Modified antibody

Abstract

This invention provides a novel antibody with improved thermal stability. [Solution] A modified antibody comprising a polypeptide having a sequence in which at least a portion of amino acid residues are deleted in the hinge region of the antibody.

Description

Technical Field

[0001] The present invention relates to modified antibodies.

Background Art

[0002] Generally, when preparing a reagent using an antibody, three properties are required: affinity, specificity, and stability. Among them, high thermal stability is important to ensure long-term stability. Therefore, conventionally, attempts have been made to improve the thermal stability of antibodies by modifying the amino acid sequence of the antibody. For example, in Patent Document 1, in the heavy chain of an antibody, at least one amino acid residue selected from the group consisting of the 8th to 11th amino acid residues based on the Kabat method and at least one amino acid residue selected from the group consisting of the 109th and 110th amino acid residues based on the IMGT method are replaced with cysteine residues, thereby obtaining an antibody with improved thermal stability.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a novel antibody with improved thermal stability.

Means for Solving the Problems

[0005] To solve the above problems, as a result of intensive research, the inventors have found that by making a polypeptide consisting of a sequence in which some amino acid residues are deleted in the hinge region of an antibody, the thermal stability of the antibody is improved.

[0006] This invention was completed based on these findings and includes the following broad embodiments of the invention. [Section 1] A modified antibody containing a polypeptide consisting of a sequence in which at least some amino acids are deleted in the hinge region of the antibody. [Section 2] The modified antibody described in item 1, which is a mouse IgG2b modified antibody. [Section 3] A modified antibody as described in item 2, comprising a polypeptide consisting of a sequence in which 5 to 10 amino acids have been deleted from the amino acid sequence of the hinge region of mouse IgG2b. [Section 4] A modified antibody according to item 2, comprising a polypeptide having a sequence in which at least a portion of any of the following amino acid sequences (i) to (iii) is deleted in the hinge region. (i) The amino acid sequence represented by Sequence ID No. 1 (EPSGPISTINPCPPCKECHKCP). (ii) An amino acid sequence in which one or more amino acids are substituted or added to the amino acid sequence represented by Sequence ID No. 1. (iii) An amino acid sequence having 85% or more identity with the amino acid sequence represented by Sequence ID No. 1. [Section 5] A modified antibody according to item 4, comprising a polypeptide having a sequence in which the second P from the N-terminus of SEQ ID NO: 1 is retained and 5 to 10 amino acids are deleted. [Section 6] A modified antibody as described in item 2, comprising one of the polypeptides (1) to (3) below in the hinge region. (1) A polypeptide containing the amino acid sequence represented by Sequence ID No. 2 (EPNPCPPCKECHKCP). (2) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted or added in the amino acid sequence represented by Sequence ID No. 2. (3) A polypeptide containing an amino acid sequence that has 85% or more identity with the amino acid sequence represented by Sequence ID No. 2. [Section 7] A modified antibody as described in any one of items 1 to 6, wherein the denaturation midpoint temperature Tm1 of the modified antibody is 3°C or higher than the denaturation midpoint temperature Tm2 of the unmodified antibody. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a novel antibody with improved thermal stability. [Brief explanation of the drawing]

[0008] [Figure 1] The amino acid sequence of the hinge region of the modified antibody in the example is shown. [Modes for carrying out the invention]

[0009] In this specification, the term "hinge region" refers to the region between the CH1 and CH2 regions in the heavy chain of an immunoglobulin. This region is rich in proline, does not form an α-helix, and contributes to the mobility of the structure consisting of the heavy chain variable region and the CH1 region. The hinge region is, for example, the region from position 226 to 243 in the constant region of the mouse IgG2b heavy chain according to Kabat numbering.

[0010] The present invention provides a modified antibody. The modified antibody of the present invention comprises a polypeptide having a sequence in which at least a portion of amino acid residues are deleted in the hinge region of the antibody. Here, an antibody having the amino acid sequence before the deletion of amino acid residues in the hinge region is referred to as an "unmodified antibody".

[0011] The unmodified antibody and the modified antibody of this embodiment may be antibodies that recognize any of the antigens. Furthermore, the unmodified antibody may be an antibody with a natural amino acid sequence (wild-type antibody) or an artificially produced antibody. An artificially produced antibody refers to an antibody whose amino acid sequence has been artificially altered by means other than heavy chain modification in this embodiment. Examples of such antibodies include antibodies with altered CDR amino acid sequences, chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0012] The unmodified antibody and the modified antibody of the present embodiment may be polyclonal antibodies or monoclonal antibodies as long as they bind to the desired antigen, but monoclonal antibodies are preferred in terms of being able to stably produce homogeneous antibodies.

