JP2021181453A - Anti-canine cd20 monoclonal antibodies - Google Patents

Abstract

To provide anti-canine CD20 monoclonal antibodies having functions not found in conventional antibodies with superior effects.SOLUTION: The present invention discloses an anti-canine CD20 monoclonal antibody which comprises a heavy chain variable region comprising a specific amino acid sequence or an amino acid sequence with deletion, substitution or addition of one to 13 amino acids in the specific amino acid sequence, and a light chain variable region comprising another specific amino acid sequence or an amino acid sequence with deletion, substitution or addition of one to 13 amino acids in the other specific amino acid sequence.SELECTED DRAWING: None

Description

The present invention relates to a monoclonal antibody against canine CD20. More specifically, the present invention relates to a monoclonal antibody against canine CD20, which has a function different from that of the existing one and has an excellent effect.

In recent years, antibody drugs have been established due to the development of medical treatment. In particular, for human B-cell lymphoma, treatment with anti-CD20 antibody is used as standard therapy in addition to CHOP therapy, which is a multidrug anticancer drug therapy.
The lifespan of pets such as dogs has been extended and the incidence of tumors has increased as in humans. There is a need to provide a variety of treatments.

The association of CD20 is also known 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 region of canine CD20 having 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 sold by Blondeless, Aratana in the United States.

In Patent Documents 3 and 4, a humanized monoclonal antibody or an antigen-binding fragment thereof that binds to CD20 containing an L-chain variable region and an H-chain variable region represented by a specific amino acid sequence, and CDR1, CDR2, and QQCDR3 are referred to. Disclosed is a chimeric or humanized monoclonal antibody or antigen-binding fragment thereof that binds to CD20 and comprises an L-chain variable region and an H-chain variable region. It was also disclosed that these antibodies could be targeted in dogs, and that dogs diagnosed with diffuse large B-cell lymphoma were treated with the 1F5 chimeric monoclonal antibody, and the traces of lymphoma disappeared. ing.

Further, Patent Documents 5 and 6 disclose an antibody or antibody fragment that recognizes canine or cat CD20 containing a hypervariable domain in a light chain containing a CDR region selected from a specific amino acid sequence and a hypervariable domain in a heavy chain. Has been done.
Although antibodies against various CD20s have been developed in this way, the effects vary depending on the antibodies, and it cannot be said that an antibody having an excellent effect has been obtained.
Therefore, in the present invention, the present inventor attempted to produce a monoclonal antibody against canine CD20, which has a function different from that of the existing one and has an excellent effect.

Japanese Unexamined Patent Publication No. 2016-13104 Special Table 2014-532649 Gazette Special Table 2006-500904 Japanese Unexamined Patent Publication No. 2009-291197 Special Table 2013-520990 Gazette Japanese Unexamined Patent Publication No. 2016-1779978

An object of the present invention is to provide a monoclonal antibody against canine CD20, which has a function different from that of the existing one and has an excellent effect.

In order to solve this problem, as a result of diligent studies, the present inventor has deleted, substituted or added 1 to 13 amino acids in the amino acid sequence set forth in SEQ ID NO: 1 or 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 an amino acid sequence in which 1 to 13 amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2. We have found that the monoclonal antibody against canine CD20 having a variable region has a function different from that of the existing one and is an excellent effect against canine CD20, and have completed the present invention.

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

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

It is a figure showing that the 4E1-7 antibody binds to Jurkat / cCD20 in a concentration-dependent manner (Example). It is a figure showing that the 4E1-7 antibody specifically recognizes a canine CD20 molecule (Example). It is a figure which showed the reactivity of 4E1-7 antibody to CLBL-1 (Example). It is a figure which showed the reactivity to the peripheral blood lymphocyte of a dog of 4E1-7 antibody (Example). It is a figure which showed the reactivity to the lymphoma cell collected from the canine lymphoma case of 4E1-7 antibody (Example). It is a figure of the result of having investigated the complement-dependent cytotoxicity activity of 4E1-7 antibody (Example). It is a figure of the result of having investigated the direct cytotoxic activity of 4E1-7 antibody (Example).

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

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

The "anti-dog 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 regions (CDRs) in the variable region are derived from non-dog animal antibodies such as rats, and the framework regions (FR) and constant regions in the variable regions other than CDR are derived from canine antibodies. It may be an antibody. A chimeric antibody or a canine antibody in which the constant region portion of the antibody is replaced with a canine antibody is preferable because it induces an immune response against a heterologous antigen and does not cause serious side effects such as anaphylactic shock.
The chimeric antibody or canine antibody is preferably IgG.

