Protein l single domain fusion antibody

Abstract

Provided is an antibody having improved heat resistance. The present invention provides a protein L single domain (PpL single domain) fusion antibody in which a PpL single domain and an antibody are linked directly or via a linker.

Description

Protein L single domain fusion antibody

[0001] This relates to a Protein L single domain fusion antibody.

[0002] Immunoassays are methods that use antibodies to specifically detect target substances. In immunoassays, for example, an antibody is directly or indirectly bound to a carrier (e.g., a solid phase such as a well or plate), and the target substance is detected by contacting the antibody with a sample. A typical immunoassay is ELISA (Non-Patent Document 1). However, because antibodies are proteins, they tend to be easily denatured by heat. Therefore, it is important to increase the heat resistance of antibodies.

[0003] ELISA kit, Cosmo Bio Co., Ltd. website (product information, ELISA kit), https: / / www.cosmobio.co.jp / product / detail / 00780001.asp?entry_id=2723

[0004] The object is to provide an antibody with improved heat resistance.

[0005] As a result of extensive research, the present inventors have found that linking a Protein L single domain (PpL single domain) to an antibody can improve the heat resistance of the antibody. They have also found that linking a PpL single domain to an antibody can further improve the production (expression) amount of soluble protein (soluble antibody), the antigen-binding activity of the antibody, and / or the binding activity of the antibody to IgG antibodies. The present invention was completed through further research based on these findings, and the present disclosure encompasses, for example, the following representative inventions. Item 1. A PpL single domain fusion antibody in which a Protein L single domain (PpL single domain) is linked to an antibody directly or via a linker. Item 2. The PpL single domain fusion antibody according to Item 1, in which an amino acid sequence encoding a PpL single domain is linked, directly or via a linker, to the C-terminus of the amino acid sequence encoding the antibody, or in which an amino acid sequence encoding a PpL single domain is linked, directly or via a linker, between the amino acid sequence encoding the heavy chain variable region and the amino acid sequence encoding the light chain variable region of the antibody. Item 3. Item 4. The PpL single domain fusion antibody according to Item 1 or 2, wherein the amino acid sequence encoding the light chain variable region is located at the C-terminus of the amino acid sequence encoding the heavy chain variable region. Item 5. The antibody is a scFv, scAb, sdAb, Fab, F(ab) ２ Item 4. The antibody according to any one of Items 1 to 3, which is a nucleotide sequence of a nucleotide sequence corresponding to a nucleotide sequence of ...

[0006] Linking a PpL single domain to an antibody can provide an antibody with improved heat resistance, and linking a PpL single domain to an antibody can improve the production (expression) amount of soluble protein (soluble antibody), the antigen-binding activity of the antibody, and / or the binding activity of the antibody to IgG antibodies.

[0007]

[0033] Figure 1 shows the amino acid sequences (SEQ ID NOS: 11 to 13) of the PpL single domains used in the test examples and the PpL single domain unfused antibodies of Comparative Examples 1 and 2. Figure 2 shows the amino acid sequences (SEQ ID NOS: 14 and 15) of the PpL single domain fusion antibodies of Examples 1 and 2. Figure 3 shows the amino acid sequences (SEQ ID NOS: 16 and 17) of the PpL single domain fusion antibodies of Examples 3 and 4. Figure 4 shows the sequence linkage order (schematic diagram) of the PpL single domains, VH, and VL in Examples 1 to 3. Figure 5 shows the results (thermostability) of the test examples. Figure 6 shows the results (amount of soluble protein produced) of the test examples. Figure 7 shows the results (antigen-binding activity) of the test examples. Figure 8 shows the results (binding activity to IgG antibodies) of the test examples. Figure 9 shows a schematic diagram of direct immobilization (physical adsorption) or indirect immobilization (immobilization via IgG antibodies) of antibodies to a carrier. Figure 10 shows the results (antigen-binding activity when antibodies are directly or indirectly immobilized to a carrier).

[0008] Hereinafter, embodiments included in the present disclosure will be described in more detail. In the present disclosure, "comprise" also means "consist essentially of" or "consist of."

[0009] The present disclosure encompasses PpL single domain fusion antibodies in which a protein L single domain (PpL single domain) and an antibody are linked directly or via a linker. In the present disclosure, PpL single domain fusion antibodies may be referred to as "fusion antibodies of the present disclosure."

[0010] Protein L Single Domain (PpL Single Domain) The PpL single domain is known as a domain that constitutes a part of Protein L. Protein L is said to be the first protein isolated from Peptostreptococcus magnus. The amino acid sequence of the PpL single domain is publicly known and can be easily determined based on various amino acid sequence databases. While not limiting the present disclosure, examples of PpL single domains include domains encoded by the amino acid sequences represented by any of SEQ ID NOS: 1 to 7. PpL single domains are broadly classified into four types: Protein L C1 domain, Protein L C2 domain, Protein L C3 domain, and Protein L C4 domain. For example, SEQ ID NOS: 1 and 2 encode the Protein L C1 domain, SEQ ID NOS: 3 and 5 encode the Protein L C2 domain, SEQ ID NOS: 4 and 5, respectively. In the present disclosure, the PpL single domain may be any single domain. The PpL single domain may be wild-type (naturally occurring) or may be a mutated wild-type. Preferred examples of the PpL single domain include the Protein L C1 domain (PpL1) and the Protein L C3 domain, more preferred examples include PpL1, and even more preferred examples include PpL1 encoded by the amino acid sequence represented by SEQ ID NO: 1.

