JP7242082B2 - Anti-canine CD20 monoclonal antibody - Google Patents

Description

The present invention relates to monoclonal antibodies against canine CD20. More specifically, it relates to a monoclonal antibody against canine CD20 that has a different function from existing ones and is highly effective.

In recent years, antibody drugs have been established due to the development of medical care. Especially for human B-cell lymphoma, in addition to CHOP therapy, which is a multidrug anticancer drug therapy, treatment with anti-CD20 antibody is used as a standard therapy.
Pets such as dogs are living longer and the incidence of tumors is increasing, as is the case with humans. There is a need to provide effective treatment methods.

CD20 is also known to be involved in canine B-cell lymphoma, and antibody drugs targeting CD20 have been developed. For example, Patent Documents 1 and 2 disclose monoclonal antibodies or antibody fragments against the extracellular domain of canine CD20, which have a heavy chain variable region consisting of a specific amino acid sequence and a light chain variable region consisting of a specific amino acid sequence. ing. It is also marketed in the United States by Blontless, Aratana.

In Patent Documents 3 and 4, a humanized monoclonal antibody that binds to CD20 comprising an L chain variable region and an H chain variable region represented by specific amino acid sequences or an antigen-binding fragment thereof, CDR1, CDR2 and QQCDR3 are disclosed. A chimeric or humanized monoclonal antibody or antigen-binding fragment thereof that binds to CD20 is disclosed, comprising a light chain variable region and a heavy chain variable region having the same structure. It was also disclosed that these antibodies can also be used in dogs, and that treatment of dogs diagnosed with diffuse large B-cell lymphoma with the 1F5 chimeric monoclonal antibody eliminated traces of lymphoma. ing.

Furthermore, Patent Documents 5 and 6 disclose an antibody or antibody fragment that recognizes canine or feline CD20, which contains hypervariable domains in the light chain and hypervariable domains in the heavy chain, which contain CDR regions selected from specific amino acid sequences. It is
Although various antibodies against CD20 have been developed in this manner, the effects vary depending on the antibody, and it cannot be said that an antibody with excellent effects has been obtained.
Therefore, in the present invention, the present inventor attempted to prepare a monoclonal antibody against canine CD20 that has a function different from that of existing antibodies and is highly effective.

Japanese Patent Application Laid-Open No. 2016-13104 Japanese Patent Publication No. 2014-532649 Japanese Patent Publication No. 2006-500904 Japanese Patent Application Laid-Open No. 2009-291197 Japanese Patent Publication No. 2013-520990 Japanese Patent Application Laid-Open No. 2016-1779978

An object of the present invention is to provide a monoclonal antibody against canine CD20 that has a function different from that of existing antibodies and is highly effective.

In order to solve this problem, the present inventors conducted intensive studies and found that 1 to 13 amino acids were deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 or in the amino acid sequence of SEQ ID NO: 1. A heavy chain variable region consisting of an amino acid sequence, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence in which 1 to 13 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 The inventors have found that a monoclonal antibody against canine CD20 having a variable region has functions different from those of existing antibodies and is a highly effective monoclonal antibody against canine CD20, thereby completing the present invention.

That is, the present invention for solving the above problems relates to monoclonal antibodies against canine CD20 shown in the following (1) to (6).
(1) A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which 1 to 13 amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and SEQ ID NO: 2 has a light chain variable region consisting of an amino acid sequence in which 1 to 13 amino acids are deleted, substituted or added in the amino acid sequence described in or the amino acid sequence of SEQ ID NO: 2, and a rat IgG2a, kappa chain monoclonal antibody against canine CD20 with
(2) A composition for reducing canine B cells, comprising the antibody of (1) above as an active ingredient.
(3) A therapeutic composition for diseases caused by an increase in canine B cells, comprising the antibody according to (1) above as an active ingredient.
(4) The therapeutic composition according to (3) above, wherein the disease is B-cell lymphoma, leukemia or autoimmune disease.
(5) A kit for detecting canine B cells, comprising the antibody according to (1) above.
(6) A diagnostic kit for diseases caused by increased canine B cells, comprising the antibody of (1) above.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a monoclonal antibody against canine CD20 that has a function different from that of existing antibodies and is highly effective. It is also possible to provide a composition for reducing canine B cells, a composition for treating diseases such as B-cell lymphoma, leukemia, or autoimmune diseases caused by an increase in canine B cells, etc., using this monoclonal antibody as an active ingredient. becomes.

