. Enrichment was performed on each of the biopanning for rhTNFα binding by ELISA. Preselected clones were evaluated for antigen binding at the end of round 2. A steady increase was observed, with a decrease in the number of monoclonal phage binders after rounds 3 and 4. The rhTNFα monoclonal binders were selected from round 0 [preselected library ] to about 6% [11 / 184] after round 1, and 99.46% [183 / 184] after round 2. increased.

[0176] A number of unique sequences were identified from the library panning. These included D1, C There are VNARs designated B4 and B5.

[0177] [ka]

[0178] Cysteine ​​(C) residues (double underlined) in CDR1 and CDR3 are type II Typical for VNARs, the marker between cysteines (single underlined) in FW1 and FW3b canonical immunoglobulin superfamily bridges It is observed that the ATP forms a second disulfide bridge in addition to the first disulfide bridge.

[0179] Expression of VNARs that bind to TNFα Preparation of soluble VNAR proteins in the cytoplasm of SHuffle cells IgNAR V-region inserts of interest identified from monoclonal phage screening The sigma-binding fragment was inserted into the expression vector pET28b( +) (Novagen). VNAR DNA was cloned into the QIAprep miniprep kit (QIAAGE). N) prepared from E. coli TG1 culture and in-house designed p Rimer pair XbaI_NARFW1_#127 (SEQ ID NO: 26) and Ec oRI_stop_myc_#129 (SEQ ID NO: 29) were used for PCR amplification, The primer SEQ ID NO: 29 introduced the cloning sites XbaI and EcoRI. c-myc (primer SEQ ID 29 incorporated c-myc), 6x histidine tag and a stop codon were introduced into the VNAR gene sequence. Purified VNAR DNA PCR The product and pET28b(+) plasmid DNA were digested with 50 U XbaI and 10 U E The digested sample was purified and ligated with coRI-HF for 2 hours at 37°C. , electrocompetent E. coli SHuffle® T7 Express cells [N New England Biolabs] and plated on TYE agar containing 50 μg / ml kanamycin. VNAR anti-hTNFα-D1, C4 and B4 fusion proteins were selected on the Expressed in the cytoplasm of SHuffle® cells upon induction with IPTG at 0°C. The cells were harvested by centrifugation and the cell pellet was soaked in Bugbuster™ Cells were lysed and soluble proteins were released by treatment with protein extraction reagent [Novagen]. VNAR soluble proteins were attached to immobilized metal affinities via the hexa-histidine tail. Purify by affinity chromatography (IMAC) in 500 mM imidazole, pH 8. Protein samples were eluted from the IMAC resin and diluted with PBS (pH 7.4) prior to use. The protein concentration was measured using an Ultraspec 6300 pro UV / Visible spectrophotometer [Ame The total purified protein was stained with Coomassie blue using a standard immunoassay (SD) [Sham Biosciences]. The purified VNAR monomer protein was visualized by S-PAGE. Migrating as a band [containing hexahistidine and c-myc tags], protein aggregates The purity was estimated to be approximately 90% based on SDS-PAGE gel.

[0180] Determination of protein integrity and purity Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] The purity and size of the purified proteins were assessed using the following method. Protein samples were diluted to 5% NuPAGE® LDS Sample Buffer containing β-mercaptoethanol The denatured protein samples were prepared in a centrifuge tube (Life Technologies) and heated to 95°C for 5 minutes. The gel was then loaded onto NuPAGE® 4-12% Bis(2-methyl-2-methyl-2-propanol) gels immersed in MES SDS running buffer. The gel was loaded onto a Tris gel [Life Technologies] and electrophoresis was carried out at 160 volts for 55 minutes. Full range recombinant protein molecular weight markers [GE Healthcare] were used as molecular weight ladder standards. The gel was washed with distilled water, stained with Coomassie blue for 1 hour, and then washed with distilled water. Destaining was carried out overnight in water.

[0181] Determining selectivity and specificity The specificity and selectivity of binding were determined using 1 μg / ml biotin-TNF and rhTNFα, or 10 μg / ml HSA, BSA, streptavidin, single-stranded DNA, syrup ELISA plates coated with either globulin or lysozyme. The ISA plate was properly blocked with 4% [w / v] milk-PBS to remove the protein. The antibody sample was loaded at a top concentration of 1 μg / ml and serially diluted. Binding was detected using a P-binding monoclonal antibody [Roche].

[0182] To obtain more accurate binding data, we also used BIACore T200 or Octe Specific molecules were measured using surface plasmon resonance with a t RED96 instrument.

[0183] BIACore TM T200 (GE Healthcare) Amine coupling is a very common approach for immobilizing ligands on chip surfaces. The chip surface is coated with N-hydroxysuccinimide (NHS) and 1-ethyl After activation with diethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Covalent capture of ligands via available primary amine groups (e.g., lysine) on the ligand Covalent capturing of carboxyl groups to form reactive succinimide esters The dextran matrix is ​​derivatized with

[0184] TNFα was diluted 1 / 10 with 10 mM sodium acetate buffer (pH 5.5) to determine the activity. The "aim for" software fixation wizard Immobilization of 200RU using the software immobilization wizard or for a specific period Furthermore, detergents probably affect ligand activity. Therefore, the running buffer was changed to P without 0.05% Tween 20. BS. A final ligand immobilization level of 202 RU was obtained.

[0185] The start-up cycle consisted of a 60-second buffer injection at a flow rate of 30 μl / min, followed by a 30-second The anti-TNFα sample cycle consisted of a 100 μl / min dissociation period followed by a 100 μl / min dissociation period. After injecting at 1000kJ / min for 120 seconds, inject 10 mM glycine (pH 2) at 30 μl / min for 60 seconds. A regeneration step was included. Finally, a baseline equilibration was allowed before starting the next cycle. A 120 second stabilization period was included at the end of each cycle to allow for accurate measurement of the variability in the variability of the variability.

