Engineered scfv conjugate

Abstract

The present invention relates to an engineered scFv comprising an albumin-binding moiety and one or more non-natural amino acids, and an engineered scFv conjugate. The engineered scFv includes non-natural amino acids having a click chemistry functional group, and can be conjugated to a substance having another click chemistry functional group.

Description

Engineered scFv junction

[0001] This application relates to a novel antibody conjugate.

[0002] This application relates to a method for producing a novel antibody conjugate.

[0003] This application relates to various uses of novel antibody conjugates.

[0004]

[0005] The antibody therapeutics market is a continuously growing sector. In particular, research on antibody conjugates—which involve attaching a payload to an antibody to utilize the characteristics of both the antibody and the payload—has been actively conducted recently. Furthermore, regarding antibody-drug conjugates, research aimed at enhancing therapeutic efficacy while reducing side effects is also becoming increasingly prevalent.

[0006] Various methods are used to attach a payload to an antibody. For example, chemical methods are used to attach the payload using a conjugating linker capable of reacting with specific residues contained in the antibody.

[0007] Among antibody conjugates, most currently FDA-approved antibody-drug conjugates utilize conjugating linkers capable of reacting with lysine or cysteine ​​residues. However, using conjugating linkers that react with lysine or cysteine ​​residues presents the problem that it is difficult to consistently control the drug-antibody ratio. Additionally, there is the issue that antibody sequence manipulation is required to avoid compromising the antibody's antigen-binding affinity.

[0008] One method used to solve the aforementioned problem is conjugation through site-specific insertion of non-natural amino acids. In particular, to solve the problem, a method of inserting a non-natural amino acid having a site-specific click chemical functional group into the antibody can be utilized. Conjugation through the insertion of site-specific non-natural amino acids has the advantage of being able to consistently regulate the payload-antibody ratio and control the effect on the antibody caused by payload conjugation.

[0009] However, according to recent research trends, non-natural amino acids are primarily inserted into the Fc region of full-length antibodies. In contrast, research on inserting non-natural amino acids into single-strand variable fragments (scFvs) has not been widely conducted.

[0010]

[0011] Addressing the disadvantages of full-length antibody-drug conjugates

[0012] The most commonly used conventional antibody conjugate is the full-length antibody-based conjugate. However, full-length antibody-based conjugates have several limitations. First, full-length antibodies have low tissue permeability due to their relatively large size. Consequently, full-length antibody-based conjugates have difficulty penetrating deep into tissues. Second, full-length antibodies are difficult to produce using microbial-based expression systems because they contain many disulfide bonds. Therefore, full-length antibody-based conjugates have the limitation of relatively high production costs. Third, an immune response caused by the Fc region may occur.

[0013] Therefore, there is a technical need to effectively deliver a target substance to a target site while solving the aforementioned problems associated with conventional full-length antibody-based antibody conjugates.

[0014]

[0015] Multifunctional scFv-based antibody conjugate

[0016] Since scFv lacks an Fc group, it cannot induce immune responses such as complement activation or immune cell-mediated cytotoxicity. Consequently, scFv itself has limitations as a therapeutic agent. Accordingly, there have been attempts to develop scFv conjugates by attaching a payload capable of exerting therapeutic effects to scFv.

[0017] However, since scFv consists of relatively short peptides, there were difficulties in attaching the payload.

[0018] Furthermore, scFvs, which correspond to antibody fragments, have the disadvantage of having a short half-life because they are peptides with a relatively short length. Additionally, scFvs have the disadvantage of being easily excreted through renal elimination. Consequently, the pharmacokinetic properties of scFv-based antibody-payload conjugates are significantly reduced. Therefore, technologies utilizing scFvs require an increase in their in vivo half-life.

[0019] Consequently, conventionally, there is no effective method to conjugate a payload to scFv while increasing its half-life. The purpose of this specification is to enable scFv to have a long half-life in the body, while effectively conjugating a payload to scFv to effectively deliver a drug to a target site (cell or tissue).

[0020]

[0021] To solve the above problem, the inventors of the present application provide a new type of engineered scFv based on scFv and an engineered scFv conjugate.

[0022] Provision of scFv-based conjugates through the insertion of non-natural amino acids and the effect of increased half-life

[0023] The inventors of the present application have developed a conjugate composition (structure) that can i) solve the problems of full-length antibody-based conjugates using scFv, ii) solve the problem of the short half-life of scFv, and iii) site-specifically bind a target payload.

[0024] Specifically, the "engineered scFv" of the present application utilizes a scFv possessing target selectivity and includes a region to which albumin can be bound to increase the half-life. Additionally, it is characterized in that one or more of the amino acids constituting the engineered scFv are non-natural amino acids having functional groups that can be used as conjugates with a payload. That is, the present specification discloses an engineered scFv that exhibits multifunctionality, which i) can specifically bind to a target site (cancer cells or specific cells) by the function of the scFv's CDR, ii) can extend the duration in the body by increasing the half-life through binding to albumin via an albumin binding moiety, and iii) can be conjugated to a payload (and a suitable linker) through the functional groups of a non-natural amino acid inserted in a site-specific manner.

[0025]

[0026] Consideration of maintaining the function of scFv-based junctions

[0027] This specification aims to provide a novel engineered scFv in which artificial additional manipulation has been performed on the scFv.

[0028] At this time, several factors must be considered when conjugating a payload to the engineered scFv. For example, although the scFv serving as the basis for the engineered scFv typically has a molecular weight of 25–30 kDa and consists of approximately 200–250 amino acids, it can attach to a target through a complementary determining region (CDR) capable of target-specific binding. Therefore, even if a payload is conjugated (or if there are non-natural amino acids for conjugation), the characteristic of the engineered scFv to attach to a target must be maintained. Additionally, the engineered scFv includes an albumin-binding region (ABM) capable of binding to albumin to increase the half-life. Therefore, as another example, even if a payload is conjugated (or if there are non-natural amino acids for conjugation), the characteristic of the engineered scFv to attach to albumin must be maintained. In addition, even if the payload is joined (or if there are non-natural amino acids for joining), it must not affect the structural safety of the engineered scFv.

[0029] Despite the difficulties mentioned above, it is difficult to predict that it is possible to develop a relatively small and effective material capable of functioning as an antibody-payload conjugate. The inventors of the present application have confirmed that the properties are maintained even when a payload is conjugated to an engineered scFv.

[0030]

[0031] This specification provides a new type of engineered scFv comprising an albumin-binding moiety and a non-natural amino acid, and a method for manufacturing the same.

[0032] This specification provides an engineered scFv comprising a non-natural amino acid at a specific position and a method for manufacturing the same.

[0033] The present specification provides an engineered scFv-payload junction in which a payload is coupled to the engineered scFv, and a method for manufacturing the same.

[0034] The present specification provides an engineered scFv-payload conjugate in which a payload is attached to an engineered scFv comprising a non-natural amino acid at the specific position, and a method for manufacturing the same.

[0035] This specification provides various uses for engineered scFv-payload conjugates. In one embodiment, the invention provides therapeutic uses for said engineered scFv-payload conjugates.

[0036] This specification provides a method for treating cancer using an engineered scFv-payload conjugate.

[0037] Figure 1 shows candidate sites on an albumabody (a variant of trastuzumab-derived 4D5 scFv with an albumin-binding moiety attached) for pAzF introduction.

[0038] Figure 2 shows candidate sites on an albubody (a variant of a trastuzumab-derived 4D5 scFv with an albumin-binding moiety attached) for pAzF introduction. Figure 2 shows the structure of the albubody rotated 90 degrees relative to Figure 1.

[0039] Figure 3 shows the composition of the [pEVOL-pAzF] plasmid. [pEVOL-pAzF] is a vector encoding an exogenous suppressor tRNA and an exogenous tRNA synthase (derived from methanococcus jannaschii) capable of linking pAzF to said exogenous suppressor tRNA.

[0040] Figure 4 shows the vector composition of [pBAD-Albu]. The [pBAD-Albu] vector is a vector that encodes the albumin-binding moiety attached to a trastuzumab-derived 4D5 scFv. It is also used as a basis for creating a vector that encodes an engineered scFv.

[0041] Figure 5 shows the results of SDS-PAGE of lysates from the cell line. BI represents the cell lysate before induction, AI represents the cell lysate after induction, and MW represents the protein ladder used as a marker. Here, induction refers to the induction of protein expression by adding pAzF and arabinose. Additionally, scFv, albubody, and engineered scFv are indicated by red arrows. G44, S87, R89, G123, S203, P234, G235, K236, and K301 represent engineered scFvs with pAzF introduced into their respective sites.

[0042] Figure 6 shows the SDS-PAGE results for an engineered scFv with pAzF introduced at positions S87, G123, S203, P234, G235, K236, and K301.

[0043] Figure 7 shows the experimental results of the MALDI-TOF mass spectra of 4D5scFv and 4D5Albu.

[0044] Figure 8 shows the SDS-PAGE results after reacting DBCO-MMAE with engineered scFv in which pAzF was introduced at positions S87, G123, S203, P234, G235, K236, and K301.

[0045] Figure 9 shows the hydrophobic interaction chromatogram results of 4D5Albu-G235pAzF and 4D5Albu-MMAE (a conjugate of 4D5Albu-G235pAzF and DBCO-MMAE).

[0046] Figure 10 shows the results of size exclusion chromatogram experiments for 4D5scFv, 4D5Albu, 4D5Albu-G235pAzF, 4D5Albu-MMAE and a calibration standard.

[0047] Figure 11 shows the experimental results of the MALDI-TOF mass spectra of 4D5Albu-G235pAzF and 4D5Albu-MMAE (a conjugate of 4D5Albu-G235pAzF and DBCO-MMAE).

[0048] Figure 12 shows the SDS-PAGE results of 4D5scFv-G178pAzF and 4D5Albu-G235pAzF and their conjugates.

[0049] Figure 13 shows the experimental results of the MALDI-TOF mass spectra of 4D5scFv-G178pAzF and 4D5scFv-MMAE.

[0050] Figure 14 shows the results of the Anti-HER2 ELISA (binding affinity analysis) experiment of 4D5Albu and 4D5Albu-MMAE with and without the presence of human serum albumin (HSA).

[0051] Figure 15 shows the results of the Anti-HSA ELISA (binding affinity analysis) experiment of 4D5Albu and 4D5Albu-MMAE.

[0052] Figure 16 shows the pharmacokinetic profiles and parameters (n=5) of 4D5scFv, 4D5scFv-MMAE, 4D5Albu, and 4D5Albu-MMAE in BALB / c mice. Here, t 1 / 2 (α) is the alpha phase half-life, t 1 / 2 (β) represents the beta phase half-life, AUC (α) represents the area under the alpha phase curve, AUC (β) represents the area under the beta phase curve, and AUC represents the total area under the curve. Additionally, concentrations were presented normalized to 100% of the initial concentration, and data were expressed as mean ± standard deviation.

[0053] Figure 17 shows the in vitro cytotoxicity of 4D5scFv-MMAE, 4D5Albu-MMAE, and free-form MMAE on HER2-positive breast cancer cell line BT-474. Cell viability was normalized to 100% relative to the control group treated with PBS (pH 7.4) and presented as mean ± standard deviation (n=4).

[0054] Figure 18 shows the in vitro cytotoxicity of 4D5scFv-MMAE, 4D5Albu-MMAE, and free-form MMAE on HER2-positive breast cancer cell line SK-BR-3. Cell viability was normalized to 100% relative to the control group treated with PBS (pH 7.4) and presented as mean ± standard deviation (n=4).

[0055] Figure 19 shows the in vitro cytotoxicity of 4D5scFv-MMAE, 4D5Albu-MMAE, and free-form MMAE on HER2-negative breast cancer cell line MDA-MB-231. Cell viability was normalized to 100% relative to the control group treated with PBS (pH 7.4) and presented as mean ± standard deviation (n=4).

[0056] Figure 20 shows the results of measuring tumor volume over time in an SK-BR-3 xenograft mouse model treated with PBS (control), 4D5scFv-MMAE, and 4D5Albu-MMAE (intravenously administered on days 0, 3, and 6).

[0057] Figure 21 shows the results of measuring tumor weight on day 14 for each group after treatment with PBS (control group), 4D5scFv-MMAE, and 4D5Albu-MMAE (intravenous administration on days 0, 3, and 6) in an SK-BR-3 xenograft mouse model.

[0058] Figure 22 shows the results of measuring the tumor volume on day 14 for each group after treatment with PBS (control group), 4D5scFv-MMAE, and 4D5Albu-MMAE (intravenous administration on days 0, 3, and 6) in an SK-BR-3 xenograft mouse model.

[0059] Figure 23 shows images of tumors excised on day 14 by group after treatment with PBS (control group), 4D5scFv-MMAE, and 4D5Albu-MMAE (intravenous administration on days 0, 3, and 6) in an SK-BR-3 xenograft mouse model.

[0060] Figure 24 shows the results of H&E staining of tumors excised on day 14 by group after treatment with PBS (control), 4D5scFv-MMAE, and 4D5Albu-MMAE in an SK-BR-3 xenograft mouse model (intravenous administration on days 0, 3, and 6) (scale = 100 μm).

[0061] Figure 25 shows the results of confirming treatment tolerance and overall health status through changes in mouse body weight during the period of treatment with PBS (control group), 4D5scFv-MMAE, and 4D5Albu-MMAE in an SK-BR-3 xenograft mouse model.

[0062] This specification provides an engineered scFv conjugate comprising an albumin-binding moiety and a non-natural amino acid:

[0063] Engineered scFv junction represented by the following [Structural Formula 1]:

[0064] [Structural Formula 1]

[0065] [Engineered scFv (S)]-[Connection (J)]-[Linker (L)]-[Payload (P)],

[0066] At this time, S includes a light chain variable region and a heavy chain variable region, and

[0067] At this time, S includes an albumin binding moiety between the light chain variable region and the heavy chain variable region, and

[0068] At this time, S includes a non-natural amino acid represented by the following chemical formula 5, and

[0069] [Chemical Formula 5]

[0070]

[0071] At this time, AA1 and AA2 refer to amino acid(s) connected to the above-mentioned non-natural amino acid, or are absent, and

[0072] At this time, J is a connection formed by a click response, and

[0073] In this case, L is a linker that connects the connection part and the payload, and

[0074] At this time, P is characterized as being a payload.

[0075]

[0076] Definition of Terms

[0077] In this specification, the term “about” means an amount, level, value, number, frequency, percentage, dimension, size, weight, or length that varies by about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% with respect to a reference amount, level, value, frequency, percentage, dimension, size, weight, or length.

[0078] In this specification, "click chemistry" collectively refers to chemical reactions in which chemical functional groups react through click reactions. In this context, a click reaction means that chemical functional groups satisfying certain conditions combine simply without the application of heat or the assistance of complex catalysts.

[0079] In this specification, a chemical functional group capable of performing a click reaction is referred to as a "click chemical functional group." The click chemical functional group may be any one selected from terminal alkyne, azide, strained alkyne, diene, dineophile, alkene, thiol, norbornene, dibenzocyclooxin-amine, trans-cyclooctene, and tetrazine, but is not limited thereto.

[0080] The above click chemical functional group requires another click chemical functional group to undergo a reaction. In this case, if a specific click chemical functional group can react with another specific click chemical functional group, the specific click chemical functional group may be referred to as the first click chemical functional group, and the other specific click chemical functional group as the second click chemical functional group. That is, the first click chemical functional group refers to a functional group capable of a click reaction with the second click chemical functional group. Furthermore, the second click chemical functional group refers to a functional group capable of a click reaction with the first click chemical functional group.

[0081] In this specification, the click reaction may refer to various reactions. For example, the click reaction may be a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

[0082] The above CuAAC may mean the following reactions.

[0083]

[0084] For example, the above click reaction may be strain-promoted azide-alkyne cycloaddition (SPAAC).

[0085] The above SPAAC may mean the following reactions.

[0086]

[0087] For example, the above click reaction may be an inverse electron-demand Diels-Alder (IEDDA) reaction.

[0088] The above IEDDA reaction may refer to the following reactions.

[0089]

[0090] In this specification, "antibody conjugate" refers to a form in which an antibody, antibody fragment, or single-strand variable fragment is conjugated to a payload. The method of conjugation to the payload may vary. For example, it may be conjugated via a linker. For example, it may be produced by inserting a non-natural amino acid having a click chemical functional group into an antibody, antibody fragment, or single-strand variable fragment and reacting it with a linker-payload having a click chemical functional group. For example, when describing an antibody, antibody fragment, or single-strand variable fragment bound to a drug, it may also be referred to as an "antibody-drug conjugate."

[0091] The "engineered scFv conjugate" of the present application refers to a conjugate in which a payload is directly or indirectly connected through a click chemical functional group of a non-natural amino acid in an engineered scFv.

[0092] In this specification, "non-natural amino acid" means an amino acid other than the 20 standard amino acids naturally used in vivo (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine). For example, in this application, "non-natural amino acid" may be a non-natural amino acid containing a click chemical functional group. For example, in this application, non-natural amino acid may mean 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet), but is not limited thereto.

[0093] In this specification, "amino acid side chain" refers to an R residue other than an amino group, a carboxyl group, and hydrogen bonded to the alpha carbon (central carbon) of an amino acid. For convenience, the amino acid side chain may be abbreviated as "side chain" or "R residue." Additionally, in this specification, the side chain of a non-natural amino acid may have a click chemical functional group.

[0094] In this specification, "amino acid residue" refers to the structure remaining in each amino acid after the water component has been removed when two or more amino acids combine to form a peptide. That is, the constituent unit that makes up a peptide may be referred to as an amino acid residue. Additionally, amino acid residues may be referred to by numbering them starting from the N-terminus of the peptide. For example, if the 44th amino acid residue from the N-terminus of a specific peptide is glycine, it may be referred to as G44. Furthermore, in this specification, "amino acid" may refer to an amino acid residue, and this should be interpreted appropriately according to the context.

[0095] In this specification, "single-chain variable fragment, scFv" refers to an antibody fragment having a configuration formed by combining a heavy chain variable region (VH)-linker-light chain variable region (VL). scFv may generally refer to a form of a single protein chain that is small in size (about 25 kDa) and contains two or fewer disulfide bonds.

[0096] In this specification, the "Complementarity-determining region (CDR)" refers to a region that directly binds to the antigen when an antibody, antibody fragment, or single-stranded variable fragment binds to the antigen, and is a portion that is complementary to the epitope structure. The said complementarity-determining region exists in the variable region of the antibody, antibody fragment, or single-stranded variable fragment.

[0097] In this specification, "variant" refers to a protein in which a base protein has been modified. The term "variant" in this application may be used to collectively refer to variants such as the scFv engineered in this application. Alternatively, the term "variant" in this application may be used to refer to a protein in which some amino acid sequences have been altered while retaining the function of the base protein.

[0098] In this specification, "conjugating linker" refers to a linker comprising a second click chemical functional group capable of binding to a first click chemical functional group. Accordingly, the conjugating linker can bind to a non-natural amino acid having a first click chemical functional group. In this specification, the conjugating linker is used to conjugate a payload to a scFv. In addition to the second click chemical functional group, various other configurations may be additionally included.

[0099] In this specification, "scFv linker" means a peptide present in a portion that connects a heavy chain variable region and a light chain variable region, such as a linker that connects a heavy chain variable region and a light chain variable region of scFv.

[0100] In this specification, "free" is used to describe a state in which a substance having a click chemical functional group is not covalently bound to a molecule or group having another click chemical functional group. That is, a free antibody having a click chemical functional group refers to an antibody having only a click chemical functional group that is not covalently bound to another click chemical functional group (e.g., a conjugating linker). The term "free" is used to emphasize that, despite the substance containing a click chemical functional group, the click chemical functional group possessed by the substance has not reacted with another click chemical functional group. For example, in this application, "free engineered scFv" means an engineered scFv that has not reacted with a conjugating linker. Additionally, "free engineered scFv" means an engineered scFv in which all possessed click chemical functional groups have not reacted with other click chemical functional groups.

[0101] In this specification, "free" may be omitted for convenience. For example, a free-engineered scFv may be referred to as "free-engineered scFv" or "engineered scFv". This may be interpreted appropriately depending on the context.

[0102] Additionally, in this specification, "freedom" may be used to distinguish the state of matter from the following "units." Further details are described below.

[0103] In this specification, "unit" refers to a substance in a state combined with another substance. In particular, it is used to distinguish it from the substance in the "free" state. Below, "free" and "unit" will be explained using free antibodies and antibody units as examples.

[0104] A free antibody having a first click chemical functional group can form an antibody conjugate by performing a click reaction with a payload connected to a conjugating linker having a second click chemical functional group. At this time, the portion of the antibody conjugate derived from the free antibody may be referred to as an "antibody unit."

[0105] However, if necessary, a portion of the part derived from the free antibody may be referred to as an antibody unit. Alternatively, a portion derived from the free antibody and a part of the conjugating linker may be integrated and referred to as an antibody unit. This is because it is impossible to accurately distinguish whether each individual component in the structure of the antibody conjugate is derived from the free antibody, or it may make the subject more ambiguous.

[0106] Below, the antibody unit is described under the assumption that a free antibody containing an azide group and a conjugating linker containing DBCO undergo a click reaction to form an antibody conjugate. This is merely an example, and it can be similarly applied even if the click chemical reactor is modified.

[0107] When a free antibody containing an azide group and a conjugating linker containing DBCO undergo a click reaction to form an antibody conjugate, the portion derived from the free antibody is as shown in the dotted box below. In this case, it is impossible to accurately distinguish and designate the portion derived from the free antibody within the antibody conjugate.

[0108]

[0109] At this time, since it is impossible to accurately distinguish and designate the portion derived from the free antibody in the antibody conjugate, the region that includes or does not include the dotted box below may be referred to as the antibody unit.

[0110]

[0111] In one specific example, the "antibody unit" of this specification may refer to the portion of the dotted box below.

[0112] , or

[0113]

[0114] As used herein, the term “pharmaceuticalally acceptable” is used to refer to materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the scope of reasonable medical judgment and corresponding to a reasonable benefit / risk ratio.

[0115] In this specification, "halogen" or "halo" refers to a substance belonging to the group of halogen elements in the periodic table.

[0116] In this specification, "hetero" refers to a compound or group comprising at least one atom that is not carbon or hydrogen.

[0117] In this specification, "alkyl" or "alkane" means a chain-like or branched, fully saturated hydrocarbon.

[0118] In this specification, "C x-y " means, for example, when used with an alkyl group, that it contains a residue comprising x to y carbons in the chain or ring. Also, "C x-y " is used to include all substituted or unsubstituted, chain-shaped alkyl groups, branched-shaped alkyl groups, or cyclic structures. In this case, CO alkyl means hydrogen.

[0119] In this specification, "alkylene" means a divalent radical derived from an alkyl group. For example, an alkylene may be -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-, but is not limited thereto. Additionally, "alkylene" is used to include all of the substituted or unsubstituted chain-shaped alkyl groups, branched-shaped alkyl groups, or cyclic structures.

[0120] In this specification, "cycloalkanes" or "cycloalkyl" groups refer to fully saturated cyclic hydrocarbons.

[0121] In this specification, the “aryl” group refers to an aromatic ring or a group derived from an aromatic hydrocarbon such as phenyl or naphthyl. Herein, “aryl” includes monocyclic and polycyclic rings.

[0122] In this specification, "payload" is a general term for a substance to be conjugated to an antibody through a covalent bond. In this case, the covalent bond refers to a bond other than a peptide bond. For example, the payload may be a term meaning a drug, a radioisotope, a fluorescent substance, an enzyme, a nanoparticle, or a chelating agent.