[0013] The unmodified antibody and the modified antibody of the present embodiment may be antibodies derived from any animal. Such animals are preferably mammals, and examples include mice, rabbits, alpacas, rats, pigs, sheep, goats, camels, cows, horses, humans, etc. Among them, mice, rabbits and alpacas are preferred, and mice are more preferred.

[0014] The class of the unmodified antibody and the modified antibody of the present embodiment may be any of IgG, IgA, IgD, but preferably IgG. As a subclass of IgG, IgG2b is preferred.

[0015] The unmodified antibody may be an antibody fragment or an antibody modification as long as it has a polypeptide in the hinge region. Also, the modified antibody of the present invention may be an antibody fragment or an antibody modification as long as it contains a polypeptide consisting of a sequence in which at least a part of the amino acid residues is deleted in the hinge region of the antibody. Such antibody fragments include, for example, F(ab’)2 and the like. Also, antibody modifications include those bound or fused to a peptide, oligopeptide, or protein, and those chemically modified. The unmodified antibody and the modified antibody of the present invention shall include both modified and unmodified antibodies modified by sugar chains or the like unless otherwise specified.

[0016] The modified antibody of the present invention has improved thermal stability compared to the unmodified antibody. The thermal stability of an antibody can generally be evaluated by a method of measuring the amount or ratio of the antibody denatured with heat stress. As the measurement method, known and commonly used methods can be used, and examples include measurement by differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), CD spectrum, fluorescence spectrum, Fourier transform infrared spectroscopy (FTIR), etc. Among these, it is preferable to evaluate by DSF.

[0017] The denaturation midpoint temperature (Tm) value of the modified antibody of the present invention is preferably at least 3°C higher than the Tm value of the unmodified antibody, more preferably at least 4°C higher, and particularly preferably at least 5°C higher. That is, when the Tm value of the modified antibody is Tm1 and the Tm value of the unmodified antibody is Tm2, it is preferable that (Tm2 - Tm1) is at least 3°C, more preferably at least 4°C, and particularly preferably at least 5°C. When there are multiple Tm values for one antibody, when comparing the lowest Tm values of the modified antibody and the unmodified antibody of the present invention, it is preferable that the above temperature difference is obtained.

[0018] Since the amino acid residues modified in the present invention are not amino acid residues in the CDR, it is considered that the affinity of the antibody for the antigen will not be reduced to such an extent that it causes practical problems. In one embodiment, the modified antibody of the present invention binds to the same antigen as the unmodified antibody, and its affinity for the antigen is also comparable to that of the unmodified antibody. The affinity of an antibody for an antigen can be evaluated, for example, by immunological measurement methods such as the ELISA method or by kinetic parameters (association rate constant, dissociation rate constant, and dissociation constant) in the antigen-antibody reaction.

[0019] In one embodiment, the modified antibody of the present invention is preferably a modified antibody of IgG2b, using IgG2b as the unmodified antibody. Although IgG2b has good affinity and specificity, it has low thermal stability and has been difficult to use as a reagent antibody or the like. However, the IgG2b modified antibody of the present invention has good thermal stability and is expected to be applied as a reagent antibody or the like.

[0020] The hinge region of IgG2b, which is the unmodified antibody, preferably contains a polypeptide consisting of any one of the following amino acid sequences (i) to (iii). (i) The amino acid sequence represented by SEQ ID NO: 1 (EPSGPISTINPCPPCKECHKCP). (ii) An amino acid sequence in which one or several amino acids are substituted or added in the amino acid sequence represented by SEQ ID NO: 1. (iii) An amino acid sequence having 85% or more identity with the amino acid sequence represented by Sequence ID No. 1.

[0021] In (i) above, Sequence ID 1 shows the amino acid sequence of the hinge region in mouse IgG2b.

[0022] In (ii) above, the number of substitutions or additions is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

[0023] In (iii) above, the identity with the amino acid sequence represented by Sequence ID No. 1 is preferably 85% or more, more preferably 90% or more, and particularly preferably 95% or more.