[0186] The concentration series and dissociation period screened were variable and were as follows: Dissociation The time was 600 seconds for all monomer domains D1, C4, B4, TNF43 and T All samples, except for NF30, were incubated at a top starting concentration of 100 nM and 120 B4 and TNF43 VNARs were assayed at 500 nM each at 0 s dissociation time. and 5 μM top starting concentration. Five blank sample cycles were performed. were used to generate a double-referenced dataset.

[0187] Binding responses to the domains were evaluated using BIACore™ T200 evaluation software. The double-referenced data were analyzed using a 1:1 Langmuir model to determine the kinetic and The affinity characteristics were obtained.

[0188] OCTET® RED96 [ForteBio®] Biolayer interferometry (BLI) was used to determine the equilibrium dissociation constant ( K D ) was determined using a dip and read streptavidin biosensor (Dip and read Rehydrate the PBS (e.g., streptavidin biosensors) in PBS (pH 7.4) for at least 30 minutes. The sensor was loaded with 20 μg / ml of biotinylated hTNF-α, and anti-TNF-α VN AR protein was serially diluted at a top concentration of 100 nM, while TNF43 and VNA The negative control was assayed at a top concentration of 1 μM. Binding association was assessed as 1 The dissociation time was monitored for 0 min, followed by 5 min. All anti-TNF-α V For NAR measurements, the curve fit data showed a complex multiphase curve, so the kinetic data The set was fitted using a two-site model, whereas the control anti-TNF -α nanobody and TNF30, 1 in the mass transport model :1 Langmuir coupling was used.

[0189] The data obtained are shown in Table 1. From Table 1, it can be seen that the monomeric VNARs tested are TNF-. have at least 500-fold lower binding affinity for TNFα compared to NAR show.

[0190] [Table 1]

[0191] In vitro neutralization assay Neutralizing capacity and ND for VNAR domains50 To determine the Cell line L929 [ATCC, CCL-1] was cultured in 10% heat-inactivated fetal bovine serum [GIBCO] and Dulbecco's modified Eagle's medium supplemented with 1 μg / ml actinomycin D [R & D systems] Each VNAR clone was grown in 5% PBS per well. 1,000 cells were plated in a 96-well plate at 37°C with 5% CO2 and humidity for 24 hours. Plates were incubated in duplicate. 50 [1x at 0.25ng / ml] and 10x LD 50 rhTNFα [2.5ng / ml] was administered to a 1000-well plate containing serially diluted VNAR proteins. The plates were then incubated at 37°C for 2 h. Incubate for 4 hours in 5% CO2 and humidity. 50 μL of 1:20 diluted WST Add 1 cell proliferation agent [Roche] and incubate at 37°C with 5% CO2 and humidity for 4-8 hours. Cytotoxicity or cell viability was measured by incubating the cells. The reading was taken at 0 nm.

[0192] TNFα in the presence of 1 μg / ml actinomycin D inhibited the growth of L929 fibrosarcoma cells. In this case, LD 50 The range is 0.25 to 0.3 ng / ml. We found that VNAR protein domains at nanomolar concentrations inhibited LDs of rhTNFα. 50 Up to The results showed that the control anti-TNFα antibody neutralized the TNFα signaling 10-fold (Figure 3). To form a bivalent molecule for comparison with MAB210, the C-terminus of the VNAR was ligated to a peptide. It was attached to the IgG Fc domain by a linker.

[0193] When measured as single domains in neutralization assays, D1 and C4 VNARs However, it was not as effective as the TNF30 VHH nanobody (Figure 2). However, as a mixture (Figure 3) or in a bivalent or When combined in a bispecific format (Figure xxxx), unexpectedly, They showed improved properties over dimeric TNF30 VHH nanobodies.

[0194] Paracellular flux assay Human epithelial colorectal adenocarcinoma cells (Caco-2) were cultured in 10% (v / v) heat-inactivated FBS. and 1% (v / v) penicillin-streptomycin (10,000 units / The cells were cultured in DMEM supplemented with 1000 μg / ml of ATP and 10,000 μg / ml of ATP. After growing in Lasco to 90% confluence, the cells were plated onto 24-well plates containing 0.4 μm semipermeable plates. Semipermeable tissue culture transwell inserts (C The cells were seeded onto a plate (Borning Inc.). 10 μl of the cell suspension was diluted with 90 μl of 0.4% trypan blue. The cells were suspended in trypan blue exclusion dye (Beckman Coulter) and the coverslip was removed. The number of viable cells was determined by carefully transferring the mixture to an attached hemocytometer.

[0195] After measuring the viable cell number, 1 x 10 cells were cultured per transwell insert. 5 10 cells 600 μl DMEM without cells was added to the outer layer, while 600 μl DMEM without cells was added to the outer layer. The transwell plate was then filled with 5% (v / v) v) Incubate at 37°C with CO2, and add used DMEM + 10% (v / v ) FBS was replaced every 48 hours. Cell proliferation was monitored until cells reached 100% confluence. (usually 5 to 7 days after seeding) and monitor under a phase contrast microscope (40x objective lens). The Caco-2 cells were grown and differentiated for an additional 21 days, during which time the spent medium The medium was changed every 48 hours until differentiation.

[0196] Polarized cells (pol) were cultured in 100 μl of DMEM containing 10% (v / v) HI-FBS. Selected insert wells containing irradiated cells (apical side) were treated with anti-TNF 10 ng / ml hTNFα, IFNγ, with or without α VNAR protein; The treated cells were incubated at 37°C in 5% (v / v) CO2 for 18 hours. After 18 hours of incubation with cytokines ± anti-TNFα VNAR, Phase contrast images of the cells were obtained, followed by imaging of the apical side (insertion side) of the Caco-2 monolayer. Add 5 μl of 10 mg / ml fluorescein isothiocyanate-labeled dextran to the well. FITC-dextran was added at 24 hours. After a period of time, the medium on the basolateral side of the transwell chamber was collected.

[0197] Fluorescence intensity was measured using a Synergy HT (BioTek®) microplate reader. Measurements were taken using a 485 nm excitation and 520 nm emission wavelengths.