[0123] In this specification, " " is used to indicate the chemical formula corresponding to the area to be explained.

[0124]

[0125] Features of the disclosure in this specification

[0126] The scFv engineered in this application is a scFv variant created based on scFv.

[0127] The engineered scFv of the present application comprises an albumin-binding moiety. The half-life of the engineered scFv can be increased by the albumin-binding moiety binding to albumin. That is, when the engineered scFv of the present application is injected into the blood, it binds to serum albumin due to the albumin-binding moiety. As a result, the half-life of the engineered scFv can be increased. Additionally, the circulation time of the engineered scFv in the blood can be increased. Furthermore, the targeting of the engineered scFv can be enhanced.

[0128] The scFv engineered in this application comprises a non-natural amino acid containing a click chemical functional group. Accordingly, it can be conjugated with a material containing a different chemical functional group. For example, it can be conjugated with a payload connected to a conjugating linker containing a different chemical functional group.

[0129] The engineered scFv of the present application may include non-natural amino acids at selected positions, taking into account the structure of the engineered scFv. Accordingly, the engineered scFv of the present application may maintain binding ability to antigens and / or albumin while including non-natural amino acids. Additionally, the engineered scFv conjugate of the present application may also maintain binding ability to antigens and / or albumin while including a drug linked via non-natural amino acids.

[0130] The engineered scFv of the present application may include non-natural amino acids at selected positions considering the structure of the engineered scFv, etc. Accordingly, the engineered scFv of the present application can be more easily conjugated to a payload connected to a linker containing chemical functional groups. Accordingly, the engineered scFv of the present application may be an engineered scFv that is easier to produce an engineered scFv-payload conjugate. Furthermore, the method for producing the engineered scFv-payload conjugate of the present application may be a method for producing an engineered scFv-payload conjugate with a higher yield.

[0131] The engineered scFv of the present application may include non-natural amino acids at selected positions considering the production (expression) of the engineered scFv. Accordingly, the engineered scFv of the present application may be an engineered scFv that is easier to produce. Additionally, the method for producing the engineered scFv of the present application may be a method for producing engineered scFv with a higher yield.

[0132] The engineered scFv-payload conjugate of the present application can selectively deliver a payload to a target. In particular, the engineered scFv-payload conjugate can appropriately release a payload (e.g., a drug) within a tumor tissue environment.

[0133] The scFv-payload conjugate engineered in this application may be used for the treatment of breast cancer. Alternatively, the scFv-payload conjugate engineered in this application may be used for the production of breast cancer therapeutic agents.

[0134] The breast cancer treatment method of the present application comprises the step of administering the engineered scFv-payload conjugate of the present application to a subject.

[0135] The scFv conjugate engineered in this application can be produced by reacting a payload comprising the scFv engineered in this application and a conjugating linker.

[0136]

[0137] Chapter 1 Engineered scFv

[0138] generalization

[0139] This specification discloses engineered scFvs. The engineered scFv of this application refers to a variant in which one or more amino acids are changed to non-natural amino acids in a scFv having an inserted albumin-binding moiety.

[0140] In this specification, a scFv with an inserted albumin-binding moiety is also referred to as an "Albubody." WO 2024 / 039238 A1 is incorporated herein by reference regarding the said Albubody structure. In this case, the engineered scFv of this application refers to an Albubody comprising one or more non-natural amino acids. In this case, the location containing said non-natural amino acids may be the albumin-binding moiety, the scFv linker, VH and / or VL. That is, said non-natural amino acid is one of the amino acids constituting the albumin-binding moiety, the scFv linker, VH and / or VL, and is located at a position that does not affect the "target selectivity (antigen binding ability) of the CDR region of the scFv" and the "albumin binding ability of the ABM."

[0141] Accordingly, in this specification, scFv variants comprising an albumin-binding moiety and one or more non-natural amino acids are referred to as "engineered scFv," "Albubody variant," or "engineered Albubody."

[0142] Unless otherwise noted, the engineered scFv described in Chapter 1 refers to a "free-engineering scFv" in which no payload, such as a drug, is bound.

[0143] The structure, composition, and characteristics of the engineered scFv are described in detail below.

[0144] Structure of an engineered scFv

[0145] The scFv engineered in this application can be represented in various structures.

[0146] For example, the above-mentioned engineered scFv (engineered albubody) may include the following structural formula:

[0147] [Heavy chain variable region (VH)]-[peptide linker containing albumin binding moiety (ABM)]-[Light chain variable region (VL)].

[0148] As a specific example, the engineered scFv (engineered albubody) may include the following structural formula. In this case, it is described in order from the N-terminus to the C-terminus:

[0149] [Heavy chain variable region (VH)]-[1st scFv linker]-[Albumin binding moiety (ABM)]-[2nd scFv linker]-[Light chain variable region (VL)].

[0150] At this time, [first scFv linker], [second scFv linker], [albumin binding moiety (ABM)], [heavy chain variable region] or / and [light chain variable region] may each include one or more non-natural amino acids.

[0151] The amino acid length of the above-mentioned engineered scFv is not limited. For example, the amino acid length of the above-mentioned engineered scFv may be 100 aa or more, 150 aa or more, 200 aa or more, 250 aa or more, 300 aa or more, 350 aa or more, 400 aa or more, 450 aa or more, 500 aa or more. For example, the amino acid length of the engineered scFv may be 150 aa or less, 200 aa or less, 250 aa or less, 300 aa or less, 350 aa or less, 400 aa or less, 450 aa or less, 500 aa or less, 550 aa or less, 600 aa or less, 650 aa or less, 700 aa or less, 750 aa or less, 800 aa or less, 850 aa or less, 900 aa or less, 950 aa or less, 1000 aa or less. For example, the amino acid length of the engineered scFv may be 300 aa or more and 350 aa or less.

[0152] The above-mentioned engineered scFv may additionally include a portion for purification at the terminal. For example, it may additionally include a His-tag portion at the C-terminus.

[0153] The above-mentioned engineered scFv may additionally include a portion for expression at the terminal. For example, it may additionally include a portion for expression at the N-terminus. Additionally, the above-mentioned engineered scFv may additionally include a portion necessary for producing a vector that expresses the engineered scFv.

[0154] The following describes in detail the components constituting the structure of the engineered scFv.

[0155] Composition (1) Heavy chain variable portion and light chain variable portion

[0156] The scFv engineered in this application comprises variable regions of heavy chain (VH) and variable regions of light chain (VL) consisting of a single chain. In this case, for example, the variable regions of heavy chain and / or the variable regions of light chain may refer to regions containing one or more non-natural amino acids.

[0157] The engineered scFv of the present application is characterized by comprising a non-natural amino acid having a click chemical reaction group. In this case, despite comprising such non-natural amino acid, the engineered scFv possesses binding affinity to an antibody. Furthermore, the heavy chain variable region and the light chain variable region included in the engineered scFv possess affinity to an antibody. A detailed explanation thereof is provided in "Characteristics of the Engineered scFv".

[0158] As described above, the heavy chain variable region and the light chain variable region have 'target selectivity' capable of recognizing an epitope of an antigen and binding specifically to the antigen. For example, to achieve such target selectivity, the heavy chain variable region and the light chain variable region each include a complementary determining region (CDR) capable of binding to a specific antigen.

[0159] The above-mentioned heavy chain variable region and light chain variable region are not limited to any specific type as long as they possess binding affinity for a specific antigen. For example, the above-mentioned heavy chain variable region and light chain variable region may be derived from a specific type of antibody. Additionally, each of the above-mentioned heavy chain variable region and light chain variable region may be identical or mutually derived from a specific type of antibody. In this case, the above-mentioned heavy chain variable region and light chain variable region may be identical to those derived from a specific type of antibody, or they may be those derived from a specific type of antibody in which one or more amino acid residues have been modified into non-natural amino acids.

[0160] For example, the heavy chain variable region and light chain variable region are Trastuzumab, Nivolumab, Cemiplimab, Dostarlimab, Retifanlimab, Toripalimab, Pembrolizumab, Naxitamab, Cetuximab, Telisotuzumab, Tisotumab, Pinatuzumab, Lifastuzumab, Indusatumab, Vandortuzumab, Sofituzumab, Vorsetuzumab, Mirvetuximab, It may be derived from an antibody selected from the group consisting of Coltuximab, Naratuximab, Indatuximab, Anetumab, Lorvotuzumab, Cantuzumab, Laprituximab, Bivatuzumab, Vadastuximab, Rovalpituzumab, Inotuzumab, Sacituzumab, Labetuzumab, Milatuzumab, Lupaltumab, Aprutumab, and OKT3 (Anti-CD3 monoclonal antibody), or one or more amino acid residues derived from it may be modified to be non-natural amino acids.

[0161] In one embodiment, the heavy chain variable region and the light chain variable region may be derived from an anti-HER2 antibody. In one embodiment, the heavy chain variable region and the light chain variable region may be derived from trastuzumab.

[0162] The sequences of the heavy chain variable region and the light chain variable region of the scFv engineered in this application may vary depending on the location in which the engineered scFv contains a non-natural amino acid. For example, if the scFv engineered in this application contains the non-natural amino acid only in a peptide liker including ABM, the sequences of the heavy chain variable region and the light chain variable region of the engineered scFv may be identical to the sequences of the heavy chain variable region and the light chain variable region of the specific antibody (or albubody) from which the engineered scFv is derived. For another example, if the scFv engineered in this application contains a non-natural amino acid in the heavy chain variable region and / or the light chain variable region, the heavy chain variable region and / or the light chain variable region of the engineered scFv may include a sequence in which a specific amino acid residue in the heavy chain variable region and / or the light chain variable region derived from the specific antibody (or albubody) is changed to a non-natural amino acid.

[0163] If the sequences of the heavy chain variable region and the light chain variable region do not contain non-natural amino acids, the sequences of the heavy chain variable region and the light chain variable region may be appropriately selected according to a specific antibody type. Additionally, if the heavy chain variable region and / or the light chain variable region contain one or more non-natural amino acids, the sequences of the heavy chain variable region and the light chain variable region may be appropriately selected according to a specific antibody type and the position of the non-natural amino acid. Detailed examples are provided in "Specific Examples of Engineered scFv".

[0164] As described above, the heavy chain variable region and the light chain variable region each include one or more complementarity determining regions (CDRs) or antigen binding regions. Preferably, the heavy chain variable region and the light chain variable region each include three complementarity determining regions. Additionally, the heavy chain variable region and the light chain variable region each include one or more framework regions (FRs). Preferably, the heavy chain variable region and the light chain variable region each include four framework regions. Accordingly, the heavy chain variable region and the light chain variable region each V H :[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4] and V L It can be expressed as [FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]. The above order refers to the direction from the N-terminus to the N-terminus. In the following description, the meaning that the variable region may include three CDRs is that, unless otherwise stated, they are included in the order of CDR1, CDR2, and CDR3. Additionally, the meaning that the variable region may include four FRs is that, unless otherwise stated, they are included in the order of FR1, FR2, FR3, and FR4. At this time, the various framework regions described above may each include one or more non-natural amino acids. This means that non-natural amino acids are included in regions with relatively less influence, rather than in CDR regions that are important for antigen binding ability.

[0165] The sequences of the heavy chain variable region and the light chain variable region may be represented as sequences having a certain degree of homology. For example, the heavy chain variable region sequence may include an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homology with a specific amino acid sequence. Additionally, the light chain variable site sequence may include an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homology with a specific amino acid sequence.

[0166] Additionally, each of the above framework regions may be represented by a sequence having a certain degree of homology. For example, the framework region may include an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homology with a specific amino acid sequence.

[0167] The above-mentioned heavy chain variable region and light chain variable region have amino acid lengths of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, respectively. 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,It may be selected from 249 or 250 aa. For example, the heavy chain variable region and the light chain variable region may each have an amino acid length of 50 to 250 aa. In one embodiment, the heavy chain variable region and the light chain variable region may each have an amino acid length of 100 to 130 aa.

[0168]

[0169] Composition (2) Peptide linker including ABM

[0170] The engineered scFv of the present application comprises a "peptide linker containing an ABM" that connects the aforementioned heavy chain variable region and light chain variable region. In this case, for example, the peptide linker containing an ABM may mean a sequence containing non-natural amino acids.

[0171] The engineered scFv of the present application is characterized by comprising a non-natural amino acid having a click chemical reaction group. In this case, despite comprising such a non-natural amino acid, the engineered scFv retains a binding affinity for albumin. Additionally, the ABM included in the engineered scFv retains a binding affinity for albumin. A detailed explanation thereof is provided in "Characteristics of the Engineered scFv".

[0172] The above-mentioned peptide linker comprises an albumin-binding moiety (ABM), a first scFv linker, and a second scFv linker. In this case, the albumin-binding moiety (ABM), the first scFv linker, and / or the second scFv linker may each include one or more non-natural amino acids. Depending on the position where the engineered scFv contains the non-natural amino acid, whether the peptide linker includes the non-natural amino acid and the position of the non-natural amino acid may differ. This is described in "Specific Examples of Engineered scFv".

[0173] The above peptide linker has a structure selected from the following. In this case, they are listed in order from the N-terminus to the C-terminus:

[0174] -[1st scFv linker]-[Albumin binding moiety (ABM)]-[2nd scFv linker];

[0175] -[1st scFv linker]-[2nd scFv linker]-[Albumin binding moiety (ABM)]-;

[0176] -[Albumin binding moiety (ABM)]- [1st scFv linker]-[2nd scFv linker]-

[0177] In a preferred example, the ABM in the linker peptide is located between the first scFv linker and the second scFv linker. In this case, the first scFv linker refers to a polypeptide or protein fragment connecting a heavy chain variable region and an albumin binding moiety. Additionally, the second scFv linker refers to a polypeptide or protein fragment connecting a light chain variable region and an albumin binding moiety.

[0178] The first scFv linker and the second scFv linker function to appropriately adjust the distance and orientation of the heavy chain variable region, the light chain variable region, and the albumin binding moiety. Through this, the first scFv linker and the second scFv linker maintain the flexibility, structural stability, and correct binding affinity to antigens and albumin of the engineered scFv.

[0179] In order to maintain the flexibility, structural stability, and proper binding affinity to antigens and albumin of the engineered scFv, the lengths of the first scFv linker and the second scFv linker can be appropriately selected.

[0180] For example, the lengths of the first scFv linker and the second scFv linker may be selected from 1aa, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, 25aa, 26aa, 27aa, 28aa, 29aa, and 30aa, respectively. In one specific example, the lengths of the first scFv linker and the second scFv linker may be integers from 3aa to 15aa.

[0181] When the lengths of the first scFv linker and the second scFv linker are each 3aa or longer, the flexibility, structural stability, binding strength to antigens, and binding strength to albumin of the engineered scFv can be adequately maintained. Accordingly, the lengths of the first scFv linker and the second scFv linker may each be independently 3aa to 20aa. In one embodiment, the lengths of the first scFv linker and the second scFv linker may be 13aa.

[0182] In order to maintain the flexibility, structural stability, and proper binding affinity to antigens and albumin of the engineered scFv, the sequences of the first scFv linker and the second scFv linker can be appropriately selected by a person skilled in the art.

[0183] For example, the sequences of the first scFv linker and the second scFv linker may each be composed of (GS)n, (GGS)n, (GTSGS)n, (GGGGS)n, or a combination thereof. The n is any natural number and may be from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The order of these links and the number of repetitions are not limited. In one embodiment, the sequence of the first scFv linker may include (GGGGS GTSGS GGS) (Sequence No. 26). In one embodiment, the sequence of the second scFv linker may include GGS GTSGS GGGGS (Sequence No. 28). Depending on the structure of the peptide linker containing ABM, when the first scFv linker and the second scFv linker are consecutive, both may be represented as a specific sequence simultaneously. For example, the first scFv linker and the second scFv linker may include an amino acid sequence represented by SEQ ID NO. 25.

[0184] In another specific example, the first scFv linker and the second scFv linker may be linkers containing non-natural amino acids.

[0185] In one sun, one or more non-natural amino acids may be included in a linker site selected from the first scFv linker and the second scFv linker.

[0186] For example, the first scFv linker may contain one non-natural amino acid, and the second scFv linker may not contain one. For another example, the second scFv linker may contain one non-natural amino acid, and the first scFv linker may not contain one. A detailed description of the type and location of the non-natural amino acid is to be provided in the “Non-natural Amino Acid” paragraph.

[0187] The above-mentioned peptide linker includes an albumin binding moiety (ABM) as described above.

[0188] The albumin-binding moiety described above includes a site capable of binding to albumin. Therefore, when the engineered scFv is administered into the body, the albumin-binding moiety binds to albumin in the body, thereby increasing the half-life of the engineered scFv. Additionally, the albumin-binding moiety binds to albumin in the body, thereby increasing the retention time of the engineered scFv in the body. Furthermore, the albumin-binding moiety binds to albumin in the body, thereby enhancing the targeting of the engineered scFv to target cells (tumors).

[0189] The above albumin binding moiety can be appropriately selected by those skilled in the art if it can bind to albumin.

[0190] The length of the albumin binding moiety is not limited. For example, the amino acid lengths of the albumin-binding moiety are 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, 25aa, 26aa, 27aa, 28aa, 29aa, 30aa, 31aa, 32aa, 33aa, 34aa, 35aa, 36aa, 37aa, 38aa, 39aa, 40aa, 41aa, 42aa, 43aa, 44aa, 45aa, 46aa, 47aa, 48aa, 49aa, 50aa, 51aa, 52aa, 53aa, 54aa, 55aa, 56aa, 57aa, 58aa, 59aa, 60aa, 61aa, 62aa, 63aa, 64aa, 65aa, 66aa, 67aa, 68aa, 69aa, 70aa, 71aa, 72aa, 73aa, 74aa, 75aa, 76aa, 77aa, 78aa, 79aa, 80aa, 81aa, 82aa, 83aa, 84aa, 85aa, 86aa, 87aa, 88aa, 89aa, 90aa, 91aa, 92aa, 93aa, 94aa, 95aa, 96aa, 97aa, 98aa, 99aa, 100aa, 101aa, 102aa, 103aa, 104aa, 105aa, 106aa, 107aa, 108aa, 109aa, 110aa, 111aa, 112aa, 113aa, 114aa, 115aa, 116aa, 117aa, 118aa, 119aa, 120aa, 121aa, 122aa, 123aa, 124aa, 125aa, 126aa, 127aa, 128aa, 129aa, 130aa, 131aa, 132aa, 133aa, 134aa, 135aa, 136aa, 137aa, 138aa, 139aa, 140aa, 141aa, 142aa, 143aa, 144aa, 145aa, 146aa, 147aa, 148aa, 149aa, 150aa, 151aa, 152aa, 153aa,154aa, 155aa, 156aa, 157aa, 158aa, 159aa, 160aa, 161aa, 162aa, 163aa, 164aa, 165aa, 166aa, 167aa, 168aa, 169aa, 170aa, 171aa, 172aa, 173aa, 174aa, 175aa, 176aa, 177aa, 178aa, 179aa, 180aa, 181aa, 182aa, 183aa, 184aa, 185aa, 186aa, 187aa, 188aa, The value may be selected from 189aa, 190aa, 191aa, 192aa, 193aa, 194aa, 195aa, 196aa, 197aa, 198aa, 199aa, and 200aa. In one embodiment, the amino acid length of the albumin-binding moiety may be 30 to 50 aa.

[0191] In addition, the type of albumin binding moiety is not limited. For example, it may be an albumin protein domain, an affibody, or a peptide.

[0192] For example, the albumin-binding moiety may include the sequence of an albumin-binding domain derived from Streptococcal protein G (Sequence No. 55). For another example, the albumin-binding moiety may include the VNAR (V domain of cartilage oligomeric matrix protein, chondrocyte-derived) sequence derived from a cartilage oligomeric matrix protein of a sea shark (Sequence No. 56). For yet another example, the albumin-binding moiety may include the sequence derived from the protein PAB of Finegoldia magna (Sequence No. 57).

[0193] As another example, a person skilled in the art may appropriately select and use known ABM sequences. For example, 10.1074 / jbc.M109943200, 10.1093 / protein / gzn028, 10.26355 / eurrev_202012_24038, or 10.1038 / s41589-021-00770-1 may be referenced.

[0194] The above albumin binding moiety is not limited to the type of albumin to which it can bind. For example, it may be human serum albumin (HSA).

[0195] The engineered scFv of the present application has an increased half-life by binding to albumin. For example, when albumin is bound to the engineered scFv of the present application, compared to the engineered scFv or scFv without albumin binding, the half-life of the engineered scFv is about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, about 120 times, about 130 times, about 140 times, about 150 times, about 160 times, about 170 times, about 180 times, about 190 times, about 200 times, about 210 times, about 220 times, about 230 times, about 240 times, about 250 times, about 260 times, about 270 times, about 280 times, about 290 times, about It can be increased by 300 times, approximately 310 times, approximately 320 times, approximately 330 times, approximately 340 times, approximately 350 times, approximately 360 times, approximately 370 times, approximately 380 times, approximately 390 times, approximately 400 times, approximately 410 times, approximately 420 times, approximately 430 times, approximately 440 times, approximately 450 times, approximately 460 times, approximately 470 times, approximately 480 times, approximately 490 times, and approximately 500 times. In one specific example, it can be increased by more than about 200 times.

[0196] The albumin binding moiety of the engineered scFv of the present application may be an albumin binding moiety containing a non-natural amino acid. In this case, the albumin binding moiety containing the non-natural amino acid is not limited to its sequence as long as it possesses binding affinity for albumin. Furthermore, depending on the position of the non-natural amino acid in the engineered scFv of the present application, whether the albumin binding moiety contains a non-natural amino acid may vary. The non-natural amino acid included therein is described in detail in “Unnatural Amino Acid (UAA).” Additionally, specific examples of an albumin binding moiety containing a non-natural amino acid are described in “Specific Examples of Engineered scFv.”

[0197]

[0198] unnatural amino acid (UAA)

[0199] As described above, the engineered scFv of the present application comprises one or more non-natural amino acids. In one embodiment, one or more sites selected from a peptide linker comprising a heavy chain variable site, a light chain variable site, and an ABM comprise a non-natural amino acid.

[0200] The number of non-natural amino acids included in the engineered scFv is not limited. For example, the engineered scFv may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural amino acids. Specifically, it may include 1 or 2 non-natural amino acids.

[0201] The non-natural amino acid included in the engineered scFv of the present application is used to concatenate a payload through a click reaction. Accordingly, the non-natural amino acid includes a click chemical functional group capable of performing a click reaction. Accordingly, the engineered scFv of the present application can perform a click reaction by utilizing the click chemical functional group of the non-natural amino acid.

[0202] The engineered scFv of the present application can be conjugated with another material comprising a corresponding click chemistry group capable of reacting with the click chemistry functional group of the non-natural amino acid. For example, a linker having a functional group capable of performing a click reaction with the functional group of the non-natural amino acid may be used to conjugate the engineered scFv with the desired payload. In this specification, the click chemistry functional group of the non-natural amino acid is referred to as the "first click chemistry functional group," and the corresponding click chemistry functional group capable of binding thereto is referred to as the "second click chemistry functional group." The second click chemistry functional group may include a linker (conjugating linker) connected to the payload used for conjugation.

[0203] The types of the above-mentioned non-natural amino acids are not limited. In addition, the types of click chemical functional groups included in the above-mentioned non-natural amino acids are not limited. For example, the above-mentioned non-natural amino acids may include click chemical functional groups such as azide or tetrazine.