[0024] In this specification, the identity between amino acid sequences can be determined using known algorithms such as BLAST. First, to determine the sequence identity between two amino acid sequences, the sequences are pre-processed to a state optimized for comparison. For example, the alignment with the other sequence is optimized by introducing a gap into one sequence. Then, the amino acid residues at each site are compared. If a site in the first sequence contains the same amino acid residue as the corresponding site in the second sequence, then those sequences are identical at that site. The identity between the two sequences is expressed as a percentage of the number of identical sites relative to the total number of sites (total amino acids or total bases). The identity of base sequences is also defined in accordance with the above.

[0025] In one embodiment, the modified antibody of the present invention is preferably an IgG2b modified antibody comprising a polypeptide consisting of a sequence in which at least a portion of the amino acid residues in the hinge region of IgG2b are deleted. The number of deleted amino acid residues in the hinge region is preferably 10 amino acids or less, more preferably 8 amino acids or less, and particularly preferably 7 amino acids. Furthermore, it is preferable that the number of deleted amino acid residues in the hinge region be 5 amino acids or more, and more preferably 6 amino acids or more. Moreover, it is more preferable that the IgG2b modified antibody comprises a polypeptide consisting of a sequence in which 5 to 10 amino acids are deleted from the amino acid sequence of the hinge region of IgG2b, even more preferable that the IgG2b modified antibody comprises a polypeptide consisting of a sequence in which 6 to 8 amino acids are deleted, and particularly preferable that the IgG2b modified antibody comprises a polypeptide consisting of a sequence in which 7 amino acids are deleted.

[0026] Here, the missing portions in the hinge region may be continuous or scattered, but it is preferable that they be continuous.

[0027] In one embodiment, the modified antibody of the present invention preferably contains a polypeptide consisting of a sequence in which at least a portion of any of the amino acid sequences (i) to (iii) above is deleted in the hinge region. More preferably, the modified IgG2b antibody contains a polypeptide consisting of a sequence in which 5 to 10 amino acids are deleted from any of the amino acid sequences (i) to (iii) above in the hinge region, even more preferably, the modified IgG2b antibody contains a polypeptide consisting of a sequence in which 6 to 8 amino acids are deleted, and particularly preferably, the modified IgG2b antibody contains a polypeptide consisting of a sequence in which 7 amino acids are deleted.

[0028] In one embodiment, it is preferable to have an IgG2b modified antibody that contains a polypeptide consisting of a sequence in which at least a portion of the amino acid residues in the hinge region of IgG2b are deleted such that the second P from the N-terminus in SEQ ID NO: 1 is retained. By retaining the second P from the N-terminus in SEQ ID NO: 1, further improvement in thermal stability can be expected. Furthermore, it is more preferable to have an IgG2b modified antibody that contains a polypeptide consisting of a sequence in which the second P from the N-terminus in SEQ ID NO: 1 is retained and 5 to 10 amino acids are deleted from any of the amino acid sequences (i) to (iii) above in the hinge region, it is even more preferable to have an IgG2b modified antibody that contains a polypeptide consisting of a sequence in which the second P from the N-terminus in SEQ ID NO: 1 is retained and 6 to 8 amino acids are deleted from any of the amino acid sequences (i) to (iii) above, and it is particularly preferable to have an IgG2b modified antibody that contains a polypeptide consisting of a sequence in which the second P from the N-terminus in SEQ ID NO: 1 is retained and 7 amino acids are deleted from any of the amino acid sequences (i) to (iii) above.

[0029] An example of a polypeptide consisting of a sequence in which at least some of the amino acid residues in the hinge region of IgG2b are deleted, leaving the second phosphorus (P) from the N-terminus in SEQ ID NO: 1, is EPNPCPPCKECHKCP (SEQ ID NO: 2). This sequence is obtained by deleting seven residues, from the 3rd to the 9th from the N-terminus, from the amino acid sequence of SEQ ID NO: 1.

[0030] In one embodiment, the modified antibody of the present invention contains one of the following polypeptides (1) to (3) in its hinge region. (1) A polypeptide containing the amino acid sequence represented by Sequence ID No. 2 (EPNPCPPCKECHKCP). (2) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, added, or deleted in the amino acid sequence represented by Sequence ID No. 2. (3) A polypeptide containing an amino acid sequence that has 85% or more identity with the amino acid sequence represented by Sequence ID No. 2.