[0198] Epithelial resistance dysfunction assay Human epithelial colorectal adenocarcinoma cells (Caco-2) were cultured in 10% (v / v) heat-inactivated FBS. and 1% (v / v) penicillin-streptomycin (10,000 units / The cells were cultured in DMEM supplemented with 1000 μg / ml of ATP and 10,000 μg / ml of ATP. After growing in Lasco to 90% confluence, 12 or 24 wells of 0. 4 μm semipermeable tissue culture transwell inserts The cells were seeded onto swell inserts (Corning Inc.). The above protocol was continued until the cells were fully differentiated. The same procedure was followed as for Le.

[0199] Polarized cells (pol) were cultured in 100 μl of DMEM containing 10% (v / v) HI-FBS. Selected insert wells containing irradiated cells (apical side) were treated with anti-TNF 10 ng / ml hTNFα with or without α VNAR protein, and IF The treated cells were incubated at 37°C with 5% (v / v) CO2 and humidity for 24 hours. Incubated with cytokines ± anti-TNFα VNAR for 24 hours. After that, transepithelial electrical resistance (TEER) was measured. llicell(R) ERS-2 Epithelial(Volt / Ohm) The apical chamber was then probed with a meter and a MERS STX01 probe (Merck Millipore). The measured resistance was normalized to the surface area under treatment.

[0200] In a 12-well tissue culture transwell insert, add 500 μL of DMEM containing 5 x 10 per well 6 Cells were seeded in the outer well (basolateral side). It is important to note that the TEER measurement was performed using a 1.5 mL DMEM solution. During the determination, the DMEM volume in the insert wells and outer wells was increased to 500 μl and 500 μl, respectively. and 1.5 ml, and volt-ohm meter electrodes ) was completely immersed in the medium without touching the bottom of the well.

[0201] B.ICOSL combined VNARS The isolation and characterization of ICOSL-binding VNARS 2D4 and CC3 are described in WO201 4 / 173975 and WO2014 / 173959.

[0202] 2. Formation of Multivalent and Multispecific VNARs A. TNF-binding domain Figure 3 shows that the combination of D1-Fc and C4-Fc molecules in a bivalent form increases the neutralizing capacity. Therefore, VNAR DNA1 and C4 and other combinations The combinations may be prepared as bivalent or bispecific fusions and when combined together as a fusion It was shown that the same improvement in neutralizing capacity was observed when

[0203] Dimer and trimer construction FIG. 4 provides a diagram of the format of bivalent and bispecific constructs.

[0204] Set up two or three separate PCR reactions using the oligonucleotide combinations listed below. Using the combination of Each oligonucleotide is amplified and has a specific sequence for easy purification and detection. / unique cloning site and / or 6xHis tag and c-Myc tag do.

[0205] [ka]

[0206] Trimer-assembled PCR oligonucleotides: here, X on the N-terminus and C-terminus, respectively an intermediate fragment flanked by the ba1 / BamH1 and APA1 / EcoR1 cloning sites; An in-house designed DNA cassette carrying the BA11 gene was used as a vector. The oligonucleotide pairs listed above, as well as the N-terminal forward oligonucleotide An oligonucleotide having both Xba1 and BssH11 sites in the oligonucleotide The PCR amplification step allows for in-house eukaryotic expression. Trimeric gene into the in-house eukaryotic expression vector, pEEE2A All other cloning was performed using pET28 carried out in the b(+) expression vector.

[0207] [ka]

[0208] 2 μl VNAR DNA (50–100 ng), 2 μl forward and reverse Oligonucleotide primers (final concentration 1 μM), 5 μl of 10X Taq polymerase 0.25 μl of Taq polymerase (final concentration 25 U / ml), 0.5 μl of PCR reaction containing dNTPs (final concentration 0.1 mM), and 38.25 μl of HO ( The final reaction volume was 50 μl. The PCR program was started at 98°C for 5 minutes. 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute were performed. A final extension of 5 min was performed at 1000 kJ / min. The amplicons were checked by agarose gel electrophoresis. The eluted DNA was purified using a QIAquick PCR purification kit. digested with endonucleases.

[0209] Dimerization and cleavage in E. coli SHuffle® T7-expressing cells Expression of α- and β-trimers Cloned into the expression vector pET28b(+) via the XbaI and EcoRI restriction enzyme sites. The cloned VNAR region and purified plasmid containing the resulting VNAR gene The resulting mixture was transferred to electrocompetent E. coli SHuffle ( le)® T7 expressing cells [NEB] were transformed and cultured in a medium containing 50 μg / ml kanamycin. Anti-hTNFα VNAR-D1, C4, and B4 fusion proteins were selected on TYE agar plates. The fusion protein was expressed in the cytoplasm of SHuffle cells upon induction with IPTG at 30°C. It was expressed.

[0210] A single colony of transformed E. coli SHuffle® T7 expressing cells was cultured at OD 60 Until the ink reaches 0.4-0.6 (usually 37°C, 250 rpm, 4-6 hours) The log-phase culture was grown in 5 mL of 2xTY-kanamycin medium. The mixture was used to inoculate 50 mL of TB medium containing kanamycin and PO4 salts, and the OD 60 The mixture was incubated overnight at 30°C and 250 rpm until the pH reached 6.0-10.0. The cells were centrifuged at 4000 rpm at 30°C for 15 minutes and then resuspended in fresh TB-kanamycin- The cells were resuspended in PO4 salts medium and allowed to recover for 1-2 hours at 30°C and 250 rpm. Induce cytoplasmic protein expression using a concentration of 1 mM and culture the cells for 12-16 h after induction, 30 Incubated at 200 rpm at 25°C. Centrifuged at 6000 rpm for 10 minutes at 25°C. The cells were harvested by centrifugation and the wet weight of the cell pellet was measured. Wet Cell Paste BugBuster™ Protein Extraction Reagent + Benzonase (registered trademark) (Novagen, UK), and the cell suspension was incubated at 10-15 rpm for 20 minutes at room temperature. The cell suspension was centrifuged at 6000 rpm for 20 minutes at 4°C. The soluble proteins separated and collected in the supernatant were subjected to immobilized metal affinity chromatography. Immobilized metal affinity chromatography (IMAC) resin (nickel- Polyhistidine tag using nitrilotriacetic acid, Ni-NTA or Ni-Sepharose The VNAR fusion proteins were prepared for affinity purification by HPLC. Elute with 0 mM imidazole (pH 8.0) and resuspend the eluate in PBS (1 L PBS / 1 ml After overnight dialysis against eluted protein (pH 7.4), the PBS was replaced and the solution was further dialyzed for 3 The protein was dialyzed for 4 hours. Protein quality was assessed by SDS-PAGE and purified using an Ultraspec 6300. Using a pro UV / visible spectrophotometer (Amersham Biosciences, GE Healthcare) Quantitative analysis was performed.