[0204] The click chemical functional group included in the above non-natural amino acid may include a bioorthogonal functional group [bioorthogonal functional group] that is not present in the natural amino acid within the cell. For example, the above non-natural amino acid may include a bioorthogonal functional group such as azide or tetrazine.

[0205] Below, the types of non-natural amino acids based on azide or tetrazine groups are explained in more detail.

[0206]

[0207] Types of non-natural amino acids

[0208] The non-natural amino acids of the present application are not limited to any specific type as long as they contain click chemical functional groups. In the following, to explain the non-natural amino acids, both the non-natural amino acid in the amino acid state and the non-natural amino acid in the amino acid residue state are illustrated.

[0209] For example, the above-mentioned non-natural amino acid may include the following click chemical functional groups:

[0210] [Chemical Formula 1-1]

[0211] (1,2,4,5 tetrazine), or

[0212] [Chemical Formula 1-2]

[0213] (Triazine).

[0214] At this time, R1 is H, halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, C 3-6 It is any one selected from heterocycloalkyl, aryl, and heteroaryl, and

[0215] At this time, the heterocycloalkyl or heteroaryl comprises one or more heteroatom groups selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O)2-, or one or more heteroatoms selected from the group consisting of O, N, and S.

[0216] In one specific example, the above non-natural amino acid may be of the following structural formula:

[0217] [Chemical Formula 2-1]

[0218]

[0219] or

[0220] ,

[0221] In this case, A1 does not exist or -A 11 -A 12 - and,

[0222] At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and

[0223] At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5It is any one selected from heteroalkylene and heteroarylene, wherein p is an integer between 1 and 3 inclusive, and

[0224] At this time, R1 is H, halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, C 3-6 It is any one selected from heterocycloalkyl, aryl, and heteroaryl, and

[0225] At this time, the heterocycloalkyl or heteroaryl comprises one or more heteroatom groups selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O)2-, or one or more heteroatoms selected from the group consisting of O, N, and S.

[0226] The above AA1 and AA2 represent amino acid residue(s) connected to the above non-natural amino acid. In this case, one of AA1 and AA2 may be absent. In this case, the absence of AA1 or AA2 should be interpreted as the non-natural amino acid being located at the end of the engineered scFv.

[0227] In one specific embodiment, the non-natural amino acid may be 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (4-(1,2,4,5-tetrazin-3-yl)phenylalanine)[frTet] represented by the following chemical formula 2-2, or 4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenylalanine (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenylalanine)[Tet_v2.0] represented by the following chemical formula 2-3.

[0228] [Chemical Formula 2-2]

[0229] ; or

[0230] ,

[0231] [Chemical Formula 2-3]

[0232] ; or

[0233] ,

[0234] In this case, AA1 and AA2 represent amino acid residues connected to the non-natural amino acid, and AA1 or AA2 may be absent. In this case, the absence of AA1 or AA2 should be interpreted as the non-natural amino acid being located at the end of the engineered scFv.

[0235] For example, the above non-natural amino acid may include a click chemical functional group as follows.

[0236] [Chemical Formula 3]

[0237] (Azaid)

[0238] In one specific example, the above non-natural amino acid may be of the following structural formula:

[0239] [Chemical Formula 4-1]

[0240] ;or

[0241] ,

[0242] In this case, A1 does not exist or -A 11 -A 12 - and,

[0243] At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and

[0244] At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is selected from heteroalkylene and heteroarylene, where p is an integer between 1 and 3.

[0245] The above AA1 and AA2 represent amino acid residues connected to the above non-natural amino acid, and AA1 or AA2 may be absent. In this case, the absence of AA1 or AA2 should be interpreted as the non-natural amino acid being located at the end of the engineered scFv.

[0246] In one specific embodiment, the non-natural amino acid may be 4-Azido-L-phenylalanine (pAzF) represented by the following chemical formula 4-2.

[0247] [Chemical Formula 4-2]

[0248] ; or

[0249] ,

[0250] In this case, AA1 and AA2 represent amino acid residues connected to the non-natural amino acid, and AA1 or AA2 may be absent. In this case, the absence of AA1 or AA2 should be interpreted as the non-natural amino acid being located at the end of the engineered scFv.

[0251] Location of non-natural amino acids

[0252] The locations in the engineered scFv of the present application that contain non-natural amino acids may vary. Additionally, the locations of non-natural amino acid locations included in the engineered scFv may be expressed in various ways. For example, the locations in the engineered scFv that contain non-natural amino acids may be expressed by specifying them as specific sites. For example, the sequence of the engineered scFv may be expressed by indicating it as a sequence containing non-natural amino acids. For another example, it may be expressed by selecting the amino acid locations in the scFv where the albumin-binding moiety is inserted that change to non-natural amino acids.

[0253] In the above-mentioned engineered scFv, the non-natural amino acid may be included at a location in one or more regions selected from a peptide linker including a heavy chain variable region, a light chain variable region, and an ABM. A detailed description thereof is as described in "Configuration (1) Heavy chain variable region and light chain variable region" and "Configuration (2) Peptide linker including ABM".

[0254] The location of the aforementioned non-natural amino acid is selected by considering various factors. First, the location of the non-natural amino acid may be selected by considering the effect that the insertion of the non-natural amino acid has on the structural stability of the engineered scFv. Accordingly, the location of the non-natural amino acid may be selected by considering the degree of change in free energy resulting from the change to the non-natural amino acid. Second, the location of the non-natural amino acid may be selected by considering its effect on the binding ability of the engineered scFv to antigens and to albumin. For example, the location of the non-natural amino acid may be selected excluding the complementarity determining site of the engineered scFv. Third, the location of the non-natural amino acid may be selected by considering the reaction with the conjugating linker when intending to link it to the payload. Accordingly, the location of the non-natural amino acid may be selected by considering solvent accessibility. Fourth, the location of the non-natural amino acid may be selected by considering the expression yield of the engineered scFv. In other words, the introduction site of the non-natural amino acid may be selected by considering factors that may affect the production efficiency of the engineered scFv.

[0255] In the engineered scFv of the present application, for various known scFv sequences that can be selected depending on the target antigen, the position for introducing a non-natural amino acid can be specified (determined) by considering the aforementioned factors.

[0256] In the above-mentioned engineered scFv, non-natural amino acids can be introduced into one or more regions selected from the heavy chain and light chain variable regions and the scFv linker.

[0257] For example, it may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural amino acids. For specific examples, it may include 1 or 2 non-natural amino acids.

[0258] In one embodiment, the engineered scFv may include one non-natural amino acid in the heavy chain variable region (VH). In one embodiment, the engineered scFv may include one non-natural amino acid in the light chain variable region (VL). In one embodiment, the engineered scFv may include one non-natural amino acid in the first scFv linker. In one embodiment, the engineered scFv may include one non-natural amino acid in the second scFv linker. In one embodiment, the engineered scFv may include one non-natural amino acid in the albumin binding moiety. In one embodiment, the engineered scFv may include one non-natural amino acid in the framework region (framework region 1, 2, 3, or 4 for the heavy chain variable region or light chain variable region).

[0259] The engineered scFv of the present application can specify (determine) the position for introducing a non-natural amino acid by considering the aforementioned factors for various known scFv sequences that can be selected depending on the target antigen.

[0260] For example, an albubody targeting antigen HER2 may be a peptide consisting of the amino acid sequence of SEQ ID NO. 1, and the engineered scFv of the present application may be a variant in which one or more amino acids selected from G44, S87, R89, G123, S203, P234, G235, K236 and K301 in the amino acid sequence of SEQ ID NO. 1 are changed to pAzF or frTet.

[0261] In one embodiment, the engineered scFv may be a fusion protein (polypeptide) comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X in SEQ ID NOs 2 to 10 is a non-natural amino acid. For example, X may be pAzF or frTet.

[0262]

[0263] Location selection criteria for non-natural amino acids

[0264] Depending on the position of the non-natural amino acid in the engineered scFv of the present application, the function and effect of the engineered scFv may vary.

[0265] For example, the binding affinity to an antigen of the engineered scFv may vary depending on the position of the non-natural amino acid included therein. In particular, the engineered scFv is a modified version in which one or more amino acids of the original albubody are changed to non-natural amino acids. Accordingly, changing one or more amino acids to non-natural amino acids can alter the binding affinity of the original albubody to an antigen. That is, the binding affinity to an antigen of the engineered scFv may vary depending on the position of the non-natural amino acid. For example, changing the amino acid of the complementarity determining region of the albubody to a non-natural amino acid can alter the binding affinity of the albubody to an antigen.

[0266] For example, the non-natural amino acid position of the engineered scFv may be selected as a position that does not significantly alter the binding affinity to the antigen of the albubody. That is, the non-natural amino acid position of the engineered scFv may be selected as a position that does not significantly interfere with the binding affinity of the engineered scFv to the antigen. For example, the position of the non-natural amino acid may be selected as a position capable of possessing about 90% or more, about 80% or more, about 70% or more, about 60% or more, about 50% or more, about 40% or more, or about 30% or more of the binding affinity to the antigen of the albubody.

[0267] The above-mentioned engineered scFv may have different binding affinities to albumin depending on the position of the included non-natural amino acid. In particular, the above-mentioned engineered scFv is a modified version in which one or more amino acids of the original albumin are changed to non-natural amino acids. Accordingly, changing one or more amino acids to non-natural amino acids can alter the binding affinity of the original albumin to albumin. That is, the above-mentioned engineered scFv may have different binding affinities to albumin depending on the position of the non-natural amino acid.

[0268] For example, the non-natural amino acid position of the engineered scFv may be selected as a position that does not significantly alter the binding affinity of the albumin of the albumin of the albumin. That is, the non-natural amino acid position of the engineered scFv may be selected as a position that does not significantly interfere with the binding affinity of the engineered scFv to the albumin of the albumin. For example, the position of the non-natural amino acid may be selected as a position capable of retaining about 90% or more, about 80% or more, about 70% or more, about 60% or more, about 50% or more, about 40% or more, or about 30% or more of the binding affinity of the albumin of the albumin of the albumin.

[0269] The degree of binding affinity of the above-mentioned engineered scFv and albumin to antibodies and albumin can be measured in various ways. For example, the binding affinity can be measured through an evaluation of binding affinity to antigens and albumin.

[0270] The engineered scFv described above is a modified version in which one or more amino acids of the original albubody are changed to non-natural amino acids. Due to the modified non-natural amino acids, the engineered scFv may have structural differences from the underlying albubody. Accordingly, changing one or more amino acids to non-natural amino acids may alter the function and structure of the original albubody. For example, it may affect the binding affinity to the antigen and the binding affinity to albumin. In this case, structural changes based on the position of the non-natural amino acids are not predicted solely by the region (position) containing the non-natural amino acids. That is, the position of the non-natural amino acids must be individually selected by considering the structure of the albubody underlying the engineered scFv, the composition of the albubody, the length of the albubody, etc.

[0271] As one method for selecting the position of the aforementioned non-natural amino acid, a natural amino acid (similar amino acid) having a structure similar to the non-natural amino acid to be modified can be used. That is, if the amino acid of the albubody forming the basis of the engineered scFv is changed to a similar amino acid, the non-natural amino acid modification position can be selected at a location with a small change in free energy.

[0272] For example, as one method for selecting the location of pAzF, tyrosine or phenylalanine having a structure similar to pAzF can be used. That is, if the amino acid of the albubody forming the basis of the engineered scFv is changed to tyrosine or phenylalanine, the location of pAzF can be selected at a position with a small change in free energy. This can be applied as is when the natural amino acid most similar to the non-natural amino acid to be inserted is tyrosine or phenylalanine. In other words, the location of pAzF selected through the above method can also be used as a location for inserting the other non-natural amino acid. For example, it can also be used as a location for inserting ftTet.

[0273] The above-mentioned engineered scFv is produced using a cell line capable of producing proteins with inserted non-natural amino acids and a vector containing a specific codon (e.g., an amber codon) encoding the engineered scFv.

[0274] At this time, even if engineered scFvs are produced based on the same albubody, the degree of expression of the engineered scFvs varies depending on where the non-natural amino acid is changed. In particular, the degree of expression in the receptor state varies. The higher the degree of expression in the receptor state, the higher the proportion of engineered scFvs that can be used without additional processing. That is, an engineered scFv containing a non-natural amino acid at a specific position may be an engineered scFv that expresses well in the receptor state compared to an engineered scFv containing a non-natural amino acid at a different position, even if the composition other than the non-natural amino acid position is identical. In this specification, the meaning of expressing well in the receptor state may also be expressed as having a high expression yield. Furthermore, the above comparison may be made in various ways according to known technology.

[0275] The above-mentioned engineered scFv contains a click chemical functional group. In this case, even if the engineered scFv has one or more non-natural amino acids modified in the same albubody, the degree to which the click chemical functional group reacts with a payload connected to a conjugating linker varies depending on the position of the non-natural amino acid. The above difference is caused by the structural characteristics of the engineered scFv and the accessibility of the click chemical functional group to the solvent, etc. Accordingly, an engineered scFv containing a non-natural amino acid at a specific position may react better with a payload connected to a conjugating linker compared to an engineered scFv containing a non-natural amino acid at a different position.

[0276] In this specification, the meaning that the conjugating linker reacts better with the connected payload may be expressed as having a higher conjugate yield. Additionally, whether the conjugating linker reacts better with the connected payload can be verified in various ways. For example, the yield may be expressed as the percentage of produced engineering scFv conjugates relative to the reacted engineering scFv after reacting the engineering scFv with the payload connected to the conjugating linker.

[0277] For example, a non-natural amino acid position of the engineered scFv may be selected as a position that reacts better with a payload to which a conjugating linker is connected. For example, after reacting the engineered scFv with the scFv to which a conjugating linker is connected, a non-natural amino acid position may be selected such that the percentage of the engineered scFv conjugate produced relative to the reacted engineered scFv is about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more. In one embodiment, a non-natural amino acid position with about 30% or more may be selected. In one embodiment, a non-natural amino acid position with about 60% or more may be selected. In one embodiment, a non-natural amino acid position with about 80% or more may be selected.

[0278] Therefore, the non-natural amino acid positions included in the above-mentioned engineered scFv may be selected differently depending on what the scFv (albubody) containing the albumin binding moiety on which the engineered scFv is based is.

[0279] For example, the expression level of a vector containing a base sequence selected in SEQ ID NOs 75 and 77 to 82 may be higher than the expression level of a vector containing a base sequence of SEQ ID NO. 73. In this case, the vector containing a base sequence selected in SEQ ID NOs 75 and 77 to 82 is a vector encoding an engineered scFv containing an amino acid sequence selected in SEQ ID NOs 3 and 5 to 9, respectively. Additionally, the vector containing a base sequence of SEQ ID NO. 73 is a vector encoding an engineered scFv containing an amino acid sequence of SEQ ID NO. 2. For another example, the expression level of a vector containing a base sequence selected in SEQ ID NOs 75 and 77 to 82 may be higher than the expression level of a vector containing a base sequence of SEQ ID NO. 73. In this case, the vector containing a base sequence selected in SEQ ID NOs 75 and 77 to 82 is a vector encoding an engineered scFv containing an amino acid sequence selected in SEQ ID NOs 3 and 5 to 9, respectively. Additionally, a vector containing the base sequence of SEQ ID NO. 76 is a vector encoding an engineered scFv containing the amino acid sequence of SEQ ID NO. 4. In other words, an engineered scFv containing a non-natural amino acid at a specific position may be an engineered scFv that is more expressed than an engineered scFv containing a non-natural amino acid at another position. For example, an engineered scFv containing the amino acid sequence selected from SEQ ID NO. 3 and 5 to 9 may be an engineered scFv that is more expressed than an engineered scFv containing the amino acid sequence of SEQ ID NO. 2. For example, an engineered scFv containing the amino acid sequence selected from SEQ ID NO. 3 and 5 to 9 may be an engineered scFv that is more expressed than an engineered scFv containing the amino acid sequence of SEQ ID NO. 4.For example, an engineered scFv in which selected positions at S87, G123, S203, P234, G235, K236, and K301 of an albubody containing the amino acid sequence of SEQ ID NO. 1 are changed to non-natural amino acids may be an engineered scFv that is better expressed than an engineered scFv in which the G44 position of an albubody containing the amino acid sequence of SEQ ID NO. 1 is changed to a non-natural amino acid. For example, an engineered scFv in which selected positions at S87, G123, S203, P234, G235, K236, and K301 of an albubody containing the amino acid sequence of SEQ ID NO. 1 are changed to non-natural amino acids may be an engineered scFv that is better expressed than an engineered scFv in which the R89 position of an albubody containing the amino acid sequence of SEQ ID NO. 1 is changed to a non-natural amino acid.

[0280] For example, an engineered scFv comprising the amino acid sequence of SEQ ID NO. 8 may be an engineered scFv in which the conjugating linker reacts better with the payload to which the conjugating linker is attached than an engineered scFv comprising the amino acid sequence selected from SEQ ID NOs. 3, 5 to 7 and 9. For example, an engineered scFv comprising the amino acid sequence of SEQ ID NO. 7 may be an engineered scFv in which the conjugating linker reacts better with the payload to which the conjugating linker is attached than an engineered scFv comprising the amino acid sequence selected from SEQ ID NOs. 3, 4, 5 and 9. For example, an engineered scFv in which the G235 position of an albubody containing the amino acid sequence of SEQ ID NO. 1 is changed to a non-natural amino acid may be an engineered scFv that reacts better with a payload connected to a conjugating linker than an engineered scFv in which the S87, G123, S203, P234, K236, or K301 positions of an albubody containing the amino acid sequence of SEQ ID NO. 1 are changed to a non-natural amino acid. For example, an engineered scFv in which the G234 position of an albubody containing the amino acid sequence of SEQ ID NO. 1 is changed to a non-natural amino acid may be an engineered scFv that reacts better with a payload connected to a conjugating linker than an engineered scFv in which the S87, G123, S203, K236, or K301 positions of an albubody containing the amino acid sequence of SEQ ID NO. 1 are changed to a non-natural amino acid.

[0281] Characteristics of engineered scFv

[0282] The engineered scFv of the present application can be manufactured based on a conventional scFv. Accordingly, the engineered scFv can possess various characteristics of the scFv.

[0283] First, engineered scFv possesses high diffusivity, vascular permeability, and tissue permeability. Consequently, engineered scFv penetrates deep into tumor tissue more easily than full-length antibodies. Second, engineered scFv can be produced in a microorganism-based expression system. As a result, engineered scFv can be produced at a relatively low production cost. Third, since engineered scFv does not contain Fc, Fc-mediated immune responses do not occur. Therefore, the engineered scFv does not have side effects caused by Fc-mediated immune responses.

[0284] Furthermore, the engineered scFv possesses several characteristics through the inclusion of an albumin-binding moiety. First, the half-life of the engineered scFv can be increased by binding albumin to the albumin-binding moiety. Second, the engineered scFv is capable of FcRn-mediated recycling by binding albumin to the albumin-binding moiety. Additionally, the engineered scFv is not renally excreted through the albumin bound to the albumin-binding moiety. Third, the engineered scFv can achieve stronger tumor targeting by binding to serum albumin. This is because albumin is more preferentially accumulated in malignant and inflammatory tissues that have highly permeable blood vessels and lack efficient lymphatic drainage. Furthermore, receptor-mediated endocytosis further promotes the accumulation of albumin within the tumor. Accordingly, if a payload is conjugated to the engineered scFv, it can serve as a means to more easily deliver the payload (e.g., a drug) to the tumor site.

[0285] Furthermore, depending on which amino acid of the albubody is changed to a non-natural amino acid, the engineered scFv may possess different characteristics. Therefore, it is important to appropriately select the location containing the non-natural amino acid in the engineered scFv. First, depending on which amino acid of the albubody is changed to a non-natural amino acid, the engineered scFv has different binding affinities for antigens and albumin. Therefore, the location of the non-natural amino acid must be selected at a position that does not significantly reduce the binding affinity for antigens and albumin. To this end, the location containing the non-natural amino acid may be selected from a position other than the complementarity determining site. Second, depending on which amino acid of the albubody is changed to a non-natural amino acid, the degree of structural similarity between the engineered scFv and the albubody varies. Therefore, even if the amino acid of the albubody is changed to a non-natural amino acid, a location that does not significantly alter the structure should be selected. Third, depending on the location where the engineered scFv contains the non-natural amino acid, the degree of expression varies. Therefore, it is important to select non-natural amino acid sites that result in high expression levels. Fourth, the degree of reaction with the payload to which the conjugating linker is attached varies depending on the location of the non-natural amino acid in the engineered scFv. Therefore, it is important to select non-natural amino acid sites that enable better reaction with the conjugating linker.

[0286]

[0287] Specific example of an engineered scFv

[0288] Hereinafter, specific examples of the scFv engineered in this application are described. The following examples are merely illustrative of the invention and the invention is not limited thereto.

[0289] The amino acid sequence of the above-mentioned engineered scFv is not significantly limited as long as it is a variant in which one or more non-natural amino acids are inserted into a scFv containing an albumin-binding moiety. In one embodiment, the above-mentioned engineered scFv may be a variant in which one or more amino acid residues are substituted with non-natural amino acids in a scFv (Sequence No. 1) containing an albumin-binding moiety including an ABM (Sequence No. 55) between the linker regions (Sequence No. 54) of the scFv (Sequence No. 51).

[0290] For example, the engineered scFv may include specific heavy chain variable regions and light chain variable regions. In one embodiment, where the specific antibody from which the engineered scFv is derived is trastuzumab, the heavy chain variable region may be an amino acid sequence represented by SEQ ID NO. 11 or 15, and the light chain variable region may be an amino acid sequence represented by SEQ ID NO. 19. The sequences refer to sequences of the heavy chain variable region or the light chain variable region that do not contain non-natural amino acids. In another embodiment, the sequence of the heavy chain variable region containing non-natural amino acids may be one amino acid sequence selected from SEQ ID NOs. 12 to 14 and 16 to 18 when derived from trastuzumab. The sequence of the light chain variable region containing non-natural amino acids may be one amino acid sequence selected from SEQ ID NOs. 20 to 24 when derived from trastuzumab. In this case, X of the amino acid sequence represented by the aforementioned sequence number is the aforementioned non-natural amino acid, and the said sequences refer to sequences of a heavy chain variable region or a light chain variable region containing a non-natural amino acid.

[0291] For example, the engineered scFv may include specific CDRs. In one embodiment, the engineered scFv may include three CDRs represented by SEQ ID NOs 29 to 31, respectively, in the heavy chain variable region. Additionally, the engineered scFv may include three CDRs represented by SEQ ID NOs 32 to 34, respectively, in the light chain variable region.

[0292] For example, the engineered scFv may include a heavy chain variable region and a light chain variable region, each comprising a specific CDR and a framework region. In one embodiment, the engineered scFv may include three CDRs represented by SEQ ID NOs 29 to 31, each comprising a heavy chain variable region including a framework region as summarized in the table below.

[0293]

[0294] In another specific example, the engineered scFv may include three CDRs represented by sequence numbers 32 to 34, respectively, and a light chain variable region including a framework region as summarized in the table below.

[0295]

[0296] For example, the engineered scFv may be an engineered scFv based on a scFv derived from a HER2 target antibody. In one specific example, it may be an engineered scFv based on a scFv derived from trastuzumab.

