JP2021151965A - Anti-podocalyxin antibodies - Google Patents

Abstract

To provide anti-podocalyxin antibodies with excellent uptake into M cells.SOLUTION: Provided is an anti-podocalyxin antibody or antigen binding fragment thereof comprising at least one CDR represented by the following (a) to (f); (a) a heavy chain CDR1 of a specific amino acid sequence; (b) a heavy chain CDR2 of a specific amino acid sequence; (c) a heavy chain CDR3 of a specific amino acid sequence; (d) a light chain CDR1 of a specific amino acid sequence; (e) a light chain CDR2 of a specific amino acid sequence; and (f) a light chain CDR3 of a specific amino acid sequence.SELECTED DRAWING: None

Description

The present invention relates to antibodies against novel podocalyxins.

Podocalixin (PCX) is a transmembrane glycoprotein with a molecular weight of about 140 kDa found in renal glomerular epithelial cells (podocytes) and has high homology with the hematopoietic stem cell marker CD34. Podocalixin is a highly sugar-chained sialomtin-type protein that has many glycosaminoglycans, O and N-linked sugar chain addition sites in the extracellular region, and thus contributes to cell adhesion and morphology. In addition, in cancer cells, sugar chains on podocalyxin act as ligands for E- / P- / L-selectin expressed on endothelial cells and are involved in cancer cell adhesion, invasion, and metastasis. Is known (Non-Patent Documents 1 and 2).

In recent years, podocalyxin is highly expressed in colon cancer, breast cancer, urinary epithelial bladder cancer, pancreatic cancer, gastric cancer, testicular tumor, prostate cancer, and ovarian cancer, and is a marker of cancer malignancy and poor prognosis. (Non-Patent Document 3), expressed in embryonic stem cells / multifunctional stem cells, which are human stem cells, and can be an undifferentiated marker of these stem cells (Non-Patent Document 4), expressed in vascular endothelial cells, It has been reported that it can be a marker for vascular dysfunction (Patent Document 1).

Therefore, anti-podocalyxin antibodies that can recognize podocalyxin molecules regardless of the presence or absence of glycosylation are widely used, including detection of normal and cancerous tissues and cells, treatment of cancer, and diagnosis of vascular dysfunction. Possible uses.

JP-A-2019-35665

Dallas MR, Chen SH, Streppel MM, Sharma S, Maitra A, Konstantopoulos K.Sialofucosylated podocalyxin is a functional E- and L-selectin ligand expressed by metastatic pancreatic cancer cells. Am J Physiol Cell Physiol. 2012 Sep 15; 303 (6) ): C616-24. Thomas SN, Schnaar RL, Konstantopoulos K. Podocalyxin-like protein is an E- / L-selectin ligand on colon cancer cells: comparative biochemical properties of selectin ligands in host and tumor cells. Am J Physiol Cell Physiol. 2009 Mar; 296 ( 3): C505-13. Cipollone JA, Graves ML, Kobel M, Kalloger SE, Poon T, Gilks CB, McNagny KM, Roskelley CD. The anti-adhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma. Clin Exp Metastasis. 2012 Mar; 29 (3): 239-52. Tateno H, Matsushima A, Hiemori K, Onuma Y, Ito Y, Hasehira K, Nishimura K, Ohtaka M, Takayasu S, Nakanishi M, Ikehara Y, Nakanishi M, Ohnuma K, Chan T, Toyoda M, Akutsu H, Umezawa A , Asashima M, Hirabayashi J. Podocalyxin is a glycoprotein ligand of the human pluripotent stem cell-specific probe rBC2LCN. Stem Cells Transl Med. 2013 Apr; 2 (4): 265-73.

In recent years, peptide / protein drugs such as antibody molecules have been increasing in place of conventional small molecule drugs, and these are often used as injections for intramuscular administration or subcutaneous administration. Since injection is painful and has a risk of developing serious side effects such as allergies and anaphylaxis, the development of oral or transmucosal administration as an alternative to injection is expected. However, since the mucosal epithelial cell layer functions as a fat-soluble barrier in the transmucosal-administered preparation, a drug having a large molecular size or a drug having a very high water solubility cannot pass through the mucosal epithelial layer and is hardly absorbed into the body. Therefore, technological development such as promotion of membrane passage, which is different from the use of conventional absorption promoters and prodrugization, is required.