[0011] In the present disclosure, the amino acid sequence encoding the PpL single domain may be encoded by an amino acid sequence in which a mutation has been introduced into a known amino acid sequence. Specifically, the amino acid sequence may contain a mutation to the extent that, when linked to an antibody to form a PpL single domain fusion antibody, the PpL single domain fusion antibody has improved heat resistance compared to an antibody not fused to a PpL single domain (hereinafter, sometimes referred to as an "unfused antibody"). More preferably, the amino acid sequence encoding the PpL single domain may contain a mutation to the extent that, when formed into a PpL single domain fusion antibody, the amount of soluble protein (soluble antibody) produced (expressed), antigen-binding activity, and / or IgG antibody-binding activity can be improved compared to the unfused antibody. Even more preferably, the amino acid sequence may contain a mutation to the extent that the heat resistance and IgG antibody-binding activity can be improved. Whether or not heat resistance is improved, whether or not the production amount of soluble protein is improved, whether or not antigen-binding activity is improved, and whether or not binding activity to an IgG antibody is improved can be determined by confirming the heat resistance, etc. under the same conditions except for whether or not the PpL single domain is fused to the antibody, preferably according to the procedure of the test example described below.

[0012] The length of the amino acid sequence encoding the PpL single domain is not limited, but may be approximately the same as that of an amino acid sequence encoding a known PpL single domain. The number of amino acid residues is preferably about 55 to 95, more preferably about 65 to 90, and even more preferably about 70 to 88.

[0013] Although the PpL single domain is not limited thereto, a preferred example of an embodiment is a PpL single domain comprising the following amino acid sequence (1) or (2): (1) the amino acid sequence represented by SEQ ID NO: 1 (2) the amino acid sequence described in (1) above in which one or more amino acids have been substituted, deleted, inserted, or added

[0014] In (2), "plural" refers to 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 or 3. In the present disclosure, the mutated amino acids may be, for example, natural amino acids or artificial amino acids. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, and sulfur-containing amino acids. Specific examples of amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Techniques for deleting, substituting, inserting and / or adding one or more amino acids in a specific amino acid sequence are known.

[0015] Furthermore, while not limiting the present disclosure, conservative substitutions are a preferred example of substitutions. In the present disclosure, conservative substitution means that an amino acid residue is substituted with an amino acid residue having a side chain with similar properties. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0016] Mutations in the amino acid sequence of SEQ ID NO: 1 are explained in the same manner as above. More preferably, the mutations in the amino acid sequence of SEQ ID NO: 1 are those that, when formed into a PpL single domain fusion antibody, improve the heat resistance to an extent equal to or greater than that of an unfused antibody to which the amino acid sequence of SEQ ID NO: 1 is linked, compared to the unfused antibody. Furthermore, the mutations in the amino acid sequence of SEQ ID NO: 1 are more preferably those that, when formed into a PpL single domain fusion antibody, improve the production amount of soluble protein, antigen-binding activity, and / or binding activity to an IgG antibody to an extent equal to or greater than that of an unfused antibody to which the amino acid sequence of SEQ ID NO: 1 is linked, compared to the unfused antibody. When the amino acid sequence of any of SEQ ID NOs: 2 to 7 is mutated instead of SEQ ID NO: 1, the mutations are explained in the same manner as for SEQ ID NO: 1.

[0017] Furthermore, in the fusion antibody of the present disclosure, only one PpL single domain may be linked, or multiple PpL single domains may be linked. Examples of multiple PpL single domains include 2 to 5, more preferably 2, 3, or 4. In the fusion antibody of the present disclosure, one type of PpL single domain may be used alone, or two or more types may be used in combination. In the fusion antibody of the present disclosure, although this does not prevent Protein L having a PpL single domain (amino acid sequence encoding Protein L) itself from being linked to the antibody, preferably Protein L itself is not linked to the antibody.

[0018] When a fusion antibody of the present disclosure comprises two or more PpL single domains, the PpL single domains may be linked to the antibody directly or via a linker in an aggregated state (i.e., a single-chain amino acid sequence in which multiple PpL single domains are linked directly or via a linker), or each PpL single domain may be linked to the antibody directly or via a linker at a separate location. For the purpose of facilitating understanding, and without limiting the present disclosure, simple examples of fusion antibodies comprising two PpL single domains in the former aggregated state include a heavy chain variable region (VH)-PpL single domain-PpL single domain-light chain variable region (VL) structure, and a VH-VL-PpL single domain-PpL single domain structure (where "-" indicates direct or linker-mediated linkage). Simple examples of the latter fusion antibody in which the PpL single domains are linked at different sites include a structure of VH-PpL single domain-VL-PpL single domain, and a structure of PpL single domain-VH-PpL single domain-VL (where "-" has the same meaning as above). Furthermore, the fusion antibody of the present disclosure may be a fusion antibody in which the former PpL single domain in an integrated state and the latter PpL single domains linked separately are mixed (for example, a structure of VH-PpL single domain-PpL single domain-VL-PpL single domain). A preferred example of the fusion antibody of the present disclosure is a fusion antibody in which the former PpL single domain in an integrated state is linked. The linker is as described below.

[0019] Antibodies in the present disclosure are not limited as long as they comprise a heavy chain variable region (VH) and / or a light chain variable region (VL). The heavy chain variable region (VH) generally comprises a heavy chain framework region (heavy chain FR) consisting of four heavy chain FR strands and a heavy chain complementarity determining region (heavy chain CDR) consisting of heavy chain CDR1 to CDR3. The light chain variable region (VL) comprises a light chain framework region (light chain FR) consisting of four light chain FR strands and a light chain complementarity determining region (light chain CDR) consisting of light chain CDR1 to CDR3. When the fusion antibody of the present disclosure comprises a VH and a VL, the VH and VL are linked directly or via a linker, or via a PpL single domain as described above. Linkers are described below in the same manner.

