HK1223107B - An anti serum albumin fab-effector moiety fusion construct, and the preparing method thereof - Google Patents

Description

Fusion construct of FAB-effector portion of anti-serum albumin and preparation method thereof

Technical Field

The present invention relates to an antigen-binding fragment (Fab) and a Fab-effector fusion protein comprising the antigen-binding fragment (Fab).

Background Art

The preparation of antigen-binding fragments (Fabs) is one of the most successful monoclonal antibody therapeutics. For example, abciximab, ranibizumab, and certolizumab pegol have been approved as drugs in many countries. Furthermore, in the European Union, polyclonal Fab preparations, including abciximab, ranibizumab, and certolizumab pegol, are commercially available.

When the exogenous effector domain forms a Fab-effector fusion with Fab, the conjugation of the exogenous effector domain can give Fab a therapeutic effect. Therefore, in fact, many antibody fragments in clinical development are conjugated to exogenous functional parts. In such a Fab-fusion protein construct (or Fab-effector part construct), the antigen binding fragment can provide target-specific delivery, and the fusion protein or (multiple) peptide (effector domain) can provide a therapeutic effect. Fusion domains derived from prokaryotic origin may include cytotoxins such as deBouganin (a deimmunized plant toxin) (see Entwistle et al., (2012) Cancer Biother Radiopharm. 27, 582-92), Staphylococcal enterotoxin (SE) (see Ilack et al., (2003) Toxicology. 185, 161-174), or mutant forms of Pseudomonas exotoxin (see Choe et al., (1994) Cancer Res. 54, 3460-3467; see Kreitman et al., (1994) Int. J. Cancer 57, 856-864). In addition, fusion domains including polypeptides from eukaryotic organisms, such as scFv (see Lu et al., (2002) J Immunolog Meth. 267, 213-226) or cytokines (see Holzer et al., (1996) Cytokine. 8, 214-221; see Sjogaard et al., (1999) Int J Oncol. 15, 873-882) can function as therapeutic agents. Although radioisotopes are usually chemically conjugated to Fab or (Fab') ₂ fragments, cytotoxins, cytokines, or enzymes are genetically fused to Fab or (Fab') ₂ . Unlike scFv, Fv or dsFv, it is known that Fab molecules can be produced in a soluble form in the periplasm of E. coli (see Humphreys et al., J. Immunol. Methods. 209, 193-202; Carter et al., Biotechnology (NY). 10, 163-167; Venturi et al., J Mol Biol. 315, 1-8; Donzeau et al., Methods Mol Biol. 378, 14-31) or even in Pseudomonas fluorescens (see Retallack et al., Prot Exp Purif. 81, 157-165), easily reaching 1-2 g/L. Currently, many commercially available biological reagents, such as rhGH, insulin, or various types of cytokines, are produced in E. coli (see Graumann and Premstaller, (2006) Biotechnol J. 1, 164-186; Chadd and Chamow, (2001) Curr Opin Biotechnol. 12, 188-194). In this regard, genetically linking therapeutic domains with Fab fragments and other therapeutic agents has great advantages in the development of new biopharmaceuticals and in improving the efficacy of current biopharmaceuticals. Furthermore, Fab molecules can be fused with other antibody fragments, such as scFv, Fv, dsFv, or dAb, to prepare bispecific or trispecific antibody molecules (see Lu et al., (2002) J Immunolog Meth. 267, 213-226). However, the expression of Fab-effector fusion proteins (wherein the effector is of eukaryotic origin) in E. coli is hampered because the effector domain has no biological function due to improper folding or lack of glycosylation in E. coli. In addition, there has been no thorough study of the optimal fusion format for producing Fab-effector fusion proteins in the periplasm of E. coli. Most serum proteins with a molecular weight of less than 50 kDa to 60 kDa, such as cytokines and growth factors, have a short half-life in vivo, for example, from a few minutes to a few hours, due to renal clearance. Therefore, extending the serum half-life of therapeutic polypeptides or proteins is one of the most actively studied areas in biopharmaceutical research (see Kontermann, (2012) Wiley, ISBN: 978-3-527-32849-9). To this end, a variety of methods have been developed, including PEGylation, polysialylation, hydroxylation (HESylation), glycosylation, or recombinant PEG analogs fused to flexible and hydrophilic amino acid chains (500-600 amino acids) (see Chapman, 2002; Adv Drug Deliv Rev. 54, 531-545; Schlapschy et al., (2007) Prot Eng Des Sel. 20, 273-283; Contermann (2011) Curr Op Biotechnol. 22, 868-876; Jevsevar et al., (2012) Methods Mol Biol. 901, 233-246). Furthermore, FcRn-mediated recycling mechanisms have also been directly or indirectly utilized to extend the in vivo half-life of therapeutic proteins. Among serum proteins, human serum albumin (HSA) and immunoglobulins (especially IgG) are known to have very long half-lives in the FcRn-mediated recycling mechanism. In humans, the serum half-life of albumin is 19 days, while the serum half-life of IgG molecules ranges from one week to almost four weeks, depending on the subclass of IgG. Therefore, these two molecules have been used as fusion partners to extend the half-life of therapeutic proteins and/or (poly)peptides.

Recombinant hGH (~19 kDa), produced in the cytoplasm or periplasm of E. coli and after an in vitro folding process, has been used clinically to treat growth hormone deficiency in infants and adults (see Blethen et al., (1997) J. Clin. Endocrinol. Metab. 82, 418-420). A major inconvenience of rhGH administration is the need for daily injections due to its short half-life (<30 minutes). In order to extend the serum half-life of hGH, chemical conjugation with polyethylene glycol (see Clark et al., (1996) J. Biol. Chem. 271, 21969-21977; Pradhananga et al., 2002 J Mol Endocrinol. 29, 1114; Cho et al., 2011; Sondergaard et al., (2011) J Clin Endocrinol Metabol. 96, 681-688) and chemical conjugation of modified hGH to the arms of humanized CovX-body IgG (CovX-Body IgG) (see Palanki et al., (2013) Bioorg. Med. Chem. Lett. 23, 402-406) have been attempted. In addition, the half-life of hGH in serum has been successfully extended by genetic fusion of human serum albumin (HSA) or a polypeptide sequence containing several hundred Pro-Ala-Ser (PAS) residues (PASylation) (see Osborn et al., 2002 Eur J Pharmacol. 456, 149-158; Anderson et al., (2011) J Biol Chem. 286, 5234-5241; Sleep et al., (2013) Biochimica et Biophysica Acta. 1830, 5526-5534; Schlapschy et al., (2013) Protein Eng Des Sel. 26, 489-501). The best studied in this category is VRS-317, a rGH genetically linked to an XTEN amino acid sequence at the N- and C-termini, which allows for a monthly dosing schedule (see Schellenberger et al., (2007) Nat Biotech. 27, 1186-1190; Cleland et al., (2012) J Pharm Sci. 101, 2744-2754; Yuen et al., (2013) J Clin Endocrinol Metab. 98, 2595-2603). In addition, hGH has been associated with vascular disease (see Thomas J Merimee et al., (1973) Diabetes, 22, 813-819) and CRETZFELDT-JAKOB disease (see John Powell-Jackson et al., 1985, Lancet, 2, 244-246). In addition, IFN-γ accelerates graft-versus-host disease (Graft-Versus-Host-Disease) (see Bruce R. Blazar et al., 2003, The Journal of Immunology, 171, 1272-1277), and IFN-α is associated with autoimmune diseases (see AImagawa et al., 1995, The Journal of clinical endocrinology & metabolism, 80, 922-926). In addition, GSCF is associated with autoimmune diseases (see Anke Franzke et al., 2003, Blood, 102, 734-739) and HCV-related liver disease (see Van Thiel DH et al., 1995, Hepato-gastroenterology, 42, 907-912).

Especially when Fab-fusion proteins are produced at low cost in microbial expression systems, Fab-fusion proteins (or polypeptides) have great potential as therapeutic agents for treating chronic diseases that require large, long-term medication doses. While the use of Fabs offers these potential advantages, the use of Fab antibodies against serum albumin (SA) has not yet been attempted in the development of protein or (poly)peptide drugs with extended in vivo half-lives. In this regard, the present inventors have completed the present invention by constructing novel Fab-effector protein (or (poly)peptide) fusion constructs against serum albumin (SA) and demonstrating that functional fusion constructs exhibit high yields in the periplasm of E. coli.

Summary of the Invention

【Technical Issues】

The technical problem to be solved by the present invention is to provide a new antigen-binding fragment (Fab) with a prolonged in vivo serum half-life.

Another technical problem to be solved by the present invention is to provide a fusion construct of Fab-effector moiety, which can be optimized for production in the periplasm of host cells.

Another technical problem to be solved by the present invention is to provide an expression vector and a host cell for producing a Fab-effector construct in a high-yield, soluble form.

Another technical problem to be solved by the present invention is to provide a pharmaceutical composition comprising the above fusion construct.

【Technical solution】

In order to solve the above problems, the present invention provides an optimized Fab-effector fusion construct (or type) for expression in the periplasm of E. coli, wherein the Fab has a heavy chain variable region bound to a heavy chain constant region 1 ( _CH1 ) and a light chain variable region bound to a light chain constant region ( _CL ).

In one embodiment of the present invention, human anti-SA Fab was selected as the antibody fragment, considering that a variety of therapeutic proteins are fused to albumin or to albumin-binding moieties such as small peptides or domain antibodies (dAbs), which extend the half-life of the therapeutic protein through an FcRn-mediated recycling mechanism (see Dennis et al., (2002) Biochimica et Biophysica Acta. 1830, 5526-5534; Sleep et al., (2013) Biochimica et Biophysica Acta. 1830, 5526-5534; Nguyen et al., (2006) Protein Eng Des Sel. 19, 291-297; Kontermann, (2011) Curr Op Biotechnol. 22, 868-876). According to previous studies, Fab fragments have an elimination half-life of 16 to 20 hours in humans after intravenous administration (see Ujhelyi and Robert, (1995) Clin Pharmacokinet. 28, 483-493) and an elimination half-life of ~3 hours in rats (see Nguyen et al., 2006 Protein Eng Des Sel. 19, 291-297). Surprisingly, the Fab of the present invention (SL335) has a half-life of 37 hours in rats, which is approximately 11 times longer than that of conventional human Fabs. Therefore, it is reasonable to assume that SL335 may have a half-life of at least 160 to 200 hours (6 to 8 days) in humans. Meanwhile, two known Vk domains, dAbr3 and dAbr16, have binding affinities for RSA of 13 nM and 1 mM, respectively, with t1 _/2 values of 53 h (dAbr3) and 43 h (dAbr16) in rats (see Holt et al., (2008) Protein Eng Des Sel. 21, 283-288). Furthermore, the Ab Fab4D5-H, which has an affinity for RSA of 92 nM, has a _t1/2b of 26.9 h (see Nguyen et al., 2006). Thus, it is suggested that the in vivo functionality of SL335 is comparable to that of previously reported dAbs and peptides specific for SA. Notably, the _VH and _VL of SL335 share only 65%-67% amino acid homology with previously reported albumin-specific dAbs at the global sequence level and ~50% amino acid homology at the complementarity determining region (CDR) level (data not shown). Specifically, in an embodiment of the present invention, the Fab specific for serum albumin (SA) comprises: a heavy chain variable region having a sequence selected from SEQ ID NO.1 (SA138VH:QVQLLQSGAE VKKPGASVKV SCKASGYTFT SYGISWVRQA PGQGLEWVGWINTYSGGTKYA QKFQGRVTMT RDTSISTVYM ELSGLKSDDTAVY YCARLGHCQRGICSDAL DTWGQGTLVTVSS), SEQ ID NO.2 (SA139VH:EVQLLQSGAE VKEPGASVKV SCKASGYTFS SYGISWVRQAPGQGLEWVGR INTYNGNTGYA QRLQGRVTMT TDTSTSIAYM EVRSLRSDDTAVY YCARLGHCQRGICSDALDTWGQGTMVT VSS), SEQ ID NO.3 (SA140VH:QVQLVQSGGG VVQTGGSLRL SCAASGFTFRNYGIHWVRQA PGKGLEWVAS ISYDGSNKYYA DSVKGRFTIS RDNSRNTVHV QMDSLRGGDTAVYYCARDVHYYGSGSYYNAF DIWGQGTLVT VSS), SEQ ID NO.4(SA141VH:QVQLVQSGGG LVQPGGSLRLSCAASGFTFS SYAMSWVRQA PGKGLEWLSV ISHDGGFQYYA DSVKGRFTVS RDNSKNTLYLQMNSLRAEDTAVY YCARAGWLRQYGM DVWGQGTLVT VSS), SEQ ID NO.5(SL18VH:EVQLVQSGTEVKKPGESLKI SCKISGYSFT AYWIAWVRQM PGKGLEWMGM IWPPDADARYS PSFQGQVTFS VDKSISTAYLQWHSLKTSDTAVY YCARLYSGSY SPWGQGTLVT VSS) and SEQ ID NO.6 (SL301, SL310 and SL335VH:QVQLVQSGGG PVKPGGSLRL SCAASGFMFR AYSMNWVRQA PGKGLEWVSS ISSSGRYIHYA DSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVY YCARETVMAGKAL DYWGQGTLVT VSS); and, a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NO.7 (SA130:ELVLTQSPSS LSASVGDRVTITCRASQSIS RYLNWYQQKP GKAPKLLIYG ASRLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSDSVPVTFGQ GTRLEIKR), SEQ ID NO.8(SA139VL:DIVLTQSPSS LSASVGDRVT ITCRASQSISSYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPPYTFGQGTKLEIKR), SEQ ID NO.9(SL18VL:ELVLTQSPGT LSLSPGERAT LSCRASQSIF NYVAWYQQKPGQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSKWPPTWTFGQGTRVDIKR), SEQ ID NO.10(SL301VL:ELVLTQSPGT LSLSPGERAT LSCRASETVSS RQLAWYQQKPGQAPRLLIYG ASSRATGIPD RFSGSGSGTD FTLTISRLEP EDSAVFYCQQ YGSSPRTFGG GTKLEIKR), SEQ ID NO. 11 (SL310VL: ELVLTQSPGT LSLSPGERAT LSCRASQSVSS SSLAWYQQKP GQAPRLLIYGASSRATGIPD RFSGSGSGTD FTLTISSLQP EDAATYYCQK YSSYPLTFGQ GTKLEIKR), and SEQ ID NO. 12 (SL335VL: ELVLTQSPGT LSLSPGETAT LSCRASQSVG SNLAWYQQKP GQAPRLLIYGASTGATGVPA RFSGSRSGTD FTLTITSLQP EDFATYYCQQ YYSFLAKTFGQ GTQLEIKR). In addition, the _VH domain of the Fab is linked to the heavy chain constant region 1 ( _CH1 domain), and the _VL domain of the Fab is linked to the light chain constant region ( _CκL domain). Further, the Fab of the present invention that is specific for serum albumin (SA) includes the amino acid sequences of SEQ ID NO.13 (CDR1) (AYSMN), SEQ ID NO.14 (CDR2) (SISSSGRYIHYADSVKG) and SEQ ID NO.15 (CDR3) (ETVMAGKALDY) in the _VH region of SL335, and the amino acid sequences of SEQ ID NO.16 (CDR1) (RASQSVGSNLA), SEQ ID NO.17 (CDR2) (GASTGAT) and SEQ ID NO.18 (CDR3) (QQYYSFLAKT) in the VL region of SL335.

