Short peptide having ph-selective binding ability to fcrn

Abstract

The present invention relates to a very short peptide sequence having pH-selective binding ability to FcRn. The peptide of the present invention is not large in size like an Fc region, thereby enabling easy penetration into target cells and / or tissues (for example, solid tumors). In addition, since N-glycosylation is not essential, the peptide can be produced in inexpensive Escherichia coli rather than animal cells. Furthermore, the peptide does not bind to FcγR and thus does not exhibit off-target toxicity, and has no possibility of forming undesired combinations, thereby maximizing production efficiency.

Description

Short peptides with pH-selective binding ability to FcRn

[0001] The present invention relates to a pH-selective binding polypeptide that exhibits pH-dependent FcRn binding ability despite its very short length, which can be utilized to extend the blood half-life of a protein or antibody therapeutic agent.

[0002]

[0003] The Fc region of the antibody mediates interaction with the neonatal receptor FcRn, and its binding recirculates antibodies intracellularly introduced from the endosome into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). Through this process, antibodies have a favorable antibody serum half-life in the range of 1 to 3 weeks, unlike other proteins that have short half-lives. The recycling of the above antibody is caused by the coordination of binding and dissociation of the Fc site to FcRn, and is due to the property of exhibiting increased binding affinity to FcRn at low pH (5.6 to 6.2) of the cell and dissociating from FcRn (neonatal Fc receptor) at high pH (7.0 to 7.8) outside the cell.

[0004] Various technologies are being developed to increase the half-life of existing protein therapeutics with short half-lives by utilizing the beneficial property of the Fc site, which is the aforementioned increase in half-life.

[0005] However, despite the aforementioned advantages, the production of antibodies or protein therapeutics containing the Fc site requires N-glycosylation and must be expressed in animal cells, resulting in relatively high production costs. Furthermore, due to the high molecular weight of the Fc protein, it is difficult to infiltrate solid tumors, and there is a problem of off-target toxicity as it possesses binding ability not only to FcRn but also to FcγR. Additionally, the production of bispecific antibodies is accompanied by the problem of unwanted combination formation, leading to increased production costs and difficulties in purification.

[0006]

[0007] Therefore, there is an urgent need to develop short peptides with Fc functions that are not large like the Fc region, making them easy to penetrate into solid tumors, can be produced in inexpensive E. coli rather than animal cells, do not possess off-target toxicity, and have no possibility of forming unwanted combinations.

[0008]

[0009] The matters described as background technology above are intended only to enhance understanding of the background of the present invention and should not be construed as an acknowledgment that they constitute prior art already known to those skilled in the art.

[0010]

[0011] Accordingly, the inventors made diligent efforts to discover a short peptide having pH-selective binding ability with FcRn, which is not large in size like the Fc region, is easy to produce, does not bind to unwanted FcγR, and can extend the half-life of target proteins or antibodies. As a result, the present invention was completed by confirming that a short peptide containing four specific amino acids (e.g., DWQW) exhibits the above properties.

[0012]

[0013] Accordingly, the object of the present invention is to provide a pH-selective binding polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the polypeptide binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8.

[0014] Another objective of the present invention is to provide a fusion protein in which an additional polypeptide is fused to the pH-selective binding polypeptide.

[0015] Another objective of the present invention is to provide a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide.

[0016] Another objective of the present invention is to provide a vector containing the nucleic acid.

[0017] Another objective of the present invention is to provide a host cell comprising the vector or transformed with the vector.

[0018] Another object of the present invention is to provide a method for preparing a pH-selective binding polypeptide or a fusion protein comprising said pH-selective binding polypeptide, comprising the following steps, wherein the pH-selective binding polypeptide binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8:

[0019] a) a step of culturing the host cells of claim 21; and

[0020] b) A step of recovering the polypeptide expressed by the host cell.

[0021] Another object of the present invention comprises the step of preparing a pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising the pH-selective binding polypeptide or the pH-selective binding polypeptide, a vector comprising the nucleic acid, or a host cell comprising the vector or transformed therefrom.

[0022] The present invention provides a pH-selective binding polypeptide that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, or a method for preparing a fusion protein comprising said pH-selective binding polypeptide.

[0023] Another objective of the present invention is to provide a composition comprising the pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising the pH-selective binding polypeptide or the pH-selective binding polypeptide, a vector comprising the nucleic acid, or an isolated host cell comprising the vector or transformed therefrom.

[0024] Another objective of the present invention is to provide a screening method for the pH-selective binding polypeptide, wherein the method comprises the step of selecting a peptide from a peptide library containing the amino acid sequence of SEQ ID NO. 1 that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8.

[0025] Another objective of the present invention is to provide a method for treating a disease comprising the step of administering to a subject in need the pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising the pH-selective binding polypeptide or the pH-selective binding polypeptide, a vector comprising the nucleic acid, or a host cell comprising or transformed therein with the vector.

[0026] Another objective of the present invention is to provide a therapeutic use of the pH-selective binding polypeptide, the nucleic acid encoding the pH-selective binding polypeptide or the fusion protein comprising the pH-selective binding polypeptide, the vector comprising the nucleic acid, or the host cell comprising or transformed therefrom the vector.

[0027]

[0028] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0029]

[0030] According to one aspect of the present invention, the present invention provides a pH-selective binding polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the polypeptide binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8.

[0031]

[0032] As used herein, the term “polypeptide” refers to a chain composed of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues within the protein may include modifications, including but not limited to glycosylation, phosphorylation, or the formation of disulfide bonds. “Protein” may include one or more polypeptides. Unless otherwise specified, the terms “polypeptide” and “protein” are used interchangeably in this specification.

[0033] The present invention relates to a polypeptide comprising an amino acid sequence that regulates binding to and dissociation for FcRn (neonatal Fc receptor).

[0034] In this specification, the term “pH-selective binding polypeptide” means a polypeptide having the characteristic of exhibiting increased binding affinity for FcRn at low pH and dissociating at high pH.

[0035] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide may have a binding affinity for FcRn at pH 5.6 to 6.2 (preferably pH 5.8 to 6.0) that is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the wild-type Fc domain, or may have a binding affinity that is 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more compared to the wild-type Fc domain.

[0036] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide may have the same or substantially unchanged degree of dissociation from FcRn (neonatal Fc receptor) at pH 7.0 to 7.8 (preferably pH 7.2 to 7.6) compared to the wild-type Fc domain.

[0037] According to one embodiment of the present invention, the pH-selective binding polypeptide of the present invention exhibited a significantly improved binding affinity under weakly acidic conditions (e.g., pH 5.8 to 6.0) compared to wild-type Fc, and exhibited the same, substantially equivalent, or greater degree of dissociation under neutral conditions (e.g., pH 7.0) (Examples 4-5).

[0038] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide of the present invention has an increased half-life compared to wild-type Fc.

[0039] The half-life of the pH-selective conjugated polypeptide of the present invention may be increased by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100% compared to the wild-type Fc domain, or increased by more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, or more than 10 times compared to the wild-type Fc domain.

[0040] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide comprises a sequence of 4 to 400, 4 to 300, 4 to 200, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 19, 4 to 18, 4 to 16, or 4 to 11 amino acids.

[0041] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus):

[0042] <General Formula 1>

[0043] DWQW-Xa

[0044] According to a preferred embodiment of the present invention, Xa in the general formula 1 may be a sequence consisting of one or more amino acid residues selected from the group comprising 20 naturally occurring standard amino acids or derivatives thereof, non-standard amino acids, artificially synthesized amino acids, or their D-isomers.

[0045] Non-standard amino acids include ① naturally derived non-standard amino acids (selenocysteine ​​(Sec, U), pyrrolicine (Pyl, O), hydroxyproline (Hyp), ornithine (Orn), citrulline (Cit), etc.), ② synthetic (artificial) amino acids (p-acetylphenylalanine (pAcF), p-azidophenylalanine (pAzF), p-benzoylphenylalanine (Bpa), L-diaminepropionic acid (Dap), N-methylleucine (NMeLeu), etc.), ③ functionally modified amino acids (phosphoserine (pSer), phosphotyrosine (pTyr), methyllysine (MeLys), acetylysine (AcLys), etc.), ④ fluorinated / halogenated amino acids (4-fluorophenylalanine, 3-chlorotyrosine, 4-bromotryptophan, etc.), and ⑤ amino acids for photoreactive or click reactions (p-benzoylphenylalanine). (Bpa), p-azidophenylalanine (pAzF), acetylysine analogs, etc.), ⑥ Fluorescence / labeling amino acids (L-4-Azido-phenylalanine, L-BODIPY-lysine, 3-Nitrotyrosine, 19F-phenylalanine, etc.), but are not limited thereto.

