KR20220141469A - NOVEL CANINE CD3ε SPECIFIC ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF - Google Patents

Abstract

The present invention relates to a canine antibody or antigen-binding fragment thereof that specifically binds to a CD3ε protein, wherein a single-stranded variable fragment (scFv) according to an embodiment of the present invention has excellent binding ability to canine CD3ε protein and T cells to stimulate T cells, thereby being usefully used for cancer treatment, immune activation, and autoimmune suppression using the same.

Description

Canine animal CD3ε-specific antibody or antigen-binding fragment thereof {NOVEL CANINE CD3ε SPECIFIC ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF}

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to the canine CD3ε protein, and a composition for cancer treatment and immune activation using the same.

According to the "People's Awareness Survey on Animal Protection" conducted by the Ministry of Agriculture, Food and Rural Affairs in 2019, about 26.4% of households owning companion animals, which translates to 22.38 million households nationwide, are raising companion animals. It is estimated The most popular companion animals are dogs, cats, and fish, and the number of dogs is estimated to reach 5.98 million.

As the number and lifespan of dogs increases, the incidence of cancer in elderly dogs is expected to increase. It was found that they visited the veterinary hospital for reasons such as vaccinations, regular check-ups, and disease treatment.

However, until now, anti-cancer drugs for dogs are only used by adjusting the dose of anti-cancer drugs used in humans, so there are few anti-cancer drugs for dogs.

In this situation, the present inventors completed the present invention by discovering a CD3ε-specific antibody capable of targeting T cells as a result of research to develop an antibody for cancer treatment and immune regulation in dogs.

The purpose of the present invention is to develop an antibody drug for treating cancer in dogs, specifically, an antibody drug that can target T cells for immune treatment. We want to develop an antibody drug in the form of a single chain variable fragment (scFv) that can act.

In order to achieve the above object, an aspect of the present invention is

(a) a heavy chain variable region comprising heavy chain complementarity determining region 1 (CDR1), CDR2 and CDR3 respectively represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and

(b) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 respectively represented by the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; a canine CD3ε-specific antibody or antigen-binding fragment thereof to provide.

The present inventors performed ELISA screening on the scFv phage library using canine CD3ε protein as an antigen. As a result, three clones (A12, B7 and C10) that significantly bind canine CD3ε protein were selected, and T cells The binding ability was further evaluated to finally select the scFv of the B7 clone ( FIGS. 1 to 4 ).

In the present invention, the term "antibody" refers to a protein molecule serving as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule having immunological reactivity with a specific antigen, polyclonal antibody, single Both clonal and complete antibodies are included. The term also includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies and tetrabodies.

In the present invention, the term "antigen binding fragment" refers to a fragment having an antigen-antibody-binding function in a complete antibody molecule, for example, Fab (fragment antigen binding), Fab', F (ab')2, scFv (single-chain Fv), Fv (variable fragment), dsFv (disulfice-stabilized Fv fragments), diabody, Fd, Fd' and BiTE (bispecific T-cell engager) to be selected from the group consisting of However, the present invention is not limited thereto.

Among the antigen-binding fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chain and the first constant region (CH1) of heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'.

Fv is a minimal antibody fragment having only a heavy chain variable region (VH) and a light chain variable region (VL), and a two-chain Fv (two-chain Fv) is a heavy chain variable region and a light chain variable region connected by a non-covalent bond. A single-stranded variable fragment (single chain Fv, scFv) is a recombinant antibody fragment reconstituted by linking only the variable regions constituting the antigen-binding site from the light chain and the heavy chain with a linker. Antibodies are large molecules of approximately 150 kDa and do not act effectively in large tumors or tissues that are difficult to penetrate. However, scFv is a molecule of approximately 30 kDa, and since it is the smallest antibody fragment that maintains a complete antigen-binding site, it can effectively act on large tumors or tissues that are difficult to penetrate while maintaining the unique antigen-binding ability of the antibody.

Fd is an antibody fragment consisting of the variable region (VH) and first constant region (CH1) domains of the heavy chain, and the bispecific T-cell engager (BiTE) refers to an antibody designed to simultaneously bind a specific antigen and a T cell surface protein, e.g. For example, it may consist of two scFv molecules that each recognize a surface antigen of a cancer cell and a CD3 of a T cell.

The antigen-binding fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the whole antibody with papain, Fab can be obtained, and F(ab')2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of a hypervariable region in the heavy and light chains of an antibody. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for the antibody to bind antigen or epitope.

According to one embodiment of the present invention, the canine CD3ε-specific antigen-binding fragment may be an scFv, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region.

In addition, the canine CD3ε-specific scFv may be one in which the light chain variable region and the heavy chain variable region are linked by a peptide linker, and the sequence of SEQ ID NO: 13 (SSRSSSGGGSSGGGGS) or SEQ ID NO: 14 (GGGGSGGGGSGGGGS) may be used as the peptide linker. . Preferably, the linker of SEQ ID NO: 13 may be used, and the canine CD3ε-specific scFv including the peptide linker may include the sequence shown in SEQ ID NO: 9.

