JP2019026625A - 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 invention provides a monoclonal antibody or antibody fragment against canine CD20, which antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence with deletion, substitution or addition of one or several amino acids in SEQ ID NO:1, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence with deletion, substitution or addition of one or several amino acids in SEQ ID NO:2.SELECTED DRAWING: None

Description

  The present invention relates to monoclonal antibodies or antibody fragments against canine CD20. More specifically, the present invention relates to a monoclonal antibody or an antibody fragment against canine CD20 having a function different from that of existing ones and excellent effect.

In recent years, antibody drugs have been established due to medical development. In particular, treatment with anti-CD20 antibody is used as a standard therapy for human B-cell lymphoma in addition to CHOP therapy, which is a multidrug anticancer drug therapy.
In pets such as dogs, the lifespan has been extended and the incidence of tumors has increased in the same way as humans. In addition to the three main therapies that have been used for surgery, radiation therapy, and chemotherapy, new useful new Provision of new treatments is required.

  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 in the United States by Blontless, Aratana.

  In Patent Documents 3 and 4, a humanized monoclonal antibody or antigen-binding fragment thereof that binds to CD20 comprising an L chain variable region and an H chain variable region represented by a specific amino acid sequence, CDR1, CDR2, and QQCDR3 A chimeric or humanized monoclonal antibody that binds to CD20 or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region are disclosed. In addition, it was disclosed that these antibodies can also be used for dogs, and that the dogs diagnosed with diffuse large B-cell lymphoma were treated with the 1F5 chimeric monoclonal antibody, so that there was no trace of lymphoma. ing.

Furthermore, Patent Documents 5 and 6 disclose an antibody or antibody fragment that recognizes a dog or cat CD20 containing a hypervariable domain in a light chain containing a CDR region selected from a specific amino acid sequence and a hyperregulatory domain in a heavy chain. Has been.
Thus, although various antibodies against CD20 have been developed, the effect varies depending on the antibody, and it cannot be said that an antibody having an excellent effect has been obtained.
Therefore, the present inventor tried to produce a monoclonal antibody against canine CD20 having a function different from that of the existing one and excellent effect in the present invention.

JP2016-13104 Special Table 2014-532649 Special Table 2006-500904 JP 2009-291197 Special Table 2013-520990 Japanese Unexamined Patent Publication No. 2016-1779978

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

  In order to solve this problem, as a result of intensive studies, the present inventors have determined that the amino acid sequence shown in SEQ ID NO: 1 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: 1 And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2, wherein one or several amino acids are deleted, substituted, or added. The inventors have found that a monoclonal antibody or antibody fragment against canine CD20 is a monoclonal antibody or antibody fragment against canine CD20 having a function different from that of existing ones and having an excellent effect, and the present invention has been completed.

That is, the present invention for solving the above problems relates to monoclonal antibodies, antibody fragments and the like against canine CD20 shown in the following (1) to (6).
(1) The heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and described in SEQ ID NO: 2 A monoclonal antibody or antibody fragment against canine CD20 having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2, wherein one or several amino acids have been deleted, substituted or added.
(2) A composition for reducing canine B cells comprising the antibody or antibody fragment according to (1) above as an active ingredient.
(3) A composition for treating a disease caused by an increase in canine B cells, comprising the antibody or antibody fragment described in (1) as an active ingredient.
(4) The therapeutic composition according to the above (3), wherein the disease is any of B cell lymphoma, leukemia or autoimmune disease.
(5) A canine B cell detection kit comprising the antibody or antibody fragment according to (1) above.
(6) A kit for diagnosis of a disease caused by an increase in canine B cells, comprising the antibody or antibody fragment according to (1) above.

  According to the present invention, it is possible to provide a monoclonal antibody or an antibody fragment against canine CD20 having a function different from that of an existing one and having an excellent effect. And this monoclonal antibody or antibody fragment as an active ingredient, a composition for reducing canine B cells, a composition for treating diseases caused by an increase in canine B cells such as B cell lymphoma, leukemia or autoimmune diseases, etc. Provision is also possible.

FIG. 4 shows that 4E1-7 antibody binds to Jurkat / cCD20 in a concentration-dependent manner (Example). FIG. 4 shows that 4E1-7 antibody specifically recognizes canine CD20 molecule (Example). It is the figure which showed the reactivity with respect to CLBL-1 of 4E1-7 antibody (Example). FIG. 4 shows the reactivity of 4E1-7 antibody to peripheral blood lymphocytes in dogs (Example). FIG. 4 shows the reactivity of 4E1-7 antibody to lymphoma cells collected from a dog lymphoma case (Example). It is a figure of the result of having investigated the complement dependence cytotoxic 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-canine CD20 monoclonal antibody or antibody fragment” of the present invention is an antibody or antibody fragment that recognizes a canine CD20 molecule, and is 1 or a number 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 comprising an amino acid sequence in which one amino acid is deleted, substituted or added, and one or several amino acids are deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2. Alternatively, it refers to an antibody or antibody fragment having a light chain variable region consisting of an added amino acid sequence. Here, several in the heavy chain variable region or the light variable region is 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, Mean 4, 3 or 2. “Several” means five, four, three or two.