[0297] For example, the engineered scFv may be an engineered scFv based on an albuma containing a scFv derived from a HER2 target antibody and an ABD derived from Streptococcal protein G. In one specific example, it may be an engineered scFv based on an albuma consisting of SEQ ID NO. 1.

[0298] In one specific example, the engineered scFv may be a variant protein comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X included in SEQ ID NOs 2 to 10 refers to a non-natural amino acid.

[0299] In one embodiment, the engineered scFv may be a variant protein comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X included in SEQ ID NOs 2 to 10 means pAzF or frTet.

[0300] In one specific example, the engineered scFv may be a variant in which one or more of the positions G44, S87, R89, G123, S203, P234, G235, K236 and K301 in the scFv having an albumin binding moiety of SEQ ID NO. 1 are changed to non-natural amino acids.

[0301] In one specific example, the engineered scFv may be a variant in which one or more of the positions G44, S87, R89, G123, S203, P234, G235, K236, and K301 in the scFv having an albumin binding moiety of SEQ ID NO. 1 are changed to non-natural amino acids. In this case, the non-natural amino acid is pAzF or frTet.

[0302] For example, regarding the engineered scFv embodiment described above, if a his-tag is included, the amino acid sequence selected from SEQ ID NOs 2 to 10 may be represented as SEQ ID NOs 59 to 67, respectively. Additionally, if an scFv with an albumin binding moiety having SEQ ID NO. 1 inserted includes a his-tag, it may be represented as SEQ ID NO. 58.

[0303]

[0304] Chapter 2 Engineered scFv-Payload Assembly

[0305] generalization

[0306] This specification discloses an engineered scFv conjugate.

[0307] The above-mentioned engineered scFv conjugate refers to a conjugate in which a payload, such as a drug, is directly or indirectly attached to the aforementioned engineered scFv. In this specification, the above-mentioned engineered scFv conjugate may also be referred to as an engineered scFv-payload conjugate. Hereinafter, the engineered scFv conjugate of the present application will be described.

[0308] The engineered scFv conjugate of the present application is not limited to any type as long as a payload is directly or indirectly conjugated to the aforementioned engineered scFv. In one example, the engineered scFv conjugate may be a conjugate in which the engineered scFv and a payload connected to a conjugating linker are conjugated through a click chemical reaction. In another example, the engineered scFv conjugate may be a conjugate in which the engineered scFv is conjugated through a click chemical reaction with a conjugating linker and reacts with a payload.

[0309] For example, the engineered scFv junction of the present application can be expressed by the following structural formula:

[0310] [engineered scFv (engineered scFv, S)]-[junction (J)]-[linker domain (L)]-[payload (P)]. When abbreviated, “SJLP”.

[0311] In the above structural formula, if the payload is a drug, the payload can be changed to a drug (Drug, D).

[0312] [Engineered scFv], which is a component of the above-mentioned engineered scFv assembly, refers to a unit in which the engineered scFv is connected to a connecting part. In this case, [Engineered scFv] as a component of the assembly may be referred to in various ways as needed in this specification as an engineered scFv, an engineered scFv unit, or an albubody variant unit.

[0313] The [linkage] above refers to a structure formed by the reaction of the side chain of a non-natural amino acid of the engineered scFV used in the production of the engineered scFV conjugate and the click chemical functional group of the conjugating linker. That is, the structure formed by the reaction of the click chemical functional group of the non-natural amino acid (first click chemical functional group) possessed by the aforementioned engineered scFV and another corresponding click chemical functional group (second click chemical functional group) is referred to as the linkage. In particular, the structure formed by the reaction of the second click chemical functional group of the conjugating linker and the side chain of the non-natural amino acid may be referred to as the [linkage].

[0314] The above [linker portion] refers to the portion of the payload connected to the conjugating linker used in the production of engineered scFv conjugates, excluding the click chemical functional group and the payload.

[0315] The above [payload] refers to a payload connected to an engineered scFv through a connection part and a linker part.

[0316] Hereinafter, each component of the “engineered scFv assembly” of the present application will be described in detail.

[0317]

[0318] Configuration (1) S (engineered scFv)

[0319] The engineered scFv conjugate of the present application comprises an engineered scFv.

[0320] In this case, the engineered scFv included in the engineered scFv assembly refers to an engineered scFv unit. Accordingly, if necessary, S on the assembly may be referred to as an engineered scFv or an engineered scFv unit. The specific configuration of the engineered scFv is included by reference in the aforementioned “Chapter 1 Engineered scFv”.

[0321] Engineered scFvs may be referred to as having or not including connections as needed.

[0322] For example, the engineered scFv unit refers to a unit that includes a linking portion and is connected to a linker portion. In this case, the engineered scFv unit refers to a form in which a second linker chemical functional group of a conjugating linker reacts with and is bonded to a free-engineered scFv. In one embodiment, the engineered scFv may be a form including a structure in which pAzF reacts with and is bonded to DBCO or BCN. In another embodiment, the engineered scFv may be a form including a structure in which frTet reacts with and is bonded to TCO (trans-cyclooctene). In one embodiment, the engineered scFv conjugate may include an amino acid sequence selected from SEQ ID NOs 2 to 10 as the engineered scFv. In this case, X may be a form including a structure in which pAzF reacts with and is bonded to DBCO or BCN. Or, X may be a form including a structure in which frTet reacts with and is bonded to TCO (trans-cyclooctene).

[0323] As another example, the above-mentioned engineered scFv unit may not include a linkage and may be expressed in a form connected to a linkage. In this case, the central carbon (alpha carbon) of one or more amino acid residues of the above-mentioned engineered scFv unit may be in a form connected to a linkage. Below, the fact that the central carbon (alpha carbon) of one or more amino acid residues of the above-mentioned engineered scFv unit is in a form connected to a linkage is described in more detail.

[0324] The engineered scFv included in the engineered scFv conjugate of the present application comprises one or more amino acid residues represented by the following structural formula. In this case, the position of the amino acid residue represented by the following structural formula is the position of the non-natural amino acid of the engineered scFv that reacted with the payload connected to the conjugating linker. Additionally, for convenience, the amino acid residue represented by the following structural formula may be referred to as a non-natural amino acid. The amino acid residue represented by the following structural formula may also be referred to as a type of non-natural amino acid of the derived free-engineered scFv.

[0325] [Chemical Formula 5]

[0326]

[0327] At this time, AA1 is the amino acid(s) to which the alpha carbon is connected to the linkage, and AA2 is the amino acid(s) to which the alpha carbon is connected to the linkage. At this time, one of AA1 and AA2 may be absent.

[0328] J is the connection part, and

[0329] L is the linker part, and

[0330] P is the payload.

[0331] At this time, the description of the above connection part, linker part, and payload is explained in a separate table of contents.

[0332] In one embodiment, the engineered scFv may be a variant protein comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X of SEQ ID NOs 1 to 10 is an amino acid residue represented by [Chemical Formula 5]. That is, X of SEQ ID NOs 1 to 10 is an amino acid residue in which an alpha carbon is connected to a linkage.

[0333] The above-mentioned engineered scFv may include additional non-natural amino acids in addition to the non-natural amino acid (amino acid residue represented by Chemical Formula 5) that reacted with the payload to which the conjugating linker is connected. The additionally included non-natural amino acid may include a side chain that did not react with the conjugating linker. For a detailed description of the non-natural amino acid, the contents of the non-natural amino acid in Chapter 1 are incorporated by reference.

[0334] If a non-natural amino acid is present at the end of the engineered scFv, the chemical formula may be as follows. The following chemical formula should be interpreted as representing the same as the absence of AA1 or AA2 in Chemical Formula 5. Additionally, the ionic forms in the following chemical formula are indicated based on physiological conditions.

[0335] [Chemical Formula 5-1]

[0336]

[0337] [Chemical Formula 5-2]

[0338]

[0339] Composition (2) J (junction)

[0340] The engineered scFv assembly of the present application includes a connection portion.

[0341] On an engineered scFv conjugate, the linkage is a structure formed by the click chemistry of the non-natural amino acid side chain of the free-engineered scFv and the second click chemical functional group of the conjugating linker.

[0342] That is, the above-mentioned linker includes elements derived from the free-engineered scFv prior to joining. Additionally, the above-mentioned linker includes elements derived from the conjugating linker prior to joining.

[0343] The above linkage is not limited in type as long as the engineered scFv conjugate of the present application is stably maintained within the blood and is not cleaved while circulating to various tissues. Additionally, the above linkage is not limited in type as long as it is a structure formed by the reaction of the non-natural amino acid side chain of the free-engineered scFv and the click chemical functional group of the conjugating linker.

[0344] Examples of connections are described below. In the chemical formulas describing the connections, only the engineered scFv connected thereto and the connections are indicated.

[0345] For example, the above linkage is a structure formed by connecting a side chain having an engineered scFv tetrazine and a click chemical functional group.

[0346] In one specific example, the above-mentioned connecting part can be represented by the following chemical formula.

[0347] [Chemical Formula 6-1]

[0348]

[0349] In this case, A1 does not exist or -A 11 -A 12 - and,

[0350] At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and

[0351] At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is any one selected from heteroalkylene and heteroarylene, wherein p is an integer between 1 and 3 inclusive, and

[0352] At this time, R1 is H, halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, C 3-6 It is any one selected from heterocycloalkyl, aryl, and heteroaryl, and

[0353] At this time, the heterocycloalkyl or heteroaryl comprises one or more heteroatom groups selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O)2-, or one or more heteroatoms selected from the group consisting of O, N, and S, and

[0354] In this case, S is an engineered scFv, and

[0355] In this case, L is a conjugating linker.

[0356] The above [Chemical Formula 6-1] may be a structure formed by the following reaction.

[0357]

[0358] In this case, S is an engineered scFv, and

[0359] At this time, L is the linker part.

[0360] In one specific embodiment, the connecting portion may have a structure represented by the following chemical formula 6-2. The structure represented by chemical formula 6-2 is a structure formed by combining frTet and TCO.

[0361] [Chemical Formula 6-2]

[0362]

[0363] In this case, S is an engineered scFv, and

[0364] At this time, L is the linker part.

[0365] For example, the above linkage is a structure formed by connecting a side chain having an azide group of an engineered scFv and a click chemical functional group.

[0366] In one specific example, the above-mentioned connecting part can be represented by the following chemical formula.

[0367] [Chemical Formula 7-1]

[0368] ,

[0369] [Chemical Formula 7-2]

[0370] ,

[0371] [Chemical Formula 7-3]

[0372] , or

[0373] [Chemical Formula 7-4]

[0374] .

[0375] In this case, A1 does not exist or -A 11 -A 12 - and,

[0376] At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and

[0377] At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is any one selected from heteroalkylene and heteroarylene, wherein p is an integer between 1 and 3 inclusive, and

[0378] In this case, S is an engineered scFv, and

[0379] At this time, L is the linker part.

[0380] The above [Chemical Formula 7-1] may be a structure formed by the following reaction.

[0381]

[0382] In this case, S is an engineered scFv, and

[0383] At this time, L is the linker part.

[0384] The above [Chemical Formula 7-2] may be a structure formed by the following reaction.

[0385]

[0386] In this case, S is an engineered scFv, and

[0387] At this time, L is the linker part.

[0388] The above [Chemical Formula 7-3] may be a structure formed by the following reaction.

[0389]

[0390] In this case, S is an engineered scFv, and

[0391] At this time, L is the linker part.

[0392] The above [Chemical Formula 7-4] may be a structure formed by the following reaction.

[0393]

[0394] In this case, S is an engineered scFv, and

[0395] At this time, L is the linker part.

[0396] In one specific embodiment, the connecting portion may have a structure represented by the following chemical formula 7-5. The structure represented by chemical formula 7-5 may be a structure formed by combining pAzF and DBCO.

[0397] [Chemical Formula 7-5]

[0398]

[0399] In this case, S is an engineered scFv, and

[0400] At this time, L is the linker part.

[0401]

[0402] Composition (3) L (linker part)

[0403] The engineered scFv conjugate of the present application includes a linker portion.

[0404] In the junction of the present application, the structure formed between the connection part and the payload is referred to as the [linker part]. The linker part of the engineered scFv junction of the present application serves to connect the engineered scFv and the payload. Additionally, the linker part of the engineered scFv junction of the present application serves to connect the connection part and the payload.

[0405] The above linker part is not limited to any structure as long as it is a structure capable of connecting the engineered scFv unit and the payload. Alternatively, the above linker part is not limited to any structure as long as it is a structure capable of connecting the connection part and the payload.

[0406] The engineered scFv conjugate of the present application can be produced by reacting a free-engineered scFv with a payload connected to a conjugating linker. In this case, the linker portion is derived from the pre-reaction conjugating linker used to conjugate the payload to the engineered scFv. Accordingly, the linker portion refers to the structure of the region existing between the linker and the payload in the region remaining after one side of the conjugating linker reacts with a non-natural amino acid to form a bonded link. Accordingly, the linker portion may also be referred to as a conjugating linker unit.

[0407] The payload connected to the above conjugating linker can be expressed by the following structural formula.

[0408] [2th click chemistry functional group (2th CC)-[Spacer]-[payload linkage linker (PL)]-[payload]

[0409] At this time, the second click chemical functional group reacts with a non-natural amino acid to form a bonded linkage, and the -[spacer]-[payload linkage linker (PL)]- region connects the linkage and the payload.

[0410] Therefore, as an example, the above linker part can be expressed by the following structural formula.

[0411] -[Spacer]-[Payload Linkage Linker (PL)]-

[0412] The [spacer] above refers collectively to the region located between the part (payload linker) that is directly connected to the payload and has a specific function in the engineered scFv conjugate and the second click chemical functional group. Depending on the needs of those skilled in the art, the spacer may include a configuration to improve solubility or a configuration to improve conjugation reactivity. In one embodiment, the spacer may include PEG (Polyethylene glycol).

[0413] In one specific example, the spacer is absent or -B1-B2-, and

[0414] At this time, B1 is non-existent or -CH2-O-, -CH2-CH2-(C=O)-, -CH2-CH2-(C=0)-NH-, -(C=0)-NH-CH2-, -(C=0)-CH2-CH2-(C=0)-, -(C=0)-CH2-CH2-NH-(C=0)-, -(C=0)-NH-CH2-CH2-CH2-NH-(C=0)-, -(C=0)-CH2-CH2-(C=0)-NH-, -(C=0)-CH2-CH2-CH2-(C=0)-NH-, -O-(C=O)-NH-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-NH-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-O-, (-CH2)n- or combinations thereof may be included, but are not limited thereto. In this case, n means a natural number greater than or equal to 1.

[0415] In this case, B2 is non-existent or -CH2-CH2-(C=O)-, -CH2-CH2-(C=O)-NH-, -CH2-CH2-NH-(C=O)-, -CH2-CH2-(C=O)-O-, -CH2-CH2-SS-, [-CH2-CH2-O]n(PEGn)-, [-CH2-CH2-O]n(PEGn)-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-(C=O)-O-, [-CH2-CH2-O]n(PEGn)-(C=O)-CH2-CH2-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-O- or combinations thereof may be included, but are not limited thereto. In this case, n means a natural number greater than or equal to 1.

[0416] The payload coupling linker mentioned above refers to a portion that is directly connected to the payload and has a specific function. For example, the payload coupling linker may be absent. For another example, the payload coupling linker may be a cleavable linker. A cleavable linker region refers to a linker that is cleaved by the target's surrounding environment when the engineered scFv is attached to the target. Accordingly, the payload attached to the cleavable linker can be effectively activated at the target. Preferably, the cleavable linker may be used when the payload is a drug, but is not limited thereto.

[0417] The above-mentioned cleavable linker is not limited by its type as long as it is cleaved by the surrounding environment of the target. For example, the above-mentioned cleavable linker may be a linker that is cleaved by enzymatic action. In one embodiment, the enzymatic action may be by a proteolytic enzyme contained in the lysosomes of a tumor cell. In one embodiment, the above-mentioned cleavable linker may be a linker comprising Val-Cit, Val-Ala, cBu-Cit, Glu-Val-Cit, or Gly-Gly-Phe-Gly. In this case, the above-mentioned cleavable linker may additionally include a PAB site. Or, the above-mentioned cleavable linker may be Val-Cit-PAB, Val-Ala-PAB, cBu-Cit-PAB, Glu-Val-Cit-PAB, or Gly-Gly-Phe-Gly-PAB. In this case, valine-citrulline (Val-Cit)- etc. may be cleaved by Cathepsin B, which is predominantly expressed in cancer cells.

[0418] For example, the cleavable linker may be a linker that is cleaved by a change in pH. In one embodiment, it may be a linker that is cleaved by a highly acidic environment by endosomes and lysosomes surrounding tumor cells. In one embodiment, the cleavable linker may be a hydrazone, semicarbazone, thiosemicabazone, cis-aconitic amide, orthoester, acetal, ketal, etc. that is cleaved by a highly acidic environment.

[0419] For example, the cleavable linker may be a linker that is cleaved by a reducing environment surrounding the tumor tissue. In one embodiment, the cleavable linker may be a linker containing disulfide bonds such as SATA (N-succinimidyl-Sacetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0420] For example, the cleavable linker may be a linker that is cleaved according to changes in glutathione concentration. In one embodiment, it may be a linker that is cleaved in dependence on high glutathione concentrations around tumor cells. In one embodiment, the cleavable linker may be a beta-glucuronide linker.

[0421] In one embodiment, the payload linker may be a cleavable linker represented by the following chemical formula.

[0422] [Chemical Formula 8-1](Val-Cit-PAB)

[0423] ;

[0424] [Chemical Formula 8-2](Val-Ala-PAB)

[0425] ;

[0426] [Chemical Formula 8-3](cBU-Cit-PAB)

[0427] ;

[0428] [Chemical Formula 8-4](Glu-Val-Cit-PAB)

[0429] ; or

[0430] [Formula 8-5](Gly-Gly-Phe-Gly-PAB)

[0431] .

[0432] In this case, B2 refers to the -B2- region of the spacer, and P refers to the payload.

[0433] In one specific example, the linker portion may be represented by the following chemical formula. In this case, when representing the linker portion, only the connected portion and the payload are indicated.

[0434] [Chemical Formula 9]

[0435] .

[0436] At this time, P is the payload and J is the connection part. Also, n is an integer greater than or equal to 0, preferably 4.

[0437]

[0438] Configuration (4) Payload

[0439] The engineered scFv conjugate of the present application includes a payload.

[0440] The payload included in the engineered scFv conjugate of the present application refers to a material in a state conjugated with the engineered scFv through the connecting portion and the linker portion. Accordingly, if necessary, P on the conjugate may be referred to as a payload or a payload unit.

[0441] The above payload is not limited to any type of material as long as it is a material conjugated to an engineered scFv. For example, the above payload may be a drug (D).

[0442] The above-mentioned drug is not limited to any type as long as it is a drug intended to act on a target site (tissue and / or cell).

[0443] For example, the aforementioned drug may be a cytotoxic drug. For instance, it may be a cytotoxic anticancer agent that kills cancer cells. Cytotoxic anticancer agents can kill cancer cells by interfering with the DNA and RNA synthesis and mitosis of rapidly proliferating cancer cells, or by exerting harmful effects on the DNA molecules themselves.

[0444] For example, the above drug may be a microtubule disrupting agent. The microtubule disrupting agent inhibits microtubule function, thereby preventing normal cell division. In one embodiment, the microtubule disrupting agent may be MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1 (Maytansinoid derivative), or DM4 (Maytansinoid derivative). In one embodiment, the above drug may be MMAE.

[0445] At this time, the chemical formula of MMAE is as follows.

[0446] [Chemical Formula 10]

[0447]

[0448] At this time, the above L refers to the linker part.

[0449] For example, the above drug may be a DNA damaging agent. The above DNA damaging agent is a substance that cuts DNA or interferes with DNA replication. Accordingly, the above DNA damaging agent prevents normal cell division. In one specific example, the above DNA damaging agent may be Calicheamicin, PBD (Pyrrolobenzodiazepine), or SN-38 (Camptothecin derivatives).

[0450] The drug used in the conjugate of the present disclosure can be appropriately selected by a person skilled in the art according to purpose or need, and can be used without limitation as long as it is a drug that can be combined via a conjugating linker.

[0451] As another example, the payload may be a material for labeling. In one embodiment, the payload may be a radioactive isotope. In another embodiment, the payload may be a fluorescent material. As another example, the payload may be a chelating agent. Thus, as a payload, a radioactive isotope, fluorescent molecule, imaging agent, etc., other than a drug may be used. That is, it will be clearly understood that the conjugate of the present disclosure may be used to deliver a target material appropriately selected as needed to the target site (cell or tissue) described above.

[0452] Characteristics of engineered scFv junctions

[0453] The engineered scFv conjugate of the present application can be produced by reacting an engineered scFv with a payload connected to a conjugating linker. Accordingly, it may include the characteristics of the engineered scFv. For details regarding the characteristics of the engineered scFv, the "engineered scFv" in Chapter 1 above is incorporated by reference.

[0454] The engineered scFv conjugate of the present application comprises a structure in which a first click chemical functional group of a free-engineered scFv is combined with a second click chemical functional group of a payload connected by a conjugating linker. That is, the engineered scFv conjugate can be formed by a click chemical reaction.

[0455] The engineered scFv conjugate of the present application can effectively deliver a payload to a target site / cell / organ, etc. This is because the engineered scFv conjugate has the ability to bind to antigens of the target site / cell / organ, etc.

[0456] The engineered scFv conjugate of the present application can selectively deliver a payload to a target site / cell / organ, etc. This is because the engineered scFv conjugate possesses selectivity toward antigens in the target site / cell / organ, etc. Additionally, the engineered scFv conjugate includes a structure capable of releasing a drug in the environment of the target site / cell / organ, etc.

[0457] The engineered scFv of the present application possesses binding ability (binding strength) to antigens and albumin, despite containing amino acid residues (non-natural amino acids) conjugated to the payload.

[0458]

[0459] Specific example of an engineered scFv conjugate

[0460] Hereinafter, specific examples of the scFv junction engineered in this application are described. The following examples are merely illustrative and are not limited thereto.

[0461] The above-mentioned engineered scFv conjugate is not limited to cases where it is formed by the reaction of a payload in which the engineered scFv of the present application and a conjugate linker are connected.

[0462] For example, the above-mentioned engineered scFv conjugate may be made using an engineered scFv based on an albuma containing a scFv derived from a HER2 target antibody and an ABD derived from Streptococcal protein G. In one specific example, it may be made using an engineered scFv based on an albuma consisting of SEQ ID NO. 1.

[0463] For example, the engineered scFv conjugate may be an engineered scFv conjugate in which a payload to which a conjugated linker is attached is conjugated to a non-natural amino acid of a variant protein comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, the non-natural amino acid is X included in SEQ ID NOs 2 to 10.

[0464] For example, the engineered scFv conjugate may be a variant protein comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X included in SEQ ID NOs 2 to 10 may be an amino acid represented by the following chemical formula 5.

[0465] [Chemical Formula 5]

[0466]

[0467] In this case, AA1 and AA2 are amino acid(s) connected to an amino acid in which the alpha carbon is connected to the linkage, and

[0468] J is the connection part, and

[0469] In this case, L is the linker part, and

[0470] In this case, P is the payload.

[0471] For example, the engineered scFv conjugate may be a variant protein containing the amino acid sequence of SEQ ID NO. 10. In this case, X included in 10 may be an amino acid represented by the following chemical formula 5-1.