According to one embodiment of the present invention, the canine CD3ε-specific scFv may include six histidine tags (6x His-tag) and an HA tag at the C-terminus for purification and tracking ( FIGS. 6 and 7 ) , SEQ ID NO: 10).

In addition, the canine CD3ε-specific scFv may bind to canine CD3ε with a dissociation constant (Kd) of 8.88 nM. The dissociation constant of an antibody indicates the binding affinity with the antigen, and the smaller the dissociation constant, the greater the binding affinity between the antibody and the antigen. The binding affinity can be determined according to a conventional method.

Another aspect of the present invention provides a polynucleotide encoding the canine CD3ε-specific antibody or antigen-binding fragment thereof, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.

According to one embodiment of the present invention, the polynucleotide encoding the canine CD3ε-specific antibody or antigen-binding fragment thereof may include the nucleic acid sequence represented by SEQ ID NO: 11, and the C- If six histidine tags (6x His-tag) and HA tags are present at the end, the nucleic acid sequence shown in SEQ ID NO: 12 may be included, but is not limited thereto.

In the present invention, the expression vector containing the polynucleotide encoding the canine CD3ε-specific antibody or antigen-binding fragment thereof is not particularly limited thereto, but bacterial cells (eg, E. coli, etc.), mammalian cells (eg, It can be a vector capable of replicating and/or expressing the polynucleotide in prokaryotic or eukaryotic cells including, for example, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells or insect cells. . Preferably, the vector may be a vector that is operably linked to an appropriate promoter so that the polynucleotide can be expressed in a host cell, and includes at least one selectable marker. For example, the polynucleotide may be introduced into a phagemid, plasmid, cosmid, mini-chromosome, virus or retroviral vector.

According to one embodiment of the present invention, the expression vector including the polynucleotide encoding the canine CD3ε-specific antibody or antigen-binding fragment thereof may be a phagemid vector (FIG. 7).

In the present invention, the transformant introduced with the expression vector is not particularly limited thereto, but bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc. transformed by introducing the expression vector; yeast cells; Fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); Or it may be a plant cell.

For the transport of the polynucleotide or the expression vector containing the same into a host cell/transformant, a transport method well known in the art may be used. As the delivery method, for example, if the host cell is a prokaryotic cell, CaCl ₂ method or an electroporation method may be used, and if the host cell is a eukaryotic cell, a microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection Methods and genetic bambadment may be used, but are not limited thereto.

Another aspect of the present invention provides a pharmaceutical composition for the treatment of cancer in a canine animal and a composition for immune activation comprising the canine CD3ε-specific antibody or antigen-binding fragment thereof as an active ingredient.

In the present invention, the term "CD3 (cluster of differentiation 3)" is one of the proteins forming a complex with the T cell receptor and is involved in activating both cytotoxic T cells and helper T cells. . CD3 consists of a CD3γ chain, a CD3δ chain, and two CD3ε chains.

Since the present inventors have demonstrated through experimental results that the canine CD3ε-specific antigen-binding fragment binds not only to CD3ε but also to CD3ε-expressing T cells ( FIG. 4 ), the CD3ε-specific antibody or antigen-binding fragment thereof is used for cancer treatment It can be usefully used as a pharmaceutical composition and a composition for immune activation.

In addition, another aspect of the present invention provides a pharmaceutical composition for improving or treating autoimmune diseases in canines comprising the canine CD3ε-specific antibody or antigen-binding fragment thereof as an active ingredient.

In the present invention, the term "autoimmune disease" refers to a disease caused by an abnormality in the immune system that protects one's body from the outside and rather attacks itself. That is, the autoimmune disease of the canine animal in the present invention is rheumatoid arthritis, lupus, autoimmune hemolytic anemia, ulcerative colitis, Crohn's disease, and Edison. It may be selected from the group consisting of bottles, but is not limited thereto.

In the present invention, the term "rheumatoid arthritis" is a disease in which the thin membrane (synovial membrane) surrounding the joint is inflamed and invades several joints throughout the body. It is known that T cells are activated by an immune response in the early stages of the disease, which triggers an inflammatory response, but as the disease progresses, the influence of T cells decreases and cartilage appears due to abnormal proliferation and activation of synoviocytes. Due to the activation of destructive enzymes and bone destructive factors, joint destruction occurs continuously.

As described above, CD3 activates T cells, which in turn activates the immune response. When the CD3ε-specific antibody of the present invention is used, the activation of T cells is inhibited, and thus an abnormal immune response such as an autoimmune disease can be suppressed. For example, visilizumab, a humanized CD3e monoclonal antibody, can potentially be used in the treatment of allograft rejection, psoriasis, and autoimmune diseases, and studies are currently underway for use in patients with ulcerative colitis and Crohn's disease.