The “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention can bind well to the extracellular region of canine CD20, and is positive for CD20 by FACS analysis, fluorescence microscope observation, etc. using living cells and tissues of dogs It can be used for analysis of cell dynamics.
Yes.

The “anti-canine CD20 monoclonal antibody or antibody fragment” 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 a rat, and the framework region (FR) and the 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 a caninized antibody in which the constant region portion of the antibody is replaced with a canine antibody is preferable because an immune response to a heterologous antigen is induced and no serious side effects such as anaphylactic shock occur.
The chimeric antibody or caninized antibody may be any of IgG, IgM, IgA, IgD, and IgE, and is particularly preferably IgG.

  The “composition for reducing canine B cells” of the present invention refers to a composition for reducing canine B cells comprising the “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention as an active ingredient. As long as it is useful for the reduction of canine B cells, it may contain other components in addition to these active ingredients, and may further contain drugs such as anticancer drugs and radionuclides. Further, a pharmaceutically acceptable additive may be contained, and examples of such an additive include a solvent such as water, a surfactant, sodium chloride, sodium citrate, anhydrous citric acid, and a pH adjuster. It is done.

The “composition for treating a disease caused by an increase in canine B cells” of the present invention is an agent for treating a disease caused by an increase in canine B cells, comprising the “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention as an active ingredient. It refers to the composition.
Examples of such diseases include canine B cell lymphoma caused by an increase in canine B cells, leukemia in which increased B cells have become tumor, autoimmune diseases caused by excessive increase of normal B cells, and the like.
For example, when the disease to be treated is B cell lymphoma, the “anti-antibacterial agent” which is an active ingredient is administered by administering the “composition for treating diseases caused by an increase in canine B cells” of the present invention to an affected dog. Antibody-Dependent-Cellular-Cytotoxicity (ADCC) activity, complement dependent by binding to CD20 that is overexpressed on the surface of B-cell lymphoma cells Canine B-cell lymphoma cells can be killed by mechanisms such as complement-dependent cytotoxicity (CDC) activity and apoptosis induction.

The “composition for treating a disease caused by an increase in canine B cells” according to the present invention may be a composition comprising an “anti-canine CD20 monoclonal antibody or antibody fragment” which is an active ingredient, in addition to these active ingredients. Further, it may further contain other components such as anticancer agents and radionuclides and drugs.
In addition, any form may be used as long as it is effective for treating a disease caused by an increase in canine B cells, and it may be provided as a liquid or powder for intravenous injection or infusion, a tablet, a capsule, or the like.
As a therapeutic method using the therapeutic composition of the present invention, for example, by intravenous injection, infusion, etc., the “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention is 1 to 10 mg / kg in one week. For example, administration may be performed at intervals of about once.

The “canine B cell detection kit” of the present invention is a kit for detecting whether a sample contains canine B cells using the “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention. That means. In addition, the “diagnosis kit for diseases caused by an increase in canine B cells” of the present invention refers to whether a dog as a target is affected by a disease caused by an increase in canine B cells such as canine B cell lymphoma, leukemia and autoimmune diseases. This means a kit for diagnosing whether or not.
These kits contain at least the “anti-canine CD20 monoclonal antibody or antibody fragment” of the present invention as one of the components.
The “anti-canine CD20 monoclonal antibody or antibody fragment” contained 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, and amylase. Examples of fluorescent substances include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Cy3, Cy5, R-phycoerythrin, B-phycoerythrin, Alexafluoro 488 or Alexafluoro 647, and radioactive substances include tritium. , Iodine 125, iodine 131 and the like. Biotin, streptavidin, or the like may also be used.

In the use of the “kit for detecting canine B cells” and “kit for diagnosing diseases caused by an increase in canine B cells” according to the present invention, it is conventionally known to be useful for detecting canine B cells and diagnosing diseases caused by an increase in canine B cells. Any reagent, device, etc. may be used.
Lymphocytes are separated from biological tissues such as blood from the dog to be diagnosed, and the “anti-canine CD20 monoclonal antibody or antibody fragment” contained in these kits is allowed to act. 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 due to increased canine B cells.
By using these kits, live cells can be detected or diagnosed, and cell fixing operations and the like are unnecessary, so that accurate or simple detection or diagnosis can be performed.