In one embodiment, the cysteine amino acids in the _CH1 domain and _CκL domain of the Fab may be deleted or substituted with serine residues. Specifically, in the aforementioned SL335, the cysteine amino acid in the _CH1 domain is at amino acid position 233 from the N-terminus of the _CH1 domain, while the cysteine in the _CκL domain is at amino acid position 214 from the N-terminus of the _CκL domain, both of which are substituted with serine residues. To avoid confusion, the H and L chains comprising the Fab are named as follows: 1) Hcys: H chain with cysteine at position 233, 2) Lcys: L chain with cysteine at position 214, 3) Hser: H chain with serine at position 233, and 4) Lser: L chain with serine at position 214.

In another embodiment of the present invention, Fab-effector fusions are constructed by genetically fusion of the effector domain to the N-terminus or C-terminus of the Fd chain or light chain of a Fab molecule. Because the folding and heterodimerization mechanisms of recombinant proteins in the periplasmic environment of E. coli are quite complex and largely unknown, it is not possible to predict which type of Fab-effector fusion is optimal for functional expression.

Furthermore, in another embodiment, a fusion construct of an antigen-binding fragment (Fab) and an effector domain (biologically active effector moiety) is provided, wherein the cysteine amino acid in the _CH1 domain and the cysteine amino acid in the _CκL domain of the Fab are deleted or substituted with serine residues; wherein the biologically active effector moiety is a protein or (poly)peptide; and wherein the Fab and the biologically active effector moiety are covalently linked by gene fusion. A peptide linker of 0 to 20 amino acids can be used to covalently link the Fab and the biologically active effector moiety by gene fusion. Among the six Fab-effector fusion types (or constructs) including hGH of the present invention, results clearly demonstrated that HserG/Lser exhibited the highest expression in E. coli. That is, according to this embodiment, removal of Cys ²³³ in the _CH1 domain and Cys ²¹⁴ in the _Cκ domain by deletion or substitution with other amino acid residues improved the soluble expression of the SL335-fusion effector construct in culture supernatant. This solves three important issues. First, fusion of the effector moiety (e.g., hGH) to the C-terminus of _CH1 is preferred over the C-terminus of _CLk . Previously, Lu et al. reported that the yield of an anti-Flt-1 scFv genetically linked to the C-terminus of an anti-KDR Fab was four times higher than when linked to the C-terminus of the _CL domain (see Lu et al., (2002) J Immunolog Meth. 267, 213-226). Although the data are not included, the present inventors performed western blot analysis using E. coli total lysate and showed that the Fd fragments of LcysG/Hcys and LserG/Hcys were almost completely degraded, resulting in no detectable soluble form of the fusion protein in the E. coli supernatant. Because _VH domains are prone to aggregation in E. coli (Dudgeon et al., (2009) Protein Eng Des Sel. 22, 217-220), it is speculated that the presence of an effector domain at the C-terminus of _CL may prevent the interaction between the _VH and _VL domains, as well as the interaction between the _CH1 domain and the _CL domain, leading to rapid aggregation and degradation of the _Fd fragment. Comparison of the soluble expression levels of LserG/Hcys and LserG/Hser revealed that the presence of Cys ²³³ in the _CH1 domain appears to accelerate this process, possibly due to the formation of an abnormal disulfide bond. After removal of Cys ²³³ in the _CH1 domain, the effector domain at the end of CH1 may have a beneficial effect on reducing _VH domain aggregation by partially blocking the hydrophobic surface of the _VH domain before _VH - _VL pairing. Second, the presence of Cys ²¹⁴ in _CLk further deteriorated the soluble production of the SL335-hGH fusion protein in an additional manner. The lower yield of HserG/Lcys compared to HserG/Lser can be explained by the tendency of the L chain to form a homodimer called the Bence Jones protein (see Kirsh et al., (2005) J Immunol Methods. 301, 173-185), in which Cys ²¹⁴ of _CLk may contribute to the stability of the homodimer or participate in the formation of abnormal disulfide bonds with other cysteine residues in the fusion protein. It is also known that the disulfide bond between _CH1 and the C-terminus of _CL in Fab is highly variable and possesses considerable flexibility (see Rothlisberger et al., (2005) J. Mol. Biol. 347, 773-789; Humphreys et al., (2007) Protein Eng Des Sel. 20, 227-234). In this regard, the present invention provides an antigen-binding fragment (Fab) comprising a heavy chain constant region 1 ( _CH1 ) lacking Cys ²³³ and a light chain constant region ( _CLk ) lacking Cys ^214. Similarly, HerGF/Lser and HserIFNb/Lser exhibit the highest expression levels in E. coli. In the fusion construct of the present invention, the molar ratio of the biologically active (poly)peptide (or protein) to the Fab is between 1:1 and 10:1, preferably between 1:1 and 4:1. Third, the two C-terminal cysteine residues present in SL335 not only inhibit expression but also restrict access to the hGH domain by anti-hGH antibodies. Interference with the interaction between the effector domain and its ligand is also crucial for the therapeutic function of the effector domain in Fab-effector fusions. The present inventors have demonstrated that the use of Fab _Δds as a fusion partner is beneficial not only for hGH, but also for other effectors such as G-CSF and IFN-β.

In another aspect of the present invention, an expression vector and a mutant E. coli SUPEX5 strain (KCTC 12657BP) as a host cell are provided to address the aforementioned technical issues. This strain was established by random chemical mutagenesis of the MC1061 E. coli strain, which was selected because it is derived from the E. coli K12 strain, a major host strain for the production of commercial biopharmaceuticals. Using the mutant SUPEX5 E. coli strain as an expression host further demonstrated a beneficial effect on HserG/Lser production compared to the parent MC1061 strain. In general, SL335-hGH fusions, as well as the combination of Fab _ds with the SUPEX5 E. coli strain, are advantageous for the soluble expression of Fab-effector fusion proteins, as clearly demonstrated by the results obtained for the SL335-GCSF fusion (SL335 _wt -GCSF vs. SL335 _Δds -GCSF), SL335-IFNβ fusion (SL335 _wt -IFNb vs. SL335 _Δds -IFNβ), EGL4-hGH fusion (EGL4 _wt -hGH vs. EGL4 _Δds -hGH), and 1β28-hGH fusion (1β28 _wt -hGH vs. 1β28 _Δds -hGH). Thus, the above results strongly support that, at least in the SUPEX5 E. coli strain, the use of Fab _Δds (a mutant form of the Fab lacking Cys ²³³ of _CH1 and Cys ²¹⁴ of _CLK ) is advantageous for soluble expression of Fab-effector fusion proteins relative to conventional Fabs. Co-expression of a chaperone or disulfide isomerase (FkpA, SurA, Skp, Sec A, Sec B, DsbA, or Dsb C) should improve the soluble and functional expression of SL335 _wt -GCSF or even SL335 _Δds -GCSF, as these fusions are known to increase the production of soluble Fab fragments in the periplasm of E. coli (see Schlapschy et al., (2006) Escherichia coli. Protein Eng Des Sel. 19, 385-390). The present inventors believe that the use of Fab _ds is advantageous, in particular when the molecular chaperones and the catalytic machinery for disulfide formation in the endoplasmic reticulum are overloaded due to high expression of the Fab-effector fusion protein in the host cell.

In one embodiment of the present invention, despite the three-fold increase in molecular size, SL335 _Δds -hGH was produced using culture flasks at a concentration of approximately 10 mg/L, which is higher than previously reported. According to previous reports, studies on soluble expression of rhGH in the periplasm of E. coli showed that the yield of pelB-hGH was 0.64 to 2.57 mg/L and the yield of ompA-hGH was 0.32 to 2.29 mg/L (see Sockolosky and Szoka, (2013) Protein Exp Purif. 87, 129-135). Furthermore, the rhGH yield was largely dependent on the promoter used and the host E. coli strain (see Soares et al., (2003) Protein Engineering. 16, 1131-1138). By simple medium optimization, the present inventors routinely achieved a yield of ∼50 mg/L in culture supernatant using culture flasks, which allowed a cell density of _OD600nm = ∼10-11 (manuscript in preparation), which could be further improved to be sufficient for industrial scale-up by fine-tuning the medium composition and fed-batch culture system.

In another aspect of the invention, the SL335 _ds -effector protein exhibits increased affinity for HSA. In one embodiment, the SL335 _ds -hGH response to HSA (human serum albumin) was enhanced, ranging from 5-9 times that of the parent SL335, and the response to RSA (rat serum albumin) was reduced, ranging from 1.3-4 times that of the parent SL335, depending on pH conditions. The genetic linkage of the antibody fragment and the effector domain can influence the antigen-binding affinity of the antibody fragment, and the variation in affinity depends largely on the nature of the antibody fragment and the effector domain, as well as how the two functional parts are linked. It is unclear whether these differences in affinity are due to the absence of interchain disulfide bonds or the presence of the hGH fusion domain. Nevertheless, compared to IFN-a2b-DOM7h-14 (whose affinity for human, mouse, and rat SA was reduced by 7.7-fold, 22.3-fold, and 15.8-fold compared to the parental DOM7h-14) (see Walker et al., (2010) Protein Eng Des Sel. 23, 271-278), the effect of the hGH fusion on the binding affinity of SL335 _Δds to the antigen appears to be negligible. Therefore, compared to domain Abs, Fabs have advantages in maintaining affinity and effector folding because the _CH1 and _CL domains provide space to reduce steric hindrance between the antigen-binding region and the effector domain bound to each ligand.

In another embodiment of the present invention, SL335 _Δds -hGH has a greatly extended serum half-life, wherein its t _1/2 (16.6 h upon intravenous administration) is similar to the t _1/2 of PEG5-hGH (250 kDa) (see Clark et al., 1996). Interestingly, the t _1/2 of SL335 _Δds -hGH is 5.6-fold greater (t _1/2 = 2.96 h), and the difference in t _1/2 between SL335 _Δds -hGH and PEG5-hGH is further extended by SC (subcutaneous) administration, reaching 16-fold (97.2 h vs. 5.93 h) (see Osborn et al., 2002), although these comparisons are circumstantial unless the experiments are performed under the same settings. Similarly, the t _1/2 of IFN-a2b-DOM7h-14 is also approximately 1.5 times that of HSA-IFN-a2b (see Walker et al., 2010). Thus, it appears that albumin-conjugate fusions provide a longer half-life than fusions with albumin, although the underlying mechanism remains to be determined. Notably, the serum t _1/2 of SL335 _Δds -hGH is similar to that of VRS-317 (t _1/2 = 15 hours) in IV administration (Cleland et al., (2012) J Pharm Sci. 101, 2744-2754). This may suggest that SL335 _Δds -hGH (called) can be administered on a longer than weekly or even monthly dosing schedule.

In another embodiment of the present invention, the pharmacodynamic effects of SL335 _Δds -hGH appear to be far superior to those of and, considering the once-weekly dosing regimen, are seven times more effective on a molar basis. Unfortunately, we had to interrupt the two-week pharmacodynamic study at day 11 because some of the hypophysectomized rats, particularly those in the vehicle-only group, died prematurely. This appears to be because the long-distance transportation from Japan to Korea following the surgery in August caused severe stress to the animals, as reflected in a 5% reduction in body weight in the vehicle-only group and a larger standard deviation than we had expected. Nevertheless, SL335 _Δds -hGH clearly appears to have great potential for development as a long-acting hGH, and from now on, we will refer to it as .

In another embodiment of the present invention, the biologically active polypeptide fused to the above Fab is any one selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins and receptors.