[0020] The amino acid sequence encoding the VH and the amino acid sequence encoding the VL may be derived from either a polyclonal antibody or a monoclonal antibody. Furthermore, the sequences may be amino acid sequences of animal origin (VH and VL sequences present in antibodies of animal origin) or non-animal origin. That is, the sequences may be naturally occurring sequences or artificial sequences produced according to procedures known in the field of genetic engineering, etc. Furthermore, in the VH and VL, both the CDR and FR may be animal-derived or non-animal-derived sequences, or only one may be animal-derived. Examples of animal origin include, but are not limited to, rabbit, mouse, rat, human, guinea pig, chicken, camel, and shark antibodies. Furthermore, the VH and VL may be derived from the same or different sources, preferably the same source. In the test examples described below, the VH and VL of an antibody targeting the antigen CRP are used, but the type of target antigen is not limited. Thus, in the present disclosure, the amino acid sequence encoding VH and / or the amino acid sequence encoding VL may be appropriately determined depending on the purpose.

[0021] In the present disclosure, antibodies include single chain antibodies (single chain Fv; scFv), scAb (single chain antibody), sdAb (single domain antibody, VHH, etc.), Fab, F(ab) ２ For example, in the case of an antibody such as Fab having a region other than VH and VL, each amino acid sequence other than VH and VL (e.g., each amino acid sequence of the heavy chain constant region (CH1-CH3), the light chain constant region (CL), etc.) may be a naturally occurring sequence or an artificial sequence, and will be explained in the same manner as above.

[0022] The PpL single domain may be linked to any part of the antibody. For example, the PpL single domain may be linked to the C-terminus or N-terminus of the antibody. Furthermore, the PpL single domain may be linked to the N-terminus or C-terminus of the VH constituting the antibody, or to the N-terminus or C-terminus of the VL, or may be linked between the VH and VL. Furthermore, when the antibody comprises a heavy chain constant region and / or a light chain constant region, the PpL single domain may be linked to the C-terminus or N-terminus of each of CH1 to CH3 and CL, or may be linked at one or more positions (e.g., 2, 3, or 4 positions) between these sequences. These are linked directly or via a linker. Linkers are described below in the same manner.

[0023] Although not limiting the present disclosure, one embodiment of the fusion antibody of the present disclosure is a fusion antibody in which a PpL single domain is linked to a single-chain antibody (PpL single-domain fused single-chain antibody). Single-chain antibodies are known as fusion proteins in which a VH and a VL are linked via a linker.

[0024] As mentioned above, the PpL single domain may be fused to any part of the single-chain antibody. Preferred examples of fusions include the following (a), (b), and (c): (a) an amino acid sequence encoding a PpL single domain is linked, directly or via a linker, to the C-terminus of the amino acid sequence encoding the single-chain antibody; (b) an amino acid sequence encoding a PpL single domain is linked, directly or via a linker, to the N-terminus of the amino acid sequence encoding the single-chain antibody; and (c) an amino acid sequence encoding a PpL single domain is linked, directly or via a linker, between the amino acid sequence encoding the VH and the amino acid sequence encoding the VL of the single-chain antibody.

[0025] More preferably, the above (a) or (c) is exemplified.

[0026] In these, the VH and VL constituting the single-chain antibody may be such that the VH is located closer to the N-terminus or C-terminus than the VL.

[0027] That is, in one embodiment, the positional relationship between the PpL single domain, VH, and VL in a PpL single domain-fused single chain antibody is exemplified as follows: (a1) From the N-terminus: VH, VL, PpL single domain (a2) From the N-terminus: VL, VH, PpL single domain (b1) From the N-terminus: PpL single domain, VH, VL (b2) From the N-terminus: PpL single domain, VL, VH (c1) From the N-terminus: VH, PpL single domain, VL (c2) From the N-terminus: VL, PpL single domain, VH

[0028] More preferably, the amino acid sequence encoding VH is located closer to the N-terminus than the amino acid sequence encoding VL. Therefore, the above (a1), (b1), and (c1) are more preferred examples. Also, the above (a1) and (c1) are more preferred examples.

[0029] In the above examples of single-chain antibodies, as described above, only one PpL single domain may be linked, or two or more PpL single domains may be linked together. Preferred examples include 1, 2, 3, or 4 PpL single domains linked in the above order.

[0030] Linker: In the present disclosure, the antibody and PpL single domain are linked directly or via a linker. The linker is not limited as long as it does not impair the effects of the present disclosure, and examples include a GS linker, a linker consisting only of glycine (G), a linker consisting only of serine (S), and an EAAAK linker. The length of the linker sequence is not particularly limited, and the number of amino acid residues in the linker is preferably 2 to 30, more preferably 3 to 25, and even more preferably 4 to 20. A preferred example of a GS linker is (GS)n, where n is an integer of 1 to 6, preferably 2 to 5, and even more preferably 2, 3, or 4. An example of an EAAAK linker is (EAAAK)n, where n is an integer of 1 to 6, preferably 2 to 5, and even more preferably 2, 3, or 4 (EAAAK is shown in SEQ ID NO: 8). A single linker may be used, or two or more types may be used in combination. In the present disclosure, the antibody and the PpL single domain are preferably linked via a linker. Furthermore, the fusion antibody of the present disclosure may have, in addition to the linker, an amino acid sequence such as a restriction enzyme site, a tag sequence (His tag, Myc tag, etc.), or a signal peptide, as long as the effect of the present disclosure is not impaired.

