Protein l single domain fusion antibodies

Abstract

To provide an antibody having improved heat resistance.SOLUTION: 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This relates to Protein L single domain fusion antibodies. [Background technology]

[0002] Immunoassays are methods that use antibodies to specifically detect target substances. In immunoassays, for example, the 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. ELISA is a typical immunoassay (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. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] ELISA kit, Cosmo Bio Co., Ltd. website (product information, ELISA kit), https: / / www.cosmobio.co.jp / product / detail / 00780001.asp?entry_id=2723 Summary of the Invention [Problem to be solved by the invention]

[0004] The object is to provide an antibody with improved heat resistance. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors have found that the heat resistance of an antibody can be improved by linking a protein L single domain (PpL single domain) to the antibody. They have also found that linking a PpL single domain to the 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) and an antibody are linked directly or via a linker. Item 2. 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; 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; A PpL single domain fusion antibody according to Item 1. Item 3. 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 C-terminally to the amino acid sequence encoding the heavy chain variable region. Item 4. The antibody according to any one of Items 1 to 3, wherein the antibody is an scFv, scAb, sdAb, Fab, F(ab)2', or full-length antibody. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0007] [Figure 1]The amino acid sequences (SEQ ID NOs: 11 to 13) of the PpL single domain used in the test examples and the PpL single domain-unfused antibodies of Comparative Examples 1 and 2 are shown. [Figure 2] 1 shows the amino acid sequences (SEQ ID NOs: 14 and 15) for the PpL single domain fusion antibodies of Examples 1 and 2. [Figure 3] 1 shows the amino acid sequences (SEQ ID NOs: 16 and 17) for the PpL single domain fusion antibodies of Examples 3 and 4. [Figure 4] 1 shows the sequence linkage order (schematic diagram) of the PpL single domain, VH and VL in Examples 1 to 3. [Figure 5] The results of the test example (heat resistance) are shown below. [Figure 6] The results of the test example (amount of soluble protein produced) are shown below. [Figure 7] The results of the test (antigen binding activity) are shown. [Figure 8] The results of the test example (binding activity to IgG antibody) are shown. [Figure 9] Schematic diagram of direct immobilization (physical adsorption) or indirect immobilization (immobilization via IgG antibody) of an antibody to a carrier. [Figure 10] The results of the test example (antigen binding activity when the antibody is directly or indirectly immobilized on the carrier) are shown. DETAILED DESCRIPTION OF THE INVENTION

[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 from various amino acid sequence databases, etc. Although not limiting to 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, 4, and 5, respectively. The PpL single domain in the present disclosure may be any single domain. The PpL single domain may be wild-type (naturally occurring) or may be a mutated version of the wild-type. Preferred examples of the PpL single domain include the Protein L C1 domain (PpL1) and the Protein L C3 domain, more preferably PpL1, and even more preferably PpL1 encoded by the amino acid sequence shown in 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 resulting 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 resulting antibody has improved production (expression) amount of soluble protein (soluble antibody), antigen-binding activity, and / or binding activity to IgG antibodies compared to the unfused antibody. Even more preferably, the amino acid sequence may contain a mutation to the extent that the resulting antibody has improved heat resistance and binding activity to IgG antibodies. Whether or not heat resistance is improved, as well as 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 and the like 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) Amino acid sequence represented by SEQ ID NO: 1 (2) An amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence described in (1) above.

[0014] In (2), "plurality" is exemplified by 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 amino acids after mutation may be, for example, either 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), valine (V), etc. Techniques for deleting, substituting, inserting, and / or adding one or more amino acids in a specific amino acid sequence are known.

[0015] Furthermore, although 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. Furthermore, 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 production amount of soluble protein, antigen-binding activity, and / or binding activity to IgG antibodies 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. 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, it is preferable that 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 each other directly or via a linker to form an aggregated structure (i.e., a single-chain amino acid sequence in which multiple PpL single domains are linked directly or via a linker), and may be linked to the antibody directly or via a linker, or each PpL single domain may be linked to a separate site on the antibody directly or via a linker. For ease of understanding, and without limiting the present disclosure, simple examples of fusion antibodies comprising two PpL single domains, which are examples of the former aggregated structure, 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] antibody In the present disclosure, antibodies 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 sequences. Examples of animal origins 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, an antibody may be any of a single chain antibody (single chain Fv; scFv), scAb (single chain antibody), sdAb (single domain antibody, VHH, etc.), Fab, F(ab)2', full-length antibody (whole antibody), etc. 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), light chain constant region (CL), etc.) may be a naturally occurring sequence or an artificial sequence, and is 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 contains 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 described above, the PpL single domain may be fused to any part of the single-chain antibody. In one embodiment, the following fusion (a), (b), or (c) is preferably exemplified: (a) An amino acid sequence encoding a PpL single domain is linked, directly or via a linker, to the C-terminus of an amino acid sequence encoding a 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. (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 either N-terminally or C-terminally relative to the VL.