[0211] For eukaryotic expression, the domains cloned into the BA11 trimer cassette were expressed as Bs Digested with sHII and EcoR1 enzymes, and transformed with pEEE2 containing the CMV promoter. A. Subcloning into a eukaryotic expression vector and transforming into an E. coli strain for plasmid propagation The isolated and purified plasmid vector containing the VNAR trimer gene was then transformed into a linearized poly(A) The plasmid DNA was incubated with polyethyleneimine (PEI) for 20 minutes at room temperature. The EI mixture contained HEK293 cells with a cell growth density of 90% confluence. The transfected HEK293 cells were then transferred to a cell culture flask containing 100 ml of PBS. The cells were incubated in a humidified atmosphere at 25°C for 5–7 days. The cell culture supernatant was collected and the IMAC resin was applied. The expressed protein was purified using and dialyzed against PBS.

[0212] Binding and TNF Neutralization Data FIG. 5 shows ELISA binding of dimeric and bispecific constructs to TNFα.

[0213] Initial ELISA data demonstrate that the bispecific D1-C4 construct inhibits TNF30 nanobody dual binding. Increased avidity (combined with binding affinity) compared to the dimeric construct It was shown that...

[0214] Some of these were subsequently tested for binding to immobilized TNF in surface plasmon resonance. and measured.

[0215] From Table 1, it can be seen that of the dimeric molecules measured, the D1-C4 bispecific molecule is the most potent TNF30 bispecific molecule. showed superior binding affinity (avidity) compared to the monovalent nanobody constructs. It is shown that:

[0216] Figure 6 shows a number of bivalent or bispecific VNAR fusions compared to bivalent TNF nanobodies. TNF neutralization data for the L929 assay are shown. When tested for saturation, the D1-C4 dimer exhibited the same activity as the TNF30 nanobody dimer construct. In this experiment, the D1-D1 dimer was superior to the D1-B4 dimer. was also inferior.

[0217] Table 1 shows the SPR binding data for the trimeric constructs tested. The introduction of an additional domain, which acts as a spacer between the α-binding domains, enhances the binding of TNFα to It has been shown that this appears to significantly improve the relative affinity (avidity) of the molecules involved. will be done.

[0218] The D1-BA11-C4 trimeric construct inhibited adal TNFα neutralization as measured by a TNFα neutralization assay. The assay was comparable to that of imumab and superior to the TNF30 nanobody construct. In this study, bivalent molecules containing the D1 domain were found to be as effective as TNF30 nanobody constructs. etc.

[0219] Figure 7 shows the results of experiments measuring the ability of different VNAR formats to neutralize TNFα function. show.

[0220] Table 2 summarizes the neutralization data. When the spacer domain is included, D1-BA11 Both D1 and D1-BA11-C4 showed a 10-fold or greater improvement in neutralizing potency, D1-BA11-C4 showed efficacy similar to that of adalimumab and MAB210. In addition, TNF30-BA11-TNF30 is more potent than the TNF30-TNF30 dimer form. The D1-C4 construct (SEQ ID NOs: 27 and 28) shows an improvement over the The GlySer linker length of (Gly4Ser)2 has been extended to (Gly4Ser)3. This resulted in improved hTNF-α neutralization ability.

[0221] Data from these experiments are shown in Table 2. Further comparative data is shown in Table 3.

[0222] [Table 2]

[0223] [Table 3-1]

[0224] [Table 3-2]

[0225] Functional activity background Human epithelial colorectal adenocarcinoma cells (Caco-2) are suitable platforms (e.g., When grown on plastic dishes or nitrocellulose filters, normal intestinal cells Furthermore, collagen-coated polycarbonate or Polyester membranes are suitable for Caco-2 monolayers as a model system for intestinal epithelial transport. It has been shown that (Wang, F., et al. Am. J. Path. 166.2 (2005): 409-419.; Hidalgo, IJ, et al Gastroenterology 1989. 96: 736-49.).

[0226] The main function of the epithelial membrane is to provide a barrier to hydrophilic solutes such as inulin and dextran. This barrier is maintained by, but not limited to, infectious, immune-mediated and idiopathic It is impaired in certain diseases that involve the intestinal epithelium, such as inflammatory bowel disease (Clayburgh, DR, et al. Lab In vest. 2004. 84(3): 282-291; Wang, F., et al. Am. J. Path. 2005. 166(2): 409-419) Intestinal barrier dysfunction (int), measured as increased paracellular permeability and decreased intestinal epithelial resistance Esophageal barrier dysfunction (ESB) is closely related to inflammatory bowel diseases (IBD), such as Crohn's disease. (Irvine EJ and Marshall JK, Gastroenterology 2000. 119.6: 1740-1744.; Wyatt et al., The Lancet 1993. 341(8858): 1437-1439.) As a result, leakage diarrhea ( The epithelial tight junction in IBD contributes to the loss of solutes that results in leak flux diarrhea. Evidence supports a decrease in epithelial tight junction proteins (Schulzke JD et al., Ann NY Acad Sci. 2009 1165:294-300; Schmitz H. et al., J Cell Sci 1999. 112(1): 137-146). Finally, interferon-γ (IFN-γ), TNFα and lipopolysaccharide (LPS) correlate with intestinal epithelial barrier dysfunction in human epithelial cell lines. It has been shown that the IL-11 receptor agonist induces IL-11 receptor agonist activity in a multiplicative manner (Wang et al., J. Cell Science 1999. 112(1): 137- 146; Schuerer-Maly CC et al., Immunology 1994. 81(1): 85).