In another embodiment of the present invention, the biologically active polypeptide is selected from the group consisting of human growth hormone, growth hormone-releasing hormone (GHRH), growth hormone-releasing peptide, interferon, interferon receptor, colony stimulating factor (CSFs), glucagon-like peptide, G protein-coupled receptor, interleukin, interleukin receptor, enzyme, interleukin binding protein, cytokine binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitory factor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor (metastasis growth factor), leukocyte antigen 2 (LEF), leukocyte antigen 3 (LEF), leukocyte antigen 4 (LEF), leukocyte antigen 5 (LEF), leukocyte antigen 6 (LEF), leukocyte antigen 7 (LEF), leukocyte antigen 8 (LEF), leukocyte antigen 9 (LEF), leukocyte antigen 10 (LEF), leukocyte antigen 21 (LEF), leukocyte antigen 12 (LEF), leukocyte antigen 13 (LEF), leukocyte antigen 14 (LEF), leukocyte antigen 15 (LEF), leukocyte antigen 16 (LEF), leukocyte antigen 17 (LEF), leukocyte antigen 18 (LEF), leukocyte antigen 22 (LEF), leukocyte antigen 23 (LEF), leukocyte antigen 24 (LEF), leukocyte antigen 25 (LEF), leukocyte antigen 26 (LEF), leukocyte antigen 27 (LEF), leukocyte antigen 28 (LEF), factor), α-1-antitrypsin, albumin, α-lactalbumin, apolipoprotein E, erythropoietin, highly glycosylated erythropoietin, angiogenin, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitors, collagenase inhibitors, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin II, bone growth factor , bone stimulating protein, calcitonin, insulin, atrial natriuretic peptide, chondroitin, elcatonin, connective tissue activating factor, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyrotropin, autotaxin, lactoferrin, myostatin, receptor, receptor antagonist, cell surface antigen, virus-derived vaccine antigen, monoclonal antibody, polyclonal antibody and antibody fragment.

In another aspect of the present invention, a pharmaceutical composition is provided, wherein the composition includes a fusion construct of the Fab-effector portion of the present invention, and a pharmaceutically acceptable excipient, and has increased persistence in vivo. The pharmaceutical composition of the present invention can be administered to the body in a variety of ways, including oral administration, transdermal administration, subcutaneous administration, intravenous administration or intramuscular administration, and more preferably, the pharmaceutical composition of the present invention can be administered as an injectable formulation. Further, the pharmaceutical composition of the present invention can be prepared using methods well known to those skilled in the art to provide rapid, lasting or delayed release of the active ingredient after administration. The above-mentioned preparations can be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, decoctions (solutions), syrups, aerosols, soft gelatin capsules and hard gelatin capsules, sterile injection solutions, sterile packaged powders, etc. Examples of suitable carriers, excipients and diluents are lactose, glucose, sucrose, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. Further, the above-mentioned preparations may additionally include fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

It should be understood that the actual amount of fusion protein or polypeptide administered should be determined based on various relevant factors, including the disease to be treated, the selected route of administration, the age, sex, and weight of the individual patient, and the severity of the patient's symptoms; as well as the type of biologically active polypeptide as the active ingredient. Because the fusion protein of the present invention has very excellent persistence in the blood, the number and frequency of administration of peptide formulations containing the fusion protein of the present invention can be significantly reduced.

As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent the terms "including," "includes," "having," "has," "with," "such as," or variations thereof are used in the specification and/or claims, such terms are not limiting and are intended to mean in a manner similar to the term "comprising."

In the present invention, "biologically active polypeptides or proteins" refer to (poly)peptides or proteins that exhibit useful biological activities when administered to mammals including humans.

In the present invention, a "Fab-effector moiety fusion construct (or type)" is a construct in which a biologically active (poly)peptide or protein is covalently linked to a Fab. Further, a "Fab-effector moiety fusion construct (or type)" is understood to include Fab-fusion proteins, Fab-fusion (poly)peptides, fusion constructs, and fusion types.

In this regard, the present invention is described in detail in the Examples. It should be noted that the description of the Examples does not limit the scope of the present invention as described in this disclosure.

Beneficial effects

The present invention provides anti-serum albumin Fab _Δds -associated (SAFA) technology as a new platform technology for the development of long-acting biotherapeutics. In this regard, compared with traditional technologies including PEGylation, Fc-fusion, AlbudAb technology and albumin-fusion, the present invention has advantages in terms of prolonged in vivo action, maintenance of effector domain conformation and binding affinity, simple production and low-cost procedures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the results of a monoclonal phage ELISA to determine the binding specificity of anti-SA Fab phage antibodies.

Figure 2 shows the determination of the antigen-binding specificity of human Fab clones by ELISA.

FIG3 shows the in vivo pharmacokinetics of SL335.

Figure 4 is a diagram of the six types of SL335-hGH fusions constructed in this study.

FIG5 shows the results of an ELISA to determine the yield and binding reactivity of the soluble SL335-hGH fusion in E. coli culture supernatants. The binding signal was visualized using TMB substrate, and the absorbance at 450 nm was measured using an ELISA plate reader. Data represent the mean ± SD of three experiments.

Figure 6 shows the results of ELISAs determining the host E. coli-dependent and temperature-dependent expression (20°C, A; 25°C, B; or 30°C, C) of SL335 and SL335-hGH variants.

FIG7 shows the results of ELISA to determine the production of soluble SL335-GCSF and SL335-IFNβ fusion constructs in E. coli culture supernatants.

FIG8 shows the results of ELISA to determine the production of soluble EGL4-hGH (A) and 1β28-hGH fusion (B) in E. coli culture supernatants.

FIG9 shows the analysis results of SL335 _wt -hGH and SL335 _ds -hGH by SDS-PAGE and Western blotting.

FIG10 shows the analysis results of HcycG/Lcys and HserG/Lser by chip-based capillary electrophoresis.

FIG11 shows the analysis results of HcycG/Lcys and HserG/Lser by MALDI-TOF mass spectrometry.

FIG. 12 is the purification of HserG/Lser by gel filtration using FPLC.

FIG13 shows the determination of hGH bioactivity in vitro of SL335 _ds -hGH by Nb2-11 cell proliferation assay.

Figure 14 shows the serum stability of SL335ds _- hGH as determined by ELISA and in vitro Nb2-11 cell proliferation assay.

FIG15 is a pharmacokinetic analysis of Growtropin or SL335 _ds -hGH in rats.

Figure 16 shows the dose-dependent increase in body weight in hypophysectomized rats treated with SL335 or SL335 _Δds -hGH. N = 3 rats/treatment group, body weight was measured once a day for each rat.

Figure 17 shows the dose-dependent increase in tibial length in rats treated with SL335 _Δds -hGH. N = 3-4 rats/treatment group, one tibial measurement per rat.

FIG18 shows the pHEKA vector of the present invention.

FIG19 shows the nucleic acid sequence of the pHEKA vector of the present invention.

FIG20 shows the deduced amino acid sequences of the VH and VL genes utilized by the anti-SA Fab clones of the present invention.

FIG21 shows the DNA sequences of the VH gene (A) and VL gene (B) utilized by the anti-SA Fab clones of the present invention.

Figure 22 shows the sequence information of the Fab-effector fusion construct of the present invention. The linker and effector domains are underlined and the CDRs are shown in bold.

DETAILED DESCRIPTION

1. Materials and Analysis

1-(1) Cloning and strains

All DNA cloning experiments were performed according to standard procedures (see Sambrook et al., (1989) Molecular cloning: A laboratory manual, 2nd ed. (New York, USA: Cold Spring Harbor Laboratory Press). Sequencing-grade oligonucleotides and codon-optimized genes for the construction of SL335-effector fusion constructs were synthesized by Bioneer in Daejeon, South Korea. PCR amplifications were performed using Pyrobest or Ex-Taq DNA polymerase (Takara, Tsu, Japan) at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min for 25 cycles, followed by 72°C for 10 min, unless otherwise indicated. Restriction enzymes, shrimp alkaline phosphatase (SIP), and T4 DNA ligase were also purchased from Takara. E. coli MC1061 strain [araD139Del(araA-leu)7697Del(lac)X74galK16galE15(GalS)λ-e14-mcrA0relA1rpsL150(strR)spoT1mcrB1hsdR2] (ATCC, Manassas, USA) was used for cloning, and recombinant protein expression was performed using E. coli SUPEX5. Recombinant phage was prepared using E. coli TG1 strain {F'[traD36proAB ⁺ lacI ^q lacZΔM15]supE thi-1Δ(lac-proAB)Δ(mcrB-hsdSM)5,(r _K ⁻ m _K ⁻ )} (Agilent Technologies, Palo Alto, USA).

1-(2) Biopanning of the HuDVFab-8L Antibody Library

Enrichment of recombinant phage binding to the target antigen was performed as previously described (see Joo et al., (2008) J. Immunol. Methods. 333, 24-37; Hur et al., (2010) Immunol Lett. 132, 24-30). Briefly, tosyl-coated magnetic beads conjugated with human, rat, or mouse serum albumin (HSA, RSA, or MSA, respectively) (Sigma-Aldrich, St. Louis, MO, USA) were mixed with 10 ¹⁰ phage from the HuDVFab-8L antibody library (AprilBio, Chuncheon, South Korea) at 4°C for 4 h and washed three times with phosphate-buffered saline containing 0.02% Tween (PBST). Phage antibodies bound to the beads were eluted using elution buffer (0.1 M glycine, pH 2). Eluted phage were used to infect fresh TG1 cells carrying the corresponding light (L) ( _VL + C _Lk ) chain and grown in 2YT medium (2×YT/ACT) containing 25 μg/ml ampicillin, 10 μg/ml carbenicillin, and 10 μg/ml tetracycline. Recombinant phage were then amplified using Ex-12 helper phage (AprilBio) for subsequent panning. After the final panning, monoclonal phage ELISA was performed to identify positive clones. Fd ( _VH + C _H1 ) genes from positive clones were subcloned into pHg3A-3 vector (AprilBio, Chuncheon, South Korea), and L chain optimization was performed using all 1.410 ^g of human naive kappa L chain in pLf1T-3 phagemid vector (AprilBio).

1-(3)-DNA sequencing analysis

pHf1g3A-2 (AprilBio) phagemid and pLf1A-3 plasmid (AprilBio) were isolated from E. coli cells producing anti-SA Fab molecules using the Wizard Plasmid Miniprep Kit (Promega, Madison, WI, USA). VH and VL genes were read using two different sequencing primers complementary to pHf1g3A-2 or pLT-2 (5'-gtgccgttctatagccatagcac-3' (SEQ ID NO: 19) and 5'-ggcactggctggtttcgctaccgtg-3' ( _SEQ ID _NO : 20), respectively. DNA sequencing was performed by SolGent in Daejeon, South Korea.

1-(4) Construction of pHEKA expression vector

DNA fragment #1 containing the Bgl II restriction site+trc promoter+g10 translation enhancer-ribosome binding site (RBS) was obtained from the pTrcHis-B vector (Invitrogen, Carlsbad, CA, USA) by PCR amplification using Pyrobest DNA polymerase and a set of PCR primers #1 (5'-gggagatcttgaaatgagctgttgacaattaatcatccg-3' (SEQ ID NO:21)) and #2 (5'-cctctttaatttttaataataaagttaatcgataattcc-3' (SEQ ID NO:22)). DNA fragment #2 containing the g10 translation enhancer + RBS + BamH I + multiple cloning site (MCS) + transcription terminator was obtained by PCR amplification from the same template as above using PCR primers #3 (5'-ggaattatcgattaactttattattaaaaattaaagaggtatatattaggatccgagc tcgagttctgca-3' (SEQ ID NO: 23)) and #4 (5'-gggcactacgtgcgaaaggcccagtctttcgact-3' (SEQ ID NO: 24)). Linked PCR was performed using Ex-Taq DNA polymerase and a set of PCR primers #1 and #4 to combine the two DNA fragments. The resulting ~520 bp DNA fragment was separated by agarose gel electrophoresis. The ligated PCR product and pET28a (Invitrogen) plasmid were then restricted using Bgl II and Dra III and ligated using T4 DNA ligase for 2 hours at RT. 3 ml of the ligation reaction solution was used to transform MC1061 electrocompetent cells, and then E. coli transformants were selected on 2YT plates containing 50 μg/ml kanamycin (Sigma-Aldrich). To subclone the Fab gene into the pHEKA vector, the Fd (VH + CHI) chain gene was PCR amplified from the pHf1g3A-2 phagemid vector using a set of PCR primers #5 (5'-ggccgcagatctgttaattaaggaggaatttaaagaattcatgaaaaaactgctgttcgcgattccgct-3' (SEQ ID NO: 25)) and #6 (5'-gggaagcttattaacaagatttgggctcaactctcttgtcc- _{3 '} (SEQ ID NO: 26)); and a set of PCR primers #7 (5'-gggggatccatgaaaaagacagctatcgcgattgcagtg-3' (SEQ ID NO: 27)) and #8 (5'-attcctccttaattaacagatctgcggccgcactcgagattaacactctcccctgttgaagctc tttgt-3' (SEQ ID NO: NO:28) The L chain gene was PCR amplified from the pLT-2 plasmid vector. Using PCR primers #6 and #7, the resulting F chain and L chain gene fragments were combined by ligation PCR, and the resulting PCR product, approximately 1.4 kbp in size, was excised from an agarose gel. The PCR product and pHEKA plasmid were then restricted using BamHI and HindIII, ligated using T4 DNA ligase at room temperature for 2 hours, and then electroporated into electrocompetent E. coli MC1061 or SUPEX5 cells. Table 1 below shows the PCR primers used in preparing the pHEKA expression vector. A schematic diagram of the pHEKA expression vector is shown in Figure 18.