4E1-7 antibody concentration-dependently binds to Jurkat/cCD20 (Example). 4E1-7 antibody specifically recognizes the canine CD20 molecule (Example). FIG. 4 is a diagram showing the reactivity of 4E1-7 antibody to CLBL-1 (Example). Fig. 10 shows the reactivity of the 4E1-7 antibody with canine peripheral blood lymphocytes (Example). Fig. 10 shows the reactivity of 4E1-7 antibody to lymphoma cells collected from canine lymphoma cases (Example). FIG. 10 shows the results of examining the complement-dependent cytotoxic activity of the 4E1-7 antibody (Example). Fig. 10 shows the results of examining the direct cytotoxic activity of the 4E1-7 antibody (Example).

The "anti-canine CD20 monoclonal antibody" of the present invention is an antibody that recognizes the canine CD20 molecule and has the amino acid sequence set forth in SEQ ID NO: 1 or one or several amino acids deleted in the amino acid sequence of SEQ ID NO: 1. , a heavy chain variable region consisting of a substituted or added amino acid sequence, and an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 An antibody having a light chain variable region consisting of Here, several in the heavy chain variable region or light variable region means 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or means two.

The "anti-canine CD20 monoclonal antibody" of the present invention can bind well to the extracellular region of canine CD20, and the kinetics of CD20-positive cells can be confirmed by FACS analysis using live canine cells and tissues, fluorescence microscope observation, etc. It can be used for analysis such as

The "anti-canine CD20 monoclonal antibody" of the present invention may be a chimeric antibody in which the constant region portion of the antibody is replaced with the constant region portion of an antibody derived from another animal. In addition, only the complementarity determining region (CDR) in the variable region is derived from a non-canine animal antibody such as rat, and the framework region (FR) and constant region in the variable region other than the CDR are derived from a canine antibody. It may be an antibody. A chimeric antibody or caninized antibody in which the constant region portion of the antibody is replaced with a canine antibody is preferred because it elicits an immune response against a heterologous antigen and does not cause serious side effects such as anaphylactic shock.
Preferably, this chimeric or caninized antibody is IgG.

The "composition for reducing canine B cells" of the present invention refers to a composition for reducing canine B cells, containing the "anti-canine CD20 monoclonal antibody" of the present invention as an active ingredient. In addition to these active ingredients, other ingredients may be included as long as they are useful for reducing canine B cells, and drugs such as anticancer agents and radionuclides may be further included. In addition, it may contain pharmaceutically acceptable additives, and examples of such additives include solvents such as water, surfactants, sodium chloride, sodium citrate, anhydrous citric acid, pH adjusters, and the like. be done.

The "composition for treating diseases caused by an increase in canine B cells" of the present invention is a composition for treating diseases caused by an increase in canine B cells, which contains the "anti-canine CD20 monoclonal antibody" of the present invention as an active ingredient. Say things.
Examples of such diseases include canine B-cell lymphoma caused by an increase in canine B cells, leukemia caused by tumorigenesis of increased B cells, and autoimmune diseases caused by an excessive increase in normal B cells.
For example, when the disease to be treated is B-cell lymphoma, administration of the "composition for treating a disease caused by increased canine B-cells" of the present invention to the affected dog results in Antibody-Dependent-Cellular-Cytotoxicity (ADCC) activity and complement-dependent cytotoxicity were induced by binding to CD20 overexpressed on the surface of B-cell lymphoma cells. (Complement-Dependent Cytotoxicity, CDC) activity, apoptosis induction and other mechanisms enable killing of canine B-cell lymphoma cells.

