JP7489057B2 - Anti-podocalyxin antibody - Google Patents

Description

The present invention relates to a novel antibody against podocalyxin.

Podocalyxin (PCX) is a single-pass transmembrane glycoprotein with a molecular weight of approximately 140 kDa that was discovered in kidney glomerular epithelial cells (podocytes) and has high homology to the hematopoietic stem cell marker CD34. Podocalyxin has many glycosaminoglycans and O- and N-linked glycosylation sites in the extracellular domain, and is a highly glycosylated sialomucin-type protein. It is known to contribute to cell adhesion and morphogenesis, and that in cancer cells, the glycans on podocalyxin act as ligands for E-/P-/L-selectin expressed on endothelial cells, and are involved in the adhesion, invasion, and metastasis of cancer cells (Non-Patent Documents 1 and 2).

In recent years, it has been reported that podocalyxin is highly expressed in colorectal cancer, breast cancer, urothelial bladder cancer, pancreatic cancer, gastric cancer, testicular tumors, prostate cancer, and ovarian cancer, and can be a marker for cancer malignancy and poor prognosis (Non-Patent Document 3), that it is expressed in human stem cells, such as embryonic stem cells and pluripotent stem cells, and can be a marker for the undifferentiation of these stem cells (Non-Patent Document 4), and that it is expressed in vascular endothelial cells and can be a marker for vascular dysfunction (Patent Document 1).

Therefore, anti-podocalyxin antibodies that can recognize podocalyxin molecules regardless of whether they are glycosylated or not may be useful in a wide range of applications, including detection of normal and cancerous tissues and cells, cancer treatment, and diagnosis of vascular dysfunction.

JP 2019-35665 A

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 carcinoma 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 and protein drugs such as antibody molecules have been increasingly used to replace conventional low molecular weight drugs, and these are often administered as injections, such as intramuscular or subcutaneous injections. Injections are painful and carry the risk of serious side effects such as allergies and anaphylaxis, so there is hope for the development of oral or transmucosal administration forms as alternatives to injections. However, in transmucosal preparations, the mucosal epithelial cell layer functions as a lipid-soluble barrier, so drugs with large molecular size or very water-soluble drugs cannot pass through the mucosal epithelial layer and are hardly absorbed into the body. This calls for the development of technologies such as membrane penetration promotion that are different from the conventional use of absorption enhancers and prodrug conversion.

In addition, it is known that mucosal immunity can induce multiple defense mechanisms, since administration of a vaccine to the mucosa can induce a localized immune response in the mucosa in addition to a systemic immune response. For this reason, mucosal vaccines are expected to be highly effective against pathogens that invade through the mucosa. However, vaccine preparations have even larger molecular and particle sizes than antibody drugs, and with the exception of some antigens with mucosal affinity and live vaccines for pathogens with mucosal invasion, existing vaccines cannot be expected to induce immunity by administering them to the mucosa. Therefore, there is a demand for technology that can promote uptake of vaccine preparations from the mucosa.

Under these circumstances, the inventors discovered that podocalyxin is highly expressed in microfold cells (M cells) in the mucosal epithelium. After further investigation, they succeeded in obtaining an anti-podocalyxin antibody that shows excellent affinity for M cells and is well taken up by the cells, thus completing 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) a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:1
(b) a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO:2
(c) a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:3
(d) a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:4
(e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:5
(f) a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:6
2) The anti-podocalyxin antibody or antigen-binding fragment thereof of 1), which has all six CDRs shown in (a) to (f).
3) An anti-podocalyxin antibody or antigen-binding fragment thereof of 1) or 2), having 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 thereto, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more identity thereto.
4) A drug compound for transmucosal administration, comprising an anti-podocalyxin antibody or an antigen-binding fragment thereof according to any one of 1) to 3) above and a drug bound thereto.

Since the antibody or antigen-binding fragment thereof of the present invention is well taken up by M cells, by binding it to a drug, the absorption efficiency of the drug via mucosal M cells is increased, making it possible to administer drugs that have previously been poorly absorbed orally or via the mucosa via mucosal administration, which is expected to improve the convenience and efficacy of taking the drug.

Assessment of anti-podocalyxin antibody uptake in M cells. Binding evaluation of 4D2 to the transmembrane domain of podocalyxin.

The following describes in detail preferred embodiments of the present invention. However, the present invention is not limited to the following embodiments.