[0046] According to a preferred embodiment of the present invention, Xa in the general formula 1 may be a sequence consisting of one or more amino acid residues selected from the group consisting of standard amino acids arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine ​​(C).

[0047] According to a preferred embodiment of the present invention, in the above general formula 1, D is aspartic acid (D), Q is glutamine (Q), and W is tryptophan (W).

[0048] According to a preferred embodiment of the present invention, Xa in the general formula 1 may be 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acid residues.

[0049] According to a preferred embodiment of the present invention, in the general formula 1, Xa is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, or 2 to 5 amino acid residues, and the residues constituting Xa may be the same or different types of amino acids.

[0050] According to one embodiment of the present invention, the amino acid sequence of General Formula 1 is DWQWH (Sequence No. 2), DWQWHY (Sequence No. 3), DWQWHYY (Sequence No. 4), DWQWHYYE (Sequence No. 5), DWQWHYYK (Sequence No. 6), DWQWHYYEC (Sequence No. 7), DWQWHYYKC (Sequence No. 8), DWQWHYYECS (Sequence No. 9), DWQWHYYKCS (Sequence No. 10), DWQWHYYECSE (Sequence No. 11), DWQWHYYKCSE (Sequence No. 12), DWQWR (Sequence No. 25), DWQWRQ (Sequence No. 26), DWQWRQH (Sequence No. 27), DWQWRQHD (Sequence No. 28), DWQWRQHQ (Sequence No. 29), It may be DWQWRQHDC (sequence number 30), DWQWRQHQA (sequence number 31), DWQWRQHDCG (sequence number 32), DWQWRQHQAA (sequence number 33), DWQWRQHDCGQ (sequence number 34), DWQWRQHQAAE (sequence number 35), DWQWRH (sequence number 36), DWQWRHE (sequence number 37), DWQWRHED (sequence number 38), DWQWRHEDT (sequence number 39), DWQWRHEDTT (sequence number 40), DWQWRHEDTTH (sequence number 41), etc., but is not limited thereto.

[0051] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide comprises an amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus):

[0052] <General Formula 2>

[0053] Xb-DWQW-Xa

[0054] According to a preferred embodiment of the present invention, Xa in the general formula 2 may be absent or consist of one or more amino acid residues.

[0055] According to a preferred embodiment of the present invention, Xb in the general formula 2 may consist of one or more amino acid residues.

[0056] As explained in the description of General Formula 1 above, Xa may be a sequence consisting of one or more amino acid residues selected from the group comprising 20 naturally occurring standard amino acids or their derivatives, non-standard amino acids, artificially synthesized amino acids, or their D-isomers. Additionally, Xb may also be a sequence consisting of one or more amino acid residues selected from the group comprising 20 naturally occurring standard amino acids or their derivatives, non-standard amino acids, artificially synthesized amino acids, or their D-isomers.

[0057] The description of the above amino acids is as described in the description of General Formula 1.

[0058] In the above general formula 2, when Xa and Xb are composed of standard amino acids, each may be a sequence comprising one or more amino acid residues independently selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine ​​(C).

[0059] In the above general formula 2, D is aspartic acid (D), Q is glutamine (Q), and W is tryptophan (W).

[0060] According to a preferred embodiment of the present invention, in the general formula 2, Xa may be 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, or 0 to 5 amino acid residues.

[0061] According to a preferred embodiment of the present invention, in the general formula 2, Xa may be 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acid residues.

[0062] According to a preferred embodiment of the present invention, in the general formula 2, Xb may be 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acid residues.

[0063] In the above general formula 2, Xa and Xb may each be the same or different amino acid residues.

[0064] According to a preferred embodiment of the present invention, in the general formula 2, Xa may be 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 1 to 8, 2 to 7, 2 to 6, or 2 to 5 amino acid residues, and the residues constituting Xa may be amino acids of the same or different types.

[0065] According to a preferred embodiment of the present invention, in the general formula 2, Xb may be 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 1 to 8, 2 to 7, 2 to 6, or 2 to 5 amino acid residues, and the residues constituting Xb may be amino acids of the same or different types.

[0066] According to one embodiment of the present invention, the amino acid sequence of General Formula 2 is DDWQW (Sequence No. 13), PDDWQW (Sequence No. 65), SPDDWQW (Sequence No. 66), GSPDDWQW (Sequence No. 67), DDWQWH (Sequence No. 68), DDWQWHY (Sequence No. 69), DDWQWHYY (Sequence No. 70), DDWQWHYYE (Sequence No. 71), DDWQWHYYK (Sequence No. 72), DDWQWHYYEC (Sequence No. 73), DDWQWHYYKC (Sequence No. 74), PDDWQWHYYECS (Sequence No. 75), DDWQWHYYKCS (Sequence No. 76), DDWQWHYYECSE (Sequence No. 77), DDWQWHYYKCSE (Sequence No. 78), PDDWQWH (Sequence No. 79), PDDWQWHY (Sequence No. 80), PDDWQWHYY (Sequence No. 14), PDDWQWHYYE (Sequence No. 81), PDDWQWHYYK (Sequence No. 82), PDDWQWHYYEC (Sequence No. 83), PDDWQWHYYKC (Sequence No. 84), PPDDWQWHYYECS (Sequence No. 85), PDDWQWHYYKCS (Sequence No. 86), PDDWQWHYYECSE (Sequence No. 87), PDDWQWHYYKCSE (Sequence No. 88), SPDDWQWH (Sequence No. 89), SPDDWQWHY (Sequence No. 90), SPDDWQWHYY (Sequence No. 91), SPDDWQWHYYE (Sequence No. 92), SPDDWQWHYYK (Sequence No. 93), SPDDWQWHYYEC (Sequence No. 94), SPDDWQWHYYKC (Sequence No. 95), SPPDDWQWHYYECS (Sequence No. 96), SPDDWQWHYYKCS (Sequence No. 97), SPDDWQWHYYECSE (Sequence No. 98), SPDDWQWHYYKCSE (Sequence No. 99), GSPDDWQWH (Sequence No. 100), GSPDDWQWHY (Sequence No. 101), GSPDDWQWHYY (Sequence No. 102), GSPDDWQWHYYE (Sequence No. 103), GSPDDWQWHYYK (Sequence No. 104),GSPDDWQWHYYEC (Sequence No. 105), GSPDDWQWHYYKC (Sequence No. 106), GSPPDDWQWHYYECS (Sequence No. 107), GSPDDWQWHYYKCS (Sequence No. 108), GSPDDWQWHYYECSE (Sequence No. 109), GSPDDWQWHYYKCSE (Sequence No. 110), QGSPDDWQWH (Sequence No. 111), QGSPDDWQWHY (Sequence No. 15), QGSPDDWQWHYY (Sequence No. 16), QGSPDDWQWHYYE (Sequence No. 17), QGSPDDWQWHYYK (Sequence No. 18), QGSPDDWQWHYYEC (Sequence No. 19), QGSPDDWQWHYYKC (Sequence No. 20), It may be QGSPDDWQWHYYECS (Sequence No. 21), QGSPDDWQWHYYKCS (Sequence No. 22), QGSPDDWQWHYYECSE (Sequence No. 23), QGSPDDWQWHYYKCSE (Sequence No. 24), etc., but is not limited thereto.

[0067] The above pH-selective binding polypeptide preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 41 and 65 to 111, and more preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 41.

[0068] According to a preferred embodiment of the present invention, one or more of the amino acids are D-type amino acids.

[0069] Unless otherwise specified, the amino acid at each position may be in the D-form or the L-form. For example, arginine may be D-arginine or L-arginine. Lysine may be D-lysine or L-lysine. Histidine may be D-histidine or L-histidine. Glutamic acid may be D-glutamic acid or L-glutamic acid. Aspartic acid may be D-aspartic acid or L-aspartic acid. Glutamine may be D-glutamine or L-glutamine. Asparagine may be D-asparagine or L-asparagine. Leucine may be D-leucine or L-leucine. Isoleucine may be D-isoleucine or L-isoleucine. Valine may be D-valine or L-valine. Methionine may be D-methionine or L-methionine. Phenylalanine may be D-phenylalanine or L-phenylalanine. Tryptophan may be D-tryptophan or L-tryptophan. Tyrosine may be D-tyrosine or L-tyrosine. Glycine may be D-glycine or L-glycine. Alanine may be D-alanine or L-alanine. Serine may be D-serine or L-serine. Threonine may be D-threonine or L-threonine. Proline may be D-proline or L-proline. Cysteine ​​may be D-cysteine ​​or L-cysteine.

[0070] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide does not substantially bind to FcγR (Fc gamma receptor) at pH 7.0 to 7.8.

[0071] According to a preferred embodiment of the present invention, the pH-selective binding polypeptide does not bind to FcγR (Fc gamma receptor) at pH 7.0 to 7.8.