[0227] Anti-TNFα treatment has been shown to restore intestinal barrier dysfunction in Crohn's disease (S Uenaert P. et al., Am J Gastroenterol 2002. 97(8): 2000-2004), therefore, we Our anti-TNF VNAR domains repair these dysfunctions induced in vitro. We tested anti-TNF VNAR domains to demonstrate their potential for bispecific / multispecific binding. The valence of VNAR domains prevents these dysfunctions compared to VNAR monomers. It was assumed to be valid.

[0228] FITC-dextran paracellular flow across polarized monolayers of Caco-2 cells (FITC- Dextran paracellular flux across polarized monolayer of Caco-2 cells) Human epithelial colorectal adenocarcinoma cells (Caco-2) were cultured in 10% (v / v) heat-inactivated FBS and and 1% (v / v) penicillin-streptomycin (10,000 units / ml each). The cells were cultured in DMEM supplemented with 1000 μg / ml of niacin and 10,000 μg / ml of niacin. After growing the cells to 90% confluence in a 24-well plate, the cells were placed in a 0.4 μm semipermeable tissue culture medium. Semipermeable tissue culture transwell inserts (Corni 10 μl of the cell suspension was added to 90 μl of 0.4% trypan blue exclusion dye. The mixture was then applied to a coverslip. The cells were carefully transferred to a hemocytometer equipped with a filter.

[0229] After measuring the viable cell number, 1 x 10 cells were cultured per transwell insert. 5 10 cells 600 μl DMEM without cells was added to the outer layer, while 600 μl DMEM without cells was added to the outer layer. The transwell plate was then filled with 5% (v / v) v) Incubate at 37°C with CO2 and humidity, adding used DMEM + 10 The % (v / v) FBS was replaced every 2 days. Cell proliferation was monitored until the cells reached 100% confluence. The cells were monitored under a phase contrast microscope (40x objective lens) until they were fully grown (usually 5-7 days after seeding). The Caco-2 cells were further grown and differentiated for 21 days, during which time the spent The medium was changed every 48 hours until differentiation.

[0230] Polarized cells (apical) were cultured in 100 μl of DMEM containing 10% (v / v) HI-FBS. The designated insert wells containing the anti-TNFα VNAR protein were placed in the wells containing the anti-TNFα VNAR protein. The treated cells were treated with 10 ng / ml hTNFα, IFNγ, or LPS with or without IFNγ. The cells were incubated for 18 hours at 37°C with 5% (v / v) CO2 and humidity. ±Phase contrast images of treated cells after 18 hours of incubation with anti-TNFα VNAR. and subsequently 5 μl of the solution was applied to the apical side (insert well) of the Caco-2 monolayer. 10 mg / ml fluorescein isothiocyanate-labeled dextran, molecular weight (3-5 kJ) 24 hours after the addition of FITC-dextran, the transwell The medium on the basolateral side of the chamber was collected.

[0231] Fluorescence intensity was measured using a Synergy HT (BioTek®) microplate reader. Measurements were taken using a 485 nm excitation and 520 nm emission wavelengths.

[0232] Figure 8 compares several TNF VNAR multidomain binding molecules. Paracellular flux across a two-cell polarized monolayer The permeability data from an experiment measuring the dimer or trimer concentrations is shown. Because the body has developed a higher level of protection for challenged cells, various It is shown that bivalent and bispecific forms exhibit improved function over monomeric forms.

[0233] Epithelial resistance dysfunction assay in polarized Caco-2 cell monolayers Human epithelial colorectal adenocarcinoma cells (Caco-2) were cultured in 10% (v / v) heat-inactivated FBS. and 1% (v / v) penicillin-streptomycin (10,000 units / The cells were cultured in DMEM supplemented with 1000 μg / ml of ATP and 10,000 μg / ml of ATP. After growing in Lasco to 90% confluence, 12 or 24 wells of 0. 4 μm semipermeable tissue culture transwell inserts The cells were seeded onto swell inserts (Corning Inc.). The cells were then allowed to stand for 10 min at 4°C until fully differentiated. The protocol was similar to that described above.

[0234] Polarized cells (pol) were cultured in 200 μl of DMEM containing 10% (v / v) HI-FBS. Selected insert wells containing irradiated cells (apical side) were treated with anti-TNF 10 ng / ml hTNFα with or without α VNAR protein, and IF The treated cells were incubated at 37°C with 5% (v / v) CO2 and humidity for 24 hours. Incubated with cytokines ± anti-TNFα VNAR for 24 hours. After that, transepithelial electrical resistance (TEER) was measured. llicell(R) ERS-2 Epithelial(Volt / Ohm) The apical chamber was then probed with a meter and a MERS STX01 probe (Merck Millipore). The measured resistance was normalized to the surface area under treatment. per well containing 500 µL of DMEM into tissue culture transwell inserts 5×10 6 1.5 mL of cells were seeded in the outer well (basolateral side). It is important to note that the DMEM contains 100% DMEM. The DMEM volumes in the inner and outer wells were adjusted to 500 μl and 1.5 ml, respectively. 1, and the volt-ohm meter electrodes The bottom of the container was completely immersed in the medium without touching it.

[0235] FIG. 9 shows an assay for measuring epithelial resistance in polarized Caco-2 cells. This experiment demonstrated that various bivalent and bispecific forms exhibit improved function over the monomeric forms. It is shown that it shows.