【Table 1】

Preparation of PCR primers for pHEKA expression vector

Establishment of 1-(5)-mutant Escherichia coli SUPEX5 strain

Chemical mutagenesis was performed essentially as described in previous work. Briefly, E. coli MC1061 cells expressing an anti-human branched-chain ketoacid dehydrogenase complex-E2 (BCKD-E2) scFv fused to alkaline phosphatase (AP) were grown in Luria Broth (LB) medium containing 50 μg/ml ampicillin to an _OD600 of ~0.3. Cells were harvested from 5 ml of culture by centrifugation at 3,000 g for 10 min and washed twice with cold 0.1 M sodium citrate buffer (pH 5.5). Cells were then resuspended in 1.9 ml of the same buffer and treated with 50 μg/ml N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Sigma-Adrich, St. Louis, MO, USA) at 37°C for 15, 30, and 45 min. After MNNG treatment, cells were mixed, rinsed twice, and resuspended in 2 ml LB culture medium. Then, colony lift assay (colony lift assay) was performed with a double membrane system as described. Briefly, a first nylon membrane (0.45 μm Nytran N nylon imprinted membrane) (GE Healthcare Life Science, Wauwatosa, WI, USA) with low protein binding capacity was used to cover LB agar plates containing 50 μg/ml ampicillin and 10 μg/ml carbenicillin. Mutated bacteria were diffused onto the membrane at a density of ¹⁰ cells/plate and grown at 37°C for 8 h. At the same time, a second nitrocellulose membrane (Bio-Trace ^™ NT nitrocellulose transfer membrane) (PALL, Port Washington, NY, USA) was placed on a fresh LB agar plate containing 50 μg/ml ampicillin, 10 μg/ml carbenicillin, and 1 mM isopropyl-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich). The first nylon membrane was removed from the LB agar plate and placed on the second membrane, which was then incubated at 37°C for 5 h. After incubation, the first membrane (with colonies) was removed and placed on a fresh LB agar plate containing 50 μg/ml ampicillin and 10 μg/ml carbenicillin, and stored at 4°C for later bacterial recovery. The second membrane was rinsed three times for 10 minutes in fresh phosphate-buffered saline containing 0.1% v/v Tween 20 (PBS/Tween) and then immersed in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Duchefa, Haarelem, The Netherlands) to visualize AP activity in E. coli colonies. E. coli colonies displaying differential AP activity were selected from the corresponding first filter, pooled, and subjected to a second round of mutagenesis and colony-picking assays. Following the second round of colony-picking assays, test-positive E. coli colonies were selected and grown in 10 ml of 2YT medium containing 50 μg/ml ampicillin and 10 μg/ml carbenicillin until an _OD600 of 0.5 was reached. IPTG was added to the culture at a final concentration of 0.1 mM, and the cells were grown overnight at 27°C. The culture supernatant was then harvested by centrifugation at 3,300 g for 20 min. To prepare periplasmic extracts, the cell pellet was resuspended in periplasmic extraction buffer (2 stocks; 200 mM Tris-HCl, 20 mM EDTA, 2 M NaCl, pH 7.4), freeze-thawed three times, and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was harvested to obtain a periplasmic extract containing the soluble anti-BCKD-AP fusion. Serial dilutions of the culture supernatant and periplasmic extract were prepared in PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich), and 50 ml of the culture supernatant or periplasmic extract sample was mixed with 100 ml of p-nitrophenylphosphate (pNPP) substrate (Roche, South San Francisco, CA, USA) in a 96-well microtiter plate (SPL, South Korea). After 5-10 min, 25 μl of 3M NaOH was added to each well to terminate the reaction, and the absorbance at 415 nm was measured using an ELISA plate reader (Bio-Rad, Hercules, CA, USA). Four mutant E. coli strains (M#5, M#7, M#54, and M#69) showing enhanced expression of the anti-BCKD-AP fusion were grown overnight at 37°C in 2YT medium without antibiotics. The cells were then plated onto LB agar plates at a density of ~ ¹⁰ cells/plate and grown overnight at 37°C. The resulting colonies were replicated onto LB agar plates with or without 50 μg/ml ampicillin. E. coli colonies that grew on LB agar plates without antibiotics but not on LB agar plates with antibiotics were selected and grown in 2YT medium without antibiotics until the OD ₆₀₀ reached ~1.0. Cell stocks were prepared by adding glycerol (20% v/v) and stored at 80° C. For use in cloning, electrocompetent cells were prepared using the mutant strains according to standard protocols and stored at 80° C. One of the mutant E. coli strains, M#5, was designated SUPEX5 (KCTC 12657BP) and used to express Fab and Fab-effector fusion proteins.

1-(6)-Enzyme-linked immunosorbent assay (ELISA)

For monoclonal phage ELISA, recombinant phage were obtained from positive E. coli clones by phage rescue and added to MaxiSorb ELISA plates (Nunc, Roskilde, Denmark) coated with 5 μg/ml HSA, RSA, MSA, or BSA at ~10 ⁸ CFU/well. Phage binding to antigen was allowed at 37°C at pH 6 or pH 7.4 for 1 hour. A goat anti-human κL Ab conjugated to HRPO (Sigma-Aldrich) was used as the secondary antibody. Binding signals were visualized using TMB substrate (BD Science, San Jose, CA, USA), and absorbance at 450 nm was measured using an ELISA plate reader (Bio-Rad, Hercules, CA, USA). Data represent the mean ± standard deviation of three experiments. For traditional ELISA, various antigens [human SA, rat SA, mouse SA, monkey SA (Alpha diagnostic Intl., San Antonio, TX, USA), canine SA (CUSABIO, Wuhan, Hubei, China), rabbit SA (Sigma-Aldrich), epidermal growth factor receptor (EGFR) (R&D systems, Minneapolis, MN, USA), epithelial cell adhesion molecule (EpCAM) (R&D systems), IL-15 receptor α (IL-15Rα) (R&D systems), IL-1β (eBioscience, San Diego, CA, USA), CD16a (R&D systems), c-MET (Sinobiological, Beijing, China)] were immobilized on microtiter plates at a concentration of 5 μg/ml, and Fab molecules were allowed to bind to the antigens and detected as above. To determine the concentration of soluble Fab or Fab-hGH fusion protein, a sandwich ELISA was performed using a mouse anti-human IgG Fd mAb (AprilBio) as a capture Ab and a goat anti-human κL chain pAb (Sigma-Aldrich) conjugated to HRPO as a detection antibody. A standard curve was drawn using human Fab fragments with known concentrations (Bethyl, Montgomery, TX, USA). The hGH domain was detected using T-20, a goat pAb specific for the C-terminus of hGH (Santacruz Biotechnology, Dallas, TX, USA) and NYThGH, a mouse mAb specific for full-length hGH (Prospec, East Brunswick, NJ, USA). Then, a rabbit anti-goat IgG pAb (Sigma-Aldrich) conjugated to HRPO or a goat anti-mouse IgG pAb (Sigma-Aldrich) conjugated to HRPO was used as a secondary antibody. Goat anti-human GCSF pAb (R&D Systems) was used to detect the G-CSF domain, and rabbit anti-human IFN-β pAb (PEPROTECH, Rocky Hill, USA) was used to detect the IFN-β domain.

Preparation of 1-(7)-soluble Fab and Fab-effector fusion proteins

Soluble Fab and Fab-hGH fusion proteins were produced by growing E. coli SUPEX5 cells at 37°C in 10 ml or 1 L of 2YT medium containing 50 μg/ml kanamycin until an OD _600nm = 0.5, followed by the addition of 0.05 mM IPTG. After incubation at 20°C with vigorous shaking for 20 h, the culture supernatant and cell pellet were separated by centrifugation at 3,300 g for 20 min. Periplasmic extracts were obtained as previously described. Purification was performed by passing the culture supernatant and/or periplasmic extract over Sepharose 4B resin immobilized with HSA (AprilBio). After thorough washing, Fab molecules bound to the resin were eluted using elution buffer (0.1 M glycine, 10% glycerol, pH 3) and immediately neutralized with Tris buffer (0.5 M Tris HCl, 2 M NaCl, pH 9.0). After affinity purification using AKTA FPLC (GE Healthcare, Wauwatosa, WI, USA), HserG/Lser was also subjected to gel filtration. Briefly, a Hiprep ^™ 16/60 Sephacryl ^™ S-200 HRP repacked column was equilibrated with equilibration buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and 5 μl of HserG/Lser (SL335 _Δds -hGH fusion) was loaded. The equilibration buffer was used at an alarm pressure of 0.35 MPa and an operating flow rate of 0.5 μl/min for elution. Fractions 13, 16, 19, and 23 were analyzed by SDS-PAGE as described below.

1-(8) Affinity measurement by biolayer interferometry

Real-time binding assays between purified SL335 and antigen (human SA, rat SA, or mouse SA) were performed using biomembrane interferometry with an Octet RED system (ForteBio, Menlo Park, CA, USA), as previously described, except that AR2G (amine reactive second-generation) sensors (Costin et al., (2013) J Virol. 87, 52-66) were used. Briefly, a predetermined concentration of SL335 was coupled to a kinetic-grade AR2G biosensor, and unbound Fab fragments were removed from the sensor surface by incubation in kinetic buffer (1 M ethanolamine, pH 8.5). The probe was then allowed to bind to predetermined concentrations of human SA, rat SA, or mouse SA at pH 6.0 or pH 7.4 (human SA concentrations at pH 6 and pH 7.4: 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM; rat SA concentrations at pH 6: 4 mM, 1 mM, 500 nM, 250 nM, and 125 nM; rat SA concentrations at pH 7.4: 4 mM, 2 mM, 1 mM, 500 nM, and 125 nM; mouse SA concentrations at pH 6 and pH 7.4: 20 mM, 10 mM, 5 mM, 2.5 mM, and 12.5 mM), followed by dissociation in PBS containing 0.1% BSA at pH 6 or pH 7.4. Association and dissociation kinetics were calculated using the Octet QK software package, which was adapted to the observed binding curves using a 1:1 binding model to calculate association rate constants. Association and dissociation rate constants were calculated using at least three different concentrations of human SA, rat SA, or mouse SA. The equilibrium dissociation constant was calculated by dividing the kinetic dissociation rate constant by the kinetic association rate constant.

1-(9) Generation of SL335-hGH fusion construct

To create SL335ds, a mutant Fd (Cys 233 Ser 233 substitution) (designated Hser) was obtained from the codon-optimized Fd chain gene of SL335 by PCR amplification using a set of PCR primers #9 (5'-ggggaatt catgaaatatctgctgcctacggcggcggcgggcctgctgctgctggctgcacaa-3' (SEQ ID NO: ²⁹ )) and #10 (5'-gggaagcttttagctgctcttcggttccacgcgtt- ³ ' (SEQ ID NO: 30)). The ~750 bp PCR product was treated with EcoR I/Hind III and ligated with pHEKA. A mutant L chain (Cys 214 → Ser 214 substitution) (designated Lser) was also obtained by PCR amplification from the codon-optimized L chain gene of SL335 using a set of PCR primers #11 (5'-gggggatccatgaaaaaaactgcgattgcgattgcggtgctggccggctttg-3' (SEQ ID NO: 31)) and #12 ( ^5' -gggctcgagttagctttcgc cgcggttaaagctctttg-3' (SEQ ID NO: ³² )), cut with BamH I/Xho I, and cloned into pHEKA containing Hser. The cloning procedure for generating the HcysG/Lcys construct was as follows: a set of PCR primers #9 and #13 (5'-agatccaggagctggtgcagaaccgcagctcttcggttccacgcgtt-3' (SEQ ID NO: 33)) was used to PCR amplify the wild-type Fd with Cys ²³³ (designated Hcys) from the codon-optimized Fd of SL335; and a set of PCR primers #14 (5'-ggttctgcaccagctcctggatcttttccgaccattccgctgagccg-3' (SEQ ID NO: 34)) and #15 (5'-gggaagcttttagaagccgcaggagccctcca-3' (SEQ ID NO: 35)) was used to PCR amplify hGH containing the linker sequence from the codon-optimized hGH gene. The Hcys and hGH genes were joined together by assembly PCR using a set of PCR primers #9 and #15, cut with EcoR I/Hind III, and cloned into pHEKA containing the wild-type L chain with Cys ²¹⁴ of SL335 (referred to as Lcys). To generate the LcysG/Hcys construct, Lcys was PCR amplified from the codon-optimized L chain of SL335 using a set of PCR primers #11 and #16 (5'-agatccaggagctggtgcagaaccgcattcgccgcggttaaagctcttt-3' (SEQ ID NO: 36)), and hGH containing the linker sequence was PCR amplified from the codon-optimized hGH gene using a set of PCR primers #14 and #17 (5'-gggctcgagttagaagccgcaggagccctcca-3' (SEQ ID NO: 37)). Using a set of PCR primers #11 and #17, the Lcys and hGH genes were linked by combinatorial PCR to generate LcysG, cut with BamH I/Xho I, and cloned into pHEKA containing wild-type Fd. To create the HserG/Lcys construct, Hser was PCR amplified from the codon-optimized wild-type Fd chain using a set of PCR primers #9 and #18 (5'-gggctcgagttagaagccgcaggagccctcca-3' (SEQ ID NO: 38)). PCR amplification of hGH containing the linker sequence, combinatorial PCR, and cloning of HserG were performed as for the HcysG/Lcys construct. To generate the LserG/Hcys construct, Lser was PCR amplified from the codon-optimized L chain of SL335 using a set of PCR primers #11 and #19 (5'-agatccaggagctggtgcagaaccgctgctcttcggttccacgcgtt-3' (SEQ ID NO: 39)). PCR amplification of hGH containing the linker sequence, combined PCR, and cloning of LserG were performed as in the creation of the LcysG/Hcys construct. To generate the HerG/Lser construct, PCR amplification of HserG and hGH, as well as combined PCR, were performed as in the creation of the HserG/Lcys construct, except that pHEKA containing Lser was used for cloning. LserG/Hser was constructed as in the creation of the LserG/Hcys construct, except that pHEKA containing Hser was used for cloning. Table 2 below shows the PCR primers used to prepare the SL335-hGH fusion construct and the SL335 _Δds -hGH fusion construct.