The "composition for treating diseases caused by an increase in canine B cells" of the present invention may be a composition comprising the active ingredient "anti-canine CD20 monoclonal antibody". It may further contain other components such as radionuclides and drugs.
In addition, the agent may be in any form as long as it is effective in treating diseases caused by an increase in canine B cells, and may be provided as a liquid, powder, tablet, capsule, or the like for intravenous injection or infusion.
Treatment methods using the therapeutic composition of the present invention include, for example, intravenous injection, infusion, etc., so that the "anti-canine CD20 monoclonal antibody" of the present invention is 1 to 10 mg/kg about once a week. and the like.

The "canine B cell detection kit" of the present invention refers to a kit for detecting whether or not a sample contains canine B cells using the "anti-canine CD20 monoclonal antibody" of the present invention. . In addition, the "diagnostic kit for diseases caused by increased canine B cells" of the present invention is used to determine whether the target dog is suffering from diseases caused by increased canine B cells, such as canine B-cell lymphoma, leukemia, and autoimmune diseases. It refers to a kit for diagnosing whether or not
These kits contain at least the "anti-canine CD20 monoclonal antibody" of the present invention as one of the components.
The "anti-canine CD20 monoclonal antibody" contained in the kit of the present invention may be unlabeled or labeled with a conventionally known labeling substance such as an enzyme, fluorescent substance or radioactive substance.

Examples of such labeling substances include enzymes such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase and amylase. Fluorescent substances include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Cy3, Cy5, R-phycoerythrin, B-phycoerythrin, Alexa fluoro 488 or Alexa fluoro 647, etc., and radioactive substances include tritium. , iodine-125, iodine-131 and the like. Alternatively, biotin, streptavidin, or the like may be used.

When using the "kit for detecting canine B cells" or the "kit for diagnosing diseases caused by an increase in canine B cells" of the present invention, conventionally known methods useful for detecting canine B cells and diagnosing diseases caused by an increase in canine B cells can be used. Any of the reagents, equipment, etc., that are used may be used.
Lymphocytes are isolated from biological tissue such as dog blood to be diagnosed, and the "anti-canine CD20 monoclonal antibody" contained in these kits is acted upon. By observing the dynamics and morphology of CD20-positive cells by FACS analysis, fluorescence microscope observation, etc., it becomes possible to detect canine B cells and diagnose diseases caused by an increase in canine B cells.
By using these kits, living cells can be detected or diagnosed, and cell fixation or the like is not required, so detection or diagnosis can be performed accurately and easily.

Examples and the like of the present invention are shown below, but the present invention is not limited to these.

1. Preparation of anti-canine CD20 monoclonal antibody 1) Establishment of canine CD20 overexpressing cells (NRK/cCD20) After cloning the previously reported canine CD20 molecule cDNA, add a FLAG tag to the C-terminus of the cloned pMx-IP vector. (provided by Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo). This was transfected into PLAT-gp cells (provided by Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo) together with pCVSVG, and the produced retrovirus was used to infect rat kidney cell line NRK. This yielded canine CD20 overexpressing cells (NRK/cCD20).

2) Immunization of rats and preparation of hybridoma SD rats were immunized with NRK/cCD20 established in 1) above. That is, NRK / cCD20 was mixed with the adjuvant Titer Max (registered trademark) Gold (CytRx), administered to the foot pad of Wistar rats (Japan SLC Co., Ltd.), and boosted 7 days and 14 days after the initial immunization. gone. Three days after the final immunization, the knee lymph nodes and inguinal lymph nodes were harvested and lymphocytes were collected. Subsequently, the collected lymphocytes and P3X63-Ag8.653 cells, a mouse myeloma cell line, were fused. The obtained cells were cultured in HAT medium to select hybridomas in which lymphocytes and mouse myeloma cells were fused.