[0031] In (2) above, the number of substitutions, additions, or deletions is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

[0032] In (3) above, the identity with the amino acid sequence represented by Sequence ID No. 2 is preferably 85% or more, more preferably 90% or more, and particularly preferably 95% or more.

[0033] In one embodiment, the modified antibody of the present invention preferably contains a polypeptide in its light chain constant region consisting of the following amino acid sequences (a1) to (a3). (a1) The amino acid sequence represented by Sequence ID No. 3. DAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (a2) An amino acid sequence in which one to several deletions, substitutions, or additions are made in the amino acid sequence represented by Sequence ID No. 3. (a3) An amino acid sequence that has 90% or more identity with the amino acid sequence represented by Sequence ID No. 3.

[0034] In (a1) above, Sequence ID 3 is the sequence of the light chain constant region of the mouse IgG2b antibody.

[0035] In (a2) above, the number of substitutions, additions, or deletions is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, particularly preferably 1 to 2, and most preferably 1.

[0036] In (a3) ​​above, the identity with the amino acid sequence represented by Sequence ID No. 3 is preferably 90% or more, more preferably 92% or more, even more preferably 94% or more, even more preferably 96% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0037] In one embodiment, the modified antibody of the present invention preferably contains a polypeptide in the CH1 region of the heavy chain constant region consisting of the following amino acid sequences (b1) to (b3). (b1) The amino acid sequence represented by Sequence ID No. 4. AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKL (b2) An amino acid sequence in which one to several deletions, substitutions, or additions are made in the amino acid sequence represented by Sequence ID No. 4. (b3) An amino acid sequence that has 90% or more identity with the amino acid sequence represented by Sequence ID No. 4.

[0038] In (b1) above, sequence number 4 is the sequence of the heavy chain CH1 region of the mouse IgG2b antibody.

[0039] In (b2) above, the number of substitutions, additions, or deletions is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, particularly preferably 1 to 2, and most preferably 1.

[0040] In (b3) above, the identity with the amino acid sequence represented by Sequence ID No. 4 is preferably 90% or more, more preferably 92% or more, even more preferably 94% or more, even more preferably 96% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0041] In one embodiment, the modified antibody of the present invention preferably contains a polypeptide in the CH2 region of the heavy chain constant region consisting of the following amino acid sequences (c1) to (c3). (c1) The amino acid sequence represented by Sequence ID No. 5. APNLEGGPSVFIFPPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIK (c2) An amino acid sequence that contains one to several deletions, substitutions, or additions in the amino acid sequence represented by Sequence ID No. 5. (c3) An amino acid sequence that has 90% or more identity with the amino acid sequence represented by Sequence ID No. 5.

[0042] In (c1) above, Sequence ID No. 5 is the sequence of the heavy chain CH2 region of the mouse IgG2b antibody.

[0043] In (c2) above, the number of substitutions, additions, or deletions is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, particularly preferably 1 to 2, and most preferably 1.

[0044] In (c3) above, the identity with the amino acid sequence represented by Sequence ID No. 5 is preferably 90% or more, more preferably 92% or more, even more preferably 94% or more, even more preferably 96% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0045] In one embodiment, the modified antibody of the present invention preferably contains a polypeptide in which the CH3 region of the heavy chain constant region consists of the following amino acid sequences (d1) to (d3). (d1) The amino acid sequence represented by Sequence ID No. 6. GLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSP (d2) An amino acid sequence that contains one to several deletions, substitutions, or additions in the amino acid sequence represented by Sequence ID No. 6. (d3) An amino acid sequence that has 90% or more identity with the amino acid sequence represented by Sequence ID No. 6.

[0046] In (d1) above, sequence number 6 is the sequence of the heavy chain CH3 region of the mouse IgG2b antibody.

[0047] In (d2) above, the number of substitutions, additions, or deletions is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, particularly preferably 1 to 2, and most preferably 1.

[0048] In (d3) above, the identity with the amino acid sequence represented by Sequence ID No. 6 is preferably 90% or more, more preferably 92% or more, even more preferably 94% or more, even more preferably 96% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0049] In a preferred embodiment, the modified antibody of the present invention comprises a polypeptide in which the sequence of the light chain constant region consists of any of the amino acid sequences (a1) to (a3) ​​above, the heavy chain CH1 region consists of a polypeptide in which the amino acid sequence (b1) to (b3) above, the heavy chain CH2 region consists of a polypeptide in which the amino acid sequence (c1) to (c3) above, and the heavy chain CH3 region consists of a polypeptide in which the amino acid sequence (d1) to (d3) above.