In the present invention, podocalyxin (PCX) refers to a type I transmembrane protein consisting of 558 amino acid residues, which is expressed in kidney glomerular epithelial cells.
The anti-podocalyxin antibody of the present invention is an antibody that has affinity for M cells and is well taken up by these cells, and has at least one of the CDRs shown in (a) to (f) below.
(a) a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:1
(b) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:2
(c) a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:3
(d) a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:4
(e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:5
(f) a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:6

The anti-podocalyxin antibody of the present invention has a structure in which two heavy chains (H chains) and two light chains (L chains) stabilized by a pair of disulfide bonds are associated with each other. The heavy chains consist of a heavy chain variable region _VH , heavy chain constant regions _CH1 , _CH2 , _CH3 , and a hinge region located between _CH1 and _CH2 . The light chains consist of a light chain variable region _VL and a light chain constant region _CL .

In the present invention, "CDR (complementarity-determining region)" refers to the region of the variable region of an antibody molecule that directly contacts an antigen. There are three CDRs in each of the light chain and heavy chain variable regions, and in the present invention, these are referred to as heavy chain CDR1-3 and light chain CDR1-3.

CDRs can be determined from DNAs encoding the heavy chain variable region _VH and the light chain variable region _VL by analysis software provided by abYsis (http://abysis.org/) or the like, and the CDRs of the anti-podocalyxin antibody of the present invention were determined from the DNA (SEQ ID NO: 7) encoding the heavy chain variable region _VH and the DNA (SEQ ID NO: 9) encoding the light chain variable region _VL of the monoclonal antibody isolated under the clone name: 4D2, as described in the Examples below. The amino acid sequences are specifically as follows, and correspond to the amino acid sequences at positions 26 to 35, 50 to 66, and 99 to 106 of the amino acid sequence of the heavy chain variable region _VH (SEQ ID NO: 8) and the amino acid sequences at positions 24 to 33, 49 to 55, and 88 to 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 or antigen-binding fragment thereof of the present invention may have at least one of the six CDRs described above, as long as it has the property of having affinity for M cells and being well taken up by the cells, preferably at a rate equivalent to the rate of uptake into M cells shown in 4D2 in the examples described below, but preferably has two or more, more preferably three or more, more preferably four or more, more preferably five or more, and most preferably has all six.

In addition, the anti-podocalyxin antibody or antigen-binding fragment thereof of the present invention preferably 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, preferably 95% or more, more preferably 98% or more identity thereto, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity thereto. The variability of the heavy chain variable region and the light chain variable region is found outside the CDR.

In other words, the anti-podocalyxin antibody or antigen-binding fragment thereof of the present invention preferably has at least one of the CDRs (a) to (f) above, and 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, preferably 95% or more, and more preferably 98% or more identity thereto, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more, preferably 95% or more, and more preferably 98% or more identity thereto.
Furthermore, the anti-podocalyxin antibody or antigen-binding fragment thereof of the present invention more preferably has all six CDRs shown in (a) to (f) above, and 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, preferably 95% or more, and more preferably 98% or more identity thereto, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more, preferably 95% or more, and more preferably 98% or more identity thereto.

Here, the identity of amino acid sequences refers to the percentage (%) of the number of positions where identical amino acid residues exist in both sequences when the two amino acid sequences are aligned, relative to the total number of amino acid residues in both sequences. Specifically, it can be calculated, for example, by the Lipman-Pearson method (Science, 227, 1435, (1985)), and can be calculated by performing analysis using the Search homology program of the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with the Unit size to compare (ktup) set to 2.

The anti-podocalyxin antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. The anti-podocalyxin antibody may be any isotype of IgG, IgM, IgA, IgD, or IgE. The anti-podocalyxin antibody may be prepared by immunizing a non-human animal such as a mouse, a rat, a hamster, a guinea pig, a rabbit, or a chicken, or may be a recombinant antibody, a chimeric antibody, a humanized antibody, or a fully humanized antibody.
Here, examples of humanized antibodies include antibodies that have heavy chain CDR1-3 and light chain CDR1-3 of an antibody produced by immunizing a mouse, and in which the framework region (FR), which is a region other than the CDRs that shows relatively few mutations, has been replaced with the corresponding region of a human antibody.