[0031] When a PpL single domain is linked between VH and VL as typified by (c) above, the VH and PpL single domain are linked directly or via a linker, and the VL and PpL single domain are linked directly or via a linker. In this case, it is also preferable that the VH and PpL single domain, and the VL and PpL single domain are both linked via a linker. While not limiting the present disclosure, an example of (c1) above where both are linked via a linker is as follows: VH, linker, PpL single domain, linker, VL from the N-terminus.

[0032] The fusion antibody of the present disclosure can be produced according to procedures known in the field of genetic engineering, etc. While not limiting the present disclosure, for example, the fusion antibody can be produced by constructing a polynucleotide containing a nucleotide sequence encoding a PpL single domain, a nucleotide sequence encoding a necessary region of the antibody (VH, VL, etc.), and, if necessary, a nucleotide sequence of a linker, etc., and transforming the polynucleotide into a host cell using a vector, and culturing and purifying the cell. In this process, the sequence of any substance, such as a restriction enzyme site, tag, or protein, may be introduced into the nucleotides as needed.

[0033] According to the present disclosure, by linking an antibody to a PpL single domain, the heat resistance of the antibody can be improved, and more preferably, the production yield, antigen-binding activity, and / or IgG antibody-binding activity of the soluble protein (solubilized antibody) can be further improved.

[0034] In the present disclosure, improved heat resistance can be determined according to the procedure described in the test example below, and as shown in the test example below, the heat treatment conditions may be 10 minutes at 80° C. Heat resistance is determined to be improved when the rate of reduction in the amount of fusion antibody (amount of desired protein) after heat treatment compared to before heat treatment is suppressed compared to the rate of reduction in the amount of antibody after heat treatment in the unfused antibody.

[0035] In the present disclosure, the production amount of soluble protein (soluble antibody) can be determined according to the procedure described in the test examples below. The supernatant and the soluble fraction (lysate) of the cells were obtained, and DCＴＭ This can be determined by quantifying the protein using a protein assay kit (manufactured by BIO-RAD) according to the manufacturer's instructions. If the amount of fusion antibody (protein amount) in the supernatant and intracellular soluble fraction of the fusion antibody is increased compared to the amount of antibody (protein amount) in the supernatant and intracellular soluble fraction of the unfused antibody, the amount of soluble protein (solubilized antibody) produced is considered to have improved.

[0036] In the present disclosure, the antigen-binding activity of an antibody can be determined according to the procedure described in the test example below, and can be determined by confirming the antigen-binding activity according to the procedure shown in the test example below (schematic diagram in FIG. 7). More specifically, the antigen-binding activity can be determined by confirming the antigen-binding activity according to the procedure shown in the test example below (schematic diagram in FIG. 7). ＴＭ Antigen activity can be confirmed by reacting the fused antibody with a fusion antibody (Thermo Fisher Scientific) followed by HRP-Mouse anti-6xHis IgG (9C11) and then by TMB. When the antigen-binding activity of the fused antibody is higher than that of the unfused antibody, the antigen-binding activity is considered to be improved.

[0037] In the present disclosure, the binding activity to an IgG antibody can be determined according to the procedure described in the test example below, and can be determined by confirming the binding activity to an IgG antibody according to the procedure shown in the test example below (schematic diagram in FIG. 8). More specifically, the binding activity to an IgG antibody can be determined by confirming the binding activity to an IgG antibody according to the procedure shown in the test example below (schematic diagram in FIG. 8). ＴＭ The binding activity to IgG antibodies can be confirmed by applying the fused antibody to a solid phase supported on a well (Thermo Fisher Scientific) followed by application of HRP-Mouse anti-6xHis IgG (9C11), and then application of TMB. If the binding activity of the fused antibody is higher than that of the unfused antibody, it is determined that the binding activity to IgG antibodies is improved.

[0038] Furthermore, as shown in the test examples described below, the present disclosure demonstrated that the desired antigen-binding activity was achieved by using the fusion antibody even when a human IgG antibody-supported solid phase was used. Thus, it can be said that the present disclosure further provides a PpL single domain fusion antibody that is useful in immunoassays using a human IgG antibody-supported solid phase.

[0039] Thus, according to the present disclosure, by linking a PpL single domain to an antibody, it is possible to provide an antibody with improved heat resistance. Furthermore, according to the present disclosure, it is possible to improve the production yield of a soluble protein (solubilized antibody), the antigen-binding activity of the antibody, and / or the binding activity of the antibody to IgG antibodies.

[0040] Since antibodies are proteins, their function as antibodies can be reduced by thermal denaturation. Improving heat resistance is useful for suppressing thermal denaturation, and therefore, the fusion antibodies of the present disclosure are easy to use in this regard.

[0041] Furthermore, in protein expression (particularly expression using host cells such as Escherichia coli), proteins are often expressed in an inactive state as proteins found not only in the resulting supernatant or soluble fraction (soluble proteins) but also in the insoluble fraction (insoluble proteins), and further denaturation treatments such as urea are required for insoluble proteins, making protein (antibody) purification and acquisition time-consuming. Improving the production (expression) amount of soluble proteins is useful for efficiently obtaining desired antibodies (fusion antibodies).