【Table 2】

PCR primers for SL335-hGH fusion construct or SL335 _Δds -hGH fusion construct

Generation of 1-(10)-SL335-GCSF fusion constructs

The cloning procedure for generating the HcysGF/Lcys construct was as follows: Hcys was PCR amplified from the codon-optimized H chain of SL335 using a set of PCR primers #9 and #20 (5'-agatccaggagctggtgcagaaccgctttcgccgcggttaaagctctttg-3' (SEQ ID NO: 40)), and G-CSF containing a linker sequence was PCR amplified from a codon-optimized G-CSF gene using a set of PCR primers #21 (5'-ggttctgcaccagctcctggatctgcgcctacctatcgcgcgagca-3' (SEQ ID NO: 41)) and #22 (5'-gggaagcttattaaggctgtgccagatggcgcag-3' (SEQ ID NO: 42)). The Hcys and G-CSF genes were joined together by combinatorial PCR using a set of PCR primers #9 and #22, cut with EcoR I/Hind III, and cloned into pHEKA containing the L chain of SL335. To generate the LcysGF/Hcys construct, Lcys was PCR amplified from the codon-optimized L chain of SL335 using a set of PCR primers #11 and #23 (5'-agatccaggagctggtgcagaaccgcattcgccgcggttaaagctcttt-3' (SEQ ID NO: 43)), and G-CSF containing the linker sequence was PCR amplified from the codon-optimized G-CSF gene using a set of PCR primers #21 and #24 (5'-taacagatctgcggccgcactcgagattaaggctgtgccagatg gcgcag-3' (SEQ ID NO: 44)). The Lcys and G-CSF genes were linked by combinatorial PCR using a set of PCR primers #11 and #25 (5'-agatccaggagctggtgcagaaccgctgc tcttcggttccacgcgtt-3' (SEQ ID NO: 45)), cut with BamH I/Xho I, and cloned into pHEKA containing the Fd of SL335. To create the HserGF/Lser construct, Hser was PCR amplified from the codon-optimized Fd of SL335 using a set of PCR primers #9 and #25. The Hser and G-CSF genes were linked by combinatorial PCR using a set of PCR primers #9 and #22, cut with EcoR I/Hind III, and cloned into pHEKA containing Lser. To generate the LserGF/Hser construct, Lser was PCR amplified from the codon-optimized L chain of SL335 using a set of PCR primers #11 and #26 (5-agatccaggagctggtgcagaaccgctttcgccgcggttaaagctctttg-3 (SEQ ID NO: 46)). G-CSF, including a linker sequence, was also PCR amplified from the codon-optimized G-CSF gene using a set of PCR primers #21 and #24. The Lcys and G-CSF genes were ligated by combined PCR using a set of PCR primers #11 and #25, cut with BamHI/XhoI, and cloned into pHEKA containing Hser. The PCR primers used to prepare the SL335-GCSH fusion construct and the SL335 _Δds -GCSF fusion construct are shown in Table 3 below.

【Table 3】

PCR primers for SL335-GCSH fusion construct or SL335 _Δds -GCSF fusion construct

1-(11) Generation of SL335-IFN-b fusion construct

The cloning procedure for generating the HcysIFNb/Lcys construct was as follows: Hcys was PCR amplified from the codon-optimized H chain of SL335 using a set of primers #9 and #27 (5'-agatccaggagctggtgcagaaccgcagctcttcggttccacgcgtt-3' (SEQ ID NO: 47)), and IFN-b containing a linker sequence was PCR amplified from the codon-optimized IFN-b1a gene using a set of PCR primers #28 (5'-ggttctgcaccagctcctggatcttcatacaacctgctgggcttcctg-3' (SEQ ID NO: 48)) and #29 (5'-gggaagcttttagttgcgcagatagccggtcag-3' (SEQ ID NO: 49)). The Hcys and IFN-b1a genes were ligated by combined PCR using a set of PCR primers #9 and #29, cut with EcoRI/Hind III, and cloned into pHEKA containing Lcys. To create the HserIFN-b/Lser construct, Hser was PCR amplified from the codon-optimized H chain of SL335 using a set of PCR primers #9 and #30 (5'-agatccaggagctggtgcagaaccgctgctcttcggttccacgcgtt-3' (SEQ ID NO: 50)). The Hser and IFN-b1a genes were ligated by combined PCR using a set of PCR primers #9 and #29, cut with EcoRI/Hind III, and cloned into pHEKA containing Lser. Table 4 below shows the PCR primers used to prepare the SL334-IFNb fusion construct and the SL335 _Δds -IFNb fusion construct.

【Table 4】

PCR primers for SL335-IFNb fusion construct or SL335 _Δds -IFNb fusion construct

1-(12) Generation of EGL4-hGH and 1b28-hGH Fusion Constructs

EGL4, a human anti-EGFR Fab, and 1b28, a human anti-IL-1b Fab, were isolated from the HuDVFab-8L antibody library (unpublished, AprilBio Co.). To create EGL4 _wt and EGL4 _Δds , Hcys and Hser were PCR amplified from the H chain gene of EGL4 cDNA using PCR primer sets #5 and #6, and #5 and #31 (5'-gggaagcttattaactagatttgggctca actctcttg-3' (SEQ ID NO: 51)), respectively. The ~750 bp PCR product was treated with EcoRI/HindIII, ligated with pHEKA, and then transformed into MC1061 competent cells. Lcys and Lser were PCR amplified from the L chain gene of EGL4 cDNA using PCR primer sets #11 and #32 (5'-gggctcgagttagcattcgccgcggttaaagctcttt-3' (SEQ ID NO: 52)), and #11 and #33 (5'-gggctcgagttagctttcgccgcggttaaagctcttt-3' (SEQ ID NO: 53), respectively. These were cut with BamHI/XhoI and cloned into pHEKA containing Hcys or Hser of EGL4, respectively. To create the EGL4 _wt -hGH fusion construct, the cloning procedure used to generate the HcysG/Lcys construct was as follows. Hcys was PCR amplified from EGL4 cDNA using a set of PCR primers #5 and #34 (5'-agatccaggagctggtgcagaaccacaagatttgggctcaactctcttgtc-3' (SEQ ID NO: 54)), and hGH containing a linker sequence was PCR amplified from a codon-optimized hGH gene using a set of PCR primers #14 and #15. The Hcys and hGH genes were ligated by combined PCR using a set of PCR primers #5 and #15, cut with EcoR I/Hind III, and cloned into pHEKA containing the Lcys of EGL4. To create the EGL4 _Δds -hGH fusion construct, Hser was PCR amplified from the H strand of the EGL4 cDNA using a set of PCR primers #5 and #35 (5'-agatccaggagctggtgcagaaccactagatttggg ctcaactctcttgtc-3' (SEQ ID NO: 55)). A further set of PCR primers was used to PCR amplify hGH containing a linker sequence from a codon-optimized hGH gene. The Hser and hGH genes were ligated by combinatorial PCR using a set of PCR primers #5 and #15, cut with EcoRI/HindIII, and cloned into pHEKA containing the Lser of EGL4 _Δds . 1b28 _wt , 1b28 Δds, 1b28 wt-hGH, and 1b28 _Δds -hGH were created using the same PCR primers as for the EGL4-hGH fusion, except that 1b28 _cDNA was used as the _PCR template. Table 5 below shows the PCR primers used to prepare the EGL4-hGH fusion construct and the 1b28-hGH fusion construct.

【Table 5】

PCR primers used to prepare EGL4-hGH fusion constructs and 1b28-hGH fusion constructs

1-(13) SDS-PAGE and Western blotting analysis

For SDS-PAGE analysis, purified SL335 _wt -hGH and SL335 _Δds -hGH proteins were resuspended in LDS sample buffer (Invitrogen) with or without sample reducing agent (Invitrogen) and loaded onto the gel at a concentration of 7 μg/well. Protein bands were visualized using Coomassie blue staining (Bio-Rad). For Western blot analysis, 500 ng of affinity-purified SL335 _wt -hGH and SL335 _Δds -hGH were loaded into each well as described above and transferred to a nitrocellulose membrane. The membrane was blocked with 3% skim milk (Bio-Rad) in PBS containing 0.01% Tween (Sigma-Aldrich) and the proteins were detected by incubation with a goat anti-human κL chain pAb conjugated to AP (Bethyl). Nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Duchefa) was added to the membrane to visualize the binding signal.

1-(14) Chip-based capillary electrophoresis

Chip-based capillary electrophoresis was performed using an Agilent 2100 bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). Protein samples were prepared according to the manufacturer's protocol and analyzed on a Protein 80 kit, which is recommended for analyzing proteins of 5 to 80 kDa. Briefly, the sample was mixed with sample buffer with or without DTT for reducing or non-reducing electrophoresis, respectively. The sample was denatured at 95°C and loaded onto a chip filled with appropriate reagents containing a fluorescent dye and a gel solution. The chip was then inserted into the system and run on the system using Expert 2100 software. The results were plotted to reflect fluorescence intensity units for protein size.

1-(15)MALDI-TOF mass spectrometry

MALDI-TOF mass spectrometry was performed on an Autoflex III Smartbeam apparatus (Bruker Daltonics, Billerica, MA, USA). Samples were mixed with an equal volume of MALDI matrix (10 mg/mL α-cyano-4-hydroxycinnamic acid) and spotted onto a MALDI target plate. External calibration was performed using a Peptide and Protein MALDI-MS Calibration Kit (Sigma-Aldrich). In positive ion mode, mass spectra were acquired in the m/z range of 15,000-160,000 for the SL335 _wt -hGH fusion and 10,000-70,000 for the SL335 _Δds -hGH fusion, respectively.

1-(16) Determination of hGH biological activity in vitro

Nb2-11 rat lymphoma cells (Sigma-Aldrich) were grown in complete DMEM supplemented with 5% horse serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Invitrogen) at 37°C in a humidified 5% _CO2 incubator (Tanaka et al., 1980). The cells were rinsed twice with DMEM, centrifuged at 1,000 g for 5 min, and resuspended at 8 × ¹⁰⁴ cells/ml in DMEM supplemented with 5% (v/v) horse serum. A 50 μg aliquot of the cell suspension was added to each well of a 96-well plate and incubated overnight. The cells were then treated with increasing concentrations (0–20 nM) of either unmodified rhGH (Dong-A Pharmaceuticals, Seoul, South Korea) or SL335 _Δds -hGH in 50 ml of DMEM supplemented with 5% horse serum for 48 h at 37°C. After incubation, 10 μl of CCK-8 (Dojindo, Mashiki-machi, Japan) was added to each well and incubated for 4 h. The absorbance at a wavelength of 450 nm was recorded on a microplate reader (Bio-Rad).

1-(17) Serum stability of SL335Δds-hGH

SL335 _wt and SL335 _Δds -hGH (10 μg / ml final concentration) were resuspended in fetal bovine serum (FBS) containing 0.03% sodium azide (Thermo Scientific, Waltham, MA, USA) and incubated at 37 ° C for 16 days. Small aliquots (50 ml) were collected daily and stored at -20 ° C until use. Binding reactivity to HSA was determined by ELISA, and hGH in vitro bioactivity was measured using Nb2-11 cells (Sigma-Aldrich) as described above.

1-(18) In vivo pharmacokinetic determination

In qualified CRO company (ChemOn, Suwon City (Suwon), South Korea), PK study was carried out. Animals were raised under the standard diet of rodent pellet feed and arbitrary drinking water conditions, and animals were kept in a room with constant humidity and constant temperature, with controlled lighting (12h lighting, then 12h darkness). Briefly, with the amount of 1mg/kg, SL335 and Neg Fab (irrelevant human Fab) were injected intravenously (IV) or subcutaneously (SC) into Sprague Dawley rats, three per group, and at several time points (5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h, 96h and 144h for IV; and 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h, 48h and 96h for SC) serum samples were obtained. The concentrations of SL335 and Neg Fab in serum samples were measured by sandwich ELISA using mouse anti-human IgG Fd mAb and HRPO-conjugated goat anti-human κL chain pAb as capture and detection antibodies, respectively. Human Fab fragments of known concentrations were also included in the assay to obtain a standard curve. The curves of (SL335 and Neg Fab) serum concentrations relative to time were fitted to a non-compartmental model using WinNonlin software and plotted using Sigma Plot software. Similarly, SL335 _Δds- hGH and SL335 Δds-hGH were injected intravenously or subcutaneously into rats, three to four per group, respectively. For IV administration, the dose of SL335 _Δds- hGH and SL335 Δds-hGH was 0.3 mg/kg, while for SC administration, the dose of SL335 Δds-hGH and SL335 _Δds -hGH was 0.6 mg/kg. Serum samples were obtained at several time points (5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h for _Δds -hGH and 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, and 144 h for SL335 Δds-hGH). The amount of Δds-hGH in the serum samples was measured using an hGH ELISA kit (Genway, San Diego, CA, USA), and the amount of SL335 _Δds -hGH was measured using a sandwich ELISA as described above. The serum concentration versus time curve was fitted to a one-compartment model using Phoenix ^™ WinNonlin software (version 6.2).