An antigen-binding fragment of an anti-podocalyxin antibody refers to a fragment of the above-mentioned anti-podocalyxin antibody of the present invention, which binds to podocalyxin and has at least one, preferably two or more, more preferably three or more, more preferably four or more, more preferably five or more, and more preferably all six of the CDRs shown in (a) to (f). Specific examples include Fab consisting of _VL , _VH , _CL , and _CH1 domains, F(ab)2 in which two Fabs are linked by a disulfide bond at the hinge domain, Fv consisting of _VL and _VH , scFv which is a single-chain antibody in which _VL and _VH are linked by an artificial polypeptide linker, as well as bispecific antibodies such as diabody type, scDb type, tandem scFv type, and leucine zipper type.

The anti-podocalyxin antibody or antigen-binding fragment thereof of the present invention can be prepared by, for example, the hybridoma method or the recombinant DNA method.
In the hybridoma method, antibody-producing cells are isolated from a non-human mammal (e.g., a mouse, a rat, a hamster, a rabbit, a monkey, a goat, etc.) that has been immunized with podocalyxin or a portion thereof, and then these are fused with myeloma cells or the like to produce hybridomas, and the antibodies produced by these hybridomas are purified to obtain the antibodies.

Examples of recombinant DNA techniques include cloning nucleic acids encoding the anti-podocalyxin antibody or functional fragments of the antibody of the present invention from hybridomas or B cells, incorporating them into an appropriate vector, and introducing them into host cells (e.g., mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.) to produce a recombinant antibody. In expressing nucleic acids encoding antibodies or antibody-binding fragments, DNAs encoding the heavy chain and light chain may be separately incorporated into expression vectors to transform host cells, or nucleic acids encoding the heavy chain and light chain may be incorporated into a single expression vector to transform host cells.

Antibodies can be isolated and purified using methods commonly used for purifying polypeptides. Isolation and purification methods include, for example, affinity columns using protein A/G/L or the like, other chromatography columns, filters, ultrafiltration, salting out, and dialysis, and these can be combined as appropriate.

The drug compound of the present invention comprises the above-mentioned anti-podocalyxin antibody or an antigen-binding fragment thereof bound to a drug.
Examples of the drug include drugs classified as antipyretics, analgesics, anti-inflammatory drugs, antirheumatic drugs, hypnotics, sedatives, anxiolytics, antipsychotics, antidepressants, antiepileptic drugs, drugs for treating Parkinson's disease, drugs for improving cerebral circulation and metabolism, muscle relaxants, drugs acting on the autonomic nervous system, antivertigo drugs, drugs for treating migraine headaches, cardiac inotropes, antianginal drugs, β-blockers, Ca antagonists, antiarrhythmic drugs, diuretics, antihypertensive drugs, antiallergic drugs, bronchodilators, drugs for treating asthma, antitussives, expectorants, drugs for treating peptic ulcers, drugs for treating gout, drugs for dyslipidemia, drugs for diabetes, hormone preparations, drugs for osteoporosis, antibacterial drugs, antiviral drugs, antiparasitic drugs, antiprotozoal drugs, immunosuppressants, anesthetics, and vaccines, and are preferably peptide or proteinaceous drugs or water-soluble polymeric drugs that are poorly absorbed when generally administered orally or mucosally.

The anti-podocalyxin antibody or its antigen-binding fragment can be bound to the drug via, for example, an amino group, a sulfhydryl group, a carboxyl group, or an aldehyde group generated on a sugar chain by reducing sugar terminal or periodate oxidation. Specifically, an amino group can be bound via a crosslinker having an N-hydroxysuccinimide ester or an isothiocyano group, a sulfhydryl group can be bound via a crosslinker having a maleimide group or a bromoacetamide group, or an aldehyde can be bound via a crosslinker having a hydrazide group. In addition, a carboxyl group can be bound by converting the carboxyl group into an active ester using an N-hydroxysuccinimide ester and reacting this active ester with a crosslinker having a primary amine, but the binding is not limited to these.