[0472] [Chemical Formula 5-1]

[0473]

[0474] In this case, AA1 refers to the amino acid(s) connected to the amino acid in which the alpha carbon is connected to the linkage, and

[0475] J is the connection part, and

[0476] In this case, L is the linker part, and

[0477] In this case, P is the payload.

[0478] For example, the above-mentioned engineered scFv assembly is represented by the following structural formula, and

[0479] [engineered scFv (engineered scFv, S)]-[junction (J)]-[linker domain (L)]-[payload (payload, P)]

[0480] At this time, S includes a light chain variable region and a heavy chain variable region capable of binding to HER2, and

[0481] At this time, S includes an albumin binding moiety between the light chain variable region and the heavy chain variable region, and

[0482] At this time, S comprises one amino acid sequence selected from SEQ ID NOs 2 to 10, and

[0483] At this time, X of sequence numbers 2 to 10 is pAzF or frTet, and

[0484] At this time, J is expressed as selected from the following chemical formulas, and

[0485] , and

[0486] ,

[0487] The above L is expressed by the following chemical formula, and

[0488]

[0489] At this time, the above n is an integer greater than or equal to 0, preferably 4, and

[0490] The above payload is MMAE and is represented by the following chemical formula.

[0491]

[0492]

[0493] Chapter 3 Applications of Engineered scFv Assemblies

[0494] This specification discloses various uses of engineered scFv conjugates.

[0495] The engineered scFv conjugate of the present application can specifically deliver a target substance for diagnosis to a target site (cell or tissue). For example, it can deliver radioisotopes, fluorescent molecules, nanoparticles, imaging agents, etc. For example, for the diagnosis of cancer, the conjugate of the present application, in which a radioisotope is attached to the engineered scFv, can be used while utilizing an engineered scFv capable of binding to a specific cancer cell antigen.

[0496] The scFv conjugate engineered in this application can specifically deliver a payload to a target site (cell or tissue). Therefore, it can be used for various applications requiring the function of specifically delivering a payload to a target site. For example, the scFv conjugate engineered in this application can be used for the treatment of various diseases. For example, the treatment may be for cancer, autoimmune diseases, infectious diseases, allergic diseases, or neurological diseases. The cancer may be diverse. For example, the cancer may be breast cancer, lung cancer, colorectal cancer, liver cancer, stomach cancer, cervical cancer, prostate cancer, leukemia, lymphoma, brain tumor, esophageal cancer, kidney cancer, thyroid cancer, oral cancer, or melanoma. Preferably, the cancer may be breast cancer.

[0497] The scFv conjugate engineered in the present application may be used for producing a disease treatment agent. The disease may be diverse. For example, the disease may be cancer, an autoimmune disease, an infectious disease, an allergic disease, or a neurological disease. In one embodiment, the disease may be breast cancer, lung cancer, colorectal cancer, liver cancer, stomach cancer, cervical cancer, prostate cancer, leukemia, lymphoma, brain tumor, esophageal cancer, kidney cancer, thyroid cancer, oral cancer, or melanoma. Preferably, the disease may be breast cancer.

[0498] Chapter 4 Pharmaceutical compositions comprising engineered scFv conjugates

[0499] This specification discloses a pharmaceutical composition comprising an engineered scFv conjugate.

[0500] In this specification, a pharmaceutical composition comprising the engineered scFv conjugate of the present invention may also be referred to as an engineered scFv conjugate composition or an engineered scFv conjugate pharmaceutical composition.

[0501]

[0502] Use of pharmaceutical compositions comprising engineered scFv conjugates

[0503] The above-described engineered scFv conjugate composition can be used for various therapeutic purposes. For example, the therapeutic use may be for treating cancer, autoimmune diseases, infectious diseases, allergic diseases, or neurological diseases. In one embodiment, the therapeutic use may be for breast cancer, lung cancer, colorectal cancer, liver cancer, stomach cancer, cervical cancer, prostate cancer, leukemia, lymphoma, brain tumor, esophageal cancer, kidney cancer, thyroid cancer, oral cancer, or melanoma. Preferably, the therapeutic use may be for breast cancer.

[0504] Additional components of a pharmaceutical composition comprising an engineered scFv conjugate

[0505] The above-mentioned engineered scFv conjugate composition may further include additional pharmaceutically acceptable components in addition to the above-mentioned engineered scFv conjugate.

[0506] The above-mentioned pharmaceutically acceptable additional components may be physiologically acceptable and act on the stabilization, absorption, dissolution, half-life, and introduction efficiency of the engineered scFv conjugate of this specification.

[0507] For example, the above-mentioned pharmaceutically acceptable additional components may be, but are not limited to, carriers, excipients, diluents, preservatives, etc.

[0508] For example, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, silicate, cellulose, distilled water, physiological saline, glycerol, ethanol, HSA (Human serum albumin) phosphate, glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salt, etc.

[0509] For example, the above preservative may be benzoic acid, sodium benzoate, sorbic acid, parahydroxybenzoic acid, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, benzalkonium chloride, benzethonium chloride, phenol, cresol, chlorocresol, or chlorobutanol, etc.

[0510] In addition, the engineered scFv conjugate composition may further include lubricants, wetting agents, emulsifiers, or suspending agents in addition to the above components.

[0511]

[0512] Formulation of a pharmaceutical composition comprising an engineered scFv conjugate

[0513] The above-described engineered scFv conjugate composition can be formulated for oral or parenteral use. In one embodiment, it can be prepared as an injectable or the like.

[0514] In the case of injectable formulations, methods known in the art may be additionally performed as needed to extend the effect of the engineered scFv conjugate; or to slow the absorption of the engineered scFv conjugate. For example, an injectable depot form may be prepared by encapsulating the active ingredient using a biodegradable polymer. As another example, an injectable depot form may be prepared by capturing the active ingredient in a liposome or microemulsion.

[0515]

[0516] Specific example of a pharmaceutical composition comprising an engineered scFv conjugate

[0517] Hereinafter, specific examples of pharmaceutical compositions comprising the engineered scFv conjugate of the present application are described. The following specific examples are merely illustrative of the invention and the invention is not limited thereto.

[0518] The engineered scFv conjugate composition of the present application comprises the engineered scFv conjugate of the present invention. In this case, the engineered scFv conjugate of the present invention is not limited.

[0519] For example, an engineered scFv conjugate composition may include an engineered scFv conjugate made based on an anti-HER2 antibody-derived scFv.

[0520] For example, an engineered scFv conjugate composition may include an engineered scFv conjugate made based on 4D5 scFv.

[0521] The engineered scFv conjugate composition may comprise an engineered scFv conjugate formed by reacting an engineered scFv comprising one amino acid sequence selected from SEQ ID NOs 2 to 10 with a payload connected to a conjugating linker. In this case, X of SEQ ID NOs 2 to 10 is a non-natural amino acid containing a click chemical functional group.

[0522] In one embodiment, the engineered scFv conjugate composition may comprise an engineered scFv conjugate formed by reacting an engineered scFv comprising one amino acid sequence selected from SEQ ID NOs 2 to 10 with an MMAE linked to DBCO-PEG4-Val-Cit-PAB. In this case, X of SEQ ID NOs 2 to 10 is pAzF.

[0523] For example, an engineered scFv conjugate composition may comprise an engineered scFv conjugate comprising an amino acid sequence selected from SEQ ID NOs 2 to 10. In this case, X included in SEQ ID NOs 2 to 9 may be an amino acid represented by the following chemical formula.

[0524]

[0525] In this case, AA1 and AA2 refer to amino acid(s) connected to the amino acid in which the alpha carbon is connected to the linkage, and

[0526] J is the connection part, where L is the linker part, and P is the payload. The connection part, linker part, and payload are described below.

[0527] For example, an engineered scFv conjugate composition may include an engineered scFv conjugate comprising the amino acid sequence of SEQ ID NO. 10. In this case, X included in 10 may be an amino acid represented by the following chemical formula.

[0528]

[0529] Here, AA1 refers to the amino acid(s) connected to the amino acid whose alpha carbon is connected to the linker, J is the linker, L is the linker, and P is the payload. The linker, linker, and payload are described below.

[0530] For example, an engineered scFv conjugate composition includes an engineered scFv conjugate represented by the following structural formula.

[0531] [engineered scFv (engineered scFv, S)]-[junction (J)]-[linker domain (L)]-[payload (payload, P)]

[0532] At this time, S includes a light chain variable region and a heavy chain variable region capable of binding to HER2, and

[0533] At this time, S includes an albumin binding moiety between the light chain variable region and the heavy chain variable region, and

[0534] At this time, S comprises one amino acid sequence selected from SEQ ID NOs 2 to 10, and

[0535] At this time, X of sequence numbers 2 to 10 is pAzF or frTet.

[0536] In this case, in the example described above, the connecting part (J) is represented as being selected from the following chemical formulas.

[0537] , and

[0538] .

[0539] In addition, the connecting part (L) is represented by the following chemical formula.

[0540] .

[0541] In addition, the payload (P) is MMAE and is expressed by the following chemical formula.

[0542]

[0543]

[0544] Chapter 5 Breast Cancer Treatment Methods

[0545] This specification discloses a method for treating breast cancer. In this specification, a method for treating breast cancer may mean a method for improving or treating breast cancer.

[0546] The above breast cancer treatment method may include the step of administering the scFv conjugate engineered in the present application to a subject. Alternatively, the above breast cancer treatment method may include the step of administering a pharmaceutical composition comprising the scFv conjugate engineered in the present application to a subject. In this case, the pharmaceutical composition comprising the engineered scFv conjugate may be as described in Chapter 4.

[0547] The subject refers to a person receiving an engineered scFv conjugate or an engineered scFv conjugate composition. In this case, the subject may be a patient diagnosed with breast cancer or having symptoms of breast cancer.

[0548] The above administration means introducing the engineered scFv conjugate of the present invention to a subject through an appropriate method.

[0549] Method for administering an engineered scFv conjugate in this application

[0550] The method of administering the engineered scFv conjugate of the present application to a subject may vary. For example, it may be introduced via parenteral administration. In this case, parenteral administration may be an injection. Furthermore, the injection may be intradermal, subcutaneous, intravenous, intramuscular, intra-arterial, transdermal, or intraperitoneal injection. In one embodiment, the method of administering the engineered scFv conjugate may be intravenous injection. In this case, the method of administration may vary depending on the patient's condition, dosage, and purpose of administration.

[0551] The application's engineered scFv conjugate dosage

[0552] The administration dose of the scFv conjugate engineered in this application can be appropriately selected according to the method of administration, the subject's body weight, the subject's gender, the subject's condition, the severity of symptoms, the form of the drug, the frequency of administration, the interval between administrations, and the duration of treatment. For example, the scFv conjugate engineered in this application may be administered at a dose of 0.2 mg / kg to 200 ml / kg per day.

[0553] Number of administrations and intervals of the engineered scFv conjugate in this application

[0554] The number and interval of administration of the scFv conjugate engineered in this application may be appropriately selected according to the administration method, the subject's body weight, the subject's gender, the subject's condition, and the treatment period. For example, the number of administrations may be 1 to 30 times per day, but is not limited thereto. For example, the administration interval may be 1 day to 1 month, but is not limited thereto.

[0555]

[0556] Chapter 6 Engineering scFv Production Method

[0557] This specification discloses a method for producing engineered scFv. Known methods for producing proteins with inserted non-natural amino acids may be appropriately applied.

[0558] The method for producing an engineered scFv according to the present application is characterized by obtaining it by expressing it in a cell line capable of producing a protein with a non-natural amino acid inserted therein. That is, the method for producing an engineered scFv according to the present application includes the step of expressing a vector for the expression of an engineered scFv in a cell line capable of producing a protein with a non-natural amino acid inserted therein.

[0559] In this specification, a cell line capable of producing a protein into which the above-mentioned non-natural amino acid is inserted may be referred to as a "producing cell-line."

[0560] Below, the detailed configuration of the above-mentioned production cell line and the above-mentioned engineered scFv production method is described.

[0561] Cell line capable of producing proteins with inserted non-natural amino acids (production cell line)

[0562] The production cell line is not limited to any specific type as long as it is capable of producing proteins with inserted non-natural amino acids.

[0563] For example, a cell line capable of producing a protein with the above-mentioned non-natural amino acid inserted may be selected from the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, Pseudomonas, Leptospira, Salmonella, Brevibacterium, Hyphomonas, Chromobacterium, Nocardia, fungi, and yeasts.

[0564] The above production cell line is characterized by being capable of producing proteins with non-natural amino acids inserted. For example, the above production cell line may be a cell line expressing a foreign-derived suppressor tRNA that recognizes a specific stop codon and a foreign-derived tRNA synthase that links the foreign-derived suppressor tRNA with a non-natural amino acid. In one specific embodiment, the above production cell line may be a cell line into which a vector encoding the foreign-derived suppressor tRNA and the foreign-derived tRNA synthase has been introduced. In one specific embodiment, the above production cell line may be an E. coli into which [pEVOL-pAzF](addgene, #31186) has been introduced.

[0565] The above-mentioned foreign-derived suppressor tRNA may have several characteristics. First, the above-mentioned foreign-derived suppressor tRNA does not react with the endogenous tRNA synthase of the production cell line. Second, the above-mentioned foreign-derived suppressor tRNA can specifically react with the foreign-derived tRNA synthase and be linked to non-natural amino acids. Third, the above-mentioned foreign-derived suppressor tRNA can recognize specific codons in the nucleotide sequence (e.g., ember codon (UAG), ocher codon (UAA), opal codon (UGA)) and insert non-natural amino acids at the corresponding positions.

[0566] For example, the foreign-derived suppressor tRNA may be a tyrosine tRNA derived from methanococcus jannaschii. In one specific example, the foreign-derived tRNA synthetase may be the tRNA represented by SEQ ID NO. 71.

[0567] The above-mentioned foreign-derived tRNA synthase may have several characteristics. First, the above-mentioned foreign-derived tRNA synthase does not react with the endogenous tRNA of the production cell line. Second, the above-mentioned foreign-derived tRNA synthase specifically reacts with the foreign-derived suppressor tRNA and can link non-natural amino acids to the foreign-derived suppressor tRNA.

[0568] For example, the foreign-derived tRNA synthase may be a variant of tyrosine tRNA synthase derived from methanococcus jannaschii. For example, the foreign-derived tRNA synthase may be Y32T, E107N, D158P, I159L, L162Q, or D286R variants of tyrosine tRNA synthase derived from methanococcus jannaschii. In one specific example, the foreign-derived tRNA synthase may be the tRNA synthase represented by SEQ ID NO. 69.

[0569] The method by which the above-mentioned production cell line expresses foreign-derived suppressor tRNA and foreign-derived tRNA synthase is not limited. For example, the above-mentioned production cell line may express foreign-derived suppressor tRNA and foreign-derived suppressor tRNA by introducing foreign-derived suppressor tRNA and a vector encoding foreign-derived suppressor tRNA. In this case, foreign-derived suppressor tRNA and foreign-derived suppressor tRNA may be encoded in separate vectors or the same vector. Additionally, foreign-derived suppressor tRNA and the vector encoding foreign-derived suppressor tRNA may be introduced simultaneously with or separately from the vector encoding engineered scFv.

[0570] In this specification, foreign-derived suppressor tRNA and combinations of foreign-derived suppressor tRNA may be referred to as orthogonal tRNA / synthetase pairs. For the production of proteins with inserted non-natural amino acids, it is necessary for a production cell line to express said orthogonal tRNA / synthetase pair.

[0571] The above-mentioned production cell line may possess additional characteristics in addition to those described above. For example, the above-mentioned production cell line may be a cell line in which the function of a release factor recognizing a specific stop codon is absent or reduced. For example, the above-mentioned production cell line may be a cell line in which an endogenous gene having a specific stop codon has been modified to have a different stop codon.

[0572] The above-mentioned production cell line may be a cell line in which the release factor recognizing a specific stop codon is non-functional or has reduced function. If the release factor recognizing a specific stop codon in the above-mentioned production cell line functions normally, it can compete with foreign-derived suppressor tRNA when expressing a vector encoding an engineered scFv. Therefore, using a production cell line in which the release factor recognizing a specific stop codon (amber codon) is non-functional or has reduced function can increase the production yield of the engineered scFv. In this case, the type of cell line in which the release factor is non-functional or has reduced function is not limited as long as the release factor can be non-functional or have reduced function. For example, the cell line in which the release factor is non-functional or has reduced function may be a cell line in which the release factor-related gene has been engineered.

[0573] For example, the production cell line may be a cell line in which one or more of the selected release factors that recognize an amber codon (UAG), an ocher codon (UAA), and an opal codon (UGA) are non-functional or have reduced function. In one specific example, the production cell line may be a cell line in which the release factor that recognizes an amber codon (UAG) is non-functional or has reduced function.

[0574] The above production cell line may be a cell line in which an endogenous gene having a specific stop codon has been modified to have a different stop codon. That is, the above production cell line may be a cell line in which there is no endogenous gene having a specific codon as a stop codon, or the number of such genes is reduced. If the above production cell line contains an endogenous gene having a specific stop codon, foreign-derived suppressor tRNA may affect the expression of said endogenous gene. Therefore, it may be necessary to use a cell line in which an endogenous gene having a specific stop codon has been modified to have a different stop codon.

[0575] For example, the above-mentioned production cell line may be a cell line in which an endogenous gene having one or more codons selected from Amber Codon (UAG), Ocker Codon (UAA), and Opal Codon (UGA) as stop codons is modified to have a different stop codon. In one specific example, the above-mentioned production cell line may be a cell line in which an endogenous gene having Amber Codon (UAG) as a stop codon is modified to have a different stop codon.

[0576] The above production cell line can express a foreign-derived suppressor tRNA that recognizes a specific stop codon and a foreign-derived tRNA synthase that links the foreign-derived suppressor tRNA with a non-natural amino acid.

[0577] In one embodiment, the production cell line may be a cell line in which a vector encoding an exogenous suppressor tRNA and an exogenous tRNA synthase is introduced into a cell line in which the function of a release factor recognizing a specific stop codon is absent or reduced, and an endogenous gene having a specific stop codon is modified to have a different stop codon. In one embodiment, the cell line may be an E. coli C321.ΔA.exp strain (addgene, #49018) into which [pEVOL-pAzF](addgene, #31186) is introduced.

[0578] Hereinafter, the method for producing scFv engineered in the present application using the above-mentioned production cell line is described.

[0579] The present application's engineered scFv production method

[0580] The method for producing engineered scFv according to the present application is characterized by expressing a nucleic acid encoding the engineered scFv in the production cell line. The method for producing engineered scFv according to the present application is not limited to the method of expressing a nucleic acid encoding the engineered scFv in the production cell line.

[0581] For example, the method for producing engineered scFv in the present application may be a method for producing engineered scFv using a production cell line having an exogenous suppressor tRNA and an exogenous tRNA synthase as endogenous genes.

[0582] In one specific example, the method may include: (a) preparing a cell line expressing a foreign-derived suppressor tRNA and a foreign-derived tRNA synthetase; (b) introducing a nucleic acid encoding an engineered scFv into the cell line; and (c) culturing the cell line into which the nucleic acid encoding the engineered scFv has been introduced in an environment where non-natural amino acids are present. In this case, the nucleic acid encoding the engineered scFv includes a specific codon, the foreign-derived tRNA synthetase is an enzyme capable of linking non-natural amino acids to the foreign-derived suppressor tRNA, the foreign-derived suppressor tRNA is a tRNA that recognizes the specific codon, and the specific codon is an ember codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0583] For example, the method for producing engineered scFv in the present application may be a method for producing engineered scFv using a production cell line created by introducing a foreign-derived suppressor tRNA and a vector encoding a foreign-derived tRNA synthase.

[0584] In one specific example, the method may include: (a) preparing a cell line comprising a nucleic acid encoding an engineered scFv, a foreign-derived suppressor tRNA, and a foreign-derived tRNA synthase; and (b) culturing the cell line in an environment where non-natural amino acids are present.

[0585] In another specific example, the method may include: (a) preparing a cell line containing a nucleic acid encoding an engineered scFv; (b) introducing a nucleic acid encoding an exogenous suppressor tRNA and an exogenous tRNA synthase into the cell line; and (c) culturing the cell line into which the nucleic acid encoding the exogenous suppressor tRNA and the exogenous tRNA synthase has been introduced in an environment where non-natural amino acids are present.

[0586] At this time, the nucleic acid encoding the engineered scFv contains a specific codon, the foreign-derived tRNA synthetase is an enzyme capable of linking a non-natural amino acid to the foreign-derived suppressor tRNA, the foreign-derived suppressor tRNA is a tRNA that recognizes the specific codon, and the specific codon is an amber codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0587] For example, the method for producing engineered scFv in the present application may be a method for producing engineered scFv using a production cell line in which a release factor recognizing a specific codon is non-functional or has reduced function.

[0588] In one specific embodiment, the method may include: (a) preparing a cell line comprising a nucleic acid encoding an engineered scFv, a foreign-derived suppressor tRNA, and a foreign-derived tRNA synthesizer, wherein a release factor recognizing a specific codon is non-functional or has reduced function; and (b) culturing the cell line in an environment where non-natural amino acids are present. In this case, the nucleic acid encoding the engineered scFv includes the specific codon, the foreign-derived tRNA synthesizer is an enzyme capable of linking non-natural amino acids to the foreign-derived suppressor tRNA, the foreign-derived suppressor tRNA is a tRNA that recognizes the specific codon, and the specific codon is an ember codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0589] For example, the method for producing engineered scFv in the present application may be a method for producing engineered scFv using a production cell line having no or reduced endogenous gene having a specific codon as a stop codon.

[0590] In one embodiment, the method may include: (a) preparing a cell line having no or reduced endogenous gene having a specific codon as a stop codon, comprising a nucleic acid encoding an engineered scFv, an exogenous suppressor tRNA, and an exogenous tRNA synthetase; and (b) culturing the cell line in an environment where non-natural amino acids are present. In this case, the nucleic acid encoding the engineered scFv includes the specific codon, the exogenous tRNA synthetase is an enzyme capable of linking non-natural amino acids to the exogenous suppressor tRNA, the exogenous suppressor tRNA is a tRNA that recognizes the specific codon, and the specific codon is an amber codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0591] For example, the method for producing engineered scFv in the present application may further include a step of determining the non-natural amino acid position of the engineered scFv.

[0592] In one specific embodiment, the method may include: (a) determining the non-natural amino acid location of the engineered scFv; (b) preparing a cell line comprising a nucleic acid encoding the engineered scFv, a foreign-derived suppressor tRNA, and a foreign-derived tRNA synthetase; and (c) culturing the cell line in an environment where the non-natural amino acid is present. In this case, the step of determining the non-natural amino acid location of the engineered scFv is characterized by selecting the non-natural amino acid location by referring to Chapter 1 of this specification, "Location of Non-Natural Amino Acid" and "Influence of Location of Non-Natural Amino Acid," wherein the nucleic acid encoding the engineered scFv includes a specific codon, the foreign-derived tRNA synthetase is an enzyme capable of linking the non-natural amino acid to the foreign-derived suppressor tRNA, the foreign-derived suppressor tRNA is a tRNA that recognizes the specific codon, and the specific codon is an ember codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0593] The method for producing engineered scFv in the present application may further include the step of extracting / purifying the expressed engineered scFv from a cell line.

[0594] In one embodiment, the extraction / purification step may include a step of extracting the cell fluid of a production cell line by one or more methods selected from enzyme treatment, freeze / thaw, ultrasonic treatment, and osmotic shock, and a step of purifying the engineered scFv from the cell fluid by one or more methods selected from centrifugation, precipitation separation, chromatography, ultrafiltration, and dialysis. In one embodiment, the engineered scFv may be purified through chromatography using a his tag.