[0027] That is, as one embodiment, the following order is exemplified as the positional relationship between the PpL single domain, VH, and VL in the PpL single domain-fused single-chain antibody. (a1) From the N-terminus, VH, VL, and PpL single domains (a2) From the N-terminus, VL, VH, and PpL single domains (b1) From the N-terminus, the PpL single domain, VH, and VL are (b2) From the N-terminus, the PpL single domain, VL, and VH (c1) From the N-terminus, VH, PpL single domain, and VL (c2) From the N-terminus, VL, PpL single domain, and 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 mentioned 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 thereof 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 linker preferably has 2 to 30 amino acid residues, more preferably 3 to 25, and even more preferably 4 to 20. A preferred example of the 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 the 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). One type of linker may be used alone, 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 also contain, in addition to the linker, amino acid sequences such as a restriction enzyme site, a tag sequence (His tag, Myc tag, etc.), or a signal peptide, as long as the effects of the present disclosure are 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, too, it is 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 VH, linker, PpL single domain, linker, and VL in this order 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 for 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 nucleotide sequence 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 binding activity to IgG antibodies 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 80°C for 10 minutes. 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 collected and subjected to DC TM This can be determined by quantifying the protein using a protein assay kit (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 using a solid phase (Nunc Maxisorp) to which the antigen rCRP is immobilized. TM Antigen activity can be confirmed by treating the fused antibody with IgG (Thermo Fisher Scientific), followed by HRP-Mouse anti-6×His 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). TMThe 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-6×His 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 has been 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. Therefore, it can be said that the present disclosure also 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 the 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, when expressing proteins (particularly those expressed using host cells such as E. coli), the proteins are often expressed not only in the resulting supernatant or soluble fraction (soluble proteins), but also in the insoluble fraction (insoluble proteins) in an inactive state, which requires further denaturation treatment using urea or the like, making protein (antibody) purification and acquisition time-consuming. Increasing the production (expression) amount of soluble proteins is useful for efficiently obtaining desired antibodies (fusion antibodies).

[0042] Furthermore, in immunoassays, for example, an antibody is supported on a support (e.g., a solid phase such as a well or plate) by directly immobilizing (physical adsorption) or indirectly immobilizing the antibody to the support, and then the antibody is further treated with 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 treated with 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 (human IgG antibody-immobilized support) is often used, and 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 and good binding activity with IgG antibodies were observed even when the antibody was directly or indirectly immobilized on a support. Therefore, the fusion antibody of the present disclosure, which has improved antigen-binding activity and / or improved binding activity with IgG antibodies, 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. [Example]

[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 antibodies 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 shown in 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 to the C-terminus of a heavy chain variable region (VH) encoded by the amino acid sequence represented by SEQ ID NO: 9 via a GS linker. 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 VH encoded by the amino acid sequence represented by SEQ ID NO: 9 is linked to the C-terminus of VL encoded by the amino acid sequence represented by SEQ ID NO: 10 via a GS linker. Example 1: PpL1-VH-VL(C2R) The PpL1-fused single-chain antibody of Example 1 is a PpL1-fused single-chain antibody in which the amino acid sequence encoding the PpL1 is linked to the N-terminus of the amino acid sequence encoding the single-chain antibody of Comparative Example 1 via an SG linker. Example 2: VH-PpL1-VL(C2R) The PpL1-fused single-chain antibody of Example 2 is a PpL1-fused single-chain antibody in which the 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 the amino acid sequence encoding PpL1 is linked, via a GS linker, to the C-terminus of VH encoded by the amino acid sequence shown in SEQ ID NO: 9, and 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 the PpL1 is linked to the C-terminus of the amino acid sequence encoding the single-chain antibody of Comparative Example 1 via an SG linker. Example 4: PpL1-VL-VH(C2R) The PpL1 fusion single-chain antibody of Example 4 is a PpL1 fusion single-chain antibody in which the amino acid sequence encoding the PpL1 is linked to the N-terminus of the amino acid sequence encoding the single-chain antibody of Comparative Example 2 via an SG linker.

[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 conventionally known general protein (antibody) expression and purification techniques. 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] ·production The nucleotide sequences of each antibody were synthesized by a contract manufacturer. Figure 4 shows a schematic diagram of the linkages 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 a custom-synthesized base sequence was added, followed by incubation 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 from (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 flask with baffles. (2) The preculture solution was added so that OD600 became 0.1, and the mixture was cultured at 30°C, 200 rpm, and 24 hours. (3) The culture medium 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 fraction and intracellular insoluble fraction] (1) 50 mL of the culture medium was centrifuged at 4°C, 13,420 g, and 20 min, and the supernatant was collected as the culture supernatant. 5 mL of 1% Triton-100-PBS was added to the resulting cells, 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 to the cell lysate, 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 to the cell lysate was added, vortexed, and centrifuged at 4°C, 20,000 g, and 15 min, and 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 soluble intracellular fraction obtained in (2). (5) 400 μL of elution buffer (250 mM imidazole, 8 M 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 resulting solution (lysate) derived from the intracellular soluble fraction (4) and the solution (insoluble protein) derived from the intracellular insoluble fraction (5) 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 the above [Preparation of Intracellular Soluble Fraction and Intracellular Insoluble Fraction] was mixed with 5 mL of 10x Binding buffer (200 mM Imidaole, 10% Triton-100, 2x PBS, pH 8.0), and centrifuged at 4°C, 10,000 g, and 20 minutes to recover the supernatant. The resulting supernatant (Supernatant) was used as a sample for the production test described below.