[0042] In immunoassays, for example, an antibody is supported on a support (e.g., a solid phase such as a well or plate) by directly (physical adsorption) or indirectly immobilizing the antibody to the support. The antibody is then further exposed to an antigen targeted by the antibody to cause an antigen-antibody reaction between the antibody and the antigen, or a substance capable of binding to the antibody is further exposed to the antibody to cause a binding reaction between the antibody and the substance, thereby detecting the antigen or target substance. In the case of indirect immobilization, a support to which a human IgG antibody has been immobilized in advance (a human IgG antibody-immobilized support) is often used. In this case, the antibody is supported on the support via the human IgG antibody. According to the present disclosure, as shown in the test examples described below, good antigen-binding activity was observed even when the antibody was directly or indirectly immobilized on a support, and good binding activity with the IgG antibody was observed. Therefore, the fusion antibody of the present disclosure, which has improved antigen-binding activity and / or improved binding activity with the IgG antibody, is useful for immunoassays, particularly immunoassays using IgG antibody-immobilized supports, which are frequently used today. Therefore, the fusion antibody of the present disclosure can be said to be a fusion antibody that can contribute to improving the sensitivity of antibody detection and immunoassay tests using various immunoassay carriers such as ELISA plates and nitrocellulose membranes. For these reasons, the fusion antibody of the present disclosure can be said to be useful for improving the detection efficiency and sensitivity of target substances such as antigens. In this disclosure, detection also includes the meaning of measurement.

[0043] Furthermore, the various characteristics (properties, structures, functions, etc.) described for each embodiment in this disclosure may be combined in any way to identify the subject matter encompassed by this disclosure, i.e., the present disclosure encompasses all subject matter consisting of any and all combinations of the combinable characteristics described herein.

[0044] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0045] Test Example 1 <Test Procedure> Production of PpL Single Domain Fusion Antibody In this test example, PpL1 was used as the PpL single domain, and a single-chain antibody was used as the antibody. The PpL1, single-chain antibody (unfused with PpL1), and PpL1-fused single-chain antibodies of Examples 1 to 4 used in this test example are as follows: Protein L Single Domain (PpL1) PpL1 encoded by the amino acid sequence represented by SEQ ID NO: 1 was used. Comparative Example 1: Unfused Single-Chain Antibody C2R (VH-VL) The single-chain antibody of Comparative Example 1 is a single-chain antibody in which a light-chain variable region (VL) encoded by the amino acid sequence represented by SEQ ID NO: 10 is linked via a GS linker to the C-terminus of a heavy-chain variable region (VH) encoded by the amino acid sequence represented by SEQ ID NO: 9. Comparative Example 2: Unfused Single-Chain Antibody C2R (VL-VH) The single-chain antibody of Comparative Example 2 is a single-chain antibody in which a VH encoded by the amino acid sequence of SEQ ID NO: 9 is linked via a GS linker to the C-terminus of a VL encoded by the amino acid sequence of SEQ ID NO: 10. Example 1: PpL1-VH-VL (C2R) The PpL1-fused single-chain antibody of Example 1 is a PpL1-fused single-chain antibody in which an amino acid sequence encoding PpL1 is linked via an SG linker to the N-terminus of the amino acid sequence encoding the single-chain antibody of Comparative Example 1. Example 2: VH-PpL1-VL (C2R) The PpL1-fused single-chain antibody of Example 2 is a PpL1-fused single-chain antibody in which an amino acid sequence encoding PpL1 is linked via a GS linker between the amino acid sequence encoding VH and the amino acid sequence encoding VL in the amino acid sequence encoding the single-chain antibody of Comparative Example 1. That is, the PpL1-fused single-chain antibody of Example 2 is a single-chain antibody in which an amino acid sequence encoding PpL1 is linked via a GS linker to the C-terminus of a VH encoded by the amino acid sequence shown in SEQ ID NO: 9, and a VL encoded by the amino acid sequence shown in SEQ ID NO: 10 is further linked via a GS linker to the C-terminus of the amino acid sequence. - Example 3: VH-VL-PpL1(C2R) The PpL1-fused single-chain antibody of Example 3 is a PpL1-fused single-chain antibody in which the amino acid sequence encoding PpL1 is linked via an SG linker to the C-terminus of the amino acid sequence encoding the single-chain antibody of Comparative Example 1.Example 4: PpL1-VL-VH(C2R) The PpL1-fused single-chain antibody of Example 4 is a PpL1-fused single-chain antibody in which the amino acid sequence encoding the single-chain antibody of Comparative Example 2 is linked to the N-terminus thereof via an SG linker with an amino acid sequence encoding the PpL1.

[0046] The PpL1, single-chain antibody (unfused with PpL1), and PpL1-fused single-chain antibody (Examples 1 to 4) were produced according to the following procedure. The following procedure follows a conventionally known method for general protein (antibody) expression and purification. The amino acid sequences of each protein produced are shown in Figures 1 to 3 (SEQ ID NOs: 11 to 17). The signal peptide and tag sequences in these figures are those used for protein purification and the like, as conventionally known in the art, and do not affect the evaluation results of heat resistance and the like, which will be described later.

[0047] The nucleotide sequences for each antibody produced were synthesized by a contract manufacturer. Figure 4 shows a schematic diagram of the linkage for the fusion antibodies of Examples 1 to 3. [Agar Culture] (1) 25 μL of C43(DE3) competent cells were thawed on ice, and 1 μL of an expression vector solution containing the contract-synthesized nucleotide sequence was added. The cells were then incubated on ice for 15 minutes. (2) Using a block incubator, the solution in (1) was heat-shocked at 42°C for 45 seconds and immediately cooled on ice. (3) The solution in (2) was plated on LB agar medium (Amp: final concentration 50 μg / mL) and cultured overnight at 37°C. [Preculture] (1) 10 mL of autoclaved 2xYT medium (Amp: final concentration 50 μg / mL) was added to a 50 mL Falcon tube. (2) A single colony on the LB agar plate was inoculated and cultured overnight at 30°C and 200 rpm to obtain a preculture solution. [Main Culture] (1) 50 mL of autoclaved Overnight Express medium (Amp: final concentration 50 μg / mL) was added to an autoclaved 500 mL baffled flask. (2) The preculture solution was added to achieve an OD600 of 0.1, and the mixture was cultured at 30°C, 200 rpm, and 24 hours. (3) The culture solution was collected in a 50 mL Falcon tube and centrifuged at 4°C, 13,420 g, and 20 minutes to separate and collect the culture supernatant and bacterial cells.