1-(19) In vivo pharmacodynamics assay

As previously described, at ChemOn, the ability of daily and weekly doses of SL335 _Δds -hGH to promote weight gain was analyzed in hypophysectomized rats using subcutaneous dosing (see Clark et al., (1996) J. Biol. Chem. 271, 21969-21977). Briefly, hypophysectomized Sprague Dawley pups (Harlan, Tokyo, Japan) were purchased and any animals that gained more than 7 g in the first 15 days after surgery were excluded from the study. Animals were randomly divided into five treatment groups (vehicle only, daily injection of 0.3 mg/kg, and weekly injections of 0.6 mg/kg, 1.2 mg/kg, or 2.4 mg/kg SL335 _Δds -hGH). Following the initial dosing regimen, body weights were recorded daily. Tibial growth was carefully measured using a bone caliper. Statistical comparisons were performed using analysis of variance followed by Dunnett's Multiple Comparison Test, and p values less than 0.05 were considered significant.

2. Experimental results

2-(1) Isolation of anti-SA Fab clones

Under the conditions of pH 6 or pH 7.4, for the magnetic beads being put together with mankind SA, rat SA or mouse SA, HuDVFab-8L antibody library is selected. After three rounds of biological panning, monoclonal phage ELISA is carried out to identify phage antibody clones that are specific to antigen. More than 60 positive clones (data not shown) are identified by ELISA, and DNA sequencing analysis is carried out to _VH and _VL genes, 8 discrete phage antibodies are identified, referred to as SA138, SA139, SA140, SA141, SL18, SL301, SL310 and SL335. By under the conditions of pH 6 or pH 7.4, monoclonal phage ELISA is carried out, the binding reactivity (Fig. 1A & Fig. 1B) of these clones and mankind SA, rat SA, mouse SA or ox SA is confirmed. Regardless of pH conditions, three phage antibody clones SA138, SA139 and SA141 are only reactive to mankind SA. SA140 only recognizes human SA at pH 7.4, but its binding reactivity disappears at pH 6. On the other hand, SL18, SL310, and SL335 bind to human SA, rat SA, and mouse SA under both pH conditions, but with slightly different intensities. SL301 has significant reactivity with human SA and rat SA at both pHs, but only weak reactivity with mouse SA at pH 7.4. All eight Fab clones have no reactivity to bovine SA. SL18, SL301, SL310, and SL335 were further characterized because they have cross-reactivity to SA from at least two different species. The Fd chain and L chain genes of the four phage antibody clones were subcloned into a pHEKA vector for periplasmic expression in E. coli, and soluble Fab fragments were prepared from the culture supernatant or periplasmic extract. After affinity purification, ELISA was performed to compare the binding reactivity of these fragments to human SA, rat SA or mouse SA under the conditions of pH 6 (FIG. 2A) and pH 7.4 (FIG. 2B). HSA, RSA, MSA or BSA at a concentration of 5 μg/ml were fixed in each well of a microtiter plate, and four purified Fab molecules (SL18, SL301, SL310 and SL335) were allowed to bind to the antigen at pH 6 (FIG. 2A) and pH 7.4 (FIG. 2B). A pAb of a goat anti-human κ L chain conjugated with HRPO was used as the second antibody. TMB substrate was used to visualize the binding signal, and an ELISA enzyme reader (Bio-Rad) was used to measure the absorbance at 450 nm. The data represent the mean standard deviation of three experiments. In combination with human SA, the order of the binding signal was SL335>SL310>SL301>SL18 at pH 6 and pH 7.4. For rat SA binding, the order was SL335 > SL310 > SL301 > SL18 at pH 6, while at pH 7.4 it was SL335 = SL310 > SL301 = SL18. For mouse SA binding, the order was SL18 > SL335 > SL310 at pH 6, while at pH 7.4 it was SL335 > SL310 > SL18. As shown in Figure 2, SL301 did not bind to mouse SA at pH 6 and only weakly bound at pH 7.4. It was found that, among the four Fab clones, SL335 bound best to both human and rat SA, regardless of pH conditions. SL335 binds to human SA twice as strongly at pH 6 as at pH 7.4 (50% binding signal is 20 ng/ml vs. 40 ng/ml), 20 times as strongly as to rat SA under the same pH conditions (50% binding signal is 20 ng/ml vs. 400 ng/ml), and four times as strongly as to rat SA at pH 7.4 (50% binding signal is 40 ng/ml vs. 160 ng/ml).

2-(2) Cross-reactivity and binding affinity of SL335

Because SL335 was the best binder among the four anti-human SA Fab clones, its cross-reactivity was further analyzed by ELISA. As shown in Figure 2, binding reactivity to human SA, rat SA, and mouse SA was reproduced. It was also found that SL335 highly recognized cynomolgus monkey SA, but bound less strongly to canine SA. In addition, SL335 did not recognize rabbit SA, nor did it recognize other unrelated antigens including EGFR, EpCAM, IL-15Ra, IL-1b, CD16a, or c-MET. The binding affinity of SL335 for human SA, rat SA, and mouse SA at pH 6 or pH 7.4 was further measured by biofilm interferometry by passing different concentrations of antigen through a biosensor coated with SL335 (see Table 6 below). The results correlate well with the ELISA data in Figure 2, where the dissociation constants of SL335 for HSA are 9 nM at pH 6 and 13 nM at pH 7.4, and the dissociation constants for RSA are 122 nM and 65 nM at pH 6 and pH 7.4, respectively. The binding affinity of SL335 for MSA is approximately 10 mM at pH 6 and approximately 1.6 mM at pH 7.4, but these data are not shown in Table 6 due to lack of reliability.

【Table 6】

Determination of the binding affinity of SL335 and HserG/Lser by biomembrane interferometry binding assay

The association and dissociation kinetics were calculated using the Octet QK software package.

2-(3) In vivo pharmacokinetics of SL335

Among all plasma proteins, HSA has an exceptionally long half-life in the FcRn-mediated recycling mechanism and is commonly used as a fusion partner for extending the half-life of therapeutic proteins. In addition, antibody fragments associated with serum albumin are known to have prolonged serum half-life. Therefore, a pharmacokinetic analysis was performed to confirm whether SL335 also has a long serum half-life. A human Fab with unknown binding specificity was used as a negative control (Neg Fab). 1 mg/kg of SL335 and Neg Fab were injected intravenously or subcutaneously into rats, three per group, and serum samples were collected at several time points (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, and 144 h for IV, and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 96 h for SC). The concentrations of SL335 and Neg Fab in serum samples were measured by sandwich ELISA using mouse anti-human IgG Fd mAb and HRPO-conjugated goat anti-human κ L chain pAb as capture and detection antibodies, respectively. Human Fab fragments of known concentrations were also used in the assay to obtain a standard curve. The serum concentration versus time curve was fitted to a one-compartment model (SL335 and Neg Fab) using WinNonlin software, and to a two-compartment model using Sigma Plot software. In intravenous administration, the terminal half-life (t _1/2 ) of SL335 was 37h, and its area under the curve (AUC _0→∞ ) was 187h mg/ml, indicating a 10-fold increase in t _1/2 and a 26-fold increase in AUC _0→∞ compared to Neg Fab (3.8h mg/ml and 7h mg/ml, respectively) ( FIG. 3A ). Subcutaneous injection of SL335 produced similar measurements, including a 9-fold increase in t _1/2 (120 h vs. 13 h) and a 44-fold increase in AUC _0→∞ (87 vs. 2 h mg/ml) compared to Neg Fab ( FIG3B ). These results clearly show that SL335 has a prolonged serum half-life and suggest that SL335 does not interfere with the interaction between RSA and FcRn in rats.

2-(4) Production of SL335-hGH Fusion

SL335 was used to create two SL335-hGH fusions and four additional SL335-hGH fusions by genetically fusing recombinant hGH (27-191 aa) to the N-terminus or C-terminus of the Fd or L chain via a short peptide linker. Recombinant hGH cDNA (27-191 aa) was fused to the C-terminus of the H or L chain of SL335 _wt in the classic Fab format via a short peptide linker, resulting in two fusion types (HcysG/Lcys and LcysG/Hcys) of the construct. Four additional fusion types (HserG/Lcys, LserG/Hcys, HserG/Lser, and LserG/Hser) were constructed as above, except that either a null form of _SL335 ( _SL335null ) or a dsFab form ( _SL335Δds ) in which Cys ²³³ at the C-terminus of CH1 and/or Cys ²¹⁴ at the C-terminus of _CLk were replaced with Ser was used. For periplasmic expression of the fusion proteins, the ompA (MKKTAIAIAVLAGFATVAQA (SEQ ID No: 56)) leader sequence was placed upstream of the L chain or L-hGH fusion, and the pelB leader sequence (MKYLLPTAAAGLLLLAAQPAMA (SEQ ID No: 57)) was placed upstream of the H chain or H-hGH fusion. In these preliminary experiments, hGH was genetically linked to the N-terminus of the Fd chain or L chain, resulting in low or no expression of soluble fusion protein. Fusion of hGH to the C-terminus of Fd also showed low expression and appeared to disrupt the folding of the hGH domain, most likely due to abnormal disulfide bonding in the SL335-hGH fusion (data not shown). It has been previously reported that removal of the interchain disulfide bonds of Fab by mutation of the C-terminal Cys residues in _CH1 and _CLk (Cys ²³³ and Cys ²¹⁴ , respectively) did not affect periplasmic production levels, stability of extraction and purification, serum stability, or serum half-life (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest; Humphreys et al., (1997) J. Immunol. Methods. 209, 193-202; Humphreys et al., (2007) Protein Eng Des Sel. 20, 227-234). By replacing both Cys ²³³ of _CH1 and Cys ²¹⁴ of _CLk with serine (Cys ²³³ Ser ²³³ and Cys ²¹⁴ Ser ²¹⁴ substitutions), we tested whether these Cys residues in SL335 regulate soluble expression and proper folding of SL335-hGH fusions. Figure 4 illustrates six SL335-hGH fusion constructs. In addition to SL335 _wt and SL335 _Δds , another SL335 variant, termed SL335 _null , was created by substituting Cys ²³³ of _CH1 or Cys ²¹⁴ of _CLk with Ser to illustrate the effect of each cysteine residue (Cys ²³³ or Cys ²¹⁴ ), respectively. The two SL335 _wt fusion derivatives are HcysG/Lcys (HCys ²³³ -hGH fusion paired with LCys ²¹⁴ ) and LcysG/Hcys (LCys ²¹⁴ -hGH fusion paired with HCys ²³³ ), the two SL335 _null fusion derivatives are HserG/Lcys (HSer ²³³ -hGH fusion paired with LCys ²¹⁴ ) and LserG/Hcys (LSer ²¹⁴ -hGH fusion paired with HCys ²³³ ). Finally, the two SL335 _Δds fusion derivatives are HserG/Lser (HSer ²³³ -hGH fusion paired with LSer ²¹⁴ ) and LserG/Hser (LSer ²¹⁴ -hGH fusion paired with HSer ²³³ ). These six SL335-hGH fusion constructs were expressed in E. coli SUPEX5 host cells, and the yield and HSA-binding reactivity of these six SL335-hGH fusion proteins in the culture supernatant were analyzed by ELISA. Under the same conditions in the presence of IPTG, E. coli clones expressing the SL335-hGH fusion protein were grown and the culture supernatant was harvested by simple centrifugation. Using a mouse anti-human Fd mAb as the capture Ab and a goat anti-human κL chain pAb conjugated to HRPO as the detection antibody, the concentration of soluble SL335-hGH fusions was measured by sandwich ELISA (Figure 5A). No soluble Fab forms from LcysG/Hcys or LserG/Hcys were detected. Although the data are not shown, Western blots performed using E. coli cell lysates showed that Cys ²³³ of Fd was responsible for heavy chain degradation and non-secretion of the Fd fragment, which is likely due to protein aggregation. The yield of HcysG/Lcys was 0.5 μg/ml, and the yields of HserG/Lcys and LserG/Hser were approximately 1.8 μg/ml and 1.4 μg/ml, respectively ( FIG. 5A ). Interestingly, the yield of HserG/Lser was approximately 4 μg/ml, eight times the yield of HcysG/Lcys. Periplasmic extracts showed the same expression pattern, but the total yield was only ˜30% of that present in the culture supernatant (data not shown). In repeated experiments, it was confirmed that the difference in yield between HcysG/Lcys and HserG/Lser was independent of clonal variation or growth rate of the E. coli clones. The binding reactivity of the SL335-hGH fusion to HSA was compared using microtiter plates coated with 5 μg/ml HSA and incubated with serial dilutions of culture supernatant containing the SL335-hGH fusion. The SL335-hGH fusion bound to HSA was then detected using a goat anti-human κL chain pAb conjugated to HRPO. As expected, the binding signal generated by detecting HSA-bound HserG/Lser using the anti-human κL pAb was 8 times that of HcysG/Lcys, and approximately 4 times that of HserG/Lcys and LserG/Hser ( FIG5B ). A similar binding signal pattern was observed when the SL335-hGH fusion was detected using T-20, a goat pAb specific for the C-terminus of hGH ( FIG5C ). However, when detected using the mouse mAb NYThGH, which is specific for full-length hGH, HserG/Lser generated a binding signal 30 times that of HserG/Lcys and LserG/Hser, and 60 times that of HcysG/Lcys ( FIG. 5D ), suggesting that the interchain disulfide bond present in SL335 interferes with the binding of NYThGH to the hGH domain of HcysG/Lcys. Since HcysG/Lcys and HserG/Lser represent the SL335-hGH fusions created using SL335 _wt and SL335 _Δds , respectively, they are hereinafter referred to as SL335 _wt -hGH fusion and SL335 _Δds -hGH fusion, respectively ( FIG. 5 ).