[0049] [Purification of PpL1-fused scFv expressed in the culture supernatant and the soluble fraction within the cells] (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 at 5 CV to equilibrate the column. (3) The sample was applied to the column and collected as FT. (4) Binding buffer was added 5 CV x 3 to wash the column, and 5 mL was collected as wash. (5) Elution buffer (2x PBS, 250 mM imidazole, pH 8.0) was added 1 CV x 5 times, and the eluate was collected. (6) A280 was measured using a Nanodrop, and fractions showing an absorption peak were collected and dialyzed overnight in 1x PBS at 4°C. [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-2×PBS, 20 mM imidazole, pH 8.0) was applied to the column three times at 5 CV to equilibrate the column. (3) The sample was applied to the column and collected as FT. (4) Binding buffer was added 5 CV x 3 to wash the column, and 5 mL was collected as wash. (5) Elution buffer (8 M urea-2×PBS, 250 mM imidazole, pH 8.0) was added 1 CV x 5 times, and the eluate was collected. (6) A280 was measured using a Nanodrop, and fractions showing an absorption peak were collected and dialyzed overnight at 4°C in 8M urea-1xPBS.

[0050] Heat resistance and soluble antibody production The heat resistance and production amount of the fusion antibody were evaluated according to the following procedure, and the results are shown in Figures 5 and 6. [Heat treatment] (1) After purification, 500 μg / mL of PpL1-fused C2R, VH-VL(C2R), and VL-VH(C2R) (Examples 1 to 4, Comparative Examples 1 and 2, culture supernatants, and soluble fractions) was diluted with 1×PBS to a concentration of 250 μg / mL. PpL1 was also purified and diluted in the same manner. (2) The solution in (1) was incubated at 80°C for 10 minutes for heat treatment. (3) Centrifugation was carried out 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, poured onto a glass plate, and then 200 μL of butanol was added on top to remove bubbles. The solution was then incubated at room temperature (25°C) for 40 minutes until the 12% separation gel solution polymerized. (2) After polymerization, the overlaying 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 minutes until the 4% stacking gel solution polymerized. (3) Each sample was incubated at 98°C for 10 minutes with a DTT-containing 2xSDS sample buffer at a volume ratio of 1:1. The samples used were Examples 1 to 4, Comparative Examples 1 and 2, and PpL1 before heat treatment, and Examples 1 to 4, Comparative Examples 1 and 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 carried out in 1× running buffer under conditions of 200 V, 2.00 A, and V constant. (5) After the electrophoresis was completed, 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 fixation solution at room temperature for 30 minutes. (7) The gel was washed with ion-exchanged water and incubated in approximately 30 mL of CBB staining solution at room temperature for 1 hour. (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 TM A 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 the solution in (1) was added to 50 μL of the sample and 0 to 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 following reaction and the results are shown in Figure 7. (1) Add 100μL of 10μg / mL rCRP-PBS to Nunc Maxisorp TM (Thermo Fisher Scientific) was added to each well (bovine serum albumin BSA block) 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 2 μg / mL was added to each well, followed by incubation 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) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0054] IgG antibody binding activity The binding activity of the fusion antibody to an IgG antibody was evaluated according to the following procedure. A schematic diagram of the reaction and the results are shown in Figure 8. (1) Add 100μL of 10μg / mL Human IgG-PBS 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 Examples 1 to 4 or Comparative Examples 1 to 2 at 0 to 10 μg / mL was added to each well, followed by incubation 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 the plate was 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 up to 15 minutes. (6) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

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

[0056] [Antigen binding activity measurement of direct immobilization (physical adsorption of PpL1-fused C2R)] (1) 100 μL of 2 μg / mL PpL1-fused C2R (Examples 1 to 3)-PBS was added to each well of Maxisorp and incubated at 4° C. overnight. (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 to 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) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, 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 Maxisorp 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 to 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) 100 μL of 0.5 M H2SO4 was added to each well to stop the reaction, and the absorbance (main wavelength: 450 nm, secondary wavelength: 650 nm) was measured using a microplate reader.

[0058] <Result> 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 have been conferred 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 in the same manner as conventionally known procedures, and in particular, the fusion antibodies shown in Examples 2 to 4 can improve the production yield of antibodies (particularly the production yield of soluble protein).

[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). 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 the IgG antibodies immobilized on the solid phase have the ability to bind to Protein L single domains. This test example suggests that the fusion antibody can be indirectly immobilized on the IgG antibody-supported solid phase through binding between the Protein L single domain linked to the antibody and the IgG antibody, thereby enabling high density and / or high orientation control of the antibody on the solid phase while maintaining the antibody's antigen-binding ability, resulting in good antigen-binding ability. 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 production yield, 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. 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; 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; The PpL single domain fusion antibody according to claim 1.

3. 2. 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 selected from the group consisting of scFv, scAb, sdAb, Fab, F(ab) ２ ' or a full-length antibody.

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