[0048] Purification [Preparation of intracellular soluble and intracellular insoluble fractions] (1) 50 mL of culture medium was centrifuged at 4°C, 13,420 g, and 20 min, and the supernatant was collected as the culture supernatant. The resulting cells were added with 5 mL of 1% Triton-100-PBS, vortexed, and then sonicated on ice at Output 3, Duty 30, and 30 min. (2) The mixture was centrifuged at 4°C, 13,420 g, and 20 min, and the supernatant was collected as the intracellular soluble fraction. (3) The precipitate obtained in (2) was washed with an equal volume of 1x PBS and centrifuged at 4°C, 20,000 g, and 10 min, and the supernatant was removed. This process was repeated once more. An equal volume of 8M urea-PBS was added to the cell lysate, vortexed, and centrifuged at 4°C, 20,000 g, and 15 min. The supernatant was collected as the insoluble fraction. (4) 400 μL of elution buffer (250 mM imidazole, 2×PBS, pH 8.0) was added to 5 mL of the intracellular soluble fraction obtained in (2). (5) 400 μL of elution buffer (250 mM imidazole, 8M urea, 2×PBS, pH 8.0) was added to 5 mL of the intracellular insoluble fraction obtained in (3). (6) The solution obtained in (4) or (5) was filtered using a syringe filter. The solution derived from the intracellular soluble fraction (4) (lysate) and the solution derived from the intracellular insoluble fraction (5) (insulable protein) were used as samples for the production test described below. [Preparation of culture supernatant] (1) 50 mL of the culture supernatant obtained in (1) of [Preparation of intracellular soluble fraction and intracellular insoluble fraction] above was mixed with 5 mL of 10x binding buffer (200 mM Imidaole, 10% Triton-100, 2x PBS, pH 8.0), and the mixture was centrifuged at 4°C, 10,000 g, and 20 minutes to recover the supernatant. The supernatant (Supernatant) obtained here was used as a sample for the production test described below.

[0049] [Purification of PpL1-fused scFv expressed in the culture supernatant and the intracellular soluble fraction] (1) An open column packed with 1 mL of Ni-NTA agarose was washed three times with 5 CV of ultrapure water. (2) Binding buffer (2x PBS, 20 mM imidazole, pH 8.0) was applied to the column three times with 5 CV to equilibrate the column. (3) The sample was applied to the column and collected as FT. (4) Binding buffer was applied three times with 5 CV, and 5 mL was collected as Wash. (5) Elution buffer (2x PBS, 250 mM imidazole, pH 8.0) was applied five times with 1 CV, and collected as Elution. (6) A280 was measured using a Nanodrop™ system. Fractions showing an absorption peak were collected and dialyzed overnight at 4°C against 1x PBS. [Purification of PpL1-fused scFv expressed in the intracellular insoluble fraction] (1) An open column packed with 1 mL of Ni-NTA agarose was washed three times with 5 CV of ultrapure water. (2) Binding buffer (8 M urea-2xPBS, 20 mM imidazole, pH 8.0) was applied to the column three times with 5 CV to equilibrate the column. (3) The sample was applied to the column and collected as FT. (4) The column was washed with 5 CV of binding buffer three times, and 5 mL was collected as Wash. (5) Elution buffer (8 M urea-2xPBS, 250 mM imidazole, pH 8.0) was applied five times with 1 CV and collected as Elution. (6) A280 was measured using a Nanodrop filter. Fractions showing an absorption peak were collected and dialyzed overnight at 4°C against 8 M urea-1xPBS.

[0050] Heat Stability and Solubilized Antibody Production Amount The heat resistance and production amount of fusion antibodies were evaluated according to the following procedure. The results are shown in Figures 5 and 6. [Heat Treatment] (1) 500 μg / mL purified PpL1-fused C2R, VH-VL(C2R), and VL-VH(C2R) (Examples 1 to 4, Comparative Examples 1 and 2, culture supernatant and soluble fraction) were diluted with 1x PBS to a concentration of 250 μg / mL. PpL1 was purified and diluted in the same manner. (2) The solution in (1) was incubated at 80°C for 10 minutes and then heat-treated. (3) The solution was centrifuged at 4°C, 20,000 g, and 10 minutes, and the supernatant was collected.