Further, in a vaccine, it is known that mucosal immunity can induce multiple defense mechanisms because it can induce an immune response locally in the mucosa in addition to a systemic immune response by administering it to the mucosa. For this reason, mucosal vaccines are expected to exert a high effect on pathogens that invade through the mucous membrane, but vaccine preparations have a larger molecular size and particle size than antibody drugs, and some of them have a larger molecular size and particle size. Immune induction cannot be expected even if an existing vaccine is administered to the mucosa, except for live vaccines of antigens having mucosal affinity and pathogens having mucosal invasion. Therefore, there is a demand for a technique for promoting uptake through mucous membranes in vaccine preparations as well.

Under such circumstances, the present inventors have discovered that podocalyxin is highly expressed in Microfold cells (M cells) of the mucosal epithelium. Then, as a result of further investigation, they succeeded in obtaining an anti-podocalyxin antibody that showed excellent affinity for M cells and was well taken up by the cells, and completed the present invention.

That is, the present invention relates to the following 1) to 4).
1) An anti-podocalyxin antibody or an antigen-binding fragment thereof having at least one of the CDRs shown in (a) to (f) below.
(A) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2.
(C) Heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3
(D) Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4.
(E) Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5.
(F) Light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.
2) The anti-podocalyxin antibody of 1) or an antigen-binding fragment thereof, which has all six CDRs shown in (a) to (f) above.
3) It has a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 10 or 90% or more identity with the amino acid sequence. An anti-podocalyxin antibody of 1) or 2) or an antigen-binding fragment thereof, which has a light chain variable region consisting of an amino acid sequence.
4) A drug compound for transmucosal administration, which comprises binding a drug to any of the anti-podocalyxin antibodies of 1) to 3) or an antigen-binding fragment thereof.

Since the antibody of the present invention or an antigen-binding fragment thereof is often taken up by M cells, by binding this to a drug, the absorption efficiency of the drug via mucosal M cells is increased, and in a drug having poor oral or mucosal absorption. However, transmucosal administration is possible, and it is expected that the convenience and effectiveness of taking the drug will be improved.

Evaluation of uptake of anti-podocalyxin antibody in M cells. Evaluation of binding of 4D2 podocalyxin to transmembrane domain.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

In the present invention, Podocalixin (PCX) refers to a type I transmembrane protein composed of 558-residue amino acids expressed in renal glomerular epithelial cells.
The anti-podocalyxin antibody of the present invention is an antibody that has an affinity for M cells and is well taken up by the cells, and has at least one of the CDRs shown in (a) to (f) below.
(A) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2.
(C) Heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3
(D) Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4.
(E) Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5.
(F) Light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

The anti-podocalyxin antibody of the present invention has a structure in which two heavy chains (H chain) stabilized by a pair of disulfide bonds and two light chains (L chain) are associated, and the heavy chain is a heavy chain variable. It consists of a region V _H , a heavy chain constant region C _H 1, C _H 2, _CH 3, and _{a hinge region located between C H} 1 and C _H 2, and the light chain is light with the light chain variable region _VL. consisting of a chain constant region _{C L.}

In the present invention, the "CDR (complementarity-determining region)" is called a complementarity determining region, and means a region of the variable region of an antibody molecule that comes into direct contact with an antigen. There are three CDRs in each of the variable regions of the light chain and the heavy chain, and in the present invention, they are referred to as heavy chain CDR1 to 3 and light chain CDR1 to 3.

The CDR can be determined from the DNA encoding the heavy chain variable region V _H and the light chain variable region _VL by the analysis software provided by abYsis (http://abysis.org/) or the like, and can be determined by the analysis software of the present invention. CDR of the anti podocalyxin antibodies, as described in the examples below, clone name: 4D2 DNA encoding the heavy chain variable region V _H of the isolated monoclonal antibody (SEQ ID NO: 7) and light chain variable regions V _L It is determined from the DNA encoding (SEQ ID NO: 9). Its amino acid sequence is as specifically below, this 26-35 th of the amino acid sequence of the heavy chain variable region _{V H} (SEQ ID NO: 8), No. 50-66, 99-106 th amino acid sequence, It corresponds to the amino acid sequences of Nos. 24-33, 49-55, and 88-97 of the amino acid sequence of the light chain variable region _{VL (SEQ ID NO: 10).}
(A) Heavy chain CDR1: GYTFTNYVLH (SEQ ID NO: 1)
(B) Heavy chain CDR2: YFYPYNDGTKYNERFKG (SEQ ID NO: 2)
(C) Heavy chain CDR3: TWDWYFDV (SEQ ID NO: 3)
(D) Light chain CDR1: SASSSVSYMH (SEQ ID NO: 4)
(E) Light chain CDR2: DTSKLAS (SEQ ID NO: 5)
(F) Light chain CDR3: QQWSSNPPIT (SEQ ID NO: 6)