Antibodies in the obtained hybridoma culture supernatant were reacted with NRK/cCD20, and hybridomas producing reactive monoclonal antibodies were screened by flow cytometry (FACS). A fluorescently labeled anti-rat IgG antibody was used as the secondary antibody.
Furthermore, antibodies in the culture supernatant of candidate hybridomas were allowed to react with canine peripheral lymphocytes, and hybridomas producing reactive monoclonal antibodies were screened by FACS. Promising hybridomas were cloned by the limiting dilution method to obtain a hybridoma cell line producing a rat monoclonal antibody (4E1-7) against canine CD20.

3) Confirmation of reactivity of 4E1-7 antibody 4E1-7 antibody diluted to various concentrations was prepared in the same manner as in 1) above and Jurkat cells overexpressing FLAG tag-canine CD20 fusion protein (Jurkat/cCD20 ). This was reacted with a secondary antibody, and fluorescence intensity was then measured by flow cytometry (Becton Dickinson, BD Accuri) to confirm that the 4E1-7 antibody bound to Jurkat/cCD20 in a concentration-dependent manner ( Figure 1). After purifying this antibody with a protein A column, it was confirmed that this 4E1-7 antibody has rat IgG2a and kappa chains.

2. Analysis of 4E1-7 antibody 1) Immunoprecipitation and Western blotting using Jurkat/cCD20
In order to confirm that the 4E1-7 antibody specifically recognizes the canine CD20 molecule, immunoprecipitation reaction using Jurkat/cCD20 and Western blotting were performed. The cell lysate obtained by lysing Jurkat/cCD20 with 1% NP40 lysis buffer was reacted with 1 μg of 4E1-7 antibody, and proteinG sepharose beads (GE healthscience) were added for immunoprecipitation, followed by centrifugation. A complex of 4E1-7 antibody and target antigen was obtained as a precipitate. The immunoprecipitate was electrophoresed as a sample for SDS-PAGE and then detected by Western blotting using an anti-FLAG tag antibody (Clone M2, SIGMA). As a result, as shown in FIG. 2, a positive band was observed near the molecular weight corresponding to the canine CD20 molecule (lane 4).
On the other hand, when a similar test was performed using Jurkat cells not expressing canine CD20 molecules as a negative control for Jurkat/cCD20, or an isotype control antibody (rat IgG2a, ebioscience) as a negative control for 4E1-7 antibody, No band corresponding to canine CD20 was detected (lane 1-3). These results confirmed that 4E1-7 specifically recognizes the canine CD20 molecule.

2) Reactivity to canine lymphoma cell lines Nine canine lymphoma cell lines (T cell types: CLC, CLK, Ema, Nody-1, EO-1, GL-1, UL-1, 17-71, CLGL-90 and B cell type: CLBL-1) was used to analyze the reactivity of the 4E1-7 antibody by flow cytometry. As a result, the 4E1-7 antibody showed reactivity only with CLBL-1, a B-cell lymphoma cell line. FIG. 3 shows the results of the reactivity of the 4E1-7 antibody to CLBL-1.

3) Reactivity to Canine Peripheral Blood Lymphocytes Lymphocytes were isolated from the peripheral blood of 4 healthy beagle dogs, and the reactivity of the 4E1-7 antibody was analyzed by flow cytometry. As a result, as shown in FIG. 4, the 4E1-7 antibody showed reactivity only with CD21-positive B cells. In addition, FIG. 4 shows the results of one of the four dogs.

4) Reactivity to lymphoma cells collected from canine lymphoma cases The reactivity of the 4E1-7 antibody to lymphoma cells (4 B-cell type and 1 T-cell type) collected from canine lymphoma cases was analyzed by flow cytometry. . As a result, as shown in FIG. 5, the 4E1-7 antibody reacted with B-cell lymphoma cells in 4 out of 4 cases, but showed no reactivity with T-cell lymphoma cells in 1 case. In addition, FIG. 5 shows the results of one animal each of the B cell type and the T cell type.