The pharmaceutical compounds of the present invention can be used for transmucosal administration since their uptake into the mucosa of body cavities by transcytosis is enhanced via the anti-podocalyxin antibody or an antigen-binding fragment thereof.
Here, transmucosal administration refers to an administration form in which the drug is absorbed via the mucous membrane of a body cavity, and specific examples thereof include nasal inhalation via the mucous membrane covering the nasal cavity, eye instillation onto the conjunctiva or cornea, pulmonary inhalation via the mucous membrane of the alveoli, inhalation onto the tracheal mucosa or bronchial mucosa, vaginal administration via the mucous membrane in the vagina, oral administration or rectal administration via the mucous membrane of the digestive tract such as the stomach, duodenum, small intestine, colon and large intestine including the rectum, and middle ear administration onto the mucous membrane of the middle ear, etc. Among these, preferred administration forms are nasal inhalation, pulmonary inhalation, tracheal or bronchial inhalation, and eye instillation.
Such drug compounds can be formulated as compositions (pharmaceutical compositions) in solid, semi-solid or liquid form for transmucosal administration together with pharma- ceutically acceptable carriers for mucosal administration.

The present invention will be described in detail below, but the present invention is not limited to these in any way.
Production Example: Preparation of anti-podocalyxin antibody (1) Establishment of clones (4A7, 4D2, and 4E5) A plasmid encoding a protein (Strep-ΔTM) in which a Strep tag and a His tag were added to the C-terminus of the extracellular domain (amino acid sequence 1 to 409) of human podocalyxin was lipofected into human embryonic kidney cells 293 (HEK293T cells) to express Strep-ΔTM. The expressed Strep-ΔTM was purified with the His tag and buffer-exchanged by ultrafiltration to serve as an immunizing antigen. This immunizing antigen was administered intraperitoneally to a BALB/c mouse (female, SLC), and the spleen was collected from the immunized mouse to prepare splenocytes. The prepared splenocytes and mouse myeloma cells were fused with polyethylene glycol, and cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine, and then in HT medium containing hypoxanthine and thymidine, and RPMI medium, respectively, and cloned by limiting dilution. In the cloning, a His tag was added to the C-terminus of the extracellular domain of human podocalyxin (amino acid sequence 1-409), and the protein (ΔTM) expressed and purified in the same manner as above was immobilized on a plate, and clones that reacted with ΔTM were selected by ELISA. The clones that showed good reactivity as a result of ELISA were named 4A7, 4D2, and 4E5.

(2) Determination of amino acid sequence, base sequence and CDR of 4D2 1×10 ⁶ cells of hybridoma (clone: 4D2) were suspended in RNAlater (SIGMA ALDRICH), and RNA was extracted from the hybridoma after suspension. Using the extracted RNA as a template, cDNA was synthesized by RT-PCR, and the _VH and _VL regions were amplified using degenerate primers. The amplified region was cloned, and the base sequences of _VH and _VL (SEQ ID NOs: 7 and 9) were determined by cycle sequencing. The obtained base sequences were converted to amino acid sequences (SEQ ID NOs: 8 and 10).
In addition, CDRs were determined from the obtained _VH base sequence (SEQ ID NO: 7) and _VL base sequence (SEQ ID NO: 9). The sequencing was outsourced to Biopeak Co., Ltd.

Example 1 Evaluation of uptake in M-like cells 220 pmol of biotin-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch) was taken as biotin, 950 μL of PBS was added thereto, and then 0.1 mg of FluoSpheres™ Streptavidin-Labeled Microspheres was added little by little. After the addition, the mixture was shaken for 1 hour in the dark to prepare fluorescent beads bound to anti-mouse IgG antibody.
200 μL of PBS was added to the fluorescent beads bound with anti-mouse IgG antibody, and 176 pmol of anti-podocalyxin antibody (clone names: 4A7, 4D2, and 4E5) obtained in the above Production Example or mouse IgG2a Isotype control (Sigma Aldrich) was added, and the mixture was shaken for 1 hour in the dark. After shaking, the mixture was centrifuged at 15,000 rpm for 30 minutes, and the precipitate was suspended in 1.3 mL of MEM (GIBCO) containing 20% bovine calf serum, 1% NEAA, and 0.0293% L-glutamine, which was used as anti-podocalyxin antibody-labeled fluorescent beads or isotype control-labeled fluorescent beads.

Cells that were induced to differentiate into M-like cells in vitro were prepared on a Transwell membrane, and the anti-podocalyxin antibody-labeled fluorescent beads or isotype control-labeled fluorescent beads prepared above were added to the lower chamber. The fluorescence intensity of the upper chamber solution was measured to evaluate the amount of beads that had migrated to the upper chamber 1, 2, and 3 hours after addition. Note that in vitro M-like cells were prepared as described below.