The anti-podocalyxin antibody of the present invention or an antigen-binding fragment thereof has a property of having an affinity for M cells and being well taken up by the cells, preferably the rate of uptake into M cells shown in 4D2 shown in the implementation described later. As long as it has the same uptake rate as, it is sufficient to have at least one of the above six CDRs, but preferably two or more, more preferably three or more, more preferably four or more, and more preferably five. As mentioned above, it is most preferable to have all six.

Further, the anti-podocalyxin antibody of the present invention or an antigen-binding fragment thereof is derived from the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence. It has a heavy chain variable region and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence. Is preferable. The variability of the heavy chain variable region and the light chain variable region is found outside the CDR.

That is, the anti-podocalyxin antibody of the present invention or an antigen-binding fragment thereof preferably has at least one of the CDRs shown in (a) to (f) above, and has the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence thereof. A heavy chain variable region consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity, and the amino acid sequence represented by SEQ ID NO: 10 or 90% or more, preferably 95% thereof. As described above, more preferably, it has a light chain variable region consisting of an amino acid sequence having 98% or more identity.
Further, the anti-podocalyxin antibody of the present invention or an antigen-binding fragment thereof more preferably has all six CDRs shown in (a) to (f) above, and has the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence thereof. A heavy chain variable region consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity, and the amino acid sequence represented by SEQ ID NO: 10 or 90% or more, preferably 95% thereof. As described above, more preferably, it has a light chain variable region consisting of an amino acid sequence having 98% or more identity.

Here, the amino acid sequence identity refers to the ratio (%) of the number of positions where the same amino acid residue exists in both sequences to the total number of full-length amino acid residues when the two amino acid sequences are aligned. Specifically, for example, it is calculated by the Lipman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)), and is calculated by the genetic information processing software Geneticx-Win (Ver.5.1.1; software development). It can be calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis program.

The anti-podocalyxin antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. Further, the anti-podocalyxin antibody may be any isotype of IgG, IgM, IgA, IgD and IgE. It may be produced by immunizing non-human animals such as mice, rats, hamsters, guinea pigs, rabbits, and chickens, or it may be a recombinant antibody, and it may be a chimeric antibody, a humanized antibody, or a fully humanized antibody. And so on.
Here, the humanized antibody has, for example, heavy chain CDR1 to 3 and light chain CDR1 to 3 of an antibody prepared by immunizing a mouse, and is a framework region (FR) which is a portion other than CDR and has relatively few mutations. ) Is replaced with the corresponding region of the human antibody.

The antigen-binding fragment of the anti-podocalyxin antibody is a fragment (fragment) of the anti-podocalyxin antibody of the present invention, and is more preferably at least one, preferably two or more of the CDRs shown in (a) to (f). Means a fragment that binds to a podocalyxin having three or more, more preferably four or more, more preferably five or more, and more preferably all six. Specifically, it _{consists of a Fab consisting of VL} , V _H , _CL , and _{CH 1 regions, and F (ab) 2, VL,} and V _H in which two Fabs are linked by disulfide bonds in the hinge region. In addition to scFv, which is a single-chain antibody in which Fv, _VL, and _VH are linked with an artificial polypeptide linker, bispecific antibodies such as diabody type, scDb type, tandemscFv type, and leucine zipper type can be mentioned.

The anti-podocalyxin antibody of the present invention or an antigen-binding fragment thereof can be produced, for example, by a hybridoma method, a recombinant DNA method, or the like.
As a hybridoma method, antibody-producing cells are isolated from non-human mammals (for example, mice, rats, hamsters, rabbits, monkeys, goats, etc.) immunized with podocalyxin or a part thereof, and these are fused with myeloma cells or the like. It can be obtained by producing a hybridoma and purifying the antibody produced by the hybridoma.