[0051] [SDS-PAGE] (1) 50 μL of APS and 5 μL of TEMED were added to the 12% separation gel solution and poured onto a glass plate. 200 μL of butanol was added to remove bubbles, and the mixture was incubated at room temperature (25°C) for 40 min until the 12% separation gel solution polymerized. (2) After polymerization, the butanol was removed, and 25 μL of APS and 5 μL of TEMED were added to the 4% stacking gel solution. The mixture was poured up to the top of the glass plate. A comb was inserted to prevent air bubbles from being trapped, and the mixture was incubated at room temperature for 40 min until the 4% stacking gel solution polymerized. (3) Each sample was incubated at 98°C for 10 min with 2x SDS sample buffer containing DTT at a volume ratio of 1:1. The samples used were Examples 1-4, Comparative Examples 1-2, and PpL1 before heat treatment, and Examples 1-4, Comparative Examples 1-2, and PpL1 after heat treatment. (4) 5 μL each of the molecular weight markers and each sample (15-well comb) was applied to the wells, and electrophoresis was performed in 1x running buffer at 200 V, 2.00 A, and constant V. (5) After electrophoresis, the gel was peeled off from the glass plate. (6) The gel was washed with ion-exchanged water and incubated in approximately 30 mL of fixative solution at room temperature for 30 min. (7) The gel was washed with ion-exchanged water and incubated in approximately 30 mL of CBB staining solution at room temperature for 1 h. (8) The gel was washed with ion-exchanged water and incubated in approximately 30 mL of destaining solution at room temperature overnight. (9) The stained and destained gel was photographed using a Typhoon FLA 9000.

[0052] [Protein quantification] BIO-RAD DC for protein quantification TMA protein assay kit was used. Microassay method: (1) 20 μL of Protein Assay Reagent S was added to 1 ml of Protein Assay Reagent. (2) 25 μL of solution (1) was added to 50 μL of sample and 0-200 μg / ml BSA solution. (3) 200 μL of Protein Assay Reagent B was added to (2) and incubated at 25°C for 15 minutes. (4) The absorbance at a wavelength of 750 nm was measured using a microplate. (5) A calibration curve was created from the measurement results of the BSA solution, and the protein concentration was quantified.

[0053] Antigen-binding activity: The antigen-binding activity of the fusion antibody was evaluated according to the following procedure. A schematic diagram of the reaction and the results are shown in Figure 7. (1) 100 μL of 10 μg / mL rCRP-PBS was added to Nunc Maxisorp TM　 (Thermo Fisher Scientific) and incubated overnight at 4°C. (2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. (3) After washing five times with PBST, 100 μL of 0.2% BSA-PBST containing 0-2 μg / mL of Examples 1-4 or Comparative Examples 1-2 was added to each well and incubated at 25°C for 1 hour. (4) After washing five times with PBST, 100 μL of 10,000-fold diluted HRP-Mouse anti-6×His IgG (9C11)-100 μg / mL Human IgG-0.2% BSA-PBST was added to each well and incubated at 25°C for 1 hour. (5) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for 15 minutes. (6) The reaction was stopped by adding 100 μL of 0.5 M H2SO4 to each well, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0054] The binding activity of the fusion antibody to IgG antibody was evaluated according to the following procedure. The reaction scheme and results are shown in Figure 8. (1) 100 μL of 10 μg / mL Human IgG-PBS was added to a Nunc Maxisorp TM　 (Thermo Fisher Scientific) and incubated overnight at 4°C. (2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. (3) After washing five times with PBST, 100 μL of 0.2% BSA-PBST containing Examples 1 to 4 or Comparative Examples 1 to 2 at 0 to 10 μg / mL was added to each well and incubated at 25°C for 1 hour. (4) After washing five times with PBST, 100 μL of 10,000-fold diluted HRP-Mouse anti-6×His IgG (9C11) (trade name: human γ-globulin derived from plasma (Fujifilm Wako, 075-06691))-0.2% BSA-PBST was added to each well and incubated at 25°C for 1 hour. (5) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for 15 minutes. (6) The reaction was stopped by adding 100 μL of 0.5 M H2SO4 to each well, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0055] Antigen-binding activity upon direct immobilization / indirect immobilization Furthermore, the antigen-binding activity of the fusion antibodies of Examples 1 to 3 was evaluated when they were directly immobilized to a solid phase and when they were indirectly immobilized (to an IgG antibody-carrying solid phase) according to the following procedure.

[0056] [Measurement of antigen binding activity of direct immobilization (physical adsorption of PpL1-fused C2R)] (1) 100 μL of 2 μg / mL PpL1-fused C2R (Examples 1 to 3) in PBS was added to each well of a Maxisorp plate and incubated overnight at 4°C. (2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. (3) After washing five times with PBST, 100 μL of 0-10 μg / mL Bt-rCRP was added to each well and incubated at 25°C for 1 hour. (4) After washing five times with PBST, 100 μL of 5000x HRP-SA-0.2%-BSA-PBST was added to each well and incubated at 25°C for 1 hour. (5) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well, and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for 15 minutes. (6) The reaction was stopped by adding 100 μL of 0.5 M H2SO4 to each well, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0057] [Antigen detection using indirect immobilization (PpL1-fused C2R indirect immobilization plate)] (1) 100 μL of 10 μg / mL human IgG-PBS was added to each well of a Maxisorp plate and incubated overnight at 4°C. (2) After washing five times with PBS, 300 μL of 2% BSA-PBS was added to each well and incubated at 25°C for 1 hour. (3) After washing five times with PBST, 100 μL of 2 μg / mL PpL1-fused C2R (Examples 1 to 3)-0.2% BSA-PBST was added to each well and incubated at 25°C for 1 hour. (4) After washing five times with PBST, 100 μL of 0-10 μg / mL Bt-rCRP-0.2% BSA-PBST was added to each well and incubated at 25°C for 1 hour. (5) After washing five times with PBST, 100 μL of 5000-fold diluted HRP-SA-0.2%-BSA-PBST was added to each well and incubated at 25°C for 1 hour. (6) After washing five times with PBST, 100 μL of pre-warmed TMB was added to each well and the absorbance (main wavelength: 650 nm, secondary wavelength: 450 nm) was measured over time using a microplate reader for 15 minutes. (7) The reaction was stopped by adding 100 μL of 0.5M H2SO4 to each well and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0058] <Results> The results of the heat resistance evaluation are shown in Figure 5. As can be seen from the values ​​of the amount after heat treatment (Conc. after heat treatment) in Figure 5, the PpL1-fused single-chain antibodies of Examples 1 to 4 (hereinafter sometimes referred to as fusion antibodies) had higher values ​​than the PpL1-unfused single-chain antibodies of Comparative Examples 1 and 2 (hereinafter sometimes referred to as unfused antibodies), indicating improved heat resistance. This demonstrates that the fusion antibodies were endowed with heat resistance.