3. Assessment of complement-dependent cytotoxicity
The ability of the 4E1-7 antibody to exert complement-dependent cytotoxicity was tested on the canine B-cell lymphoma cell line CLBL-1 as a target cell line. 4E1-7 antibody (Figure 6: ●) or an isotype control (rat IgG2a, ebioscience (Figure 7: ○)) diluted to various concentrations from 0 to 10 µg/mL was added to target cells seeded in a 96-well plate. Rabbit complement (Low-Tox-M, CEDARLANE) was added to each well at a final concentration (1:40) and incubated at 37°C for 90 minutes. Cell viability was then measured by the trypan blue method. As a result, as shown in FIG. 6, no decrease in viability was observed regardless of the concentration of the 4E1-7 antibody, and no induction of complement-dependent cytotoxic activity was observed.

4. Assessment of direct cytotoxic activity
The ability of the 4E1-7 antibody to induce cell death directly without relying on complement or cytotoxic cells was tested with CLBL-1 as the target cell line. Add 4E1-7 (Figure 7: ●) or isotype control (rat IgG2a, ebioscience (Figure 7: ○)) diluted to various concentrations of 0 to 10 μg/mL to target cells seeded in a 96-well plate and incubate at 37°C. for 72 hours. Then, the cell proliferation rate was measured using cell counting kit-8 (DOJINDO). As a result, as shown in FIG. 7, when the 4E1-7 antibody was added, a concentration-dependent decrease in cell proliferation rate was observed, confirming that it exhibits direct cytotoxic activity.

5. Identification of heavy and light chain variable region amino acid sequences
Total RNA was extracted from the hybridoma producing the 4E1-7 antibody, and reverse transcription was performed using Superscript (registered trademark) III (Invitrogen) and primers specific to the constant region of rat antibody (IgG) as a template. bottom.
As primers, YTM1224 (SEQ ID NO: 3) was used for light chain amplification and YTM172 (SEQ ID NO: 4) was used for heavy chain amplification. Using the resulting cDNA as a template, YTM166 (SEQ ID NO: 5) and YTM1224 (SEQ ID NO: 3) for the light chain, and YTM166 (SEQ ID NO: 5) and YTM172 (SEQ ID NO: 4) for the heavy chain as primers A primary PCR was performed using The PCR products were then gel purified and either YTM170 (SEQ ID NO: 6) and YTM173 (SEQ ID NO: 7) or YTM170 (SEQ ID NO: 6) and YTM174 (SEQ ID NO: 6) were used to amplify the entire variable region of the light or heavy chain. No. 8) was used to perform nested PCR. By cloning the nested PCR product into the pBluescript SK(-) vector, the nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence were determined. The amino acid sequence of the heavy chain variable region is shown in Sequence Listing SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in Sequence Listing SEQ ID NO: 2.

1 above. ~ 5. A monoclonal antibody against canine CD20 having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO:2 was obtained by the above steps.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a monoclonal antibody against canine CD20 that has a function different from that of existing antibodies and is highly effective. Using this monoclonal antibody as an active ingredient, it is also possible to provide compositions for reducing canine B cells, and compositions for treating diseases such as B-cell lymphoma, leukemia, and autoimmune diseases caused by an increase in canine B cells. Become.

Claims (6)

Canine CD20 having a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2, and having rat IgG2a and kappa chains monoclonal antibody against. A composition for reducing canine B cells, comprising the antibody of claim 1 as an active ingredient. A therapeutic composition for diseases caused by increased canine B cells, comprising the antibody of claim 1 as an active ingredient. 4. The therapeutic composition according to claim 3, wherein the disease is either B-cell lymphoma, leukemia or autoimmune disease. A kit for detecting canine B cells, comprising the antibody of claim 1. A kit for diagnosing a disease caused by increased canine B cells, comprising the antibody of claim 1.

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