As a recombinant DNA method, for example, the anti-podocalyxin antibody of the present invention or a nucleic acid encoding a functional fragment of the antibody is cloned from a hybridoma, B cell, or the like, incorporated into an appropriate vector, and this is incorporated into a host cell (for example, a mammalian cell line). , Escherichia coli, yeast cells, insect cells, plant cells, etc.) and produced as a recombinant antibody. In the expression of a nucleic acid encoding an antibody or antibody-binding fragment, DNA encoding a heavy chain or a light chain may be separately incorporated into an expression vector to transform a host cell, encoding the heavy chain and the light chain. Nucleic acid may be integrated into a single expression vector to transform host cells.

For the separation and purification of the antibody, the method used in the conventional purification of a polypeptide can be used. Examples of the isolation and purification method include an affinity column using protein A / G / L or the like, other chromatography columns, filters, ultrafiltration, salting out, and dialysis, which can be appropriately combined.

The drug compound of the present invention is obtained by binding a drug to the above-mentioned anti-podocalyxin antibody or an antigen-binding fragment thereof.
Here, the drugs include antitussives, analgesics, anti-inflammatory drugs, anti-rheumatic drugs, hypnotics, sedatives, anti-anxiety drugs, anti-psychiatric drugs, antidepressants, anti-epileptic drugs, Parkinson's disease therapeutic drugs, and cerebral circulation. Metabolism improver, muscle relaxant, autonomic nervous system agent, anti-disease drug, migraine drug, cardiotonic drug, anti-angina drug, β-blocker, Ca antagonist, antiarrhythmic drug, diuretic drug, antihypertensive drug, Anti-allergic drug, bronchial dilator, asthma drug, antitussive drug, expectorant drug, digestive ulcer drug, gout drug, dyslipidemia drug, diabetes drug, hormone preparation, osteoporosis drug, antibacterial drug, antiviral drug, anti Drugs classified into parasite drugs, antigenic insect drugs, antiarrhythmic drugs, anesthesia and vaccines can be mentioned, but they are peptide or protein drugs and water-soluble polymer drugs that are poorly absorbed by general oral administration or mucosal administration. Is preferable.

The binding of the anti-podocalyxin antibody or its antigen-binding fragment to the above drug is, for example, via an amino group, a sulfhydryl group, a carboxyl group, a reducing sugar terminal, or an aldehyde group generated in a sugar chain by oxidation with periodic acid. Be done. Specifically, in the case of an amino group, a cross-linking agent having an N-hydroxysucciimide ester and an isothiocyano group, in the case of a sulfhydryl group, a cross-linking agent having a maleimide group or a bromoacetamide group, and in the case of an aldehyde, a hydrazide group. It can be bound via a cross-linking agent having. Further, if it is a carboxyl group, it can be bonded by using an N-hydroxysucciimide ester to make the carboxyl group an active ester and reacting this active ester with a cross-linking agent having a primary amine. Not limited.

The drug compound of the present invention can be used for transmucosal administration because it enhances uptake into the coelomic mucosa by transcellular transport via an anti-podocalyxin antibody or an antigen-binding fragment thereof.
Here, the transmucosal administration refers to an administration form that is absorbed through the mucosa of the body cavity, and specifically, nasal inhalation via the mucosa covering the nasal cavity, instillation on the conjunctival or corneal membrane, and alveolar vesicles. Transpulmonary inhalation via the mucosa, inhalation into the tracheal mucosa and bronchial mucosa, transvaginal administration via the mucosa in the vagina, oral administration via the gastrointestinal mucosa such as the large intestine including the stomach, duodenum, small intestine, colon and rectum. And rectal administration, and middle ear administration to the middle ear mucosa. Of these, preferred administration forms are nasal inhalation, pulmonary inhalation, tracheal or bronchial inhalation, and eye drops.
Such a drug compound can be formulated as a transmucosal-administered drug in solid, semi-solid or liquid form as a composition (pharmaceutical composition) with a pharmaceutically acceptable carrier for mucosal administration.