[0050] The modified antibody of the present invention can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide containing the coding sequence of the modified antibody of the present invention, and recovering a fraction containing the antibody of the present disclosure.

[0051] In this specification, "polynucleotide" may include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, etc. Furthermore, polynucleotide refers to both double-stranded and single-stranded molecules.

[0052] A polynucleotide containing the coding sequence of the modified antibody of the present invention preferably contains the coding sequence of the modified antibody of the present invention in a state capable of expressing the modified antibody of the present invention. The polynucleotide may contain other sequences in addition to the coding sequence. Examples of other sequences include secretion signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, replication bases, and drug resistance gene coding sequences, which are positioned adjacent to the antibody coding sequence of this disclosure. Furthermore, the polynucleotide may be a linear polynucleotide or a cyclic polynucleotide (vector, etc.).

[0053] The polynucleotide containing the coding sequence of the modified antibody of the present invention can be synthesized, for example, by reverse transcription and RACE (Rapid Amplification of cDNA ends) using RNA extracted from a hybridoma that produces the unmodified antibody (which can be produced by known methods such as the method described in Kohler and Milstein, Nature, vol.256, pp.495-497, 1975). Specifically, the polynucleotide encoding the heavy chain is amplified by PCR using a primer for modifying the heavy chain to obtain the polynucleotide encoding the modified heavy chain. The obtained polynucleotide, together with the polynucleotide encoding the light chain of the unmodified antibody, is incorporated into an expression vector known in this technology to obtain an expression vector containing the polynucleotide encoding the modified antibody of the present invention. Here, the polynucleotide encoding the light chain and the polynucleotide encoding the heavy chain may be incorporated into one expression vector or into two separate expression vectors. The type of expression vector is not particularly limited and may be an expression vector for mammalian cells or an expression vector for E. coli. The obtained expression vector can be used to transform or transfect suitable host cells (e.g., mammalian cells or E. coli) to produce modified antibodies.

[0054] Once the modified antibody of the present invention is produced by recombinant expression, it can be purified by methods known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, protein A chromatography, gel filtration column chromatography), centrifugation, and salting out. Furthermore, the modified antibody of the present invention can be fused with known heterologous polypeptide sequences to facilitate purification. For example, the modified antibody of the present invention can be purified by recombinantly adding a polyhistidine tag (His tag), a FLAG tag, a hemagglutinin tag (HA tag), or a myc tag.

[0055] In one embodiment, a diagnostic reagent containing the modified antibody of the present invention is provided. This diagnostic reagent can be used, for example, in a diagnostic method that includes the step of measuring the amount or concentration of an antigen bound to the modified antibody of the present invention in a biological sample collected from a subject.

[0056] There are no particular restrictions on the species of organisms used as subjects; for example, various mammals such as humans, monkeys, rabbits, mice, rats, dogs, and cats can be used.

[0057] The biological sample is not particularly limited as long as it can contain an antigen that binds to the modified antibody of the present invention. Examples of biological samples include whole blood, serum, plasma, saliva, cerebrospinal fluid, synovial fluid, urine, tissue fluid, sweat, tears, saliva, and other bodily fluids, as well as samples derived from these bodily fluids. Samples derived from bodily fluids are not particularly limited as long as they are prepared from bodily fluids, and examples include samples obtained by concentrating and purifying the antigen to which the modified antibody of the present invention binds from bodily fluids. The biological sample may be used alone or in combination of two or more types.

[0058] Biological samples can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by blood collection using a syringe or the like. Serum is the portion of blood from which blood cells and certain blood clotting factors have been removed, and can be obtained, for example, as the supernatant after blood has been coagulated. Plasma is the portion of blood from which blood cells have been removed, and can be obtained, for example, as the supernatant after centrifugation under conditions that do not cause blood to coagulate.

[0059] A testing method using the diagnostic reagent includes a step of measuring the amount or concentration of an antigen bound to the modified antibody of the present invention, and the method is not particularly limited. For example, an immunoassay can be used. Immunoassays can be widely employed, regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive methods. More specifically, examples of immunoassays include ELISA (e.g., direct, indirect, sandwich, competitive methods, etc.), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, latex immunoturbidimetry, immunochromatography, Western blotting, immunoprecipitation, slot or dot blotting assays, immunohistochemistry, fluorescent immunoassays, immunoassays using avidin-biotin or streptavidin-biotin systems, and immunoassays using surface plasmon resonance (SPR).