[0236] B ICOSL binding domain Construction and efficacy data of multivalent forms of the construct 2D4 and CC3 Fc fusions Figure 10 shows the ICOSL VNAR (and human IgG Fc, which is a further improvement For multivalent and multispecific VNARs of the invention incorporating The format is shown.

[0237] method PCR amplify the desired VNAR monomer domain and subclon it into a eukaryotic expression vector. This cloning was carried out by cloning a DNA fragment encoding human IgG1 Fc ( The IgG1 Fc fragment also contains the five-ply IgG1 Fc fragment that normally disulfide bridges the light chain to a serine-mutated IgG1 Fc fragment. The full-length human IgG1 hinge sequence containing the 5 prime most Cys residues was cloned. The primer was then placed on the 5' end of the primer (loaded).

[0238] During PCR amplification, oligonucleotides are used to identify the carboxyl groups of the VNAR domains. Amino acids that insert a linker sequence between the terminal end of the IgG1 and the N-terminal residue of the human IgG1 hinge region residues, as well as restriction endonuclease sites compatible with mammalian vector expression systems. The linker sequence introduced by this process consisted of the underlined RT amino acid residues. The nucleic acid sequence encoding introduces a BsiW1 restriction endonuclease site GGGGSGG GG RT or the codon usage of the underlined GADQ amino acid residues introduces the Bcl1 site. EnterGGGGSGG GADQ Both of these sites were either Fc nuclei or Compatible with cloning sites in different versions of biological expression vectors. Introducing a unique BssHII site at the 5' end of the plicon, making it compatible with eukaryotic vector construction do.

[0239] DNA sequence for introducing linker-VNAR domain fusions at the carboxyl terminus of Fc The sequence was designed and the synthesis of these intermediate fragments was carried out by GeneArt (Invitrogen). The N-terminus of these fragments is the naturally occurring BsrGI region within the CH3 region of human IgG1. The EcoR1 site in the vector was utilized. Between the carboxyl terminus and the amino terminus of the VNAR domain is the amino acid sequence TAAAAT AAAATAAAATA AAA The underlined triangles in the linker are The use of codons in the heavy alanine region allows for the introduction of a NotI restriction site, This will allow for subsequent cloning efforts to assemble additional bispecific VNAR constructs. It can be used in

[0240] PEI-mediated transcription in HEK 293 cell suspensions using serum-free medium Post PEI-mediated transfection and transient expression The expression levels of the NAR Fc fusion protein were measured by ELISA. After an initial 0.2 μm filtration clarification step to remove Protein A affinity chromatography was performed to purify the product. - A second chromatography step to complete the purified protein is optionally performed ion exchange chromatography or size exclusion chromatography by exchanging the buffer between Proteins were concentrated using an Amicon ultrafiltration unit and purified using U The final protein concentration was measured by V spectroscopy. Analytical SEC and SDS-PAGE was used to determine the integrity of the final purified protein.

[0241] ICOSL neutralization assay Ligand-receptor neutralization assays were performed as follows: Human ICOS receptor CHO cells expressing β-glucan were cultured in 96-well cell culture plates (Greiner, Bio-One) in DMEM. The cells were grown in F12 + 5% FBS medium until confluent. A total of 20 μl of rmB7-H2 / Fc (158-B7, R&D Systems) was added to DM with 40 μl of serially diluted anti-ICOSL-VNAR-Fc in EM / F12 + 2% FBS After pre-incubation at 16°C for 1 hour, the solution was added to the cells. Afterwards, the cells were gently washed three times with DMEM / F12 + 2% FBS and diluted 1:1 with the same medium. Incubate with goat anti-human Fc-HRP (SIGMA) diluted to 100,000 at 16°C for 40 minutes. The cells were washed and developed with TMB substrate.

[0242] Figure 11 shows ELISA binding data demonstrating that ICOSL VNARS bind to these formats. These results demonstrate that the antibodies bind to their cognate antigens in vitro.

[0243] Figure 12 shows a combination of TNF R1 domain, ICOSL VNAR and human IgG Fc. The present invention provides multivalent and multispecific VNAR formats incorporating these It provides improved functional properties.

[0244] Figure 13 shows a combination of TNF R1 domain, ICOSL VNAR and human IgG Fc. We present efficacy data for multivalent and multispecific VNARs of the present invention incorporating provides further improved functional properties.

[0245] These data suggest that the fusions are linked to TNF via TNFR1 and to the VNAR domain. It has been shown that it can bind to mICOSL and hICOSL via ATP. These constructs express mICOSL or hICOSL in the CHO-expressed receptors, which are their cognate receptors. It can inhibit binding to

[0246] These data suggest that VNARs combined in a multivalent format can bind to their targets. and the molecules can exhibit improved properties over monomeric VNARs. It is shown that:

[0247] In vivo preclinical studies Research Background The Tg197 mouse model of rheumatoid arthritis expresses wild-type and 3'-modified human tumor necrosis factor (TNF) It is a transgenic mouse line that carries and expresses a human tumor necrosis factor (hTNF-α) transgene. These transgenic mice rely on the overexpression of bioactive human TNF-α to Chronic polyarthritis develops at 4-7 weeks of age with a 100% incidence rate (Keffer et al. 1991, EMB O J., Vol. 10, pp. 4025-4031). The first anti-TNF-α therapeutic antibody, Remicad Exemplary of the therapeutic efficacy of IFN-γ and other anti-TNF-α biologics is the Tg197 mouse model. (Shealy et al., 2002, Arthritis Research & Therapy, 4(5) , p.R7; Shealy et al., 2010, MAbs (Vol. 2, No. 4, pp. 428-439). Taylor & Francis ).

[0248] The purpose of this study was to investigate the effects of arthritis on the Tg197 transgenic mouse model of arthritis. Anti-TNF-α D1-Fc compared to Humira® in preventing symptoms The objective of this study was to evaluate the therapeutic effect of C4 (Quad-X™).