[0072] In this specification, the terms “FcγR” or “Fc gamma receptor” refer to receptors that bind to the Fc region of immunoglobulin. FcRs that bind to IgG antibodies include receptors of the Fcγ family, as well as allelic variants and other spliced ​​forms of these receptors. The Fcγ family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Human IgG1 binds to most human Fc receptors and yields the strongest Fc effector function. The types of activating Fc receptors to which human IgG1 binds are considered equivalent to those of murine IgG2a. In contrast, human IgG4 yields minimal Fc effector function (see Vidarsson Get al., Front Immunol. 5:520 (published online October 20, 2014)).

[0073] The constant region may be manipulated, for example by recombinant techniques, to eliminate one or more effector functions. "Effector function" refers to the interaction between the antibody Fc region and the Fc receptor or ligand, or the biochemical event arising therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and the downregulation of cell surface receptors (e.g., B cell receptors; BCR). These effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain). Therefore, the term "constant region without Fc function" includes a constant region in which one or more effector functions mediated by the Fc region are reduced or absent.

[0074] The effector function of an antibody can be reduced or bypassed by different approaches. The effector function of an antibody can be reduced or bypassed by using antibody fragments lacking the Fc region (e.g., Fab, F(ab')2, short-chain Fv(scFv), or sdAb composed of monomer VH or VL domains). Alternatively, so-called aglycosylated antibodies can be produced by removing a sugar connected to a specific residue in the Fc region to reduce the effector function of the antibody while retaining other valuable properties of the Fc region (e.g., extended half-life and heterodimerization). Glycosylated antibodies can be produced, for example, by deleting or altering sugar-attached residues, by enzymatically removing sugar, by generating antibodies in cells cultured in the presence of a glycosylation inhibitor, or by expressing antibodies in cells that cannot glycosylate proteins (e.g., bacterial host cells) (see, for example, U.S. Patent Publication No. 20120100140). Another approach involves using the Fc region from IgG subtypes with reduced effector function, for example, IgG2 and IgG4 antibodies are characterized by having a lower level of Fc effector function than IgG1 and IgG3. The residue closest to the hinge region in the CH2 domain of the Fc portion is responsible for the antibody's effector function and contains significantly overlapping binding sites for C1q (complement) and IgG-Fc receptors (FcγR) on effector cells of the innate immune system (Vidarsson G et al., Front Immunol. 5:520 (published online on October 20, 2014)).Accordingly, antibodies with reduced or absent Fc effector function may be produced by generating, for example, a chimeric Fc region comprising a CH2 domain from an IgG4 isotype IgG antibody and a CH3 domain from an IgG1 isotype IgG antibody, or a chimeric Fc region comprising a hinge region from IgG2 and a CH2 region from IgG4 (see, e.g., Lau C et al., J Immunol. 191:4769-4777 (2013)), or an altered Fc effector function, for example, an Fc region having a mutation with reduced or absent Fc function. Such Fc regions having mutations are known in the art. For example, see U.S. Patent Publication No. 20120100140 and the U.S. and PCT applications cited therein and An et al., mAbs1:6, 572-579 (2009), the disclosures of which are incorporated herein by reference in their entirety.

[0075] As demonstrated in the embodiments of the present invention (Example 6), the pH-selective binding polypeptide disclosed in the present specification has almost no binding ability with FcγR, so concerns regarding off-target toxicity, such as the unwanted effector function, can be prevented during application.

[0076]

[0077] According to another aspect of the present invention, the present invention provides a fusion protein in which an additional polypeptide is fused to the pH-selective binding polypeptide.

[0078]

[0079] As used herein, the terms “fusion,” “linkage,” or “conjugation” refer to the joining of two or more molecules. Such linkage may be covalent or non-covalent. Such linkage may also be genetic (i.e., fusion by recombination). Such linkage may be achieved using various recognized techniques, such as chemical conjugation and recombinant protein production.

[0080] As used herein, the term "fusion protein" means a single polypeptide in which two or more different polypeptide sequences are connected or combined directly or via a linker sequence.

[0081] The fusion protein may include additional polypeptides in addition to the pH-selective binding polypeptide (e.g., all polypeptides of any amino acid sequence according to the desired purpose, such as antibodies or their antigen-binding fragments, protein therapeutics, functional protein domains, tag sequences, signal sequences, or the Fc region of immunoglobulins).

[0082] According to a preferred embodiment of the present invention, the additional polypeptide is fused with the pH-selective binding polypeptide through a linker.

[0083] As used herein, the term "linker sequence" refers to an amino acid sequence for linking two or more polypeptide sequences or protein domains. The linker sequence may be arbitrarily designed to improve the structural flexibility and stability of the fusion protein, enhance the function of the coupled polypeptide, or improve expression efficiency. The linker sequence may include, but is not limited to, a flexibility linker composed of amino acids such as glycine (Gly) and serine (Ser) to impart flexibility; a rigid linker comprising an α-helix forming sequence (e.g., EAAAK repeat sequence) to maintain a fixed structure; an enzymatically cleavable linker for the purpose of separating each protein after fusion (e.g., ENLYFQG (TEV protease site), VPRGS (thrombin site)); a disulfide-inducing linker for structural stabilization (e.g., CGGGC); and, if necessary, non-natural amino acids, peptidomimetic, or chemical spacers.

[0084] According to a preferred embodiment of the present invention, the additional polypeptide is fused to the N-terminus, C-terminus, or both ends of the pH-selective binding polypeptide.

[0085] According to a preferred embodiment of the present invention, the additional polypeptide fused to the N-terminus and the additional polypeptide fused to the C-terminus of the pH-selective binding polypeptide have different sequences.

[0086] For example, a unique bispecific antibody structure can be created and utilized by fusing different types of scFvs to the N-terminus and C-terminus of the pH-selective binding polypeptide.

[0087] According to a preferred embodiment of the present invention, the additional polypeptide does not contain Fc or a functional fragment of Fc (a function having the ability to selectively bind to hFcRn at pH 5.6 to 6.2 and the ability to dissociate from FcRn at pH 7.0 to 7.8).

[0088] In this specification, the terms “Fc region (fragment crystallizable region),” “Fc domain,” or “Fc” refer to the C-terminal region of the heavy chain of an antibody that mediates the binding of immunoglobulin to host tissues or factors, including binding to an Fc receptor located on various cells of the immune system (e.g., effector cells) or to a first component (C1q) of the authentic complement system. Accordingly, the Fc region comprises the constant region of the antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region comprises two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In the case of IgG, the Fc region includes the immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of the immunoglobulin heavy chain vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position C226 or P230 (or an amino acid between these two) to the carboxy-terminus of the heavy chain, where the numbering follows the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from approximately amino acid 231 to approximately amino acid 340, and the CH3 domain is located on the C-terminal side of the Cm domain in the Fc region, i.e., it extends from approximately amino acid 341 to approximately amino acid 447 of IgG. The Fc region may be a spontaneous sequence Fc containing any allotype variant, or a variant Fc (e.g., a non-spontaneous Fc). Additionally, Fc refers to a region in an isolated state, or a region associated with an Fc-containing protein polypeptide, such as a "binding protein containing an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesion).

[0089] In this specification, the term “functional fragment of Fc” means a fragment of Fc having the function of selectively binding to hFcRn in acidic conditions (e.g., pH 5.6 to 6.2) and dissociating from FcRn in neutral conditions (e.g., pH 7.0 to 7.8).

[0090] According to a preferred embodiment of the present invention, the additional polypeptide is a polypeptide comprising the amino acid sequence of an antibody or an antigen-binding fragment thereof.

[0091] The above antibody may be a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a chimeric antibody, an antibody conjugate, a human antibody or a humanized antibody, or an antigen-binding fragment thereof.

[0092] The above antigen-binding fragment may be Fab, Fab', F(ab')2, Fv, short-chain Fv(scFv), scAb, or sdAb.

[0093] In this specification, the term “antibody” is a term of the art and may be used interchangeably herein, and refers to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the term includes the whole antibody and any antigen-binding fragment (i.e., “antigen-binding portion”) or short chains thereof. In some embodiments, “antibody” refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or the antigen-binding portion thereof. In other embodiments, “antibody” refers to a short-chain antibody comprising a single variable domain, e.g., a VHH domain. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region consists of three domains CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain CL.

[0094] The VH and VL regions can be further subdivided into a supervariable region called the complementation determining region (CDR), which is interspersed with more conserved regions called the framework region (FR). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino-terminus toward the carboxy-terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the authentic complement system.