As described in a literature article (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 x ¹⁰⁵ Caco-2 cells were seeded on the membrane of a Transwell (Corning, pore size: 3 μm, 24 wells) and left to stand overnight, after which the Transwell membrane was immersed in a 24-well plate supplemented with Eagle's MEM (20% FBS, 0.1 mM NEAA EMEM) containing 20% calf serum and 0.1 mM non-essential amino acids. The Transwell was transferred to a 24-well plate containing fresh 20% FBS and 0.1 mM NEAA EMEM approximately every 3 days and cultured for 21 days to form a Caco-2 monolayer. After 21 days of culture, 1 x ¹⁰⁶ Raji B cells were added to the upper chamber of the Transwell and cultured for an additional 3 days to promote differentiation into M-like cells, which were used as M-like cells.

The results of evaluation of fluorescent substance uptake in M-like cells are shown in Figure 1. In the uptake up to 1 hour (0h-1h), promotion of uptake was confirmed for all fluorescent beads modified with anti-podocalyxin antibodies compared to the isotype control, and in particular, 4D2 was shown to have the highest uptake efficiency. In addition, when comparing uptake up to 2 and 3 hours, 4A7 and 4E5 were almost the same as the isotype control, but 4D2 maintained a high uptake efficiency and was significantly higher than fluorescent beads modified with other antibodies.
Thus, rapid uptake from M cells was confirmed with any of the anti-podocalyxin antibodies, but in the case of 4D2, the promotion of uptake continued for a long period of time, and the promotion effect of higher uptake efficiency was observed 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-terminus of the amino acid sequence from 229 to 506 of podocalyxin was expressed in E. coli, and the purified product was adjusted to 1 μg/10 μL. An equal amount of SDS-PAGE sample buffer (8% SDS, 40% glycerol/250 mM Tris-HCl Buffer pH 6.8) was mixed thereto and reacted at 95° C. for 5 minutes, and this was used as a sample for Western blotting.
For Western blotting, the specimen was electrophoresed on a 12.5% polyacrylamide gel (ePAGEL, manufactured by Atto Co., Ltd.), and a transfer reaction to a PVDF membrane was performed using a semi-dry transfer device (manufactured by Atto Co., Ltd.). The PVDF membrane after transfer was immersed in 75 mL of blocking buffer (TBS containing 10% skim milk) and a masking reaction was performed at room temperature for 4 hours. After the reaction, the PVDF membrane was washed three times with an appropriate amount of TBS, and then the PVDF membrane was immersed in 4D2 solution and reacted at 4°C for about 16 hours (primary antibody reaction). After the primary antibody reaction, the PVDF membrane was washed five times with TBS containing Tween 20 (trade name), and a solution of HRP-labeled anti-mouse antibody (Jackson Immuno Research Laboratories, Inc.) was added and reacted at room temperature for 60 minutes (secondary antibody reaction). After the secondary antibody reaction, the PVDF membrane was washed five times with Tween 20 (trade name)-containing TBS, and the binding of the antibody to podocalyxin was detected using Super Signal (trade name) Western Lightning-Plus ECL (trade name, manufactured by Perkin Elmer).
In Fig. 2, the left lane shows Dr. Western (Oriental Yeast), a molecular weight marker, and the right lane shows a recombinant protein in which glutathione-S-transferase is fused to the N-terminus of the amino acid sequence from positions 229 to 506 of podocalyxin. As shown here, 4D2 bound to a protein having the amino acid sequence from positions 229 to 506 of podocalyxin. Positions 229 to 506 are the amino acid sequence near the transmembrane domain of podocalyxin. Therefore, it was thought that 4D2 recognizes and binds to an amino acid sequence near the membrane, and that an antibody that binds to this region is highly efficiently taken up by M cells.

Claims (3)

An anti-podocalyxin antibody or an antigen-binding fragment thereof having the CDRs shown in the following (a) to (f):
(a) a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:1
(b) a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO:2
(c) a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:3
(d) a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO:4
(e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:5
(f) a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:6 The anti-podocalyxin antibody or its antigen-binding fragment according to claim 1, having 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 thereto, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more identity thereto. A drug compound for transmucosal administration, comprising the anti-podocalyxin antibody or antigen-binding fragment thereof according to claim 1 or 2 bound to a drug.