[0595] Yield of the engineered scFv production method of the present application

[0596] The method for producing engineered scFv in the present application is characterized by expressing a nucleic acid encoding an engineered scFv in a production cell line.

[0597] At this time, the yield of the method for producing the engineered scFv varies depending on the sequence of the nucleic acid encoding the engineered scFv. That is, the yield of the method for producing the engineered scFv varies depending on the sequence of the engineered scFv to be expressed.

[0598] For example, the yield of the method for producing engineered scFv may vary depending on where the nucleic acid encoding the engineered scFv contains a specific codon. In this case, the specific codon is a codon recognized by a foreign-derived suppressor tRNA. In one specific example, the nucleic acid encoding the engineered scFv containing a specific codon at a specific location can express the engineered scFv with a higher yield by a value selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10 times, or between two selected values, compared to the nucleic acid encoding the engineered scFv containing a specific codon at a different location.

[0599] In one specific embodiment, a nucleic acid encoding an engineered scFv comprising a nucleic acid sequence selected from SEQ ID NOs. 42 and 44 to 49 can express the engineered scFv with a yield 2 to 4 times higher than a nucleic acid encoding an engineered scFv comprising the nucleic acid sequence of SEQ ID NO. 41.

[0600] In one specific embodiment, a nucleic acid encoding an engineered scFv comprising a nucleic acid sequence selected from SEQ ID NOs 42 and 44 to 49 can express the engineered scFv with a yield 2 to 4 times higher than a nucleic acid encoding an engineered scFv comprising the nucleic acid sequence of SEQ ID NO. 43.

[0601] Specific example of the engineered scFv production method of the present application

[0602] Hereinafter, specific examples of the engineered scFv production method of the present application will be described. The following specific examples are merely illustrative of the invention and the invention is not limited thereto.

[0603] For example, the above method for producing engineered scFv may include: (a) preparing a cell line comprising a nucleic acid encoding the engineered scFv, an exogenous suppressor tRNA, and an exogenous tRNA synthetase; and (b) culturing the cell line in an environment where non-natural amino acids are present. In this case, the nucleic acid encoding the engineered scFv may include an amber codon (UAG), and the nucleic acid encoding the engineered scFv may include a nucleic acid sequence selected from SEQ ID NOs 74 to 82 (or SEQ ID NOs 86 to 94 if a histag is included), the exogenous tRNA synthetase may be an enzyme capable of linking a non-natural amino acid to the exogenous suppressor tRNA, the exogenous suppressor tRNA may be a tRNA that recognizes the amber codon (UAG), and the non-natural amino acid may be pAzF or frTet.

[0604] For example, the method may include: (a) preparing a cell line comprising a nucleic acid encoding an engineered scFv, an alien-derived suppressor tRNA, and an alien-derived tRNA synthetase, wherein a release factor recognizing an amber codon (UAG) is non-functional or has reduced function, and an endogenous gene having an amber codon (UAG) as a stop codon is non-functional or reduced; and (b) culturing the cell line in an environment where non-natural amino acids are present. In this case, the nucleic acid encoding the engineered scFv may include an amber codon (UAG), and the nucleic acid encoding the engineered scFv may include a nucleic acid sequence selected from SEQ ID NOs 74 to 82, the alien-derived tRNA synthetase is an enzyme capable of linking a non-natural amino acid to the alien-derived suppressor tRNA, the alien-derived suppressor tRNA is a tRNA that recognizes the amber codon (UAG), and the non-natural amino acid may be pAzF or frTet.

[0605] For example, the method may include: (a) preparing a cell line in which a release factor recognizing an amber codon (UAG) is non-functional or has reduced function, and an endogenous gene having an amber codon (UAG) as a stop codon is non-functional or reduced; (b) introducing a nucleic acid encoding an engineered scFv and a nucleic acid encoding a foreign-derived suppressor tRNA and a foreign-derived tRNA synthetase into the cell line; and (c) culturing the cell line into which the nucleic acid has been introduced in an environment where non-natural amino acids are present. At this time, the nucleic acid encoding the engineered scFv includes an amber codon (UAG), the nucleic acid encoding the engineered scFv includes a nucleic acid sequence selected from SEQ ID NOs 74 to 82, the foreign-derived tRNA synthetase is an enzyme capable of linking a non-natural amino acid to the foreign-derived suppressor tRNA, the foreign-derived suppressor tRNA is a tRNA that recognizes the amber codon (UAG), and the non-natural amino acid may be pAzF or frTet.

[0606]

[0607] Chapter 7 Method for Producing Engineered scFv Assemblies

[0608] This specification discloses a method for producing an engineered scFv assembly.

[0609] The method for producing an engineered scFv conjugate of the present application includes a reaction in which a payload is connected to the engineered scFv of the present application using a conjugating linker. At this time, the payload may be connected to the conjugating linker first, and then reacted with the engineered scFv in a form (structure) in which the payload is connected to the conjugating linker. The form (structure) is referred to as a "payload connected to a conjugating linker."

[0610] Accordingly, in one specific embodiment, the method for producing the engineered scFv assembly may include the following:

[0611] a) react the payload with the conjugating linker; and

[0612] b) A payload connected to a conjugating linker is reacted with an engineered scFv, wherein the first click functional group of a non-natural amino acid contained in the engineered scFv and the second click functional group contained at one end of the conjugating linker are combined through a click reaction.

[0613] Hereinafter, the payload connected to the conjugating linker is described, and the method for producing the engineered scFv of the present application is described in detail.

[0614]

[0615] Payloads linked to a conjugating linker (Drugs linked to a conjugating linker)

[0616] The method for producing an engineered scFv conjugate of the present application may include preparing a payload connected to a conjugating linker.

[0617] In this case, one end of the conjugating linker has a second click chemical functional group, and the other end has a functional group capable of binding to the payload. The payload connected to the conjugating linker can be prepared by the functional group located at the other end, rather than the second click chemical functional group, reacting with the payload.

[0618] The click chemical functional group (second click chemical functional group) included in the payload connected to the conjugating linker may vary. For example, it may be a click chemical functional group containing an alkyne group. For another example, it may be a click chemical functional group containing cyclooctyne. For another example, it may be a click chemical functional group such as DIFO (Difluorinated cyclooctyne), DBCO (Dibenzocyclooctyne), or BNC (Bicyclononyne). For another example, it may be a click chemical functional group such as TCO (trans-cyclooctene). The click chemical functional group (second click chemical functional group) of the conjugating linker may react with the click chemical functional group (first click chemical functional group) of the scFv engineered in this application. Additionally, the click chemical functional group of the conjugating linker may combine with the side chain of the non-natural amino acid of the scFv engineered in this application to form a link.

[0619] For example, a payload connected to the above conjugating linker can be expressed by the following structural formula.

[0620] [2th click chemistry functional group (2th CC)-[spacer]-[payload linkage linker (PL)]-[payload (P)]

[0621] For example, the [second click chemical functional group] may include an alkyne group or a cyclooctyne. For another example, the [second click chemical functional group] may include DBCO, TCO, or BCN.

[0622] Accordingly, the payload connected to the conjugating linker of the present application can be represented by one of the following chemical formulas.

[0623] [Chemical Formula 11-1]

[0624] ;

[0625] [Chemical Formula 11-2]

[0626] ; or

[0627] [Chemical Formula 11-3]

[0628] .

[0629] The [spacer] above refers collectively to the region located between the part (payload linker) that is directly connected to the payload and has a specific function in the engineered scFv conjugate and the second click chemical functional group. Depending on the needs of those skilled in the art, the spacer may include a configuration to improve solubility or a configuration to improve conjugation reactivity. In one embodiment, the spacer may include PEG (Polyethylene glycol).

[0630] In one specific example, the spacer is absent or -B1-B2-, and

[0631] At this time, B1 is non-existent or -CH2-O-, -CH2-CH2-(C=O)-, -CH2-CH2-(C=0)-NH-, -(C=0)-NH-CH2-, -(C=0)-CH2-CH2-(C=0)-, -(C=0)-CH2-CH2-NH-(C=0)-, -(C=0)-NH-CH2-CH2-CH2-NH-(C=0)-, -(C=0)-CH2-CH2-(C=0)-NH-, -(C=0)-CH2-CH2-CH2-(C=0)-NH-, -O-(C=O)-NH-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-NH-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-O-, (-CH2)n- or combinations thereof may be included, but are not limited thereto. In this case, n means a natural number greater than or equal to 1.

[0632] In this case, B2 is non-existent or -CH2-CH2-(C=O)-, -CH2-CH2-(C=O)-NH-, -CH2-CH2-NH-(C=O)-, -CH2-CH2-(C=O)-O-, -CH2-CH2-SS-, [-CH2-CH2-O]n(PEGn)-, [-CH2-CH2-O]n(PEGn)-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-(C=O)-O-, [-CH2-CH2-O]n(PEGn)-(C=O)-CH2-CH2-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-O- or combinations thereof may be included, but are not limited thereto. In this case, n means a natural number greater than or equal to 1.

[0633] The payload coupling linker mentioned above refers to a portion that is directly connected to the payload and has a specific function. For example, the payload coupling linker may be absent. For another example, the payload coupling linker may be a cleavable linker. A cleavable linker region refers to a linker that is cleaved by the target's surrounding environment when the engineered scFv is attached to the target. Accordingly, the payload attached to the cleavable linker can be effectively activated at the target. Preferably, the cleavable linker may be used when the payload is a drug, but is not limited thereto.

[0634] The above-mentioned cleavable linker is not limited by its type as long as it is cleaved by the surrounding environment of the target. For example, the above-mentioned cleavable linker may be a linker that is cleaved by enzymatic action. In one embodiment, the enzymatic action may be by a proteolytic enzyme contained in the lysosomes of a tumor cell. In one embodiment, the above-mentioned cleavable linker may be a linker comprising Val-Cit, Val-Ala, cBu-Cit, Glu-Val-Cit, or Gly-Gly-Phe-Gly. In this case, the above-mentioned cleavable linker may additionally include a PAB site. Or, the above-mentioned cleavable linker may be Val-Cit-PAB, Val-Ala-PAB, cBu-Cit-PAB, Glu-Val-Cit-PAB, or Gly-Gly-Phe-Gly-PAB. In this case, valine-citrulline (Val-Cit)- etc. may be cleaved by Cathepsin B, which is predominantly expressed in cancer cells.

[0635] For example, the cleavable linker may be a linker that is cleaved by a change in pH. In one embodiment, it may be a linker that is cleaved by a highly acidic environment by endosomes and lysosomes surrounding tumor cells. In one embodiment, the cleavable linker may be a hydrazone, semicarbazone, thiosemicabazone, cis-aconitic amide, orthoester, acetal, ketal, etc. that is cleaved by a highly acidic environment.

[0636] For example, the cleavable linker may be a linker that is cleaved by a reducing environment around the tumor tissue. In one embodiment, the cleavable linker may be a linker containing disulfide bonds such as SATA (N-succinimidyl-Sacetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0637] For example, the cleavable linker may be a linker that is cleaved according to changes in glutathione concentration. In one embodiment, it may be a linker that is cleaved in dependence on high glutathione concentrations around tumor cells. In one embodiment, the cleavable linker may be a beta-glucuronide linker.

[0638] In one embodiment, the payload linker may be a cleavable linker represented by the following chemical formula.

[0639] [Chemical Formula 8-1](Val-Cit-PAB)

[0640] ;

[0641] [Chemical Formula 8-2](Val-Ala-PAB)

[0642] ;

[0643] [Chemical Formula 8-3](cBU-Cit-PAB)

[0644] ;

[0645] [Chemical Formula 8-4](Glu-Val-Cit-PAB)

[0646] ; or

[0647] [Formula 8-5](Gly-Gly-Phe-Gly-PAB)

[0648] .

[0649] In this case, B2 refers to the -B2- region of the spacer, and P refers to the payload.

[0650] The above [spacer] and [payload linker] may be collectively referred to as the [linker domain (L)].

[0651] In one embodiment, the [linker domain (L)] can be represented by the following chemical formula.

[0652]

[0653] In this case, the 2nd CC means [2nd click chemical functional group], and P means [payload]. Also, n is a natural number greater than or equal to 1.

[0654] Regarding the above payload, the contents of the payload of the configuration (4) in Chapter 2 are included by reference. In this case, when the above payload is expressed by a chemical formula, it may be abbreviated and referred to as P.

[0655] In one specific example, the payload connected to the conjugating linker of the present application can be represented by the following chemical formula.

[0656] [Chemical Formula 12]

[0657]

[0658] Method for producing an engineered scFv assembly in this application

[0659] The method for producing an engineered scFv conjugate of the present application is not limited to any specific type, provided that it is produced by reacting a payload in which an engineered scFv and a conjugating linker are connected.

[0660] For example, the above-mentioned method for producing an engineered scFv assembly may include the following:

[0661] a) react the payload with the conjugating linker; and

[0662] b) The payload connected to the conjugating linker is reacted with the engineered scFv, wherein the first click chemical functional group of the non-natural amino acid contained in the engineered scFv and the second click chemical functional group of the payload connected to the conjugating linker are combined through a click reaction.

[0663] At this time, the method of reacting the payload and the conjugating linker can be properly performed through known techniques.

[0664] As another example, the above-mentioned method for producing an engineered scFv assembly may include the following.

[0665] a) Prepare a payload connected to a conjugating linker; and

[0666] b) The payload connected to the conjugating linker is reacted with the engineered scFv, wherein the first click chemical functional group of the non-natural amino acid contained in the engineered scFv and the second click chemical functional group of the payload connected to the conjugating linker are combined through a click reaction.

[0667] As another example, the above-mentioned method for producing an engineered scFv assembly may include the following.

[0668] a) Prepare the payload and engineered scFv connected to the conjugating linker; and

[0669] b) The payload connected to the conjugating linker is reacted with the engineered scFv, wherein the first click chemical functional group of the non-natural amino acid contained in the engineered scFv and the second click chemical functional group of the payload connected to the conjugating linker are combined through a click reaction.

[0670] The above-mentioned engineered scFv can be prepared by the "method for producing engineered scFv" of this specification.

[0671] Yield of the method for producing an engineered scFv assembly of the present application

[0672] The yield of the scFv conjugate production method of the present application may be affected by various factors.

[0673] For example, the location of non-natural amino acids in the engineered scFv can affect the yield of the scFv conjugate production method.

[0674] The engineered scFv of the present application may exhibit differences, such as accessibility to the payload connected to the conjugating linker, depending on the position of the non-natural amino acid. Accordingly, an engineered scFv containing a non-natural amino acid at a specific position may react better with the payload connected to the conjugating linker as an engineered scFv containing a non-natural amino acid at a different position. In other words, a method for producing an engineered scFv conjugate using a specific engineered scFv may exhibit a higher yield than a method for producing an engineered scFv conjugate using a different engineered scFv.

[0675] For example, a method for producing engineered scFv junctions using a specific engineered scFv can yield about 2, 3, 4, 5, 6, 7, 8, 9, and 10 times higher than a method for producing engineered scFv junctions using a different engineered scFv. In this case, the yield can be expressed in various ways. For example, the yield can be expressed as the percentage of engineered scFv junctions produced relative to the engineered scFv reacted after reacting the engineered scFv with a payload connected to a conjugating linker.

[0676] For example, a method for producing an engineered scFv conjugate using an engineered scFv containing the amino acid of SEQ ID NO. 8 may show a higher yield than a method for producing an engineered scFv conjugate using an engineered scFv containing one amino acid sequence selected from SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 10.

[0677] For example, a method for producing an engineered scFv conjugate using an engineered scFv containing the amino acid of SEQ ID NO. 7 may show a higher yield than a method for producing an engineered scFv conjugate using an engineered scFv containing one amino acid sequence selected from SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 9 and SEQ ID NO. 10.

[0678]

[0679] Specific example of the method for producing an engineered scFv assembly of the present application

[0680] Hereinafter, specific examples of the method for producing an engineered scFv assembly according to the present application will be described. The following specific examples are merely illustrative of the invention and the invention is not limited thereto.

[0681] For example, the above-mentioned method for producing an engineered scFv assembly may include the following:

[0682] a) Prepare a payload connected to an engineered scFv and a conjugating linker; and

[0683] b) Reacting a payload connected to a conjugating linker with an engineered scFv, wherein the first click chemical functional group of pAzF contained in the engineered scFv and the second click chemical functional group contained at one end of the conjugating linker are combined through a click reaction,

[0684] At this time, the engineered scFv comprises one amino acid sequence selected from SEQ ID NOs 2 to 10, wherein X of SEQ ID NOs 2 to 10 is pAzF; and

[0685] At this time, the payload connected to the above conjugating linker is expressed by the following structural formula.

[0686]

[0687]

[0688] Possible embodiments of the invention

[0689] Hereinafter, embodiments of the present invention will be described. The following embodiments are intended to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these embodiments.

[0690] Engineered scFv

[0691] [Example 1]

[0692] An engineered single-chain variable fragment (scFv) comprising an albumin-binding moiety and one or more non-natural amino acids.

[0693] [Example 2]

[0694] In Example 1, the albumin binding moiety is characterized by being included between the heavy chain variable region and the light chain variable region of the single chain variable fragment, and

[0695] At this time, the above-mentioned non-natural amino acid is an engineered single-strand variable fragment (scFv) that is not included in the complementarity determining portion (CDR) of the single-strand variable fragment.

[0696] [Example 3]

[0697] In Example 1, the non-natural amino acid is an engineered single-chain variable fragment (scFv) located at a position capable of maintaining binding ability to albumin.

[0698] [Example 4]

[0699] In Example 1, the non-natural amino acid is an engineered single-chain variable fragment (scFv) located at a position capable of maintaining binding ability to the antigen.

[0700] [Example 5]

[0701] In Example 1, the non-natural amino acid is an engineered single-chain variable fragment (scFv) that is not located at the position forming the disulfide bond of the scFv.

[0702] [Example 6]

[0703] In Example 1, the engineered single-chain variable fragment further comprises a configuration for a His-tag, and

[0704] The above-mentioned non-natural amino acid is an engineered single-chain variable fragment (scFv) that is not located at the His-tag.

[0705] [Example 7]

[0706] As an engineered single-strand variable fragment (scFv) comprising one amino acid sequence selected from SEQ ID NOs 2 to 10,

[0707] X of the above sequence numbers 2 to 10 is characterized as being a non-natural amino acid having a click chemical functional group.

[0708] [Example 8]

[0709] An engineered single-strand variable fragment (scFv) comprising a sequence in which one or more of the positions G44, S87, R89, G123, S203, P234, G235, K236, and K301 in the amino acid sequence of SEQ ID NO. 1 are changed to non-natural amino acids.

[0710] [Example 9]

[0711] In one of Examples 1 to 8, the non-natural amino acid is an engineered single-chain variable fragment (scFv) which is 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet).

[0712]

[0713] Engineered scFv junction

[0714] [Example 10]

[0715] As an engineered single-chain variable fragment (scFv) conjugate comprising an albumin-binding moiety and one or more non-natural amino acids,

[0716] At this time, the above-mentioned non-natural amino acid is characterized by being conjugated to the payload through a click reaction.

[0717] [Example 11]

[0718] In Example 10, the conjugate is characterized in that the non-natural amino acid is located at a position capable of maintaining the binding ability to albumin of the albumin binding moiety.

[0719] [Example 12]

[0720] In Example 10, the conjugate is characterized in that the non-natural amino acid is located at a position capable of maintaining binding ability to the antigen of the engineered scFv.

[0721] [Example 13]

[0722] A conjugate characterized in that, in Example 10, the non-natural amino acid is not located at the position forming the disulfide bond of the engineered scFv and at the His-tag position of the engineered scFv.

[0723] [Example 14]

[0724] A conjugate characterized in that, in Example 10, the non-natural amino acid is 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet).

[0725] [Example 15]

[0726] An engineered single-chain variable fragment (scFv) conjugate comprising at least one non-natural amino acid represented by the following [Formula 5] and an albumin-binding moiety:

[0727] [Chemical Formula 5]

[0728]

[0729] At this time, AA1 and AA2 refer to amino acid(s) connected to the above-mentioned non-natural amino acid, or are absent, and

[0730] At this time, J is a connection formed by a click response, and

[0731] In this case, L is a linker that connects the connection part and the payload, and

[0732] At this time, P is characterized as being a payload.

[0733] [Example 16]

[0734] Engineered scFv junction represented by the following [Structural Formula 1]:

[0735] [Structural Formula 1]

[0736] [Engineered scFv (S)]-[Connection (J)]-[Linker (L)]-[Payload (P)],

[0737] At this time, S includes a light chain variable region and a heavy chain variable region, and

[0738] At this time, S includes an albumin binding moiety between the light chain variable region and the heavy chain variable region, and

[0739] At this time, S contains non-natural amino acids, and

[0740] At this time, J is a connection formed by a click response, and

[0741] In this case, L is a linker that connects the connection part and the payload, and

[0742] At this time, P is characterized as being a payload.

[0743] [Example 17]

[0744] In Example 15 or 16, the non-natural amino acid is an engineered scFv conjugate located at a position capable of maintaining the binding ability to albumin of the albumin binding moiety.

[0745] [Example 18]

[0746] In Example 15 or 16, the non-natural amino acid is an engineered scFv conjugate located at a position capable of maintaining the binding ability to the antigen of the engineered scFv.

[0747] [Example 19]

[0748] In Example 15 or 16, the connecting portion is characterized by being selected from the following chemical formula, and

[0749] [Chemical Formula 6-1]

[0750] ,

[0751] [Chemical Formula 7-1]

[0752] ,

[0753] [Chemical Formula 7-2]

[0754] , or

[0755] [Chemical Formula 7-3]

[0756] ,

[0757] In this case, A1 does not exist or -A 11 -A 12 - and,

[0758] At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and

[0759] At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is any one selected from heteroalkylene and heteroarylene, wherein p is an integer between 1 and 3 inclusive, and

[0760] At this time, R1 is H, halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, C 3-6 It is any one selected from heterocycloalkyl, aryl, and heteroaryl, and

[0761] At this time, the heterocycloalkyl or heteroaryl comprises one or more heteroatom groups selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O)2-, or one or more heteroatoms selected from the group consisting of O, N, and S, and

[0762] At this time, S is [engineered scFv (S)], and

[0763] In this case, L is an engineered scFv junction that is the linker part.

[0764] [Example 20]

[0765] An engineered scFv conjugate according to Example 15 or 16, wherein the linker portion comprises a cuttable linker region.

[0766] [Example 21]

[0767] In Example 15 or 16, the linker portion is represented by [Structural Formula 2] as follows, and

[0768] [Structural Formula 2]

[0769] -[Spacer]-[payload linkage linker (PL)]-,

[0770] At this time, the above spacer is non-existent or -B1-B2-, and

[0771] At this time, B1 is non-existent or -CH2-O-, -CH2-CH2-(C=O)-, -CH2-CH2-(C=0)-NH-, -(C=0)-NH-CH2-, -(C=0)-CH2-CH2-(C=0)-, -(C=0)-CH2-CH2-NH-(C=0)-, -(C=0)-NH-CH2-CH2-CH2-NH-(C=0)-, -(C=0)-CH2-CH2-(C=0)-NH-, -(C=0)-CH2-CH2-CH2-(C=0)-NH-, -O-(C=O)-NH-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-NH-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-O-, (-CH2)p- or a combination thereof, including

[0772] In this case, B2 is non-existent or -CH2-CH2-(C=O)-, -CH2-CH2-(C=O)-NH-, -CH2-CH2-NH-(C=O)-, -CH2-CH2-(C=O)-O-, -CH2-CH2-SS-, [-CH2-CH2-O]n(PEGn)-, [-CH2-CH2-O]n(PEGn)-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-(C=O)-O-, [-CH2-CH2-O]n(PEGn)-(C=O)-CH2-CH2-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-O- or a combination thereof, comprising

[0773] At this time, the above p and n independently represent natural numbers greater than or equal to 1; and

[0774] The above payload linking linker is an engineered scFv conjugate that is a Val-Cit-PAB, Val-Ala-PAB, cBu-Cit-PAB, Glu-Val-Cit-PAB, Gly-Gly-Phe-Gly-PAB, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, SATA(N-succinimidyl-Sacetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene) or beta-glucuronide linker.