[0095] The antibody may have any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any type (e.g., IgD, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subtype (e.g., IgG1, IgG2, IgG3, and IgG4 in humans; and IgG1, IgG2a, IgG2b, and IgG3 in mice). Immunoglobulins, e.g., IgG1, exist in several allotypes, which differ from each other by up to a few amino acids. The antibody disclosed herein may be derived from any of the commonly known isotypes, types, subtypes, or allotypes. In certain embodiments, the antibody disclosed herein is an IgG1, IgG2, IgG3, or IgG4 subtype or any hybrid thereof.

[0096] In this specification, the term “antigen-binding fragment of an antibody” refers to one or more fragments of an antibody having the ability to specifically bind to an antigen (e.g., human Her2). Such “fragments” are, for example, about 8 to about 1,500 amino acid lengths, suitably about 8 to about 745 amino acid lengths, suitably about 8 to about 300, about 8 to about 200 amino acids, or about 10 to about 50, or 100 amino acid lengths. It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the “antigen-binding portion” of an antibody, for example, the anti-Her2 antibody disclosed herein, are a Fab fragment which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; a F(ab')2 fragment which is a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region; and a Fd fragment consisting of VH and CH1 domains. It includes an Fv fragment composed of VL and VH domains of a single arm of an antibody, and a disulfide-linked Fvs (sdFv); a dAb fragment composed of a VH domain (Ward et al., (1989) Nature 341: 544-546); and a combination of two or more isolated CDRs that can be selectively linked by an isolated complementarity determining region (CDR) or a synthetic linker. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using a recombination method, thereby forming a single protein chain (referred to as a single-chain Fv (scFv)) in which the VL and VH regions are paired to form a monovalent molecule, which can be a scAb in which a cKappa sequence is fused to the scFv.

[0097] According to a preferred embodiment of the present invention, the additional polypeptide is a polypeptide comprising the amino acid sequence of a therapeutic protein.

[0098]

[0099] According to another aspect of the present invention, the present invention provides a nucleic acid encoding a pH-selective binding polypeptide or a fusion protein comprising said pH-selective binding polypeptide.

[0100] As used herein, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably and are intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded and may be cDNA. The nucleic acid may exist in whole cells, cell lysates, or in a partially purified or substantially pure form. In some aspects, the nucleic acid is a DNA sequence and / or an RNA sequence (e.g., mRNA). In some aspects, the nucleic acid comprises a modified nucleotide analog. Nucleic acid is “isolated” or “substantially purified” when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA linked to DNA isolated from nature) or proteins using alkali / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other standard techniques widely known in the art (see F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York). In some aspects, nucleic acid molecules may or may not contain intron sequences. In some aspects, said nucleic acid is a cDNA molecule. The nucleic acid described herein may be obtained using standard molecular biology techniques known in the art.

[0101] According to another aspect of the present invention, the present invention provides a vector comprising the nucleic acid.

[0102] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of carrying other nucleic acids. Such a vector may be a vector capable of replicating and / or expressing said nucleic acid molecule in eukaryotic or prokaryotic cells, including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.), and preferably may be a vector comprising at least one selection marker that is operably linked to a suitable promoter so that said nucleic acid molecule can be expressed in a host cell. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be attached. Another type of vector is a viral vector, to which additional DNA fragments can be attached to a viral genome. Some vectors are capable of autonomous replication within the introduced host cell (e.g., bacterial vectors having a bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episosome mammalian vectors) can be incorporated into the host cell's genome after being introduced into the host cell and thus replicate along with the host genome. Additionally, some vectors can direct the expression of operatively linked genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful for recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0103] In this specification, the term "expression vector" refers to a nucleic acid structure containing elements necessary for the transcription and translation of an inserted coding sequence, and in the case of an RNA viral vector, contains elements necessary for replication and translation when introduced into a suitable host cell. Expression vectors may include plasmids, phages, viruses, and derivatives thereof.

[0104] In this specification, “viral vector” includes, but is not limited to, the nucleic acid sequences of the following viruses: retroviruses (e.g., Moloney’s rat leukemia virus, Harvey’s rat sarcoma virus, rat mammary tumor virus and Rous’s sarcoma virus); lentiviruses; adenoviruses; adeno-associated viruses; SV40 type virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccina virus; poliovirus; and RNA viruses such as retroviruses. Some viral vectors are based on non-cytogenetic eukaryotic viruses in which non-essential genes are replaced by genes of interest. Non-cytogenetic viruses include retroviruses, the life cycle of which involves the reverse transcription of genomic viral RNA into DNA and the subsequent incorporation of proviral DNA into host cell DNA.

[0105] Other vectors include plasmid vectors. In recent years, plasmid vectors have been found to be particularly advantageous for delivering genes to in vivo cells because they cannot replicate or integrate within the host genome. However, these plasmids, equipped with promoters compatible with host cells, can express peptides from genes operatively encoded within the plasmid. Commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript.

[0106]

[0107] According to another aspect of the present invention, the present invention provides a host cell comprising the vector or transformed with the vector.

[0108] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell containing nucleic acids that are not naturally present within it, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that this term refers not only to a specific cell but also to its offspring. Although these offspring may not actually be identical to the parent cell because specific modifications may occur in subsequent generations due to mutations or environmental influences, these cells are still included within the scope of the term "host cell" as used herein.

[0109] The above host cell may be a eukaryotic or prokaryotic cell including human cells and non-human cells (mammalian cells (e.g., monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.)).

[0110] In this specification, the term "transformation" refers to a method of delivering a vector comprising a nucleic acid molecule encoding the pH-selective binding polypeptide or a fusion protein containing the pH-selective binding polypeptide to a host cell. Such transformation may be performed by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Additionally, transformation involves delivering a target into a cell using viral particles by means of infection. Furthermore, the vector may be introduced into a host cell by means of gene bombardment, etc. In the present invention, transformation may be used interchangeably with transfection or transduction. The transformation may be performed in vitro or ex vivo.

[0111] It is preferable that the host cell of the present invention be an 'isolated' host cell.

[0112]

[0113] According to another aspect of the present invention, the present invention provides a method for preparing a pH-selective binding polypeptide or a fusion protein comprising said pH-selective binding polypeptide, which binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, comprising the following steps:

[0114] a) a step of culturing the above host cells; and

[0115] b) A step of recovering the polypeptide expressed by the host cell.

[0116]

[0117] According to a preferred embodiment of the present invention, a method for preparing a fusion protein comprising the pH-selective binding polypeptide further comprises a step (step c) of checking whether the polypeptide recovered in step b) binds to FcRn at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8.

[0118]

[0119] According to another aspect of the present invention, the present invention comprises the step of preparing a pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising said pH-selective binding polypeptide or said pH-selective binding polypeptide, a vector comprising said nucleic acid, or a host cell comprising said vector or transformed therein.

[0120] A method for preparing a pH-selective binding polypeptide that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, or a fusion protein comprising said pH-selective binding polypeptide.

[0121]

[0122] According to another aspect of the present invention, the present invention provides a composition comprising the pH-selective binding polypeptide, a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide, a vector comprising the nucleic acid, or a host cell comprising the vector or transformed therein.

[0123]

[0124] According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition for the prevention or treatment of disease.

[0125]

[0126] According to another aspect of the present invention, the present invention provides a method for treating a disease comprising the step of administering to a subject in need the pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising the pH-selective binding polypeptide or the pH-selective binding polypeptide, a vector comprising the nucleic acid, or an isolated host cell comprising or transformed therefrom the vector.

[0127]

[0128] According to another aspect of the present invention, the present invention provides a therapeutic use of the pH-selective binding polypeptide, a nucleic acid encoding a fusion protein comprising the pH-selective binding polypeptide or the pH-selective binding polypeptide, a vector comprising the nucleic acid, or an isolated host cell comprising or transformed therefrom the vector.

[0129]

[0130] As used herein, the term “object” includes both human and non-human animals. The term “non-human animal” includes all vertebrates excluding humans, such as non-human primates, mammals and non-mammals like sheep, dogs, cattle, chickens, amphibians, and reptiles.

[0131] In this specification, the term "prevention" may refer to any act of suppressing or delaying the onset of a disease or disorder of a subject by administering the above composition. Additionally, in this invention, the term "treatment" may refer to any act of improving or beneficially altering the symptoms of a disease or disorder of a subject by administering the above composition.

[0132] As used herein, the term “therapeutically effective amount” means an amount of drug that is effective in “treating” a disease or disorder of a subject, or in combination with other therapeutic agents, or in reducing the risk, potential, likelihood, or occurrence of a disease or disorder (e.g., cancer or tumor). “Therapeutically effective amount” includes an amount of drug or therapeutic agent that provides some degree of improvement or benefit to a subject who has a disease or disorder (e.g., cancer or tumor) or is at risk of developing it. Accordingly, “therapeutically effective” amount means an amount that reduces the risk, potential, likelihood, or occurrence of a disease, alleviates or mitigates a disease or disorder to some degree, reduces at least one indicator, or reduces at least one clinical symptom of a disease or disorder.