[0059] The results of the evaluation of production yield are shown in Figure 6. For the evaluation of production yield, samples before heat treatment were used. As shown in Figure 6, the fusion antibodies of Examples 1 to 3 could be produced even when an expression system using E. coli was used. In particular, the production yield of soluble protein was improved in Examples 2 and 3 compared to Comparative Example 1 (Soluble protein (Supernatant + Lysate)). Furthermore, although not shown in Figure 6, the production yield of soluble protein was also improved in Example 4 compared to Comparative Example 2. Thus, it was found that PpL1-fused single-chain antibodies can be expressed and produced using procedures similar to those known in the art, and that the production yield of antibodies (particularly the production yield of soluble protein) can be improved for the fusion antibodies shown in Examples 2 to 4.

[0060] The results of evaluating the antigen-binding activity of the fusion antibodies are shown in Figure 7. As shown in Figure 7, antigen-binding activity was observed in all of Examples 1 to 4. In particular, antigen-binding activity was improved in Examples 2 and 3 (VH-PpL1-VL, VH-VL-PpL1 in the figure) compared to Comparative Example 1 (C2R(VH-VL) in the figure). From this, it was inferred that, in order to enhance antigen-binding activity, it is desirable to link an amino acid sequence encoding a PpL single domain between the amino acid sequence encoding the heavy chain variable region and the amino acid sequence encoding the light chain variable region of the antibody, as in Example 2, or to link an amino acid sequence encoding a PpL single domain to the C-terminus of the amino acid sequence encoding the antibody, as in Example 3.

[0061] The results of evaluating the binding activity to IgG antibodies are shown in Figure 8. As shown in Figure 8, Comparative Example 1 (C2R(VH-VL) in the figure) did not exhibit binding activity to IgG antibodies, whereas the fusion antibodies of Examples 1 to 4 exhibited binding activity to human IgG antibodies. Conventionally, immunoassays often utilize supports to which IgG antibodies have been immobilized in advance (human IgG antibody-immobilized supports). For example, a simple procedure involves allowing a desired antibody to act on a human IgG antibody-immobilized support to bind to the human IgG antibody, and then further allowing an antigen targeted by the desired antibody to act on the support to induce an antigen-antibody reaction between the desired antibody and the antigen. As described above, the fusion antibodies of Examples 1 to 4 exhibited binding activity to IgG antibodies, demonstrating that PpL single domain fusion antibodies are useful for detecting target substances in immunoassays.

[0062] Furthermore, we investigated whether the fusion antibody maintained its antigen-binding activity when bound to a solid phase (Figures 9 and 10), and as shown in Figure 10, the antigen-binding activity of the fusion antibody was observed when bound to a solid phase, both in the case of direct immobilization (physical adsorption) and indirect immobilization (human IgG binding). This also demonstrated that the PpL1 fusion antibody is useful in immunoassays.

[0063] 8 and 10 , it is known that some human IgG antibodies have the ability to bind to Protein L. Therefore, it can be said that IgG antibodies immobilized on a solid phase have the ability to bind to Protein L single domains. This test example suggests that the binding of the IgG antibody to a Protein L single domain linked to the antibody allows the fusion antibody to be indirectly immobilized on an IgG antibody-supported solid phase, thereby enabling high-density and / or highly oriented antibody immobilization on the solid phase while maintaining the antibody's antigen-binding ability, resulting in favorable antigen-binding activity. This suggests that fusing a PpL single domain to an antibody is a useful means for immobilizing a desired antibody on an IgG antibody (especially a human IgG antibody) immobilization support, which is frequently used in immunoassays. This also indicates that PpL single domain fusion antibodies are useful for increasing antibody density and maintaining high antigen-binding activity through oriented adsorption immobilization.

[0064] Furthermore, although not shown in the results, a PpL1-fused single-chain antibody was similarly produced using a single-chain antibody C1R with different VH and VL amino acid sequences instead of the single-chain antibody C2R. This PpL1-fused single-chain antibody also showed improved heat resistance, and similar trends in improved productivity, antigen-binding activity, and IgG-binding activity.

Claims

1. A PpL single domain fusion antibody in which a protein L single domain (PpL single domain) and an antibody are linked directly or via a linker.

2. The PpL single domain fusion antibody according to claim 1, wherein an amino acid sequence encoding a PpL single domain is linked, either directly or via a linker, to the C-terminus of the amino acid sequence encoding the antibody, or wherein an amino acid sequence encoding a PpL single domain is linked, either directly or via a linker, between the amino acid sequence encoding the heavy chain variable region and the amino acid sequence encoding the light chain variable region of the antibody.

3. The PpL single domain fusion antibody according to claim 1, wherein the amino acid sequence encoding the light chain variable region is located C-terminally to the amino acid sequence encoding the heavy chain variable region.

4. The antibody is an scFv, scAb, sdAb, Fab, F(ab) ２ ' or a full-length antibody.

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