[0775] [Example 22]

[0776] In Example 15 or 16, the payload is an engineered scFv conjugate that is MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1 (Maytansinoid derivative), DM4 (Maytansinoid derivative), Calicheamicin, PBD (Pyrrolobenzodiazepine), or SN-38 (Camptothecin derivatives).

[0777] [Example 23]

[0778] In any one of Examples 10 to 22, the engineered single-strand variable fragment (scFv) conjugate comprises one amino acid sequence selected from SEQ ID NOs 2 to 10, and

[0779] At this time, X of sequence numbers 2 to 10 is characterized as being the above-mentioned non-natural amino acid.

[0780]

[0781] Applications of Engineered ScFV Conjugates

[0782] [Example 24]

[0783] Cancer treatment use of any one of the engineered scFv conjugates of Examples 10 to 23.

[0784] [Example 25]

[0785] Use of any one of the engineered scFv conjugates of Examples 10 to 23 for the production of cancer therapeutic agents.

[0786]

[0787] Pharmaceutical composition comprising an engineered scFv conjugate

[0788] [Example 26]

[0789] A pharmaceutical composition for cancer treatment comprising any one of the engineered scFv conjugates of Examples 10 to 23.

[0790]

[0791] Cancer treatment methods

[0792] [Example 27]

[0793] A method for treating cancer comprising administering any one of the engineered scFv conjugates of Examples 10 to 23 to a subject.

[0794]

[0795] Engineered scFv production method

[0796] [Example 28]

[0797] Engineered scFv production method including the following steps:

[0798] (a) A step of preparing a cell line comprising a nucleic acid encoding an engineered scFv, a foreign-derived suppressor tRNA, and a foreign-derived tRNA synthase;

[0799] At this time, the nucleic acid encoding the engineered scFv includes a specific codon, and

[0800] The above-mentioned foreign-derived tRNA synthase is an enzyme capable of linking a non-natural amino acid to the above-mentioned foreign-derived suppressor tRNA, and

[0801] The above-mentioned foreign-derived suppressor tRNA is a tRNA that recognizes the above-mentioned specific codon; and

[0802] (b) a step of culturing the cell line in an environment where the non-natural amino acid is present,

[0803] Accordingly, the engineered scFv is expressed from the nucleic acid encoding the engineered scFv, and the non-natural amino acid included in the engineered scFv corresponds to the position of the embercodon included in the nucleic acid encoding the engineered scFv.

[0804] [Example 29]

[0805] In Example 28, the method for producing engineered scFv comprises one nucleic acid sequence selected from SEQ ID NOs 74 to 82, wherein the nucleic acid encoding the engineered scFv comprises an engineered scFv.

[0806] [Example 30]

[0807] A method for producing engineered scFv in Example 28, wherein the non-natural amino acid is 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet).

[0808] [Example 31]

[0809] A method for producing an engineered scFv in Example 28, wherein the specific codon is an emer codon (UAG), an ocher codon (UAA), or an opal codon (UGA).

[0810]

[0811] Method for producing engineered scFv junctions

[0812] [Example 32]

[0813] Method for producing an engineered scFv-conjugate comprising the following steps:

[0814] (a) Prepare a payload connected to an engineered scFv and a conjugating linker; and

[0815] (b) Inducing a reaction of the conjugated linker connected to the payload and the engineered scFv,

[0816] At this time, the first click chemical functional group of the non-natural amino acid included in the engineered scFv and the second click chemical functional group of the conjugating linker are combined through a click reaction.

[0817] [Example 33]

[0818] In Example 32, the engineered scFv is one of the engineered scFvs described in Examples 1 to 9, and the method for producing an engineered scFv composite.

[0819] [Example 34]

[0820] A method for producing an engineered scFv composite in Example 32, wherein the payload to which the conjugating linker is connected includes a cuttable linker region.

[0821] [Example 35]

[0822] In Example 32, the conjugating linker is an engineered scFv production method represented by the following structural formula 3:

[0823] [Structural Formula 3]

[0824] [2th click chemistry functional group (2th CC)-[spacer]-[payload linkage linker (PL)]-[payload].

[0825] [Example 36]

[0826] In Example 35, the second click chemical functional group is an alkyne group

[0827] Method for producing an engineered scFv conjugate that is cyclooctyne, DIFO (Difluorinated cyclooctyne), BCO (Dibenzocyclooctyne), or TCO (trans-cyclooctene).

[0828] [Example 37]

[0829] In Example 35, the spacer is absent or -B1-B2-, and

[0830] At this time, B1 is non-existent or -CH2-O-, -CH2-CH2-(C=O)-, -CH2-CH2-(C=0)-NH-, -(C=0)-NH-CH2-, -(C=0)-CH2-CH2-(C=0)-, -(C=0)-CH2-CH2-NH-(C=0)-, -(C=0)-NH-CH2-CH2-CH2-NH-(C=0)-, -(C=0)-CH2-CH2-(C=0)-NH-, -(C=0)-CH2-CH2-CH2-(C=0)-NH-, -O-(C=O)-NH-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-NH-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-O-, (-CH2)p- or a combination thereof, including

[0831] In this case, B2 is non-existent or -CH2-CH2-(C=O)-, -CH2-CH2-(C=O)-NH-, -CH2-CH2-NH-(C=O)-, -CH2-CH2-(C=O)-O-, -CH2-CH2-SS-, [-CH2-CH2-O]n(PEGn)-, [-CH2-CH2-O]n(PEGn)-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-(C=O)-O-, A method for producing an engineered scFv conjugate comprising [-CH2-CH2-O]n(PEGn)-(C=O)-CH2-CH2-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-O- or a combination thereof, wherein p and n independently mean natural numbers greater than or equal to 1.

[0832] [Example 38]

[0833] In Example 35, the payload linking linker is an engineered scFv conjugate that is a Val-Cit-PAB, Val-Ala-PAB, cBu-Cit-PAB, Glu-Val-Cit-PAB, Gly-Gly-Phe-Gly-PAB, hydrazone, semicarbazone, thiosemicazone, cis-aconitic amide, orthoester, acetal, ketal, SATA (N-succinimidyl-Sacetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene) or beta-glucuronide linker. Production method.

[0834]

[0835] ingredient

[0836] Escherichia coli (E. coli) TOP10 competent cells (#C404010) were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). E. coli C321.ΔA.exp (#49018) and the [pEVOL-pAzF] plasmid (#31186) were obtained from Addgene (Watertown, MA, USA). Bactotryptone (#211705) and yeast extract (#212750) were provided by BD Biosciences (San Jose, CA, USA). 4-Azido-L-phenylalanine (pAzF; #06162) was purchased from Chem-Impex International (Wood Dale, IL, USA). Nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (#30210) was obtained from Qiagen (Hilden, Germany). Disposable PD-10 desalination column (#GE17-0851-01) was purchased from Cytiva (Uppsala, Sweden). DBCO-MMAE (#BP-25659) and MMAE (#BP-22278) were purchased from BroadPharm (San Diego, CA, USA). Steel target plate (#8280781) and Protein Standard II (#8207234) were provided by Bruker (Billerica, MA, USA). ZipTip (#ZTC04S008) fitted with 0.6 μL C4 resin was purchased from Millipore (Merck, Darmstadt, Germany). Chloramphenicol and trifluoroacetic acid (TFA; 99%) were provided by Daejung Chemicals & Metals (Gyeonggi, South Korea). 96-well area Immunoplates (#32396) and 96-well area Cell Culture Plates (#30096) were purchased from SPL Life Sciences (Pocheon, South Korea).Recombinant human HER2 (#10004-HCCH) was purchased from Sino Biological (Beijing, China). Tween-20 (#1610781) was provided by Bio-Rad (Hercules, CA, USA), and skim milk (#SKI400) was provided by BioShop (Burlington, Ontario, Canada). His-tag antibody (#2365) and HRP-linked anti-rabbit IgG antibody (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). Hydrochloric acid (HCl; #001_00122) was provided by Duksan Central Science (Seoul, South Korea). L-(+)-arabinose (#A11921) and disposable polypropylene columns (#29924) were purchased from Thermo Fisher Scientific. RPMI 1640 medium (HEPES, #22400105), fetal bovine serum (FBS; qualified, #26140079), and penicillin-streptomycin (10,000 U mL). 1 ; #15140122) was provided by Gibco (Thermo Fisher Scientific). Cell Counting Kit-8 (#CK04) was obtained from Dojindo Laboratories (Kumamoto, Japan). Ampicillin (#A0166), cinnafied acid (SA; #D7927), human serum albumin (HSA; #A3782), and 3,3',5,5'-tetramethylbenzidine (TMB; #860336) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were purchased from Sigma-Aldrich unless otherwise noted.

[0837] Experimental method

[0838] Matrix-Assisted Laser Desorption / Ionization Time-of-Flight (MALDI-TOF) Analysis

[0839] Purified proteins (4D5Albu, 4D5Albu-G235pAzF, 4D5scFv, 4D5scFv-G178pAzF) and conjugates (4D5Albu-MMAE, 4D5scFv-MMAE) were prepared in TA70 buffer (70% acetonitrile, 0.1% TFA in distilled water) using ZipTip C4 and mixed in a 1:1 (v / v) ratio with a SA matrix solution saturated in TA30 (30% acetonitrile, 0.1% TFA in distilled water). The sample-matrix mixture was deposited onto a steel target pre-coated with a matrix solution saturated in ethanol. Molecular weight analysis was performed using an Autoflex Speed ​​MALDI-TOF mass spectrometer (Bruker) with Protein Standard II as the calibration standard.

[0840] Enzyme-linked immunosorbent assay (ELISA)

[0841] The antigen binding affinities of 4D5Albu and 4D5Albu-MMAE were compared using anti-HER2 ELISA. Recombinant HER2 antigen (0.5 μg mL) 1100 μL of the sample was dissolved in PBS (pH 7.4), placed in a 96-well immunoassay plate, and incubated overnight at 4°C. Subsequently, the plate was washed with PBST (0.05% Tween 20 in PBS, pH 7.4) and blocked using 5% skim milk in PBST. The albumin binding effect of 4D5Albu was investigated by incubating the purified protein (1 μM) in PBS (pH 7.4) at room temperature for 30 minutes in the presence or absence of 2 μM human serum albumin (HSA). Sample mixtures were prepared in blocking buffer, and the initial concentration of 50 nM was diluted threefold for analysis. 100 μL of the diluted sample was incubated in each well at room temperature. After removing unbound proteins, rabbit anti-His-tag antibody (1:1,500 dilution, 100 μL in blocking buffer) was added and incubated to bind to the protein. After removing unbound antibodies, HRP-linked anti-rabbit IgG antibody (1:3,000 dilution, 100 μL in blocking buffer) was added and incubated. After the final wash, 100 μL of TMB substrate was added and incubated until color appeared. After stopping the reaction with 2 M HCl, the bound protein was quantified by measuring the absorbance at 450 nm. To evaluate the anti-HSA binding efficiency of the protein and conjugate, 0.5 μg mL 1 The HSA was dissolved in PBS (pH 7.4) and coated onto a plate, and the remaining conditions and procedures were performed as described above.

[0842] Size Exclusion Chromatography (SEC)

[0843] Purified proteins (4D5Albu, 4D5Albu-G235pAzF, 4D5scFv) and conjugates (4D5Albu-MMAE) were characterized by rapid protein liquid chromatography (FPLC) using SEC. Samples prepared in PBS (pH 7.4) were injected into a Superdex 75 Increase 10 / 300 GL column (Cytiva), and 0.2 mL min 1 It was eluted at a flow rate. Molecular weight standards for conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa) were used in the Gel Filtration Calibration Kit LMW (Cytiva). Analysis was performed using an NGC Quest 10 chromatography system (Bio-Rad).

[0844] Hydrophobic Interaction Chromatography (HIC)

[0845] HIC was used to analyze the purified protein (4D5Albu-G235pAzF) and the conjugate (4D5Albu-MMAE). Samples were prepared in a solution containing PBS (pH 7.4) and 1 M ammonium sulfate and loaded onto a TSKgel Butyl-NPR, 2.5 μm, 4.6 mm ID x 10.0 cm L column (Tosoh Bioscience, King of Prussia, PA, USA). Samples were first run with 1 M ammonium sulfate and 25 mM sodium phosphate buffer (pH 7.0), with the flow rate set to 0.4 mL / min. Elution was performed as a stepwise gradient; the mobile phase was first changed to 50% elution buffer (25 mM sodium phosphate buffer, pH 7.0) and maintained for 10 minutes, then changed to 80% elution buffer and continued for an additional 10 minutes. The analysis was performed using a 1260 Infinity II LC system (Agilent, Santa Clara, CA, USA).

[0846] In vitro cytotoxicity analysis

[0847] BT-474 cells were purchased from the Korean Cell Line Bank (Seoul, South Korea). SK-BR-3 cells and MDA-MB-231 were provided by the laboratory. All cells were sterilized in 10% FBS, 100 units mL 1 Penicillin, 100 μg mL 1 Cells were cultured in RPMI 1640 medium containing streptomycin. BT-474 cells were cultured in 5,000 cell wells of 96-well culture plates. 1 , other cell lines 3,000 cell wells 1 Cells were inoculated at a density of [value] and cultured for 24 hours at 37 °C in a 5% CO2 environment. 4D5scFv, 4D5Albu-MMAE, and MMAE were diluted twofold in PBS (pH 7.4) and mixed with the same medium at a 1:3 (v / v) ratio. After removing the existing medium from the adhered cells, the prepared sample-medium mixture was added. After 3 days of culture, the medium in each well was replaced with 100 μL of fresh medium and 10 μL of CCK-8 reagent mixture. After 1 hour of incubation, cytotoxicity was evaluated by measuring absorbance at 450 nm using a SynergyH1 microplate reader (BioTek, Agilent).

[0848] In vivo pharmacokinetics study

[0849] All animal studies were conducted in accordance with the guidelines of the Animal Ethics Committee (GIST-2021-092). The serum half-lives of 4D5scFv-MMAE, 4D5Albu-MMAE, and their unconjugated forms (4D5scFv, 4D5Albu) were investigated through pharmacokinetic studies in mice. Purified 4D5Albu, 4D5Albu-MMAE, 4D5scFv, and 4D5scFv-MMAE (4 μM) were prepared in 200 μL of PBS (pH 7.4) and injected into the tail veins of 7-week-old female BALB / c mice (n=5). Blood samples were collected via orbital blood sampling at 5 minutes (initial time point), 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, and 120 hours after injection. Serum was separated from blood by centrifugation at 1,500 g for 10 minutes at 4 °C and stored at -80 °C until analysis. Protein concentration in serum was determined by ELISA at each time point, and the concentration was calculated by correcting the values ​​using a standard calibration curve.

[0850] In vivo anticancer efficacy in an SK-BR-3 xenograft model

[0851] In vivo anticancer efficacy studies were conducted in accordance with the guidelines of the Animal Ethics Committee (GIST-2022-054). An SK-BR-3 xenograft model was established using 6-week-old male BALB / c nude mice (n=5 per group). Each mouse was injected subcutaneously with 2,000,000 SK-BR-3 cells suspended in 100 μL of PBS (pH 7.4). When the tumor size reached approximately 50 mm³, 28 μM of 4D5scFv-MMAE or 4D5Albu-MMAE (prepared in 200 μL) was administered intravenously at 3-day intervals for a total of 3 doses. The control group was treated with PBS.

[0852] Tumor volume and body weight were measured on days 0, 3, 6, 8, 10, 12, and 14. Tumor volume was calculated using the following formula: Tumor volume (mm³) = Width² × Length / 2. The study was terminated on day 14, and the tumor was excised. At the end of the study, the tumor volume was calculated using the following formula: Tumor volume (mm³) = 4 / 3ð × (Width / 2) × (Length / 2) × (Height / 2). The tumor was fixed overnight in 4% formaldehyde, followed by dehydration with stepwise ethanol and embedding in paraffin. Sections were cut to a thickness of 5 μm and stained with hematoxylin and eosin (H&E) for histological analysis. Slides were observed under a microscope at 20x magnification to evaluate the degree of necrosis and cell morphology.

[0853] Statistical analysis

[0854] Results were presented as mean ± standard deviation (SD) values. The statistical significance of differences between groups was determined using Tukey's test following one-way analysis of variance (ANOVA). Differences with a p-value of less than 0.05 or 0.01 were considered statistically significant. Data visualization and statistical analysis were performed using OriginPro (Version 2023, OriginLab Corporation, Northampton, MA, USA).

[0855] Experimental Example 1: Design of an engineered scFv (Selection of non-natural amino acid insertion sites)

[0856] To site-specifically conjugate the cytotoxic drug MMAE to 4D5Albu via the SPAAC reaction, the optimal site for modifying an amino acid at a specific position in 4D5Albu to pAzF was identified through computer analysis of the model structure of 4D5Albu (trastuzumab-derived scFv-based albumabody, SEQ ID NO. 1). This analysis focused on selecting a side chain that could ensure high conjugation efficiency and stable protein expression upon the introduction of pAzF.

[0857] Below, the model structure of 4D5Albu was predicted using ColabFold, a web server for protein structure prediction utilizing AlphaFold2. Additionally, solvent accessibility was determined using PyMOL (Version 2.4.1, Schrφdinger, New York, NY, USA), and mutation stability was evaluated by the estimated free energy change due to Y and W mutations via PyRosetta (PyRosetta4, The PyRosetta Team at Johns Hopkins University, Baltimore, MD, USA).

[0858] First, solvent accessibility was evaluated, and residues with high solvent accessibility were prioritized. This is because residues with high solvent accessibility are more favorable for an efficient reaction with DBCO.

[0859] Second, using the Rosetta energy function, we obtained the estimated change in free energy (the absolute value of the difference in free energy between 4D5Albu with the Y or W mutation introduced and wild-type 4D5Albu) when amino acids at specific positions in 4D5Albu were mutated into pAzF analogs Y and W, and compared this with the energy of wild-type 4D5Albu. Through this, we were able to identify the most stable mutation that minimizes disruption to the structural stability of 4D5Albu.

[0860] Finally, residues located in secondary structural elements or functional regions (CDR, disulfide bond, His-tag, etc.) were excluded. Additionally, residues located in linker regions that could interfere with binding to ABD were excluded. However, residues in linker regions far from ABD were included.

[0861] As a result, nine candidate sites for pAzF introduction were identified: G44, S87, R89, G123, S203, P234, G235, K236, and K301 (Figures 1 and 2). Additionally, the estimated free energy changes and solvent accessibility for each of these Y and W mutations are listed in [Table 1] below.

[0862] [Table 1]

[0863]

[0864] Among these, G44, S87, and S89 were located in the heavy chain variable region (VH), G123 in the linker region, and G203, P234, P235, K236, and K301 in the light chain variable region (VL). In particular, S87, R89, K236, and K301 showed relatively low solvent accessibility (50–60%), while G44 and G235 exhibited higher mutation energy scores (7 or higher) than other residues. K301 was located near the C-terminus and immediately preceding the His-tag sequence. Since all candidate sites were located away from the CDR, the possibility of antibody activity being hindered was minimized.

[0865] Experimental Example 2: Fabrication of an Engineered scFv

[0866] Engineered scFv was expressed by co-transforming [pEVOL-pAzF], a vector encoding engineered scFv, into E. coli C321.ΔA.exp cells.

[0867] At this time, the E. coli C321.ΔA.exp cell is a cell line in which the gene is modified to have a different stop codon with an amber codon (UAG) as the stop codon, and the release factor that recognizes the amber codon (UAG) is functionally removed. In addition, [pEVOL-pAzF] is a vector encoding an exogenous suppressor tRNA and an exogenous tRNA synthetase (derived from methanococcus jannaschii) capable of linking pAzF to the exogenous suppressor tRNA (Fig. 3).

[0868] 2-1. Preparing Vectors

[0869] The vector encoding the engineered scFv was prepared by mutating the vector encoding 4D5Albu ([pBAD-4D5Albu], FIG. 4) to the amber codon through position-specific mutation induction using primers. Specifically, the [pBAD-4D5Albu-XAmb] vector was constructed using the primer pairs listed in [Table 2] below (where 4D5Albu-XpAzF refers to an engineered scFv in which the amino acid at the X-position of the wild-type 4D5Albu is changed to pAzF).

[0870] [Table 2]

[0871]

[0872] In addition, 4D5scFv (trastuzumab-derived scFv) and 4D5Albu used in the experiment were expressed by surfcloning the nucleic acids encoding each into the pBAD plasmid ([pBAD-4D5scFv] and [pBAD-4D5Albu]).

[0873] In addition, to express 4D5scFv (4D5scFv-G178) with pAzF introduced into the G178 site, a vector encoding 4D5scFv-G178 was prepared using the primers in [Table 3] below, similar to the method of the vector encoding the engineered scFv above ([pBAD-4D5scFv-G178Amb]).

[0874] [Table 3]

[0875]

[0876] 2-2. Expression

[0877] To express wild-type 4D5scFv and 4D5Albu, E. coli TOP10 cells were transformed with the [pBAD-4D5scFv] and [pBAD-4D5Albu] plasmids, respectively. The transformed cells were 100 μg mL⁻¹. 1 Incubated overnight in 2xYT medium containing ampicillin at 37 °C with shaking at 200 rpm. After overnight incubation, inoculated into fresh medium to obtain an optical density (OD) at 600 nm. 600 ) was grown until it reached 0.5. Then, after induction with 0.2% (w / v) L-(+)-arabinose, culture was continued for an additional 24 hours at 23 °C.

[0878] For the expression of the 4D5scFv-G178pAzF and 4D5Albu-pAzF variants, each pBAD expression vector containing an amber codon mutation was co-transfected into E. coli C321.ΔA.exp cells with [pEVOL-pAzF] and cultured under similar conditions. When the OD600 reached 0.5, protein expression was induced by adding 1 mM pAzF and 0.4% (w / v) L-(+)-arabinose. Cells were harvested by centrifugation at 8,000 rpm and 4 °C, and cell culture samples before (BI) and after (AI) induction were collected by SDS-PAGE for expression analysis.

[0879] 2-3. Extraction and Purification

[0880] The harvested cell pellet is 1 mg mL 1The cells were resuspended in a lysis buffer containing lysozyme (10 mM imidazole, 50 mM NaH2PO₄, 300 mM NaCl, pH 8.0). Cells were lysed by sonocation using a 5 mm sonocation tip at 32% amplitude (500 W, 20 kHz) in a 1-second pulse, 2-second rest cycle. The lysate was centrifuged at 10,000 g for 20 minutes at 4 °C, and the supernatant was incubated with Ni-NTA agarose resin at 4 °C. The protein-bound resin was loaded onto a polypropylene column equipped with a filter. The resin was washed with a wash buffer (20 mM imidazole), and the protein was eluted using an elution buffer (250 mM imidazole). The purified protein was stored at 4 °C and analyzed by SDS-PAGE.