[0133] There are no limitations on the types of diseases or disorders intended to be prevented or treated in this specification, and it includes all types of diseases or disorders that can bring about benefits by increasing the half-life of antibodies or therapeutic proteins in the body, such as enhancing the effect of prevention or treatment, increasing the maintenance period, or reducing side effects by lowering the dosage.

[0134] According to a preferred embodiment of the present invention, the disease is cancer or a tumor.

[0135] According to a preferred embodiment of the present invention, the composition further comprises a pharmaceutically acceptable carrier.

[0136] In this specification, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components; additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions.

[0137] According to another aspect of the present invention, the present invention provides a screening method for pH-selectively binding polypeptides, wherein the method comprises the step of selecting a peptide from a peptide library comprising the amino acid sequence of SEQ ID NO. 1 that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8.

[0138]

[0139] The features and advantages of the present invention are summarized as follows:

[0140] (i) The present invention provides a peptide of a very short sequence (e.g., 13 amino acids) having pH-selective binding ability to FcRn.

[0141] (ii) The peptide of the present invention has the advantage of being able to penetrate target cells and / or tissues (e.g., solid tumors) because it is not large in size like the Fc region, and can be produced in inexpensive E. coli rather than animal cells because N-glycosylation is not essential, and does not have off-target toxicity because it does not bind to FcγR, and has the advantage of maximizing production efficiency because there is no possibility of combination formation.

[0142]

[0143] Figure 1 shows a peptide library with a random loop capable of phage display constructed to discover specific peptide sequences that selectively bind to human FcRn at pH 6.0 and dissociate at pH 7.4.

[0144] Figure 2 shows the results of selecting only clones from a selected library pool that have no binding affinity to B2M and GST under pH 6.0 conditions and exhibit absorbance only in human FcRn-GST-B2M.

[0145] Figure 3 shows the results confirming that the selected clones do not bind to human FcRn recombinant protein, B2M, and GST under pH 7.4 conditions.

[0146] Figure 4 shows the results of gene sequencing analysis for the two finally selected clones.

[0147] Figure 5 shows the three-dimensional structure of Trastuzumab scAb-ECSE predicted using an artificial intelligence-based protein structure prediction algorithm.

[0148] Figure 6 shows the SDS-PAGE analysis results of purified Trastuzumab scAb-ECSE.

[0149] Figure 7 shows the results of measuring the binding affinity between Tastuzumab IgG1, Trastuzumab scAb, Trastuzumab scAb-ECSE, and FcRn using ELISA under pH 6.0 and pH 7.4 conditions.

[0150] Figure 8 shows the association constant (K) for IgG antibody and Trastuzumab scAb-ECSE antibody using OCTET Data Analysis software. on ), dissociation constant (K dis ) and equilibrium dissection constant (equilibrium dissection, K D Shows the results of the analysis of ).

[0151] Figure 9 shows the results of verifying the binding affinity between Trastuzumab IgG1, Trastuzumab scAb, and Trastuzumab scAb-ECSE (PepFc) antibodies and FcγR1A using ELISA under pH 6.0 and pH 7.4 conditions.

[0152] Figure 10 shows the results of verifying the binding affinity between Trastuzumab scAb and Trastuzumab scAb-ECSE (PepFc) antibodies and FcγR2A, FcγR2B, FcγR3A, and FcγR3B using ELISA under pH 6.0 and pH 7.4 conditions.

[0153] Figure 11 shows the results of verifying the binding affinity between Trastuzumab IgG1, Trastuzumab scAb, and Trastuzumab scAb-ECSE (PepFc) antibodies and the human Her2 receptor using ELISA.

[0154] Figure 12 shows the results of using a flow cytometer to determine whether Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc) bind to human breast cancer cell line SK-BR-3 that overexpresses human Her2.

[0155] Figure 13 shows fluorescence images obtained using a live cell real-time analyzer for the analysis of the internalization activity of Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc) on human Her2-overexpressing human breast cancer cell line SK-BR-3.

[0156] Figure 14 shows the results of real-time fluorescence analysis performed using a live cell real-time analyzer to analyze the internalization activity of Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc) on human Her2-overexpressing human breast cancer cell line SK-BR-3.

[0157] Figure 15 shows the results of in silico protein molecular modeling and docking simulations performed to elucidate the structural and functional mechanisms of selective binding of the PepFc sequence (QGSPDDWQWHYYECSE) to human FcRn (hFcRn) at pH 6.0.

[0158] Figure 16 shows the results of producing Trastuzumab-scAb-PepFc variant proteins by fusing a total of five types of alanine substituents (D370A, D371A, W372A, Q373A, W374A), including the four important residues of PepFc and D370, respectively (left side of Figure 16) and the results of measuring the average ELISA absorbance for FcRn at pH 6.0 when the D370A, D371A, W372A, Q373A, and W374A variants were treated at the same molar concentration (right side of Figure 16) in order to experimentally verify the simulation results of Figure 15. It was experimentally confirmed that the four residues of D371, W372, Q373, and W374 are important.

[0159] Figure 17 shows the results of newly fabricating a peptide library capable of phage display, including D371, W372, Q373, and W374, which were identified as major residues in Figure 16.

[0160] Figure 18 shows the results of selecting clones from the library of Figure 17 that bind to human FcRn recombinant protein at pH 6.0 and dissociate at pH 7.4.

[0161] Figure 19 shows the amino acid sequences of the clones selected in Figure 18.

[0162]

[0163] The present invention will be described in more detail below through embodiments. These embodiments are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments according to the gist of the invention.

[0164]

[0165] Examples

[0166] <Example 1> Preparation of a bacteriophage peptide library having a peptide sequence specifically binding to the human neonatal fragment crystallizable receptor (FcRn)

[0167] To discover specific peptide sequences that selectively bind to human FcRn at pH 6.0 and dissociate at pH 7.4, a peptide library with random loops capable of phage display was constructed. The amino acid sequence arrangement constituting the library was designed by referring to the paper by Lee, PS et al. (2022) 'Improved therapeutic index of an acidic pH-selective antibody', mAbs, 14(1). The library constructed for selective binding at pH 6.0 and binding dissociation at pH 7.4 includes histidine (H), aspartic acid (D), and glutamic acid (E), and was constructed using degenerated codons to arrange various amino acids. The design of DNA primers for antibody library production using the PCR technique, DNA gene sequence amplification, and library construction were based on the paper by Manfred T Reetz & Jose´ Daniel Carballeira (Manfred T Reetz & Jose´ Daniel Carballeira, Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes, Nature Protocols, 2(4), 891-903.) and the book by Frances H. Arnold & George Georgiou (Directed Evolution Library Creation: Methods and Protocols, Humana press, 2010.).

[0168] More specifically, primers composed of degenerated codons NNK (20 amino acids), NRB (12 amino acids), and HRB (9 amino acids) were designed, and peptide library plasmid DNA was constructed using gene amplification and cloning techniques. Subsequently, to utilize phage display technology, a screening method for selecting antibodies that selectively bind to human FcRn, the plasmid DNA of the peptide library with random loops was transformed into E. coli TG1, which is specialized for bacteriophage production, to construct a peptide library with bacteriophage random loops, and the results of sequence analysis of colonies randomly selected from it are shown in Figure 1 and Table 1.

[0169]

[0170] Peptide Library (Sequence List) Library SEQ ID NO SequenceLibrary-142GGQCSCRSHRRLELibrary-243QGRRRRCNWRRGELibrary-344LWGRNHCQNLHRQDLibrary-445TGHCWHCWRVCFTDLibrary-546IGRRWQQRYMCILLibrary-647WTSQSYRSCRRIELibrary-748WGQRRSRSSARWRLibrary-849AGGWNSRSYFRKLLibrary-950WLGHYRYQRWRSGLibrary-1051WSHQWHRSQHQTNLibrary-1152GCWRRHRQIWRDRLibrary-1253PGNNSQNYWVWWWLibrary-1354LVSRHNNNWCKENLibrary-1455FDSCQRNCYWRWD

[0171] <Example 2> Phage screening for the discovery of peptides that selectively bind to human FcRn at pH 6.0 and dissociate at pH 7.4

[0172] Screening was performed to select peptides from the constructed random-loop peptide library that specifically bind to human FcRn at pH 6.0 and dissociate at pH 7.4. The peptide library used for phage screening was approximately 2 x 10 10 Approximately 1 x 10⁶ phages amplified in a solution displaying peptides with random loops, comprising a library of varying sizes 12 Using several pieces of phage, phage screening was performed in each round in the following manner.