[0060] In the step of measuring the amount or concentration of an antigen bound to the modified antibody of the present invention, the type of label used in the labeling substance (e.g., labeled antibody) for detecting the antigen is not particularly limited. Examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Among these, enzyme labeling such as peroxidase and alkaline phosphatase is more preferred from the viewpoint of safety, economy, and detection sensitivity.

[0061] The method for measuring the amount of antigen bound to the modified antibody of the present invention is not particularly limited, and an appropriate embodiment can be selected depending on the type of test method. The measurement can be performed, for example, by quantifying the signal derived from the label of the labeling agent used. More specifically, for example, the measurement can be performed by contacting the antigen bound to the modified antibody of the present invention with an antibody labeled against the antigen, and quantifying the signal derived from the label of the bound labeled antibody.

[0062] Based on the obtained signal amount, the amount of antigen bound to the modified antibody of the present invention can be calculated. For example, in the case of a non-competitive method, the obtained signal amount can be used directly as the amount of antigen bound to the modified antibody of the present invention. Alternatively, in the case of a competitive method, the obtained signal amount and the amount of antigen bound to the modified antibody of the present invention are inversely proportional, so the amount of antigen bound to the modified antibody of the present invention can be calculated from the obtained signal amount based on this relationship.

[0063] A diagnostic reagent containing the modified antibody of the present invention may also be in the form of a composition containing the modified antibody of the present invention. The composition may optionally contain other components. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like. Alternatively, a diagnostic reagent containing the modified antibody of the present invention may also be in the form of a kit containing the modified antibody of the present invention. The kit may contain instruments, reagents, etc., that can be used to carry out the testing method of this disclosure.

[0064] In one embodiment, a pharmaceutical composition comprising the modified antibody of the present invention is provided. The pharmaceutical composition may contain a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers that can be used include phosphate-buffered saline, water, emulsions such as oil-water emulsions, and various types of wetting agents, which are standard pharmaceutical carriers known in the art. The pharmaceutical composition may also contain pharmaceutically acceptable additives. Such additives can be appropriately selected from additives known in the art. Examples include excipients, lubricants, binders, disintegrants, coating agents, capsule bases, plasticizers, colorants, solvents, stabilizers, preservatives, buffers, analgesics, bases, emulsifiers, suspending agents, flavoring agents, sweeteners, adsorbents, solubilizers, pH adjusters, thickeners, isotonic agents, dispersants, preservatives, wetting agents, flavoring agents, antioxidants, and the like.

[0065] The formulation form of the pharmaceutical composition is not particularly limited and may be any formulation known to those skilled in the art, such as solid dosage forms, semi-solid dosage forms, liquid dosage forms, injections, suppositories, etc. Specific dosage forms include, but are not limited to, tablets, pills, granules, powders, capsules, lozenges, injections, liquids, elixirs, syrups, limonades, suppositories, ointments, suspensions, emulsions, liniments, lotions, transdermal formulations, patches, poultices, aerosols, etc. [Examples]

[0066] The present invention will be described in detail below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0067] Example 1: Preparation of modified antibodies Both antibody 1 and antibody 2 are mouse IgG2b antibodies. Antibody 1: Anti-DPP4 antibody Antibody 2: Antibody against the glycoprotein of the rabies virus

[0068] [Table 1]

[0069] The DNA fragments encoding the antibody heavy chain and light chain were inserted into separate vectors (pcDNA3.1(+) or pcDNA3.4), respectively, and transfected into ExpiCHO-S cells for expression. After culturing for the period recommended by Thermo Fisher Scientific, the culture supernatant was collected, filtered, and purified with Protein A.

[0070] Through the above procedure, two modified antibodies, antibody 1-EPNP and antibody 2-EPNP, were obtained. A schematic of the obtained modified antibodies is shown in Figure 1.

[0071] The amino acid sequence of the hinge region of antibody 1-EPNP and antibody 2-EPNP is EPNPCPPCKECHKCP (SEQ ID NO: 2). This sequence is a sequence in which 7 residues from the 3rd to the 9th residues from the N-terminal side are deleted from the sequence EPSGPISTINPCPPCKECHKCP (SEQ ID NO: 1) of the hinge region of unmodified antibody 1, antibody 2 and antibody 3.