Hereinafter, the present invention will be specifically described, but the present invention is not limited thereto.
Production example Preparation of anti-podocalyxin antibody (1) Establishment of clones (4A7, 4D2 and 4E5) To human fetal kidney cells 293 (HEK293T cells), C of the extracellular region of human podocalyxin (1st to 409th amino acid sequence) A plasmid encoding a protein (Strep-ΔTM) having a Strep tag and a His tag added to the terminal was lipofected to express Strep-ΔTM. The expressed Strept-ΔTM was purified with a His tag, and the buffer exchanged by ultrafiltration was used as an immune antigen. This immune antigen was intraperitoneally administered to BALB / c mice (female, SLC), and spleens were collected from immunized mice to prepare spleen cells. The prepared splenocytes and mouse myeloma cells were fused using polyethylene glycol, cultured in a HAT medium containing hypoxanthine, aminopterin, and thymidine, and then sequentially cultured in an HT medium containing hypoxanthine and thymidine, and RPMI medium. Then, cloning was performed by the limit dilution method. In cloning, a His tag is added to the C-terminal of the extracellular region of human podocalyxin (amino acid sequences 1 to 409), and the protein (ΔTM) expressed and purified in the same manner as described above is immobilized on a plate and ΔTM. Clones that react with the above were selected by the ELISA method. As a result of ELISA, the clones showing good reactivity were designated as 4A7, 4D2 and 4E5.

(2) Determination of Amino Acid Sequence, Nucleotide Sequence and CDR of ^{4D2 A hybridoma (clone: 4D2) of 1 × 10 6} cells was suspended in RNAlater (SIGMA ALDRICH), and RNA was extracted from the hybridoma after suspension. Extracted RNA as a template to synthesize cDNA by RT-PCR, to amplify the region of the _{V H} and _{V L} using degenerate primers. The amplified region was cloned and _{the nucleotide sequences of V H} and _VL (SEQ ID NOs: 7 and 9) were determined by the cycle sequencing method. Further, the obtained base sequence was converted into an amino acid sequence (SEQ ID NOs: 8 and 10).
In addition, the _{CDR was determined from the obtained V H} base sequence (SEQ ID NO: 7) and _VL base sequence (SEQ ID NO: 9). These sequencings were outsourced to Biopeak Co., Ltd.

Example 1 Evaluation of uptake in M-like cell Biotin-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch) was collected as biotin in 220 pmol, 950 μL of PBS was added thereto, and 0.1 mg of FluoSphereTM Streptavidin-Labeled Microcells was added little by little. Added. After the addition, the mixture was shaded and shaken for 1 hour to prepare fluorescent beads to which an anti-mouse IgG antibody was bound.
200 μL of PBS was added to the fluorescent beads to which the anti-mouse IgG antibody was bound, and 176 pmol of the anti-podocalyxin antibody (clone name: 4A7, 4D2 and 4E5) or mouse IgG2a Isotype control (Sigma Aldrich) obtained by the above production example was further added. , Shake for 1 hour with shading. After shaking, centrifuge at 15,000 rpm for 30 minutes, and suspend by adding 1.3 mL of 20% calf serum, 1% NEAA, and MEM (GIBCO) containing 0.0293% L-glutamine to the precipitate. These were designated as anti-podocalyxin antibody-labeled fluorescent beads or Isoprecipitol-labeled fluorescent beads.

Cells that promoted differentiation into M-like cells in vitro were prepared on the membrane of Transwell, and the anti-podocalyxin antibody-labeled fluorescent beads or Isotype control-labeled fluorescent beads prepared above were added to the chamber below the cells. The fluorescence intensity of the upper chamber solution was measured to evaluate the amount of beads transferred to the upper chamber after a few hours. The in vitro M-like cell was prepared as described below.

Literature (Kai H, Motomura Y, Saito S, Hashimoto K, Tatefuji T, Takamune N, Misumi S. Royal jelly enhances antigen-specific mucosal IgA response. Food Sci Nutr. 2013 Mar 6; 1 (3): 222-227. ^{), 3 × 10 5} Caco-2 cells were seeded on a membrane of Transwell (Corning, pore size: 3 μm, 24 wells), allowed to stand overnight, and then 20% calf serum, Transwell membranes are immersed in 24-well plates supplemented with Eagles MEM (20% FBS, 0.1 mM NEAA EMEM) containing 0.1 mM non-essential amino acids. Incubate for 21 days while transferring Transwell to a 24-well plate supplemented with fresh 20% FBS, 0.1 mM NEAA EMEM approximately every 3 days to form a monolayer of Caco-2. After culturing for 21 days, 1 × 10 ⁶ Razi B cells were added to the upper chamber of Transwell, and further culturing for 3 days promoted differentiation into M-like cells, which were used as M-like cells. ..