[0173] The human FcRn used in this phage screening was prepared by transforming HEK293 animal cells with recombinant expression vector DNA composed of the human FcRn-GST (Glutathione S-transferase)-B2M (β2 microglobulin) genetic information to express the antigen, and then purifying the DNA using GST resin for screening.

[0174] To select peptides that have selective binding at pH 6.0 and dissociation at pH 7.4 to human FcRn, a negative selection process was performed on recombinant GST protein and recombinant B2M in each round. Prior to peptide screening, each recombinant protein, GST and B2M, was immobilized on magnetic beads and mixed with a peptide-labeled phage construct solution to perform negative selection under pH 6.0 conditions. After reacting the FcRn-GST-B2M recombinant purified protein immobilized on magnetic beads with the negatively selected phage solution under room temperature pH 6.0 conditions, the protein was washed with a 25 mM sodium phosphate, 150 mM NaCl (pH 6.0, 0.05% Tween-20) solution and then screened to elute phages displaying peptides bound to the human FcRn-GST-B2M protein using a pH 7.4 PBS solution to select a library pool that binds to the human FcRn recombinant protein at pH 6.0 and dissociates at pH 7.4. Individual phages obtained from a single colony in a selected library pool were subjected to ELISA for human FcRn recombinant protein, and only clones that showed no binding affinity to B2M and GST under pH 6.0 conditions and exhibited absorbance only in human FcRn-GST-B2M were selected (Fig. 2). Subsequently, after confirming that the selected clones did not bind to human FcRn recombinant protein, B2M, and GST under pH 7.4 conditions (Fig. 3), gene sequencing analysis was performed on the clones, and the amino acid sequences obtained from the two gene sequences are shown in Fig. 4.

[0175]

[0176] <Example 3> Conversion and expression purification of a single-chain antibody fragment (scAb) conjugated with a peptide that selectively binds to excavated human FcRn at pH 6.0 and dissociates at pH 7.4

[0177] After constructing a library phage using KM13 Helper phage with the constructed antibody library, peptides that bind specifically to human FcRn with pH selectivity were discovered through biopanning using magnetic beads under pH 6.0 and pH 7.4 conditions and their gene sequences were analyzed. Based on the results, a Trastuzumab scAb antibody was constructed in which a human cKappa sequence was fused to a single chain fragment variable (scFv) having the CDR region of Trastuzumab (Herceptin). Additionally, a Trastuzumab scAb-ECSE antibody expression vector was constructed so that the ECSE peptide among the discovered peptide sequences was expressed at the C-terminus of the Trastuzumab scAb constructed using a glycine serine linker sequence. For the conversion to the scAb form for the expression, purification, and stabilization of the discovered antibody in E. coli, an expression vector for E. coli cells was prepared and expressed and purified in E. coli by referring to the paper by Jennifer A. Maynard (Maynard, J., Maassen, C., Leppla, S. et al. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat Biotechnol 20, 597-601 (2002).). The three-dimensional structure of the Trastuzumab scAb-ECSE antibody formed at this time was predicted using an artificial intelligence-based protein structure prediction algorithm, and the predicted structure is shown in Fig. 5. In addition, the results of SDS-PAGE analysis of the protein actually expressed in E. coli and purified to high purity by affinity chromatography are shown in Fig. 6.

[0178]

[0179] <Example 4> Verification of Human FcRn Binding Ability of pH-Selective ECSE Peptide Using ELISA

[0180] The binding affinity to human FcRn was verified using ELISA under pH 6.0 and pH 7.4 conditions using the protein purified from the Trastuzumab scAb-ECSE antibody prepared in Example 3 described above and expressed in E. coli cells. Specifically, the human FcRn-GST-B2M antigen was diluted with PBS (Phosphate-Buffered Saline, pH 7.4) buffer solution and bound to ELISA plates (Corning, USA) at a concentration of 4 μg / well for 16 hours at 4°C, and then blocked for 1 hour at room temperature with a pH 7.4 PBS solution containing 5% skim milk or 25 mM sodium phosphate and 150 mM sodium chloride at pH 6.0. After removing the skim milk, the Trastuzumab scAb-ECSE expressed and purified in the above examples was serially diluted in pH 7.4 PBS solution or pH 6.0, 25 mM Sodium phosphate, 150 mM NaCl solution at a rate of 30 μl / well and reacted at room temperature for 1 hour on ELISA plates coated with human FcRn-GST-B2M antigen. Subsequently, after washing four times with 150 μl / well PBST (Phosphate-buffered saline with 0.05% Tween-20) solution or 25 mM sodium phosphate, 150 mM NaCl (pH 6.0, 0.05% Tween-20), Goat anti-cKappa-HRP conjugate was diluted at a ratio of 1:5,000 in pH 7.4 PBS solution containing 5% skim milk or pH 6.0, 25 mM sodium phosphate, 150 mM NaCl solution and reacted on each ELISA plate for 1 hour at room temperature. After removing the above solutions, 150 μl / well PBST solution or 25 mM sodium phosphate, 150 mM NaCl (pH 6.0, 0.After washing four times with a 0.5% Tween-20 solution, the reaction was carried out with 30 μl / well of TMB (Thermo Scientific), and after 20 minutes, the reaction was terminated with 2N H2SO4. The absorbance was measured at 450 nm, and the results are shown in Figure 7.

[0181]

[0182] <Example 5> Verification of human FcRn binding ability to pH-selective Trastuzumab scAb-ECSE (PepFc) using Octet

[0183] The binding affinity to human FcRn was verified using an OCTET RH16 (Sartorius AG, Germany) system under pH 6.0 and pH 7.4 conditions, utilizing the protein purified from the expression of the Trastuzumab scAb-ECSE antibody prepared in Example 3 described above in E. coli cells. Specifically, an AR2G (Amine Reactive 2nd Generation) sensor was used to immobilize molecules containing amine groups (-NH2) on the chip surface via covalent bonding. The human FcRn-GST-B2M antigen was diluted with a 10 mM acetate (pH 5.0) buffer solution to a concentration of 100 nM and immobilized on the sensor, followed by quenching with a 1 M ethanolamine (pH 8.5) buffer solution. Subsequently, a baseline was established by flowing a pH 6.0, 25 mM sodium phosphate, and 150 mM NaCl solution for 120 seconds. Then, IgG antibodies and Trastuzumab scAb-ECSE antibodies were serially diluted starting from 200 nM and incubated for 60 seconds in the pH 6.0, 25 mM sodium phosphate, and 150 mM NaCl solution for association. Finally, dissociation was performed by flowing a pH 6.0, 25 mM sodium phosphate, and 150 mM NaCl solution for 300 seconds. OCTET Data Analysis software was used to determine the association constant (K) for the antibodies. on ), dissociation constant (K dis ) and equilibrium dissection constant (equilibrium dissection, K DThe results of the analysis of ) are shown in Figure 8. The binding affinity to human FcRn under pH 7.4 PBS solution conditions was analyzed in the same manner as the pH 6.0 buffer conditions described above, but the binding affinity analysis was performed only under 200 nM antibody conditions.

[0184]

[0185] <Example 6> Verification of Non-binding of Trastuzumab scAb-ECSE (PepFc) Antibody to Human FcγR

[0186] The binding affinity of the Trastuzumab scAb-ECSE antibody used in Example 5 described above to human FcγRs (Fig. 9: FcγR1A, Fig. 10: FcγR2A, FcγR2B, FcγR3A, and FcγR3B) was verified using ELISA under pH 7.4 conditions using the protein purified from expression in E. coli cells. Specifically, each human FcγR antigen was diluted with PBS (Phosphate-Buffered Saline, pH 7.4) buffer solution and bound to an ELISA plate (Corning, USA) at a concentration of 4 μg / well for 16 hours at 4°C, and then blocked with pH 7.4 PBS containing 5% skim milk for 1 hour at room temperature. After removing the skim milk, the Trastuzumab scAb-ECSE expressed and purified in the above example was serially diluted in pH 7.4 PBS solution at a rate of 30 μl / well and reacted at room temperature for 1 hour on ELISA plates coated with human FcγR receptor antigens. Then, after washing four times with 150 μl / well PBST (Phosphate-buffered saline with 0.05% Tween 20) solution, the Goat anti-cKappa-HRP conjugate was diluted at a ratio of 1:5,000 in pH 7.4 PBS solution containing 5% skim milk and reacted at room temperature for 1 hour on each ELISA plate. After removing the above solution, the sample was washed four times with 150 μl / well of PBST (0.05% Tween-20 in PBS pH 7.4) solution, reacted with 30 μl / well of TMB (Thermo Scientific), and after 20 minutes, the reaction was terminated with 2N H2SO4. The absorbance was measured at 450 nm, and the results are shown in Figures 9 and 10.