[0249] method A total of 40 mice were assigned to each of five test groups, G1 to G5 (Table 4). For the purposes of the study, the transgenic mice were assigned to groups of 8 mice. , respectively, with test compound or vehicle buffer (phosphate buffered saline, PBS, pH 7.4) was administered subcutaneously twice a week from the age of 3 weeks before the establishment of arthritis. The transgenic mice (2 males and 2 females) were cultured for 7 weeks until they reached 10 weeks of age. An additional group of untreated animals (14 females) was compared with 3-week-old control mice for histopathological status. The animals were used as a control group and necropsied before the first dose.

[0250] Before the first arthritis scoring, mice were divided into groups based on age and sex. A balanced study included G1–G2 mice pooled from different litters of matings synchronized at weaning. It consisted of 8 heterozygous Tg197 mice (4 males and 4 females) in 5 groups. The allocation of mice to experimental groups was determined to ensure equal distribution of body weights among the different groups at the start of the study. In vivo arthritis scores were assessed as described in Table 5.

[0251] At 10 weeks of age, all animals were necropsied and blood from each animal was collected (serum was isolated and stored at -80°C). and two ankle joints were collected. The ankle joints of all experimental animals were dissected, calcified, and further Histopathological evaluation of arthritis was performed after treatment. Histopathology of the ankles was performed using the compositions described in Table 6. Evaluated by microscopy according to the Histopathology Scoring System, representative images only was included in the results section.

[0252] [Table 4]

[0253] [Table 5]

[0254] In vivo arthritis scores with group mean scores are shown as graphs in the results section. show.

[0255] [Table 6]

[0256] Histopathological scores with group mean scores are presented as bar graphs in the results section. Exemplary histopathological images at 25x magnification are also shown in the Appendix. is presented to.

[0257] result Anti-hTNF-α D1-Fc-C4 (Quad-X) in the Tg197 arthritis model The efficacy of fluticasone-400 (Trademark) and Humira® was evaluated according to a prophylactic dosing regimen. , i.e., mice developed mild evidence of in vivo arthritic pathology and early histopathological lesions. Treatment began at 3 weeks of age in the indicated mice. By 10 weeks of age, the vehicle-treated control group G1 In vivo arthritis scores in 3-week-old untreated animals were dramatically increased compared to untreated animals at the same age, but At age 1, the histopathological lesions observed in G1 animals were not observed in the 3-week-old control mice. were statistically more severe than expected.

[0258] In vivo and histopathological and body weight responses of test substance D1-Fc-C4 anti-hTNFα Efficacy evaluation of treatment for arthritis symptoms The 3, 10, and 30 mg / kg dose regimens of the D1-Fc-C4 test article resulted in G1 in vivo and histopathological arthritis pathology compared to vehicle-treated mice. A statistically significant and robust inhibition of Tg197 was observed. More specifically, administration of the drug twice a week for 7 weeks resulted in a significant reduction in Tg197 expression. After administering 14 doses over a period of 2 weeks, the dosing regimen produced the following results: Approximately 88% of G2 animals in vivo after 3 mg / kg D1-Fc-D4 test article treatment and approximately 86% inhibition of arthritis histopathology scores. Approximately 88% of G5 animals were in vivo treated with 10 mg / kg of D1-Fc-C4 test article. % significant inhibition and approximately 83% inhibition of arthritis histopathological score Approximately 88% of G3 animals were in vivo treated with 30 mg / kg of D1-Fc-C4 test article. % significant inhibition and approximately 86% inhibition of arthritis histopathological score Statistical significance was only achieved for the 3mg / kg and 10mg / kg dose regimens However, there was more weight gain at all dose levels compared to vehicle-treated mice in G1. Similar findings were observed in the mean body weight curves of D1-Fc-C4 test article-treated mice. was observed.

[0259] Dose-response efficacy evaluation of the D1-Fc-C4 test article Treatment with the D1-Fc-D4 test article at doses of 3, 10, and 30 mg / kg resulted in In vivo arthritis assessment and body weight scores, and all doses performed similarly and The therapeutic effect of No dose-dependent response was observed.

[0260] Humira® in vivo and histopathological and arthritic symptoms Efficacy evaluation of treatment effects Vehicle treatment in G1 with a 10 mg / kg dose regimen of Humira® Compared to mice, Tg197 showed robust in vivo and histological arthritis pathology. More specifically, a 14-week, twice-weekly, 7-week period resulted in a statistically significant inhibition. After administering the dose, we observed the following: Approximately 82% of G4 animals showed in vivo Humira® treatment at 10 mg / kg. % inhibition and approximately 86% inhibition of arthritis histopathology scores Similar findings were observed in G1 mice, where there was more weight gain compared to vehicle-treated mice. Observed in the mean weight curves of Humira®-treated mice.

[0261] Dose-Response Comparison of D1-Fc-C4 Test Article with Humira® Inhibition of D1-Fc-C4 test substance with Humira® at a dose of 10 mg / kg Comparison of adverse effects included body weight, in vivo arthritis scores, and histopathological evaluation. All evaluation parameters were found to be statistically indistinguishable.

[0262] D1-Fc-C4 test article and Humira® versus 3-week-old control animals Histopathological comparison of effects 3, 10 and 30 mg / Kg of D1-Fc-C4 test article and 10 mg / Kg The inhibitory effect of Humira resulted in a reduction in histopathological lesions at 10 weeks compared with 3 weeks of age. Statistically distinct from untreated control animals.

[0263] [Table 7]

[0264] [Table 8]

[0265] [Table 9]

[0266] Additionally, the VNAR S17 Quad-X™ construct targets mouse TNF-α A second example of in vitro potency enhancement using the compound was performed.

[0267] VNAR S17 is a specific antibody that does not have binding or neutralizing activity against human TNF-α. VNAR S17-Fc has an in vitro potency of approximately 8 nM (ND 50) is a potent neutralizer of murine TNF-α. TM Construct (S1 When designed as a IgG antibody (Fc-S17), the in vitro neutralizing potency was reduced to 0.2 nM by approximately 4. 0 times improvement (Figure 30).