To determine whether high yields of soluble SL335 _Δds -hGH fusions depend on the removal of interchain disulfide bonds in SL335, the host E. coli strain, or the induction temperature, SL335 _wt , SL335 _Δds , SL335 wt-hGH fusions, and SL335 _Δds- hGH fusions were expressed in parental MC1061 and mutant SUPEX5 cells at 20°C ( FIG. 6A ), 25°C ( FIG. 6B ), or _30° C ( FIG. 6C ), and the amount of Fab molecules in the culture supernatant was measured by ELISA. The yield of SL335 _wt expressed in the MC1061 strain was 1 μg/ml at 20°C, which is approximately three times the yield at 25°C and 30°C. This suggests that induction of SL335 _wt at temperatures below 25°C is particularly advantageous when using MC1061 as a host strain. Similar results were obtained using the SUPEX5 strain. In the case of SL335 _Δds , the yield at 20°C was approximately 1.3 μg/ml regardless of the host E. coli strain and induction temperature. These results show that the presence or absence of interchain disulfide bonds in the Fab does not significantly affect the yield of soluble Fab production at 20°C, regardless of the host E. coli strain and induction temperature. The yield of SL335 _wt -hGH fusion was approximately 0.3-0.5 μg/ml, regardless of the host E. coli strain and induction temperature. On the other hand, the yield of the SL335 _Δds -hGH fusion expressed in the MC1061 strain was 1.8 μg/ml at both 20°C and 25°C, and 1.5 μg/ml at 30°C, indicating less temperature dependence. However, the yield of the SL335 _Δds -hGH fusion expressed in the SUPEX5 strain was 4.0 μg/ml at both 20°C and 25°C, and 3.5 μg/ml at 30°C. These results indicate that the yield of the SL335-hGH fusion protein obtained using the SL335 _Δds form and the E. coli SUPEX5 strain was 12 times that obtained using the combination of the SL335 _wt form and the E. coli MC1061 strain.

2-(5) Generation of SL335-GCSF, SL335-IFNb, EGL4-hGH, and 1b28-hGH Fusion Constructs

To demonstrate the beneficial effects of the Fab _Δds format and the SUPEX5 strain on improving soluble expression of Fab-effector fusion proteins, various Fab-effector fusion constructs were generated. First, two SL335-GCSF fusion variants (designated HcysGCSF/Lcys for SL335 _{wt- GCSF} and HserGF/Lser for SL335 _Δds -GCSF) and two SL335-IFNb fusion variants (designated HcysIFNb/Lcys for SL335 _wt- IFNb and HserIFNb/Lser for SL335 _Δds -IFNb) were created in the same manner as for the generation of SL335 wt-hGH and SL335 _Δds -hGH fusions to determine the impact of the effector domain. The induction temperature was set to an optimal 20°C, and the expression levels of these fusion proteins in E. coli culture supernatants were compared by ELISA. The production of SL335 _wt -GCSF in MC1061 and SUPEX5 was 0.3 mg/ml and 0.6 mg/ml, respectively, and the production of SL335 _Δds -GCSF in MC1061 and SUPEX5 was 0.6 mg/ml and 1.5 mg/ml, respectively ( FIG7A ). In addition, the production of SL335 _wt -IFNb in MC1061 and SUPEX5 was approximately 0.16 mg/ml, and the production of SL335 _Δds -IFNb in MC1061 and SUPEX5 was 0.2 mg/ml and 0.5 mg/ml, respectively ( FIG7B ). Thus, the combination of the SL335 _Δds -GCSF fusion and the SUPEX5 strain produced 5-fold more SL335-GCSF fusion than the combination of the SL335 _wt -GCSF fusion and the MC1061 strain, and the combination of the SL335 _Δds -IFNb fusion and the SUPEX5 strain produced approximately 3-fold more SL335-IFNb fusion than the combination of the SL335 _wt -IFNb fusion and the MC1061 strain. Secondly, we also created two Fab-hGH fusion constructs using EGL4 (human anti-EFGR Fab) and 1b28 (human anti-IL-1b Fab) to determine the impact of Fab. In the same manner as for the generation of the SL335 _wt -hGH and SL335 _Δds -hGH fusions, the two EGL4-hGH fusion constructs were an HcysG/Lcys type EGL4 _wt -hGH fusion and an HserG/Lser type EGL4 _Δds -hGH fusion. Similarly, the 1b284-hGH fusion construct is a HcysG/Lcys-type 1b28 _wt -hGH fusion and a HserG/Lser-type 1b28 _Δds -hGH fusion. The yield of the EGL4 _wt -hGH fusion in the MC1061 and SUPEX5 strains was 8090 ng/ml, and the yield of the EGL4 _Δds -hGH fusion in the MC1061 strain was 140 ng/ml, while the yield in the SUPEX5 strain was 220 ng/ml ( FIG. 8A ), indicating that the combination of the EGL4 _Δds -hGH fusion and the SUPEX5 host cells produced 2.4 times the amount of EGL4-hGH fusion protein in the culture supernatant as the combination of the EGL4 _wt -hGH fusion and the MC1061 host cells. In the case of the 1b28-hGH fusion construct, the yield of the 1b284 _wt -hGH fusion was 50 ng/ml in the MC1061 strain and 100 ng/ml in the SUPEX5 strain, and the yield of the 1b28 _Δds -hGH fusion was 900 ng/ml in the MC1061 strain and 4 mg/ml in the SUPEX5 strain ( Figure 8B ), indicating that the combination of the 1b28 _Δds -hGH fusion and SUPEX5 host cells produced 800-fold more 1b28-hGH fusion form in the culture supernatant than the combination of the 1b28 _wt -hGH fusion and MC1061 host cells.

2-(6) Molecular characteristics of SL335wt-hGH and SL335 _Δds -hGH

The molecular characteristics of the SL335 _wt -hGH and SL335 _Δds -hGH fusions are further described. The fusion proteins in the culture supernatant were affinity purified through a resin coated with HSA and analyzed by SDS-PAGE and Western blotting under reducing and non-reducing conditions. HcysG/Lcys (lane 1) and HserG/Lser (lane 2) were affinity purified from the culture supernatant using HSA-immobilized agarose beads and subjected to SDS-PAGE using 4-12% Bis-Tris gels under reducing or non-reducing conditions. Protein bands were visualized using Coomassie blue staining (Figure 9A). The proteins from each SDS-PAGE were transferred to a nitrocellulose membrane and Lcys and Lser were detected using a goat anti-human κL Ab conjugated to AP (Figure 9B). The binding signal was visualized using NBT/BCIP substrate. In SDS-PAGE analysis, under reducing conditions, both SL335 _wt -hGH and SL335 _Δds -hGH produced two major protein bands of 46 kDa and 23 kDa, corresponding to the Fd-hGH fusion and the L chain, respectively. As expected, under non-reducing conditions, SL335 _Δds -hGH produced two identical protein bands due to the lack of interchain disulfide bonds. In the case of SL335 _wt -hGH, a major 70 kDa protein band corresponding to the correct heterodimeric form of SL335 _wt -hGH was visible. However, numerous SL335 _wt -hGH derivatives of varying sizes were also observed, including four distinct unidentified protein bands ranging from 24 to 45 kDa and a few faint protein bands corresponding to 100 kDa and 135 kDa. Proteins of 15 kDa and 12.5 kDa were observed in all samples. Western blot analysis was then performed using anti-human Fd mAb, anti-κL chain pAb, and anti-hGH pAb T-20. Under non-reducing and reducing conditions (data not shown), blots using anti-human Fd mAb detected only HcysG and HserG, each sized at 46 kDa. On the other hand, under non-reducing conditions ( FIG. 9B ), the anti-κL chain pAb detected all four protein bands of 24 to 45 kDa in the SL335 _wt -hGH sample, as well as those larger than 70 kDa. This result indicates that Cys ²¹⁴ of the L chain is responsible for the formation of multiple multimeric L chains, at least through the formation of an abnormal disulfide bond. Under non-reducing conditions ( Figure 9C ), blotting with T-20 anti-hGH pAb correctly recognized the 70 kDa heterodimeric form of SL335 _wt -hGH and the ~45 kDa monomeric HerG of SL335 _Δds -hGH. Proteins of 15 kDa and 12.5 kDa were not detected by any of these antibodies, suggesting that they are degradation products from the fusion or contaminants from E. coli host proteins.

Chip-based capillary electrophoresis confirmed the SDS-PAGE analysis. HcysG/Lcys ( FIG. 10A ) and HserG/Lser ( FIG. 10B ) were prepared using sample buffers for reducing or non-reducing electrophoresis with or without DTT, and chip-based capillary electrophoresis was performed using the Protein 80 kit on an Agilent 2100 Bioanalyzer system according to the manufacturer's protocol. Results were plotted as fluorescence intensity units relative to protein size. Under non-reducing conditions, SL335 _wt -hGH produced SL335 _wt -hGH derivatives ranging in size from 27.1 kDa to 52.4 kDa, many of which disappeared under reducing conditions in the presence of DTT ( FIG. 10A ). SL335 _Δds -hGH produced nearly identical protein peaks under non-reducing and reducing conditions ( FIG. 10B ), except for minor changes in molecular weight.

SL335 _wt -hGH and SL335 _Δds -hGH were further analyzed using MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometry was performed on an Autoflex III Smartbeam apparatus (Bruker Daltonics, Billerica, MA, USA). Affinity-purified HcysG/Lcys ( FIG. 11A ) and HserG/Lser ( FIG. 11B ) were mixed with MALDI matrix and spectra were acquired in positive ion mode in the m/z range of 10,000 to 150,000 Da. A mass spectrum of SL335 _Δds -hGH was acquired in the m/z range of 10,000 to 70,000 Da. A mass spectrum was obtained for SL335 _wt -hGH in the m/z range of 15,000 to 160,000, as the SL335 _wt -hGH sample showed a protein band greater than 70 kDa, as shown in FIG8A . The molecular masses of Lcys, HcysG, and SL335 _wt -hGH were identified as 23,226 Da, 46,226 Da, and 69,837 Da, respectively ( FIG11A ). The sizes of three discrete proteins larger than the correct SL335 _wt -hGH were 92,824 Da, 117,455 Da, and 139,347 Da. In the case of SL335 _Δds -hGH, the molecular masses of Lser and HserG were identified as 23,334 Da and 46,667 Da, respectively ( FIG11B ). The low peak for HserG compared to Lser may indicate a lower ionization efficiency of the larger molecule or the presence of a lower molar ratio of HserG compared to Lser in the sample.

By using FPLC, by SephacrylS-200HR column, further purification of affinity-purified SL335 _Δds -hGH. After affinity purification using Sephacryl ^™ S-200HR pre-filled column and AKTA FPLC (GE Healthcare, Wauwatosa, WI, USA), gel filtration of HserG/Lser was performed. Equilibration buffer (20mM HEPES pH 7.4 containing 150mM NaCl) was used to balance the column and load affinity-purified HserG/Lser. Equilibration buffer was used to elute at a flow rate of 0.5ml/min. The arrow indicates the components selected for SDS-PAGE analysis (Figure 12A). Under reducing conditions, components #13, #16, #19 and #23 (Figure 12B) obtained from two different peaks were analyzed by 4-12% Bis-Tris gel. Coomassie blue staining was used to visualize the protein bands. Two peaks corresponding to approximately 66 kDa and 25 kDa were visualized from fractions #12 to #27 ( FIG9A ). Four fractions (fractions #13, #16, #19, and #23) were then analyzed by SDS-PAGE under reducing conditions to determine the protein content of the fractions ( FIG9B ). The results showed that the fractions from the 66 kDa peak (fractions #13, #16, and #19) contained heterodimeric SL335 _Δds -hGH, while the fraction from the 25 kDa peak (fraction #23) contained primarily monomeric Lser.

2-(7) In vitro functional characterization of SL335Δds-hGH

To determine whether the removal of interchain disulfide bonds in hGH fusions and SL335 _wt affects the binding affinity for HSA or RSA, biomembrane interferometry assays were performed using SL335 _Δds -hGH at pH 6 and pH 7.4 (see Table 6 below). The dissociation constants of SL335 _Δds -hGH for HSA were 1.7 nM at pH 6 and 1.5 nM at pH 7.4, indicating that the affinity of SL335 _Δds -hGH for HSA was 5-fold and 8.7-fold greater than that of SL335, respectively. The dissociation constants of SL335 _Δds -hGH for RSA were 499 nM at pH 6 and 83.6 nM at pH 7.4, indicating that the affinity of SL335 _Δds -hGH for RSA was reduced by 4.2-fold and 1.3-fold, respectively, relative to SL335.

The in vitro hGH activity of SL335 _ds -hGH was also measured using Nb2-11 rat lymphoma cells, which were treated with hGH to proliferate in a concentration-dependent manner. Nb2-11 rat lymphoma cells were resuspended at a concentration of 8× ¹⁰ cells/ml in DMEM containing 5% (v/v) horse serum, and 50 μl aliquots of the cell suspension were added to each well of a 96-well plate and incubated overnight. The cells were then treated with increasing concentrations of either HserG/Lser in DMEM containing 5% (v/v) horse serum for 48 hours at 37°C. Following incubation, 10 μl of CCK-8 solution was added to each well, and the cells were incubated for 4 hours. The absorbance at a wavelength of 450 nm was recorded on a microplate reader. Data represent the mean SD of three experiments. In the absence of HSA, SL335 _Δds -hGH was able to stimulate the growth of Nb2-11 cells with a significant _EC50 of 50 pM (3.5 ng/ml) ( FIG13A ). This value represents a 6.7-fold decrease compared to the effectiveness of the rhGH standard (7.5 pM). In the presence of 10 mM HSA, the potency of SL335 Δds-hGH and SL335 _Δds -hGH was largely unaffected, although SL335 _Δds -hGH exhibited an approximately 5-fold decrease in potency compared to SL335 Δds-hGH ( FIG13B ). SL335, used as a negative control, did not show any proliferative effect. These results clearly demonstrate that SL335 _Δds -hGH possesses functional hGH biological activity.