[0881] 2-4. Production Verification

[0882] In SDS-PAGE analysis, a protein band corresponding to the molecular weight of the engineered scFv was observed (approx. 33 kDa) (Fig. 5). Additionally, the engineered scFv was obtained with high purity through purification using the Ni-NTA / His-tag system and matched the expected molecular weight (Fig. 6). Furthermore, consistent with previous studies, 4D5scFv and 4D5Albu were successfully produced in E. coli TOP10 cells, which was confirmed by SDS-PAGE (Figs. 5 and 6) and MALDI-TOF (Fig. 7).

[0883] Most engineered scFv is 4–8 mg L 1 Although a production yield of was shown, 4D5Albu-G44pAzF and 4D5Albu-R89pAzF were 2 mg L 1 It was excluded from further experiments due to showing a much lower yield than the above. On the other hand, 4D5scFv and 4D5Albu were approximately 22 mg L 1 It was produced stably with a considerably high yield.

[0884] Experimental Example 3: Preparation of Engineered scFv-Drug Conjugate

[0885] All variants of the purified engineered scFv, excluding 4D5Albu-G44pAzF and 4D5Albu-R89pAzF, were reacted with the DBCO-PEG4-Val-Cit-PAB-MMAE (DBCO-MMAE) linker-drug conjugate (conjugate represented by Formula 12 of this specification) in a molar ratio of 1:4 to prepare the conjugate through the SPAAC reaction.

[0886] At this time, the DBCO-MMAE was dissolved in DMSO and stored at -20 °C until use, and the engineered scFv and DBCO-MMAE were mixed in the elution buffer at final concentrations of 20 μM and 80 μM, respectively, and reacted overnight at 4 °C while rotating. Additionally, after the reaction, unreacted DBCO-MMAE was removed, and the buffer was replaced with PBS (pH 7.4) using a PD-10 desalting column. The resulting product was analyzed by SDS-PAGE.

[0887] DBCO-MMAE has a molecular weight of approximately 1.5 kDa, allowing unconjugated engineered scFv (approx. 33 kDa) and the conjugated product (approx. 34.5 kDa) to be distinguished on the gel. After reaction with DBCO-MMAE, clearly separated protein bands were observed in all engineered scFvs, whereas no conjugated bands appeared in wild-type 4D5scFv or 4D5Albu (Fig. 8) (each band was quantified and analyzed using the volume tool function of Image Lab software (Bio-Rad)).

[0888] Experimental Example 4: Confirmation of engineered scFv-drug conjugate conjugation yield

[0889] The bonding yield of each engineered scFv showed significant differences (Fig. 8) (each bonding yield was calculated by dividing the volume of the bonded product band by the total volume of the bonded and unbonded bands).

[0890] Among them, 4D5Albu-G235pAzF exhibited a conjugation yield of over 80% according to protein gel image analysis, while 4D5Albu-P234pAzF recorded a yield of approximately 60%. These results are consistent with computer analysis, in which 4D5Albu-G235pAzF and 4D5Albu-P234pAzF demonstrated relatively high solvent accessibility exceeding 80%. This suggests that at least 80% solvent accessibility is required for efficient conjugation. In contrast, the other five engineered scFvs (4D5Albu-S87pAzF, 4D5Albu-G123pAzF, 4D5Albu-S203pAzF, 4D5Albu-K236pAzF, and 4D5Albu-K301pAzF) all showed low conjugation yields of 30% or less. These low conjugation efficiencies may largely be related to low solvent accessibility (50-60%), and with the exception of G123, although it has 80% solvent accessibility, the presence of ABD may explain the low conjugation yields, as actual accessibility is likely lower than expected. The differences in conjugation efficiency among variants highlight the importance of thorough computer analysis.

[0891] Subsequently, the 4D5Albu-G235pAzF variant was selected as the final candidate because it showed the highest conjugation yield. In subsequent experiments, the MMAE conjugate produced from the 4D5Albu-G235pAzF variant was named 4D5Albu-MMAE.

[0892] To further confirm the conjugation yield of 4D5Albu-MMAE, hydrophobic interaction chromatography (HIC) analysis was performed. The chromatogram separated 4D5Albu-G235pAzF and 4D5Albu-MMAE based on differences introduced by the conjugation of MMAE molecules (Fig. 9). In the HIC analysis of 4D5Albu-MMAE, two distinct peaks were observed. The unconjugated form was primarily eluted at the 50% elution buffer step, whereas the conjugated form was eluted at the 80% elution buffer step, indicating an increase in hydrophobicity after MMAE conjugation. The conjugation yield was confirmed to be 85% through the calculation of the area under the curve (AUC) for each peak, which was consistent with the SDS-PAGE results. Additionally, the oligomeric states of 4D5Albu and 4D5Albu-MMAE were analyzed by size-exclusion chromatography (SEC) under physiological conditions (Fig. 10). The chromatograms showed that 4D5Albu and 4D5Albu-MMAE existed mostly as monomers, whereas 4D5scFv existed as a mixture of monomers and dimers. This difference may be due to the preference for the monomeric form of scFv as the linker between the VH and VL regions is lengthened by ABD insertion.

[0893] The synthesis of 4D5Albu-MMAE was confirmed by MALDI-TOF mass spectrometry (Fig. 11). The average mass of 4D5Albu-G235pAzF was measured to be 32,702 Da, which is close to the theoretical average mass of 32,890 Da for single positive ionization. In the analysis of 4D5Albu-MMAE, a relatively small peak corresponding to the unconjugated 4D5Albu-G235pAzF and a larger peak representing the conjugated product were observed. The measured average mass of 4D5Albu-MMAE was 34,499 Da, which is in good agreement with the expected mass of 34,548 Da.

[0894] As a control, a 4D5scFv variant (4D5scFv-G178pAzF) in which pAzF was introduced at the G178 position corresponding to the G235 position of 4D5Albu was prepared and conjugated with DBCO-MMAE to generate 4D5scFv-MMAE, which was then analyzed by SDS-PAGE (Fig. 12) and MALDI-TOF (Fig. 13). The conjugation yield was found to be 85-90%, similar to that of 4D5Albu-MMAE.

[0895] Experimental Example 5: Evaluation of Engineered scFv-Drug Function_Confirmation of Binding Affinity

[0896] The binding affinity of 4D5Albu and 4D5Albu-MMAE to HER2 was evaluated in vitro (Fig. 14). Conjugated 4D5Albu-MMAE was found at a half-maximal effective concentration (EC₀) of 2.38 ± 0.07 nM. 50 It showed ), which is a value comparable to 4D5Albu (1.27 ± 0.04 nM). When the two molecules were pre-incubated with HSA, the EC 50 The values ​​increased slightly, with 4D5Albu-MMAE and 4D5Albu showing 5.71 ± 0.34 nM and 3.57 ± 0.13 nM, respectively. In addition, the binding affinity of 4D5Albu and 4D5Albu-MMAE for HSA was also measured (Fig. 15). 4D5Albu showed an EC of 1.22 ± 0.03 nM. 50 While [it] showed [that], 4D5Albu-MMAE showed a slightly higher EC of 2.77 ± 0.05 nM. 50 It was shown to maintain a strong binding affinity for HSA.

[0897] In conclusion, 4D5Albu-MMAE largely retained its in vitro binding affinity characteristics, indicating that the site-specific binding of MMAE had minimal effect on the overall structure.

[0898] Experimental Example 6: Evaluation of Engineered scFv-Drug Function_Verification of Pharmacokinetic Profile

[0899] The pharmacokinetic profiles of 4D5scFv, 4D5scFv-MMAE, 4D5Albu, and 4D5Albu-MMAE in mouse serum were evaluated to confirm albumin-mediated serum half-life prolongation (Fig. 16). The concentrations of 4D5scFv and 4D5scFv-MMAE decreased rapidly after intravenous injection, exhibiting serum half-lives of 0.26 hours and 0.25 hours, respectively, which appear to be primarily due to renal removal and intracellular degradation. In contrast, 4D5Albu and 4D5Albu-MMAE exhibited beta-phase half-lives (t 1 / 2 (α)) showed significantly improved pharmacokinetic profiles of 59 hours and 53 hours, respectively. The area under curve (AUC) for 4D5scFv and 4D5scFv-MMAE was calculated to be 35 hours x % and 34 hours x %, respectively, while the AUC values ​​for 4D5Albu and 4D5Albu-MMAE were 4,645 hours x % and 4,041 hours x %, respectively. The strong binding affinity of ABD to serum albumin and the high concentration of serum albumin in the blood (~500 μM) suggest that engineered scFv and albu bodies can circulate while tightly bound to serum albumin. These interactions enable engineered scFv and albu bodies to evade cell degradation and avoid renal filtration through FcRn-mediated recycling. 4D5Albu-MMAE compared to 4D5scFv-MMAE t 1 / 2 (α) was found to have increased 212-fold and AUC 118-fold.

[0900] In conclusion, 4D5Albu-MMAE largely retained its in vitro binding affinity and in vivo pharmacokinetic properties, indicating that the site-specific binding of MMAE had minimal effect on the overall structure.

[0901] Experimental Example 7: Evaluation of Engineered scFv-Drug Function_Confirmation of In Vitro Cytotoxicity

[0902] The in vitro cytotoxicity of 4D5Albu-MMAE was evaluated in HER2-positive breast cancer cell lines (BT-474 and SK-BR-3) and HER2-negative breast cancer cell lines (MDA-MB-231), and compared with the cytotoxicity of 4D5scFv-MMAE and MMAE (Figs. 17, 18, and 19). Treatment with 4D5Albu-MMAE exhibited high cytotoxicity in HER2-positive cells, showing a trend similar to that of MMAE (Figs. 17 and 18). Specifically, 4D5Albu-MMAE showed half-maximal inhibitory concentrations (IC1) of 2.7 ± 0.5 nM in BT-474 cells and 13.5 ± 2.2 nM in SK-BR-3 cells. 50 ) was shown, and MMAE had ICs of 1.0 ± 0.4 nM and 1.5 ± 0.2 nM, respectively. 50 It showed values. Notably, there was no significant difference in cytotoxicity between 4D5scFv-MMAE and 4D5Albu-MMAE, and the IC50 of 4D5scFv-MMAE 50 The values ​​were found to be 2.9 ± 1.4 nM in BT-474 cells and 22.4 ± 6.6 nM in SK-BR-3 cells. This suggests that the fusion of ABD did not significantly affect the cellular antigen targeting and internalization activities of the ADC. On the other hand, the cytotoxicity of 4D5Albu-MMAE was significantly reduced in the HER2-negative cell line MDA-MB-231 (Fig. 19). IC of MMAE 50While the value was 1.8 ± 0.3 nM, 4D5scFv-MMAE and 4D5Albu-MMAE did not exhibit toxicity within the experimental concentration range. Considering that the drug-antibody ratio (DAR) of 4D5Albu-MMAE is 0.85, these results indicate that 4D5Albu-MMAE exhibits potent and highly specific cytotoxicity against HER2-positive cancer cells.

[0903] Experimental Example 8: Evaluation of Engineered scFv-Drug Function_Confirmation of In Vivo Anticancer Effect

[0904] The anticancer effects of 4D5scFv-MMAE and 4D5Albu-MMAE were evaluated in an SK-BR-3 xenograft model using BALB / c nude mice (n=5), which are HER2-positive models frequently used in ADC xenograft studies.

[0905] All groups received their respective therapeutic agents intravenously on days 0, 3, and 6, and tumor volume was measured for 2 weeks (Fig. 20). After the initial first administration, tumor growth inhibition was observed in both the 4D5scFv-MMAE and 4D5Albu-MMAE groups, and there was no significant difference in tumor volume between the two groups. However, after the second injection on day 3, the therapeutic effect of 4D5Albu-MMAE was more pronounced compared to 4D5scFv-MMAE. On day 14, the average tumor volume of the 4D5Albu-MMAE group (133 ± 69 mm³) was significantly smaller than that of the control group (1,155 ± 246 mm³) and the 4D5scFv-MMAE group (668 ± 464 mm³) (p < 0.001). These results indicate that 4D5Albu-MMAE demonstrated a much more potent anticancer effect than 4D5scFv-MMAE.

[0906] Tumor weight and volume allowed for the additional evaluation of anticancer effects at the end of the study (Figs. 21, 22, and 23). The average tumor weight of the control group was 0.74 ± 0.16 g and the tumor volume was 653 ± 87 mm³, while the 4D5scFv-MMAE group showed reduced results with a tumor weight of 0.33 ± 0.20 g and a tumor volume of 320 ± 170 mm³. In particular, the 4D5Albu-MMAE group showed the most significant tumor suppression effect, with a tumor weight of 0.04 ± 0.03 g and a tumor volume of 41 ± 25 mm³. These data clearly demonstrate that 4D5Albu-MMAE showed superior efficacy compared to the control group and the 4D5scFv-MMAE group (p < 0.001).

[0907] Hematoxylin and eosin (H&E) staining of tumor sections on day 14 revealed clear histological differences between the treatment groups (Fig. 24). In the control group, viable tumor cells were densely packed, and with intact nuclei, they exhibited basal staining. Only micronecrosis was observed, indicating rapid tumor proliferation. In contrast, extensive necrotic regions were observed in the 4D5Albu-MMAE group; these were large eosinophilic zones devoid of living tumor cells, highlighting tumor cell death. Tumors treated with 4D5scFv-MMAE showed a mixture of necrotic and viable tumor cell regions, reflecting partial anticancer activity. These histological patterns are consistent with differences in tumor volume and weight, indicating that 4D5Albu-MMAE demonstrated the most pronounced tumor suppression effect, likely due to longer cycle times and sustained cytotoxic effects.

[0908] Furthermore, the anticancer effect of the aforementioned 4D5Albu-MMAE is particularly impressive considering the administration regimen used. In this study, 4D5Albu-MMAE with a DAR of 0.85 was administered three times at 3-day intervals at a dose of 28 μM, which corresponds to 170 ng kg of MMAE.1 This corresponds to administering a smaller amount less frequently compared to the dosing regimen in a previous study using Fab-MMAE with a DAR of 1 (G. Badescu et al, Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug. Chem. 2014, 25, 1124-1136.). Despite relatively low cumulative doses and DARs, 4D5Albu-MMAE achieved significant tumor regression, which is likely due to improved pharmacokinetics. These results demonstrate the potential of the albubody scaffold as an effective platform for treating solid tumors, and further enhancement of anticancer activity may be possible by optimizing the dosing regimen.

[0909] The superior efficacy of albubody-based ADCs compared to scFv-based ADCs is primarily attributed to their extended serum half-lives. As circulation times lengthen, the likelihood of drug accumulation in the tumor microenvironment increases. Introducing ABD into scFv-drug conjugates may provide an opportunity to utilize scFv-based therapies for the treatment of a wider range of solid tumors.

[0910] Body weight analysis showed no significant difference between the control and treatment groups during the study period, indicating that all treatments were well tolerated and did not cause systemic toxicity (Fig. 25). The absence of weight loss suggests that 4D5scFv-MMAE and 4D5Albu-MMAE were safe at the administered doses and that there were no off-target effects or general health deterioration.

[0911] Experimental Example 9: Fabrication of an Engineered scFv Using frTet

[0912] Based on Experimental Examples 1 and 2, an engineered scFv using frTet can be fabricated.

[0913] Even when using frTet, mutation stability will be evaluated by the change in estimated free energy due to Y and W mutations via PyRosetta (PyRosetta4, The PyRosetta Team at Johns Hopkins University, Baltimore, MD, USA), and the location will be selected. Accordingly, the non-natural location identified in Experimental Examples 1 and 2 will be the same even when using frTet.

Claims

1. Engineered scFv-drug conjugate represented by the following [Structural Formula 1]: [Structural Formula 1] [Engineered scFv (S)]-[Connection (J)]-[Linker (L)]-[Payload (P)], At this time, S comprises one amino acid sequence selected from SEQ ID NOs 2 to 10, and At this time, X of sequence numbers 2 to 10 is a non-natural amino acid represented by the following chemical formula, and [Chemical Formula 5] At this time, AA1 and AA2 are each amino acids in which the alpha carbon is connected to an amino acid residue linked to J, or are absent, and J is a structure formed by a click response, and L includes a cuttable linker region, and P is characterized as being a drug.

2. In claim 1, the above S is an engineered scFv-drug conjugate comprising the nucleotide sequence of SEQ ID NO.

8.

3. The engineered scFv-drug conjugate in which the non-natural amino acid is 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet).

4. In paragraph 3, J is an engineered scFv-drug conjugate represented by the following chemical formula: In this case, A1 does not exist or -A 11 -A 12 - and, At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is any one selected from heteroalkylene and heteroarylene, wherein p is an integer between 1 and 3 inclusive, and At this time, R1 is H, halogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, C 3-6 It is any one selected from heterocycloalkyl, aryl, and heteroaryl, and At this time, the heterocycloalkyl or heteroaryl comprises one or more heteroatom groups selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O)2-, or one or more heteroatoms selected from the group consisting of O, N, and S.

5. The engineered scFv-drug conjugate in which the non-natural amino acid is 4-Azido-L-phenylalanine (pAzF).

6. In paragraph 5, J is an engineered scFv-drug conjugate represented by one of the following chemical formulas. [Chemical Formula 7-1] , [Chemical Formula 7-2] , [Chemical Formula 7-3] , or [Chemical Formula 7-4] In this case, A1 does not exist or -A 11 -A 12 - and, At this time, A 11 is non-existent, or C 1-5 It is an alkylene, and At this time, A 12 is non-existent, or [Arilen] p , -[Arilen] p -C 1-5 Alkylene-, -[arylene] p -C 1-5 Heteroalkylene-, -arylene-C 1-5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene] p , -[Hetero-Arilene] p -C 1-5 Alkylene-, -[heteroarylene] p -C 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene-, -heteroarylene-C 1-5 Heteroalkylene-arylene-, and -heteroarylene-C 1-5 It is selected from heteroalkylene and heteroarylene, where p is an integer between 1 and 3.

7. In paragraph 1, the above L is expressed by [Structural Formula 2] as follows, and [Structural Formula 2] -[Spacer]-[payload linkage linker (PL)]-, At this time, the above spacer is non-existent or -B1-B2-, and At this time, B1 is non-existent or -CH2-O-, -CH2-CH2-(C=O)-, -CH2-CH2-(C=0)-NH-, -(C=0)-NH-CH2-, -(C=0)-CH2-CH2-(C=0)-, -(C=0)-CH2-CH2-NH-(C=0)-, -(C=0)-NH-CH2-CH2-CH2-NH-(C=0)-, -(C=0)-CH2-CH2-(C=0)-NH-, -(C=0)-CH2-CH2-CH2-(C=0)-NH-, -O-(C=O)-NH-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-NH-, -O-(C=O)-NH-CH2-CH2-CH2-(C=O)-O-, (-CH2)p- or a combination thereof, including In this case, B2 is non-existent or -CH2-CH2-(C=O)-, -CH2-CH2-(C=O)-NH-, -CH2-CH2-NH-(C=O)-, -CH2-CH2-(C=O)-O-, -CH2-CH2-SS-, [-CH2-CH2-O]n(PEGn)-, [-CH2-CH2-O]n(PEGn)-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-(C=O)-O-, [-CH2-CH2-O]n(PEGn)-(C=O)-CH2-CH2-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH2-CH2-NH-(C=O)-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-NH-, [-CH2-CH2-O]n(PEGn)-CH-CH-(C=O)-O- or a combination thereof, comprising At this time, the above p and n independently represent natural numbers greater than or equal to 1; and An engineered scFv-drug conjugate characterized in that the payload linker is a region containing a cleavable linker.

8. In claim 1, the engineered scFv-drug conjugate in which the cleavable linker is Val-Cit-PAB, Val-Ala-PAB, cBu-Cit-PAB, Glu-Val-Cit-PAB or Gly-Gly-Phe-Gly-PAB.

9. The engineered scFv-drug conjugate of claim 1, wherein the drug is MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1 (Maytansinoid derivative), DM4 (Maytansinoid derivative), Calicheamicin, PBD (Pyrrolobenzodiazepine), or SN-38 (Camptothecin derivatives).

10. A pharmaceutical composition for treating breast cancer comprising one of the engineered scFv-drug conjugates of claims 1 to 9.

11. Use of one of the engineered scFv-drug conjugates of claims 1 to 9 for producing a breast cancer treatment.

12. A method for treating breast cancer comprising administering one of the engineered scFv-drug conjugates of claims 1 to 9 to a subject.

13. Comprising one amino acid sequence selected from SEQ ID NOs 2 to 10, and X of SEQ ID NOs 2 to 10 is an engineered scFv that is a non-natural amino acid having a click chemical functional group.

14. In paragraph 13, the non-natural amino acid having the click chemical functional group is an engineered scFv that is 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet).

15. In claim 13, an engineered scFv comprising one amino acid sequence selected from SEQ ID NO. 3 and SEQ ID NOs. 5 to 10.

16. A method for producing an engineered scFv-drug conjugate comprising the following: (a) Prepare a drug connected to an engineered scFv and a conjugating linker. At this time, the engineered scFv is prepared by culturing a cell line containing a nucleic acid encoding the engineered scFv, an exogenous suppressor tRNA, and an exogenous tRNA synthase in an environment where non-natural amino acids are present, and The nucleic acid encoding the above-mentioned engineered scFv includes an emer codon (UAG), and The nucleic acid encoding the above-mentioned engineered scFv comprises one nucleic acid sequence selected from SEQ ID NOs 74 to 82, and The above-mentioned foreign-derived tRNA synthase is an enzyme capable of linking the above-mentioned non-natural amino acid to the above-mentioned foreign-derived suppressor tRNA, and The above-mentioned foreign-derived suppressor tRNA is a tRNA that recognizes the above-mentioned amber codon (UAG), and The above-mentioned non-natural amino acid is 4-Azido-L-phenylalanine (pAzF) or 4-(1,2,4,5-tetrazin-3-yl)phenylalanine (frTet); (b) Inducing a reaction between the drug connected to the conjugating linker and the engineered scFv to obtain an engineered scFv-drug conjugate, At this time, the first click chemical functional group of pAzF or frTet included in the engineered scFv and the second click chemical functional group of the conjugating linker are combined through a click reaction.

17. A method for producing an engineered scFv-drug conjugate in which the foreign-derived suppressor tRNA and the foreign-derived tRNA synthase are derived from methanococcus jannaschii.

18. A method for producing an engineered scFv-drug conjugate according to claim 16, wherein the cell line is a cell line in which the release factor recognizing the amber codon (UAG) is non-functional or has reduced function, and the endogenous gene having the amber codon (UAG) as a stop codon is non-functional or has reduced function.

19. A method for producing an engineered scFv-drug conjugate comprising, in which the drug to which the conjugating linker is connected comprises one click chemical functional group selected from cyclooctyne, DBCO, alkyne, BCN, and TCO.

20. A method for producing an engineered scFv-drug conjugate according to claim 16, wherein the conjugating linker comprises a cleavable linker region.

21. A method for producing an engineered scFv-drug conjugate according to claim 16, wherein the drug is MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), DM1 (Maytansinoid derivative), DM4 (Maytansinoid derivative), Calicheamicin, PBD (Pyrrolobenzodiazepine), or SN-38 (Camptothecin derivatives).

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