[0187]

[0188] <Example 7> Verification of the binding ability of Trastuzumab scAb-ECSE (PepFc) to the target antigen Her2 receptor using ELISA

[0189] The binding affinity of the Trastuzumab scAb-ECSE antibody produced in the previously described examples to the target antigen, the human Her2 receptor, was verified by ELISA using the purified protein expressed in E. coli cells. Specifically, the human Her2 receptor antigen was diluted with PBS (Phosphate-Buffered Saline, pH 7.4) buffer solution and bound to an ELISA plate (Corning, USA) at a concentration of 4 μg / well for 16 hours at 4°C, and then blocked for 1 hour at room temperature in a pH 7.4 PBS solution containing 5% skim milk. After removing the skim milk, the Trastuzumab scAb-ECSE, Trastuzumab IgG1, and Trastuzumab-scAb expressed and purified in the above examples were serially diluted in PBS (Phosphate-Buffered Saline, pH 7.4) solution at a rate of 30 μl / well and incubated at room temperature for 1 hour on ELISA plates coated with human Her2 receptor antigen. Subsequently, after washing four times with 150 μl / well PBST (0.05% Tween-20 in PBS), the Goat anti-cKappa-HRP conjugate was diluted at a ratio of 1:5,000 in a pH 7.4 PBS solution containing 5% skim milk and incubated at room temperature for 1 hour on each ELISA plate. After removing the above solution, the sample was washed four times with 150 μl / well PBST solution, reacted with 30 μl / well TMB (Thermo Scientific), and after 20 minutes, the reaction was terminated with 2 N H2SO4. The absorbance was measured at 450 nm, and the results are shown in Figure 11.

[0190]

[0191] <Example 8> Verification of the binding ability of Trastuzumab scAb-ECSE (PepFc) to the target antigen Her2 receptor using flow cytometry

[0192] To confirm the specific binding ability to the human Her2 receptor, fluorescent labels (Alexa Fluor) were applied to Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc). ® After attaching the antibodies (488), cyto-binding ability analysis was performed on the human breast cancer cell line SK-BR-3, which overexpresses human Her2, using a flow cytometer. The antibodies used in the experiment were fluorescently labeled according to the manufacturer's protocol using the Thermo Fisher Scientific Alexa Fluor® 488 Protein Labeling Kit (e.g., Cat# A10235). After protein quantification and buffer exchange, the labeling reagent was reacted with the protein at a specific molar ratio and stirred under dark conditions at room temperature for 1 hour. After the reaction, unreacted fluorescent dyes were removed through a desalting process using a spin column, and the concentration of the fluorescently labeled protein and labeling efficiency were measured and used for analysis. The cells used for the analysis were Her2-overexpressing SK-BR-3 cells, which were cultured in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% Penicillin-Streptomycin at 37°C under 5% CO2 conditions. Cells in the logarithmic growth phase were harvested, washed twice with PBS (Phosphate-Buffered Saline, pH 7.4), and then suspended in PBS + 1% BSA (Bovine Serum Albumin) at approximately 1 x 10⁻⁶ 6The concentration was adjusted to cells / 100 μL, and blocking was achieved through a 1-hour reaction at room temperature. Subsequently, fluorescently labeled antibody proteins were added to the cells at a final concentration of 2 μg / mL, and the reaction was carried out under dark conditions at room temperature for 1 hour. After the reaction, the cells were washed twice with PBS + 1% BSA to remove non-specific binding. The cell samples were resuspended in PBS, and the results of analysis using a flow cytometer are shown in Figure 12.

[0193]

[0194] <Example 9> Verification of internalization activity of Trastuzumab scAb-ECSE (PepFc) in Her2 receptor overexpressing cell lines

[0195] To verify the internalization activity in human Her2 receptor-expressing cell lines, fluorescent labels (pHrodo Green maleimide) were attached to Trastuzumab IgG and Trastuzumab scAb-ECSE (PepFc), and the internalization activity was analyzed against the human breast cancer cell line SK-BR-3, which overexpresses human Her2, using a live cell real-time analyzer. The antibodies used in the experiment were fluorescently labeled according to the manufacturer's protocol using pHrodo Green Maleimide from Thermo Fisher Scientific (e.g., Cat# P35370). After protein quantification and buffer exchange, the labeling reagent was reacted with the proteins at a specific molar ratio and stirred under dark conditions at room temperature for 1 hour. After the reaction, unreacted fluorescent dyes were removed through a desalting process using a spin column, and the concentration of the fluorescently labeled protein and the labeling efficiency were measured and used for analysis. The cells used for the analysis were Her2-overexpressing SK-BR-3 cells, cultured in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% Penicillin-Streptomycin at 37°C under 5% CO2 conditions. Approximately 2 x 10⁻¹⁰ 4 Cells were seeded into a 96-well cell culture plate to form cells / well, and then fluorescently labeled antibody proteins were added to the cells at a final concentration of 30 nM or 100 nM. The cells were cultured at 37°C and 5% CO2 conditions, and the fluorescence images taken 24 hours after antibody treatment using a live cell real-time analyzer are shown in Fig. 13, and the results of real-time fluorescence analysis are shown in Fig. 14.

[0196]

[0197] <Example 10> Structural and Functional Verification of Key Amino Acid Residues in PepFc

[0198] To elucidate the structural and functional mechanisms by which the PepFc sequence (QGSPDDWQWHYYECSE) selectively binds to human FcRn (hFcRn) at pH 6.0, in silico protein molecular modeling, docking simulations, and alanine substitution analysis were performed. First, using BIOVIA Discovery Studio (ZDOCK module), molecular docking simulations were performed after ligand removal, utilizing the Trastuzumab-scAb-PepFc protein and human FcRn (β2 microglobulin complex, PDB ID: 3M17) registered in the RCSB Protein Data Bank as the basic structures. PepFc stably bound to the groove region of the junction surface of the FcRn α2-α3 domain, specifically to the Lys-rich patch region of the α2 helix, and it was analyzed that the Asp370, Trp372, Gln373, and Trp374 residues of PepFc interact with Trp176, Lys177, Glu178, His256, Gly258, etc. of FcRn (Fig. 15). To experimentally verify these simulation results, Trastuzumab-scAb-PepFc variant proteins were produced by fusing a total of five alanine substituents (D370A, D371A, W372A, Q373A, W374A), including the four key residues of PepFc and D371 which is consecutive to D370, to the C-terminal PepFc region. All variants were E. After expression in coli, it was purified by chromatography using KappaSelect resin and showed a single band with an expected molecular weight (approx. ~42 kDa) on SDS-PAGE (Fig. 16 left). Using the purified protein, ELISA coated with human FcRn-GST-B2M antigen was performed under pH 6.0 and pH 7.4 conditions, respectively. Under pH 6.0 conditions, the average absorbance of the wild-type Trastuzumab-scAb-PepFc was 3.The average absorbance was 46, and the average absorbances of the D370A, D371A, W372A, Q373A, and W374A variants were 2.16, 0.97, 1.73, 0.96, and 1.72, respectively (Fig. 16 right). Accordingly, the relative binding affinities compared to the wild type were calculated to be approximately 62%, 28%, 50%, 28%, and 50%, respectively. In other words, as the decrease in binding affinity of the alanine substituents of the binding major residues predicted in the in silico simulation results was confirmed, it was confirmed that the binding major residues predicted in the simulation are very important sites for FcRn binding in PepFc at pH 6.0. ELISA was performed under pH 7.4 conditions using the same method, but no absorbance signal was detected in any of the samples. Therefore, through in silico simulations and actual protein ELISA experiments, it was confirmed that Asp370, Asp371, Trp372, Gln373, and Trp374 residues of PepFc form a stable complex with FcRn at pH 6.0 and contribute to a pH-dependent binding mechanism in which this bond dissociates at pH 7.4.

[0199]

[0200] <Example 11> Preparation of a bacteriophage peptide library having shorter peptide sequences that specifically bind to the human neonatal fragment crystallizable receptor (FcRn) and are conferred pH selectivity

[0201] Based on the PepFc amino acid sequence and binding major residues obtained from the previous example, a novel peptide library capable of phage display with conserved major residues (DWQW preserved by removing one consecutive D from DDWQW) was constructed to discover a shorter peptide sequence that selectively binds to human FcRn at pH 6.0 and dissociates at pH 7.4. The library constructed for selective binding at pH 6.0 and binding dissociation at pH 7.4 includes histidine (H), aspartic acid (D), and glutamic acid (E), and the library was constructed using degenerated codons to arrange various amino acids. The design of DNA primers for antibody library production using the PCR technique, DNA gene sequence amplification, and library construction were based on the paper by Manfred T Reetz & Jose´ Daniel Carballeira (Manfred T Reetz & Jose´ Daniel Carballeira, Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes, Nature Protocols, 2(4), 891-903.) and the book by Frances H. Arnold & George Georgiou (Directed Evolution Library Creation: Methods and Protocols, Humana press, 2010.).