[0072] Example 2: Thermal stability evaluation The thermal stability of each antibody prepared in Example 1 was evaluated. For the thermal stability evaluation, differential scanning fluorimetry (DSF) was used. Each antibody was diluted with PBS to prepare a sample (1 mg / mL). Then, using Tycho NT.6 (manufactured by Nano Temper Technologies), the midpoint temperature Tm value of each antibody was measured. The measurement conditions are as follows. <DSF measurement conditions> · Sample volume: 10 μL · Measurement range: 35 - 95 °C · Heating rate: 30 °C / min

[0073] The results are shown in Table 2. In Table, ΔTm (°C) indicates the difference between the Tm value of each modified antibody and the Tm value of the unmodified antibody.

[0074]

Table 2

[0075] As shown in Table 2, it was revealed that in the modified antibodies of any of the antibodies, the Tm value increased compared to the unmodified antibody, and the thermal stability was improved.

[0076] Example 3: Affinity of the modified antibody for the antigen DPP4 was used as the antigen for antibody 1, and the glycoprotein of the rabies virus was used as the antigen for antibody 2. Each antigen was dissolved in PBS to a concentration of 5 μg / mL, and 100 μL was added to the wells of a plate (Nunc-Immuno Module plate (flat bottom)). The antigens were immobilized in the plate wells by shaking for 90 minutes. After removing the antigen solution, 250 μL of a PBS solution containing 1% BSA and 0.1% NaN3 (blocking solution) was added to each plate well, and blocking was performed by shaking for 90 minutes and then standing overnight at 4°C. After removing the blocking solution, 100 μL of antibody sample solution was added to each well, and the antigen-antibody reaction was performed by shaking for 1 hour. Antibody sample solutions were prepared by diluting each antibody to 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 1000 ng / mL using a PBS solution containing 1% BSA and 0.1% NaN3. After the antigen-antibody reaction, the antibody sample solution was removed, and 100 μL of anti-mouse IgG-HRP was added to each well as the detection antibody, and the mixture was shaken for 1 hour. After removing the detection antibody, 100 μL of 1-Step® Ultra TMB-ELISA Substrate Solution was added to each plate well as the ELISA substrate, and the mixture was allowed to stand for 10-15 minutes. After determining that the color change of the ELISA substrate was appropriate, 100 μL of EP stop solution was added to each plate well. Subsequently, the absorbance at 450 nm and 650 nm was measured. The results are shown in Table 3.

[0077] [Table 3]

[0078] As shown in Table 3, the affinity of each modified antibody for the antigen was comparable to that of the corresponding unmodified antibody.

Claims

1. A modified antibody containing a polypeptide consisting of a sequence in which at least some amino acids are deleted in the hinge region of the antibody.

2. The modified antibody according to claim 1, which is a mouse IgG2b modified antibody.

3. The modified antibody according to claim 2, comprising a polypeptide consisting of a sequence obtained by deleting 5 to 10 amino acids from the amino acid sequence of the hinge region of mouse IgG2b.

4. The modified antibody according to claim 2, comprising a polypeptide having a sequence in which at least a portion of any of the following amino acid sequences (i) to (iii) is deleted in the hinge region. (i) The amino acid sequence represented by Sequence ID No. 1 (EPSGPISTINPCPPCKECHKCP). (ii) An amino acid sequence in which one or more amino acids are substituted or added to the amino acid sequence represented by Sequence ID No.

1. (iii) An amino acid sequence having 85% or more identity with the amino acid sequence represented by Sequence ID No.

1.

5. The modified antibody according to claim 4, comprising a polypeptide having a sequence in which the second P from the N-terminus of SEQ ID NO: 1 is retained and 5 to 10 amino acids are deleted.

6. The modified antibody according to claim 2, wherein the hinge region contains any of the polypeptides (1) to (3) below. (1) A polypeptide containing the amino acid sequence represented by Sequence ID No. 2 (EPNPCPPCKECHKCP). (2) A polypeptide comprising an amino acid sequence in which one or more amino acids are substituted or added in the amino acid sequence represented by Sequence ID No.

2. (3) A polypeptide containing an amino acid sequence having 85% or more identity with the amino acid sequence represented by Sequence ID No.

2.

7. Midpoint temperature Tm of modified antibody denaturation １ However, the denaturation midpoint temperature Tm of unmodified antibodies ２ A modified antibody according to claim 1, which is 3°C or more higher than the specified temperature.

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