SL335 _Δds -hGH was then incubated at 37°C for 16 days to determine serum stability. FBS (rather than human serum) was used to resuspend the samples because the binding ability of SL335 _Δds -hGH and SL335 to HSA in human serum would complicate subsequent experiments. Samples were collected once a day, and HSA binding reactivity and in vitro bioactivity were measured by ELISA ( FIG14A ) and Nb2-11 cell proliferation assay ( FIG14B ), respectively. SL335 was also used as a control. Similar to SL335, even after 16 days of incubation at 37°C, SL335 _Δds -hGH showed no changes in HSA binding reactivity and Nb2-11 proliferation activity, demonstrating that despite the lack of interchain disulfide bonds, SL335 _Δds -hGH is as stable as SL335.

2-(8) Pharmacokinetic and pharmacodynamic studies in rats

Because SL335 _Δds -hGH has been shown to be a promising candidate for long-acting hGH, in vivo efficacy studies were conducted. First, the pharmacokinetics of SL335 _Δds -hGH in rats were compared by measuring serum levels of each analog as a function of time after a single intravenous or subcutaneous injection. Each group of four rats was injected subcutaneously (Figure 15A) with a single dose of 0.6 mg/kg of Growtropin or SAFAtropin, or intravenously (Figure 15B) with a single dose of 0.3 mg/kg of Growtropin or SAFAtropin. Depending on the protein, serum samples were collected at intervals over a period of up to 144 hours. As described above, serum samples were analyzed by ELISA at the indicated times. Pharmacokinetic parameters are shown in Table 7.

【Table 7】

Pharmacokinetic parameters of Growtropin or SAFAtropin after single intravenous or subcutaneous injection in rats

The values shown are means with standard deviations. The abbreviations are as follows: Cmax: maximum concentration; t _1/2 : terminal half-life; AUC _0→∞ : area under the concentration-time curve extrapolated to infinity; Cl/f: apparent total plasma clearance.

Regardless of the route of administration, SL335 _Δds -hGH showed a significant prolongation of t _1/2 . SL335 _Δds -hGH increased t _1/2 83-fold (16.6 h vs. 0.2 h) compared to Growtropin by intravenous administration and 68-fold (97.2 h vs. 1.4 h) by subcutaneous administration.

Regardless of the route of administration, the AUC _0→∞ of SL335 _Δds -hGH increased by 10-fold and the clearance (Cl/f) decreased by more than 10-fold compared to SL335 Δds -hGH. Each group of four rats was administered a single dose of 0.6 mg/kg Growtropin or SAFAtropin by subcutaneous injection or a single dose of 0.3 mg/kg Growtropin or SAFAtropin by intravenous injection. Serum samples were collected at intervals extending to 144 hours, depending on the protein. Serum samples were analyzed by ELISA at the indicated times, as described above. Interestingly, the _Cmax values of SL335 _Δds -hGH decreased by 6-fold and 3-fold, respectively, depending on the route of administration.

Next, the growth rate of hypophysectomized rats was compared over 10 days following daily SC dosing or vehicle buffer control (vehicle only), or weekly SC dosing of SL335 _ds -hGH. Hypophysectomized rats were treated with vehicle only or daily at 0.3 mg/kg, or with increasing doses on days 0 and 7. The solid line indicates the mean percent change in body weight. Error bars represent standard deviation. Rats treated with vehicle showed approximately 5% weight loss. However, rats receiving daily injections (0.3 mg/kg) showed a 5% weight gain, which resulted in a total weight gain of 10% relative to the vehicle only group. Weekly injections of SL335 _Δds -hGH produced a dose-dependent weight gain, with a dose of 2.4 mg/kg producing a 15% weight gain and a dose of 0.6 mg/kg producing a 3.5% weight gain. An equimolar SL335 _Δds -hGH (1.2 mg/kg) dosing regimen resulted in a 5% body weight gain, which is comparable to that achieved with daily injections.

Figure 17 shows that once-weekly administration of 0.6 mg/kg SL335 _Δ ds-hGH achieved an increase in tibia length equivalent to daily administration. Solid bars indicate the mean of the measured tibia lengths. Error bars indicate standard deviation.

Industrial Applicability

Because the fusion constructs of the present invention can be prepared to include various types of effector moieties (including human growth hormone, interferon, erythropoietin, colony stimulating factor or derivatives thereof, and antibody derivatives, etc.), the present invention can be used to develop biologically active protein or polypeptide therapeutics.

The teachings of all patents, published applications, or references cited herein are incorporated by reference in their entirety.

Claims (29)

1. An antigen-binding fragment targeting serum albumin (SA), wherein the antigen-binding fragment comprises: (a) The heavy chain variable region V _H domain, having an amino acid sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6; and (b) The light chain variable region V _L domain has an amino acid sequence selected from the group consisting of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12; Among them, the cysteine amino acid is missing, or is replaced by any other amino acid residue other than cysteine. Among them, the deleted or substituted cysteine is used for disulfide bonds between the heavy chain and the light chain in the _CH1 domain, or for disulfide bonds between the heavy chain and the light chain in the _CκL domain, or for disulfide bonds between the heavy chain and the light chain in the _CH1 domain and between the heavy chain and the light chain in the _CκL domain. The antigen-binding fragment specifically binds to serum albumin. 2. An antigen-binding fragment that binds to serum albumin (SA), wherein the antigen-binding fragment comprises: (a) The amino acid sequences of SEQ ID NO. 13 (CDR1), SEQ ID NO. 14 (CDR2), and SEQ ID NO. 15 (CDR3) that determine the CDRs of the V _H domain; and (b) Determine the amino acid sequences of SEQ ID NO.16 (CDR1), SEQ ID NO.17 (CDR2), and SEQ ID NO.18 (CDR3) of the CDRs of the V _L domain; Among them, the cysteine amino acid is missing, or is replaced by any other amino acid residue other than cysteine. Among them, the deleted or substituted cysteine is used for disulfide bonds between the heavy chain and the light chain in the _CH1 domain, or for disulfide bonds between the heavy chain and the light chain in the _CκL domain, or for disulfide bonds between the heavy chain and the light chain in the _CH1 domain and between the heavy chain and the light chain in the _CκL domain. The antigen-binding fragment specifically binds to serum albumin. 3. The antigen-binding fragment according to claim 1 or 2, wherein the _VH domain is connected to the heavy chain constant region _1CH1 domain, and the _VL domain is connected to the light chain constant region _CκL domain. 4. The antigen-binding fragment according to claim 1 or 2, wherein the _VH domain has the amino acid sequence of SEQ ID NO. 6, and the _VL domain has the amino acid sequence of SEQ ID NO. 12. 5. A fusion construct of a bioactive effector portion and an antigen-binding fragment according to claim 1 or 2, wherein the bioactive effector portion is a protein or polypeptide; and wherein the antigen-binding fragment and the bioactive effector portion are covalently linked by gene fusion. 6. An expression vector comprising: (a) a promoter; (b) a first nucleic acid sequence encoding an antigen-binding fragment as claimed in claim 2; and (c) a second nucleic acid sequence encoding a bioactive polypeptide or protein and optionally a linker, wherein the promoter, the first nucleic acid sequence, and the second nucleic acid sequence are operatively linked together. 7. A host cell comprising the expression vector of claim 6. 8. The host cell according to claim 7, wherein the host cell is Escherichia coli (E. coli). 9. The host cell according to claim 8, wherein the host cell is SUPEX5 KCTC 12657BP. 10. A method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of Escherichia coli, comprising introducing the expression vector of claim 6 into Escherichia coli. 11. The method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of Escherichia coli according to claim 10, wherein the Escherichia coli is SUPEX5 KCTC 12657BP. 12. A method for increasing the in vivo half-life of a bioactive protein or polypeptide, the method comprising linking the bioactive protein or polypeptide to an antigen-binding fragment as described in claim 1 or 2 by gene fusion. 13. The method for increasing the in vivo half-life of a bioactive protein or polypeptide according to claim 12, wherein the bioactive protein or polypeptide is linked to an antigen-binding fragment via a peptide linker having 0 to 20 amino acids. 14. The method for increasing the in vivo half-life of a bioactive protein or polypeptide according to claim 12, wherein the bioactive protein or polypeptide is selected from human growth hormone (hGH), growth hormone-releasing hormone (GHRH), growth hormone-releasing peptide, interferon (IFN), colony-stimulating factor (CSF), glucagon-like peptide, interleukin, enzyme, macrophage activating factor, macrophage peptide, B cell kinase, T cell kinase, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, angiopoietin, hemoglobin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIII, factor IX, factor XIII, plasminogen activator, fibrin-binding peptide, etc. Hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone-stimulating protein, calcitonin, insulin, atrial natriuretic peptide, chondroitin-inducing factor, calcitonin-dependent, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatostatin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotoxin, lactoferrin, myostatin, receptor, receptor antagonist, cell surface antigen, virus-derived vaccine antigen, monoclonal antibody, polyclonal antibody, and antibody fragment. 15. The method for increasing the in vivo half-life of a bioactive protein or polypeptide according to claim 12, wherein the bioactive protein or polypeptide is selected from the group consisting of α1-antitrypsin, thrombin, urokinase, streptokinase, superoxide dismutase, factor VIIa, interferon receptor, G protein-coupled receptor, interleukin receptor, cytokine-binding protein, highly glycosylated erythropoietin, and granulocyte colony-stimulating factor (GCSF). 16. The method for increasing the in vivo half-life of a bioactive protein or polypeptide according to claim 12, wherein the bioactive protein or polypeptide is an interleukin-binding protein. 17. The antigen-binding fragment according to claim 1 or 2, wherein any amino acid residue other than cysteine is serine. 18. The fusion construct according to claim 5, wherein any amino acid residue other than cysteine is serine. 19. The fusion construct according to claim 5, wherein the antigen-binding fragment and the bioeffects portion are covalently linked by a peptide linker having 0 to 20 amino acids via gene fusion. 20. The fusion construct according to claim 5, wherein the bioactive effector portion is selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, and receptors. 21. The fusion construct according to claim 5, wherein the bioactive effector portion is selected from human growth hormone (hGH), growth hormone-releasing hormone (GHRH), growth hormone-releasing peptide, interferon (IFN), colony-stimulating factor (CSF), glucagon-like peptide, interleukin, enzyme, macrophage activating factor, macrophage peptide, B cell kinase, T cell kinase, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, angiopoietin, hemoglobin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIII, factor IX, factor XIII, plasminogen activator, fibrin-binding peptide, hirudin, and protein C. The group consisting of C-reactive protein, renin inhibitor, collagenase inhibitor, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone-stimulating protein, calcitonin, insulin, atrial natriuretic peptide, chondroitin-inducing factor, calcitonin-dependent natriuretic peptide, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatostatin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotoxin, lactoferrin, myostatin, receptor, receptor antagonist, cell surface antigen, virus-derived vaccine antigen, monoclonal antibody, polyclonal antibody, and antibody fragment. 22. The fusion construct according to claim 5, wherein the bioactive protein or polypeptide is selected from the group consisting of α1-antitrypsin, thrombin, urokinase, streptokinase, superoxide dismutase, factor VIIa, interferon receptor, G protein-coupled receptor, interleukin receptor, cytokine-binding protein, highly glycosylated erythropoietin, and granulocyte colony-stimulating factor (GCSF). 23. The fusion construct according to claim 5, wherein the bioactive protein or polypeptide is an interleukin-binding protein. 24. The fusion construct according to claim 5, wherein the molar ratio of the bioactive effector portion to the antigen-binding fragment is between 1:1 and 10:1. 25. The method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of Escherichia coli according to claim 10, wherein the bioactive protein or polypeptide is selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcription factors, blood factors, vaccines, structural proteins, ligand proteins, and receptors. 26. The method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of *Escherichia coli* according to claim 10, wherein the bioactive protein or polypeptide is selected from human growth hormone (hGH), growth hormone-releasing hormone (GHRH), growth hormone-releasing peptide, interferon (IFN), colony-stimulating factor (CSF), glucagon-like peptide, interleukin, enzyme, macrophage activating factor, macrophage peptide, B cell kinase, T cell kinase, protein A, allergy inhibitory factor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, albumin, α-lactalbumin, apolipoprotein-E, erythropoietin, angiopoietin, hemoglobin, thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIII, factor IX, factor XIII, plasminogen activator. The group consisting of one of the following: fibrin-binding peptide, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone-stimulating protein, calcitonin, insulin, atrial natriuretic peptide, chondroitin-inducing factor, calcitonin-dependent, connective tissue activating factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, growth regulator, insulin-like growth factor, adrenocortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotoxin, lactoferrin, myostatin, receptor, receptor antagonist, cell surface antigen, virus-derived vaccine antigen, monoclonal antibody, polyclonal antibody, and antibody fragment. 27. The method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of Escherichia coli according to claim 10, wherein the bioactive protein or polypeptide is selected from the group consisting of α1-antitrypsin, thrombin, urokinase, streptokinase, superoxide dismutase, factor VIIa, interferon receptor, G protein-coupled receptor, interleukin receptor, cytokine-binding protein, highly glycosylated erythropoietin, and granulocyte colony-stimulating factor (GCSF). 28. The method for improving the soluble expression of bioactive proteins or polypeptides in the periplasm of Escherichia coli according to claim 10, wherein the bioactive protein or polypeptide is an interleukin-binding protein. 29. The fusion construct of claim 24, wherein the molar ratio of the bioactive effector portion to the antigen-binding fragment is between 1:1 and 4:1.