The "composition for reducing dog B cells" of the present invention refers to a composition for reducing dog B cells containing the "anti-dog CD20 monoclonal antibody" of the present invention as an active ingredient. If it is useful for the reduction of canine B cells, it may contain other components in addition to these active ingredients, or may further contain a drug such as an anticancer agent or a radionuclide. Further, it may contain a pharmaceutically acceptable additive, and examples of such an additive include a solvent such as water, a surfactant, sodium chloride, sodium citrate, anhydrous citric acid, a pH adjuster and the like. Be done.

The "composition for treating a disease caused by an increase in canine B cells" of the present invention is a composition for treating a disease caused by an increase in canine B cells, which comprises the "anti-dog CD20 monoclonal antibody" of the present invention as an active ingredient. That means.
Examples of such diseases include canine B cell lymphoma caused by an increase in canine B cells, leukemia in which increased B cells become tumors, and autoimmune diseases caused by an excessive increase in normal B cells.
For example, when the disease to be treated is B-cell lymphoma, the active ingredient "antibody" can be administered by administering the "composition for treating a disease due to an increase in canine B cells" of the present invention to a affected dog. Antibody-Dependent-Cellular-Cytotoxicity (ADCC) activity and complement-dependent cytotoxicity by binding to CD20, which is overexpressed on the surface of B-cell lymphoma cells, by the canine CD20 monoclonal antibody. (Complement-Dependent Cytotoxicity, CDC) The mechanism of activity, induction of apoptosis, etc. enables the killing of canine B-cell lymphoma cells.

The "composition for treating a disease caused by an increase in canine B cells" of the present invention may be a composition comprising an active ingredient "anti-canine CD20 monoclonal antibody", and in addition to these active ingredients, an anti-cancer agent, It may further contain other components such as radionuclides and drugs.
Further, any form of the agent may be used as long as it is effective in treating a disease caused by an increase in canine B cells, and may be provided as a liquid or powder for intravenous injection or infusion, tablets, capsules or the like.
As a therapeutic method using the therapeutic composition of the present invention, for example, by intravenous injection, infusion, etc., the "anti-dog CD20 monoclonal antibody" of the present invention is about once a week so as to be 1 to 10 mg / kg. For example, it may be administered at intervals of.

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

Examples of such a labeling substance include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, amylase and the like as enzymes. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Cy3, Cy5, R-phycoerythrin, B-phycoerythrin, Alexafluoro488 or Alexafluoro647, and examples of the radioactive substance include trithium. , Iodine 125, iodine 131 and the like. Moreover, biotin, streptavidin and the like may be used.

In using the "kit for detecting canine B cells" and "kit for diagnosing diseases caused by an increase in canine B cells" of the present invention, conventionally known useful for detecting canine B cells and diagnosing diseases caused by an increase in canine B cells. Any of the following reagents, devices, etc. may be used.
Lymphocytes are isolated from living tissues such as dog blood to be diagnosed, and the "anti-dog CD20 monoclonal antibody" contained in these kits is allowed to act on them. By observing the dynamics and morphology of CD20-positive cells by FACS analysis, fluorescence microscope observation, etc., it is possible to detect canine B cells and diagnose diseases due to an increase in canine B cells.
By using these kits, living cells can be detected or diagnosed, and no cell fixing operation or the like is required, so that detection or diagnosis can be performed accurately and easily.

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

1. 1. Preparation of anti-canine CD20 monoclonal antibody 1) Establishment of canine CD20 overexpressing cells (NRK / cCD20) After cloning the previously reported cDNA of canine CD20 molecule, FLAG tag is added to this C-terminal and pMx-IP vector. (Distributed by Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo). This was gene-introduced into PLAT-gp cells (distributed by Dr. Toshio Kitamura, Institute of Medical Science, University of Tokyo) together with pCVSVG, and then infected with the rat kidney cell line NRK using the produced retrovirus. As a result, canine CD20 overexpressing cells (NRK / cCD20) were obtained.

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

The antibody in the culture supernatant of the obtained hybridoma was reacted with NRK / cCD20, and the hybridoma producing a reactive monoclonal antibody was screened by flow cytometry (FACS). As the secondary antibody, a fluorescently labeled anti-rat IgG antibody was used.
Furthermore, the antibody in the culture supernatant of the candidate hybridoma was reacted with canine peripheral lymphocytes, and the hybridoma producing a reactive monoclonal antibody was screened by FACS. A promising hybridoma was 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 Jurkat cells (Jurkat / cCD20) overexpressing the fusion protein of FLAG tag and canine CD20 prepared by the same method as 1) above with 4E1-7 antibody diluted to various concentrations. ) And incubated. By reacting this with a secondary antibody and then measuring the fluorescence intensity by flow cytometry (Becton Dickinson, BD Accuri), it was confirmed that the 4E1-7 antibody binds to Jurkat / cCD20 in a concentration-dependent manner ( Figure 1). Then, after purifying this antibody with a protein A column, it was confirmed that this 4E1-7 antibody had rat IgG2a and kappa chains.