The evaluation results of the uptake of the fluorescent substance in the M-like cell are as shown in FIG. 1. However, in the uptake up to 1 hour (0h-1h), the fluorescent beads modified with any anti-podocalyxin antibody were compared with the Isotype control. The promotion of uptake was confirmed, and it was shown that 4D2 had the highest uptake efficiency. Comparing the uptake up to 2 and 3 hours, 4A7 and 4E5 are almost the same as Isotype control, but 4D2 maintains high uptake efficiency, even when compared with fluorescent beads modified with other antibodies. The value was significantly higher.
Therefore, rapid uptake from M cells was confirmed with any of the anti-podocalyxin antibodies, but in the case of 4D2, the uptake was promoted for a long time, showing a higher uptake efficiency promoting effect with the passage of time.

Example 2 Confirmation of podocalyxin binding site of 4D2 A recombinant protein in which glutathione-S-transferase was fused to the N-terminal side of the amino acid sequence at positions 229 to 506 of podocalyxin was expressed in Escherichia coli, and the purified product was 1 μg / 10 μL. It was adjusted to be. An equal amount of sample buffer for SDS-PAGE (8% SDS, 40% glycerol / 250 mM Tris-HCl Buffer pH 6.8) was mixed therewith, and the mixture was reacted at 95 ° C. for 5 minutes to prepare a sample for Western blotting.
For Western blotting, a sample was electrophoresed on a 12.5% polyacrylamide gel (ePAGE manufactured by Ato), and a transfer reaction was carried out on a PVDF membrane using a semi-dry transfer device (Ato). The post-transcriptional PVDF membrane was immersed in 75 mL of blocking buffer (TBS containing 10% skim milk), and a masking reaction was carried out at room temperature for 4 hours. After the reaction, the PVDF membrane was washed 3 times with an appropriate amount of TBS, and then the PVDF membrane was immersed in a 4D2 solution and reacted at 4 ° C. for about 16 hours (primary antibody reaction). After the primary antibody reaction, the PVDF membrane was washed 5 times with TBS containing Tween 20 (trade name), an HRP-labeled anti-mouse antibody (Jackson Immuno Research Laboratories) solution was added, and the reaction was carried out at room temperature for 60 minutes (secondary). Antibody reaction). After the secondary antibody reaction, the PVDF membrane was washed 5 times with TBS containing Tween 20 (trade name), and the antibody to podocalyxin was washed with Super Signal (trade name) Western Lighting-Plus ECL (trade name, manufactured by Perkin Elmer). A bond was detected.
In FIG. 2, the left lane is a molecular weight marker Dr. Western (Oriental yeast), the right lane is a recombinant protein in which glutathione-S-transferase is fused to the N-terminal side of the amino acid sequence at positions 229 to 506 of podocalyxin. As shown here, 4D2 bound to a protein having the amino acid sequence of positions 229-506 of podocalyxin. The 229 to 506 positions are amino acid sequences near the transmembrane domain of podocalyxin. Therefore, it is considered that 4D2 recognizes and binds to the amino acid sequence near the membrane, and the antibody that binds to this region is considered to have high uptake efficiency by M cells.

Claims (4)

An anti-podocalyxin antibody or an antigen-binding fragment thereof having at least one of the CDRs shown in (a) to (f) below.
(A) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2.
(C) Heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3
(D) Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4.
(E) Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5.
(F) Light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6. The anti-podocalyxin antibody or antigen-binding fragment thereof according to claim 1, which has all six CDRs shown in (a) to (f) above. A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more identity with the amino acid sequence. The antipodocalyxin antibody according to claim 1 or 2, or an antigen-binding fragment thereof, which comprises a light chain variable region comprising. A drug compound for transmucosal administration, which comprises binding a drug to the anti-podocalyxin antibody according to any one of claims 1 to 3 or an antigen-binding fragment thereof.

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