  Examples 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 cDNA of canine CD20 molecule, a FLAG tag was added to this C-terminal and pMx-IP vector (Distributed by Dr. Toshio Kitamura, Institute of Medical Science, The University of Tokyo). This gene was introduced into PLAT-gp cells (provided by Dr. Toshio Kitamura of the Institute of Medical Science, the 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 production of hybridoma SD rats were immunized with NRK / cCD20 established in 1) above. Specifically, NRK / cCD20 was mixed with Titer Max (registered trademark) Gold (CytRx) as an adjuvant and administered to the foot pad of Wistar rats (Japan SLC Co., Ltd.), and boosted 7 days and 14 days after the first immunization. went. Three days after the final immunization, the sub-knee and inguinal lymph nodes were collected and lymphocytes were collected. Subsequently, the recovered lymphocytes and the mouse myeloma cell line P3X63-Ag8.653 cells were fused. The obtained cells were cultured in a HAT culture medium, and hybridomas in which lymphocytes and mouse myeloma cells were fused were selected.

The antibody in the culture supernatant of the obtained hybridoma was reacted with NRK / cCD20, and a 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.
Further, antibodies in the culture supernatant of candidate hybridomas were reacted with canine peripheral lymphocytes, and hybridomas producing reactive monoclonal antibodies were screened by FACS. A promising hybridoma was cloned by a 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 in 1) above, with 4E1-7 antibody diluted to various concentrations ). This was reacted with a secondary antibody, and then the fluorescence intensity was 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). Then, after this antibody was purified with a protein A column, it was confirmed that this 4E1-7 antibody had 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 and Western blotting using Jurkat / cCD20 were performed. The cell lysate obtained by dissolving Jurkat / cCD20 in 1% NP40 lysis buffer and 1 μg of 4E1-7 antibody were reacted, and protein G 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 around the molecular weight corresponding to the canine CD20 molecule (lane 4).
On the other hand, if Jurkat cells that do not express canine CD20 molecules as a negative control of Jurkat / cCD20, or the same test using an isotype control antibody (rat IgG2a, ebioscience) as a negative control of 4E1-7 antibody, A band corresponding to canine CD20 was not detected (lane1-3). From these results, it was confirmed that 4E1-7 specifically recognizes the canine CD20 molecule.

2) Reactivity against 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 The reactivity of 4E1-7 antibody was analyzed by flow cytometry using B cell type: CLBL-1). As a result, the 4E1-7 antibody was reactive only with CLBL-1, which is a B cell lymphoma cell line. FIG. 3 shows the results of reactivity of the 4E1-7 antibody to CLBL-1.

3) Reactivity to peripheral blood lymphocytes of dogs Lymphocytes were isolated from 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 was reactive only with CD21-positive B cells. FIG. 4 shows the results for one of the four 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 reacted with B cell type lymphoma cells in 4 out of 4 cases and showed no reactivity with 1 type of T cell type lymphoma. FIG. 5 shows the results for one B cell type and one T cell type.

3. Evaluation 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 lymphoma cell line CLBL-1 as a 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 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. Thereafter, the cell viability was measured by the trypan blue method. As a result, as shown in FIG. 6, no decrease in the survival rate was observed regardless of the concentration of the 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 induce cell death directly without relying on complement or cytotoxic cells. Add 4E1-7 (Figure 7: ●) or isotype control (rat IgG2a, ebioscience (Figure 7: ○)) diluted to various concentrations from 0 to 10 µg / mL to the target cells seeded in a 96-well plate, and add 37 ° C Incubated for 72 hours. Thereafter, 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 exhibited direct cytotoxic activity.

5). Identification of amino acid sequences of variable regions of heavy and light chains
Total RNA was extracted from the hybridoma producing the 4E1-7 antibody, and this was used as a template to perform reverse transcription using primers specific to the constant region of Superscript (registered trademark) III (Invitrogen) and rat antibody (IgG). did.
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 The primary PCR was performed. The PCR product is then gel purified and YTM170 (SEQ ID NO: 6) and YTM173 (SEQ ID NO: 7), or YTM170 (SEQ ID NO: 6) and YTM174 (sequence) are used to amplify all variable regions of the light or heavy chain. Nested PCR was performed using No. 8). The nested PCR product was cloned into the pBluescript SK (-) vector to determine the nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence. 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. Through this step, a monoclonal antibody or antibody fragment 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.

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

Claims (6)

A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and the amino acid sequence set forth in SEQ ID NO: 2 Alternatively, a monoclonal antibody or antibody fragment against canine CD20 having a light chain variable region comprising 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. A composition for reducing canine B cells, comprising the antibody or antibody fragment according to claim 1 as an active ingredient. A composition for treating a disease caused by an increase in canine B cells, comprising the antibody or antibody fragment according to claim 1 as an active ingredient. The therapeutic composition according to claim 3, wherein the disease is any of B-cell lymphoma, leukemia or autoimmune disease. A kit for detecting a canine B cell, comprising the antibody or antibody fragment according to claim 1. A kit for diagnosis of a disease caused by an increase in canine B cells, comprising the antibody or antibody fragment according to claim 1.