2.4 E1-7 antibody analysis 1) Immunoprecipitation reaction and Western blotting using Jurkat / cCD20
To confirm that the 4E1-7 antibody specifically recognizes the canine CD20 molecule, immunoprecipitation reaction and Western blotting using Jurkat / cCD20 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 protein G Sepharose beads (GE health science) were added for immunoprecipitation, followed by centrifugation. A complex of 4E1-7 antibody and target antigen was obtained as a precipitate. The immunoprecipitate was run 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 dog CD20 molecule (lane 4).
On the other hand, when a similar test was performed using Jurkat cells expressing no canine CD20 molecule 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 (lane1-3). From these results, it was confirmed that 4E1-7 specifically recognizes the canine CD20 molecule.

2) Reactivity to canine lymphoma cell lines 9 canine lymphoma cell lines (T cell type: CLC, CLK, Ema, Nody-1, EO-1, GL-1, UL-1, 17-71, CLGL-90 and Using B cell type: CLBL-1), the reactivity of 4E1-7 antibody was analyzed by flow cytometry. As a result, the 4E1-7 antibody showed reactivity only with CLBL-1, which is a B cell type lymphoma cell line. Figure 3 shows the results of the reactivity of the 4E1-7 antibody with CLBL-1.

3) Reactivity to peripheral blood lymphocytes in dogs Lymphocytes were isolated from the peripheral blood of 4 healthy beagle dogs, and the reactivity of 4E1-7 antibody was analyzed by flow cytometry. As a result, as shown in FIG. 4, the 4E1-7 antibody showed reactivity only on CD21-positive B cells. Figure 4 shows the results of 1 out of 4 animals.

4) Reactivity to lymphoma cells collected from canine lymphoma cases The reactivity of 4E1-7 antibody to lymphoma cells (4 B cell types 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 responded to B-cell lymphoma cells in 4 of 4 cases and did not respond to T-cell lymphoma in 1 case. In addition, FIG. 5 shows the results of one B cell type and one T cell type.

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

4. Evaluation of direct cytotoxic activity
CLBL-1 was tested as a target cell line for the ability of the 4E1-7 antibody to directly induce cell death without dependence on complement or cytotoxic cells. Add 4E1-7 (Fig. 7: ●) or isotype control (rat IgG2a, ebioscience (Fig. 7: ○)) diluted to various concentrations of 0 to 10 μg / mL to the target cells seeded on a 96-well plate, and add 37 ° C. Incubated for 72 hours. After that, the cell proliferation rate was measured using the 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 the cell proliferation rate was observed, and it was confirmed that the cell proliferation activity was directly exerted.

5. Identification of amino acid sequences in the variable regions of heavy and light chains
Total RNA was extracted from hybridomas that produce 4E1-7 antibody, and reverse transcription reaction was performed using this as a template using primers specific for the constant region of Superscript® III (Invitrogen) and rat antibody (IgG). bottom.
As primers, YTM1224 (SEQ ID NO: 3) was used for amplification of the light chain, and YTM172 (SEQ ID NO: 4) was used for the amplification of the heavy chain. Using the obtained 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 are used as primers. Primary PCR was performed using. The PCR product is then gel purified to amplify all variable regions of the light or heavy chain, YTM170 (SEQ ID NO: 6) and YTM173 (SEQ ID NO: 7), or YTM170 (SEQ ID NO: 6) and YTM174 (SEQ ID NO: 6). Nested PCR was performed using No. 8). The nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence were determined by cloning the nested PCR product into the pBluescript SK (-) vector. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

Above 1. ~ 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.

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to provide a monoclonal antibody against canine CD20, which has a function different from that of the existing one and has an excellent effect. Then, using this monoclonal antibody as an active ingredient, it is possible to provide a composition for reducing canine B cells and a composition for treating diseases caused by an increase in canine B cells such as B cell lymphoma, leukemia or autoimmune diseases. Become.

Claims (6)

The amino acid sequence set forth in SEQ ID NO: 1 or the heavy chain variable region consisting of an amino acid sequence in which one or more and 13 or less amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2. Canine CD20 having 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 or the amino acid sequence of SEQ ID NO: 2 and having rat IgG2a, kappa chains. Monochrome antibody against. A composition for reducing canine B cells, which comprises the antibody according to claim 1 as an active ingredient. A composition for treating a disease caused by an increase in canine B cells, which comprises the antibody according to claim 1 as an active ingredient. The therapeutic composition according to claim 3, wherein the disease is either B-cell lymphoma, leukemia or an autoimmune disease. A kit for detecting canine B cells, which comprises the antibody according to claim 1. A kit for diagnosing a disease caused by an increase in canine B cells, which comprises the antibody according to claim 1.

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