[0202] More specifically, primers composed of degenerated codons MRM (K, N, Q, H), SAN (D, E, Q, H), DSB (C, G, R, S), and RSC (G, S) were designed, and peptide library plasmid DNA was constructed using gene amplification and cloning techniques. Subsequently, to utilize phage display technology, a screening method for selecting antibodies that selectively bind to human FcRn, the plasmid DNA of the peptide library with random loops was transformed into E. coli TG1, which is specialized for bacteriophage production, to construct a peptide library with bacteriophage random loops, and the results of sequencing analysis of colonies randomly selected from it are shown in Figure 17 and Table 2.

[0203]

[0204] Peptide library using PepFc-Core (Sequence list) Library SEQ ID NO Sequence PepFc-Core-A.A56DWQWHYYECSEPepFc_Lib-157DWQWRHQHSTDPepFc_Lib-258DGQWRQDHGSHPepFc_Lib-359DWQWREDQRAEPepFc_Lib-460DWQWRHQDTTDPepFc_Lib-661DWQWRDDHTAQPepFc_Lib-762DWQWREHQCADPepFc_Lib-863DWQWQHHQGSDPepFc_Lib-964DWQWHHHEGAH

[0205] <Example 12> Phage Screening for the Discovery of Short Peptides Selectively Binding to Human FcRn at pH 6.0 and Dissociating at pH 7.4 Screening was performed to select peptides that specifically bind to human FcRn at pH 6.0 and dissociate at pH 7.4 from the peptide library with random loops constructed in Example 11. The peptide library used for phage screening was approximately 2 x 10 10Approximately 1 x 10⁶ phages amplified in a solution displaying peptides with random loops, comprising a library of varying sizes 12 Phage screening was performed in each round using dogs as follows. The human FcRn used in this phage screening was prepared by transforming HEK293 animal cells with recombinant expression vector DNA composed of the human FcRn-GST (Glutathione S-transferase)-B2M (β2 microglobulin) genetic information to express the antigen, and then purifying it using GST Resin for screening. To select peptides exhibiting selective binding at pH 6.0 and dissociation at pH 7.4 for human FcRn, a negative selection process was performed on recombinant GST protein and recombinant B2M in each round. Prior to peptide screening, the respective recombinant proteins, GST and B2M, were immobilized on magnetic beads and mixed with a solution of peptide-labeled phage constructs under pH 6.0 conditions to conduct negative selection. After reacting the purified FcRn-GST-B2M recombinant protein immobilized on magnetic beads with the negatively selected phage solution under room temperature pH 6.0 conditions, the samples were washed with a solution of 25 mM sodium phosphate and 150 mM NaCl (pH 6.0, 0.05% Tween-20). Subsequently, screening was performed using pH 7.4 PBS solution to elute phages displaying peptides bound to the human FcRn-GST-B2M protein. Clones that bound to the human FcRn recombinant protein at pH 6.0 and dissociated at pH 7.4 were selected. Individual phage ELISA was then performed using only the selected clones to identify clones exhibiting a high signal to human FcRn-GST-B2M under pH 6.0 conditions. Genetic analysis was performed on the clones (Figs. 18 and 19).

[0206]

[0207] The sequences of the short peptides having Fc function discovered in this example are listed in Table 3 below.

[0208]

[0209] Short peptides with Fc function (Sequence List) SEQ ID NO Number of amino acids sequence

[0210] Although embodiments of the present invention have been described above, those skilled in the art may modify and change the present invention in various ways by adding, changing, deleting, or adding components, etc., without departing from the spirit of the invention as described in the claims, and such modifications and changes are also to be included within the scope of the rights of the present invention.

Claims

1. A polypeptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the polypeptide binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, a pH-selective binding polypeptide.

2. The pH-selective binding polypeptide of claim 1, wherein the pH-selective binding polypeptide comprises a sequence of 4 to 400, 4 to 300, 4 to 200, 4 to 100, 4 to 90, 4 to 80, 4 to 70, 4 to 60, 4 to 50, 4 to 40, 4 to 30, 4 to 20, 4 to 19, 4 to 18, 4 to 16, or 4 to 11 amino acids.

3. The pH-selective binding polypeptide according to claim 1, wherein the pH-selective binding polypeptide comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus): <General Formula 1> DWQW-Xa In the above general formula 1, Xa is a sequence consisting of one or more amino acid residues selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine ​​(C).

4. A pH-selective binding polypeptide according to claim 3, wherein Xa in general formula 1 is 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acid residues.

5. The pH-selective binding polypeptide of claim 1, characterized in that the pH-selective binding polypeptide comprises the amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus): <General Formula 2> Xb-DWQW-Xa In the above general formula 2, Xa is absent or consists of one or more amino acid residues selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine ​​(C); and Xb is a sequence consisting of one or more amino acid residues selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), and cysteine ​​(C).

6. A pH-selective binding polypeptide according to claim 5, wherein in the above general formula 2, Xa and Xb are each independently 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5 amino acid residues.

7. The pH-selective binding polypeptide of claim 1, wherein the pH-selective binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 41 and 65 to 111.

8. A pH-selective binding polypeptide according to claim 1, wherein one or more of the amino acids are D-type amino acids.

9. In claim 1, the pH-selective binding polypeptide is a pH-selective binding polypeptide that does not substantially bind to FcγR (Fc gamma receptor) at pH 7.0 to 7.

8.

10. A fusion protein in which an additional polypeptide is fused to the pH-selective binding polypeptide of claim 1.

11. In paragraph 10, the additional polypeptide is a fusion protein fused with the pH-selective binding polypeptide through a linker.

12. In paragraph 10, the additional polypeptide is a fusion protein fused to the N-terminus, C-terminus, or both ends of the pH-selective binding polypeptide.

13. A fusion protein in which, in paragraph 10, the additional polypeptide fused to the N-terminus and the additional polypeptide fused to the C-terminus of the pH-selective binding polypeptide are different sequences.

14. In paragraph 10, the additional polypeptide is a fusion protein that does not contain Fc or a functional fragment of Fc having the ability to selectively bind to hFcRn at pH 5.6 to 6.2 and to dissociate from FcRn at pH 7.0 to 7.

8.

15. In paragraph 10, the additional polypeptide is a fusion protein comprising an amino acid sequence of an antibody or an antigen-binding fragment thereof.

16. In paragraph 15, the antibody is a fusion protein that is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a chimeric antibody, an antibody conjugate, a human antibody or a humanized antibody, or an antigen-binding fragment thereof.

17. In paragraph 15, the fusion protein wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, short-chain Fv(scFv), scAb, or sdAb.

18. In paragraph 10, the additional polypeptide is a fusion protein that is a polypeptide comprising the amino acid sequence of a therapeutic protein.

19. A nucleic acid encoding the pH-selective binding polypeptide of claim 1 or a fusion protein comprising said pH-selective binding polypeptide.

20. A vector containing the nucleic acid of claim 19.

21. Isolated host cells containing the vector of claim 20 or transformed with said vector.

22. A method for preparing a pH-selective binding polypeptide or a fusion protein comprising said pH-selective binding polypeptide, comprising the following steps, which binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8: a) a step of culturing the host cells of claim 21; and b) A step of recovering the polypeptide expressed by the host cell.

23. A step comprising preparing a pH-selective binding polypeptide of claim 1, a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide, a vector comprising the nucleic acid, or a host cell comprising the vector or transformed therefrom. A pH-selective binding polypeptide that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.8, or a method for preparing a fusion protein comprising said pH-selective binding polypeptide.

24. A composition comprising the pH-selective binding polypeptide of claim 1, a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide, a vector comprising the nucleic acid, or an isolated host cell comprising the vector or transformed therein.

25. A composition according to claim 24, characterized in that the composition further comprises a pharmaceutically acceptable carrier.

26. In paragraph 25, the composition is a pharmaceutical composition for the prevention or treatment of cancer.

27. A screening method for pH-selectively binding polypeptides of claim 1, the screening method comprising the step of selecting a peptide from a peptide library containing the amino acid sequence of SEQ ID NO. 1 that binds to FcRn (neonatal Fc receptor) at pH 5.6 to 6.2 and dissociates from FcRn at pH 7.0 to 7.

8.

28. A method for treating a disease comprising the step of administering to a subject in need the pH-selective binding polypeptide of claim 1, a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide, a vector comprising the nucleic acid, or an isolated host cell comprising or transformed therein with the vector.

29. The pH-selective binding polypeptide of claim 1, a nucleic acid encoding the pH-selective binding polypeptide or a fusion protein comprising the pH-selective binding polypeptide, a vector comprising the nucleic acid, or a therapeutic use of an isolated host cell comprising or transformed with the vector.

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