[0268] In addition, the S17 Quad-X TM and D1-C4 Quad-X TM The structure is TN It was shown to recognize different species of F-α (Figure 31).

[0269] Discussion and Conclusions The results of this study indicate that the reference Humira® and D1-Fc-C4 (Qua dX™) and D1-BA11-C4 anti-hTNF-α agents in Tg197 animals inhibited the arthritic phenotype observed in mice treated with guanidinium nitrite, thereby reducing body weight gain compared to vehicle-treated animals. It has been shown that the activity of IL-14 is increased and arthritis pathology is reduced in vivo and histopathologically.

[0270] The therapeutic effect of the reference Humira® was evaluated at a dose of 10 mg / kg (Figure 29 ~ Figure 32), in vivo arthritis and ankle histopathology compared to vehicle-treated mice In Figure 33, 1 mg / kg Humi ra® shows significant disease breakthrough at 8 weeks. Humira® dosing at 3 mg / kg, 10 mg / kg, and 30 mg / kg Previous Tg197 mouse model studies using regimens of 1 mg / kg (Figure 33) or Groups of mice treated with either 3 mg / kg Humira® (Figure 35) These groups of mice showed significant improvement in disease compared with the untreated group. Similar time-dependent disease progression was observed in the in vivo arthritis (AS) and histopathological scores (HS). ) were treated with either 10 mg / kg or 30 mg / kg Humira® This was significantly higher than that of the control group (Figure 33).

[0271] The D1-Fc-C4 (Quad-X™) test article was administered at all doses evaluated, i.e. i.e., 0.5, 1, 3 mg / kg, 10 mg / kg and 30 mg / kg were used to completely eliminate the disease. showed similar and statistically indistinguishable treatment effects with controls, thus demonstrating a dose-dependent response. Furthermore, in vivo arthritis and histopathological evaluations demonstrated that the 3 mg / kg The therapeutic effect of the D1-Fc-C4 test article was compared with that of 10 mg / kg Humira® The therapeutic effect of 3 mg / kg at 10 weeks of age was statistically comparable to that of In D1-Fc-C4 treated mice, no signs of disease improvement were observed, and the 0.5 and 8-week-old No significant difference was observed for D1-Fc-C4 at 1 mg / kg or 1 mg / kg.

[0272] Therefore, we further investigated whether the D1-Fc-C4 anti-hTNF-α domain could be used as a standard treatment. in vitro (L929 and Caco2 dextromethorphan) than Humira®, a therapeutic in vitro (Figs. 2, 3, 6-8, 20, 23, and 28) and in vivo (Figs. 29-35) We also demonstrated that the effect of TNF-α on the non-F D1, a non-Fc-based tandem multivalent VNAR The in vivo efficacy of -BA11-C4 was demonstrated (Figures 33 and 34).

[0273] Finally, we demonstrate that anti-TNF VNAR(D1-Fc) also works in an Fc-only format. When administered systemically, it has similar efficacy to dexamethasone in a rat model of inflammatory eye disease. We were able to demonstrate that uveitis can be controlled and treated (by using a combination of acetaminophen and acetaminophen) (Figure 36).

Claims

1. A TNF-α specific VNAR binding domain comprising a combination of CDRs and hypervariable regions (HVs) selected from: a) CDR1 having HCATSS (SEQ ID NO: 68), HV2 having TNEESISKG (SEQ ID NO: 71), HV4 having SGSKS (SEQ ID NO: 72), and CDR3 having ECQYGLAEYDV (SEQ ID NO: 1); or b) CDR1 having NCGLSS (SEQ ID NO: 69), HV2 having TNEESISKG (SEQ ID NO: 71), HV4 having EGSKS (SEQ ID NO: 73) and CDR3 having SWWTQNWRCSNSDV (SEQ ID NO: 6).

2. 2. The TNF-α-specific VNAR binding domain of claim 1, comprising the amino acid sequence of SEQ ID NO: 2 or 7, or a functional variant thereof having at least 90% sequence identity, wherein the functional variant comprises amino acid sequences identical to the CDR1, the HV2, the HV4 and the CDR3.

3. The TNF-α-specific VNAR binding domain of claim 1 or 2, wherein the TNF-α-specific VNAR binding domain is humanized or deimmunized.

4. The TNF-α-specific VNAR binding domain of claim 3, wherein the humanized or deimmunized TNF-α-specific VNAR binding domain has the following amino acid sequence: 【Chemistry 1】

5. A multidomain specific binding molecule comprising at least one TNF-α-specific VNAR binding domain according to any one of claims 1 to 4.

6. 10. An isolated nucleic acid comprising a polynucleotide sequence encoding a TNF-α-specific VNAR binding domain according to any one of claims 1 to 4 or a multidomain specific binding molecule according to claim 5.

7. A method for preparing a TNF-α-specific VNAR binding domain of any one of claims 1 to 4 or a multidomain-specific binding molecule of claim 5, comprising culturing or maintaining a host cell comprising the isolated nucleic acid of claim 6 under conditions such that the host cell produces the TNF-α-specific VNAR binding domain or multidomain-specific binding molecule.

8. 8. The method of claim 7, further comprising isolating the TNF-α-specific VNAR binding domain or multidomain-specific binding molecule.

9. A pharmaceutical composition comprising a TNF-α-specific VNAR binding domain according to any one of claims 1 to 4, or a multidomain specific binding molecule according to claim 5.

10. 10. The pharmaceutical composition of claim 9, further comprising at least one pharmaceutically acceptable carrier.

11. A TNF-α-specific VNAR binding domain according to any one of claims 1 to 4, or a multidomain specific binding molecule according to claim 5, for use in therapy.

12. 10. The TNF-α-specific VNAR binding domain of any one of claims 1 to 4, or the multidomain-specific binding molecule of claim 5, for use in a method for treating a condition mediated by TNFα, comprising administering a therapeutically effective amount of the TNF-α-specific VNAR binding domain of any one of claims 1 to 4, or the multidomain-specific binding molecule of claim 5.

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