Since the single-stranded variable fragment (scFv) that specifically binds to the canine CD3ε protein according to an embodiment of the present invention has excellent binding ability to the canine CD3ε protein and T cells, it can stimulate T cells. It can be usefully used for therapeutic, immune activation, and autoimmune suppression purposes.

1 is a result of confirming a phage clone that binds to the CD3ε protein of a canine animal using an scFv phage library.
2 is a result confirming the binding level of phage clones binding to CD3ε protein in canines.
3 is a result of confirming the dissociation constant of a phage clone that binds to the CD3ε protein of canine animals.
4 is a result of confirming whether a phage clone that binds to CD3ε protein of a canine animal binds to a T cell of a canine animal.
5 is a result of expression and isolation of the scFv of the B7 clone that specifically binds to the canine CD3ε protein.
6 is a schematic diagram of a construct for expressing the scFv of the B7 clone.
7 is a schematic diagram of a phagemit vector into which the scFv construct of the B7 clone is introduced.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1: Anti-CD3εscFv discovery of canine animals

Prepare a phage library in which 2x10 ¹⁰ chicken-derived single-chain antibody library phage (scFv phage library; Bioneer, Korea) and 1.9x10 ¹⁰ human-derived single-chain antibody library phage are mixed, and perform ELISA screening using canine CD3ε protein as an antigen did.

As a result, three clones (A12, B7, and C10) that significantly bind to the canine CD3ε protein were selected, and it was confirmed that the B7 clone was derived from chickens, and the A12 and C10 clones were derived from humans ( FIGS. 1 and FIG. 1 ). 2). As a result of analyzing the single-chain antibody library phage of A12 and C10 clones, it was confirmed that the same clone was used, and only the A12 clone was used in subsequent experiments.

Example 2: Construction of canine anti-CD3εscFv

2-1. Confirmation of CD3ε binding ability of selected clones

The A12 and B7 clones selected in Example 1 were amplified, diluted by 1/2 at a starting concentration of 200 nM, and dispensed in a 96-well plate. Then, after reacting with canine CD3ε protein (50 ng/well), absorbance was measured.

As a result, the dissociation constant (Kd) values of the A12 and B7 clones were 16.45 nM and 8.88 nM, respectively, indicating that both clones bind to canine CD3ε protein at a similar level ( FIG. 3 ).

2-2. Confirmation of T cell binding ability of the selected clones

Since CD3ε is overexpressed in T cells, the binding ability of the A12 and B7 clones selected in Examples to canine T cells was confirmed. Canine peripheral blood mononuclear cells (PBMC) were dispensed on the plate at a concentration of 4x10 ⁶ cells/well, and reacted with A12 and B7 clones at various concentrations, respectively. Thereafter, Dylight-650-conjugated anti-His Tag antibody was treated to confirm the degree of binding between A12 and B7 clones and canine T cells, respectively.

As a result, it was confirmed that the C10 clone did not bind to canine T cells, but the B7 clone bound to T cells (Kd=48.19 nM), and finally B7 clones were selected with canine anti-CD3εscFv (Fig. 4). ).

Example 3: Preparation of canine anti-CD3εscFv construct

The final selected B7 clone was introduced into the E. coli TG1 strain, and the E. coli was cultured at 30° C. for 16 hours to express scFv. At the end of the culture, the E. coli pellet was recovered by centrifugation, the pellet was crushed, and the scFv was purified with Ni-NTA. Imidazole was used in the range 0-400 mM. As a result of SDS-PAGE analysis of each fraction after purification, it was found that the canine anti-CD3εscFv expressed in the B7 clone had a size of about 28 kDa ( FIG. 5 ).

The amino acid sequence of the purified anti-CD3εscFv was analyzed to confirm each CDR sequence, and for purification and tracking of the anti-CD3εscFv, six histidine tags (6x His-tag) and an HA tag were introduced at the end to construct the anti-CD3εscFv construct. devised (Fig. 6). In addition, the construct was introduced into the phagemid vector pComb3xTT (Fig. 7).

Claims (13)

a heavy chain variable region comprising a heavy chain complementarity determining region 1 (CDR1), CDR2 and CDR3 represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and
a light chain variable region comprising light chain CDR1, CDR2 and CDR3 respectively represented by the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7;
A canine CD3ε-specific antibody or antigen-binding fragment thereof comprising a. According to claim 1, wherein the canine CD3ε-specific antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8 Including, canine CD3ε-specific antibody or antigen-binding fragment thereof. According to claim 1, wherein the antigen-binding fragment Fab (fragment antigen binding), Fab', F(ab')2, Fv (variable fragment), dsFv (disulfice-stabilized Fv fragments), scFv (single-chain Fv) , diabody, Fd, Fd' and BiTE (bispecific T-cell engager) selected from the group consisting of, canine CD3ε-specific antibody or antigen-binding fragment thereof. The canine CD3ε-specific antibody or antigen-binding fragment thereof according to claim 3, wherein the scFv has a light chain variable region and a heavy chain variable region connected by a peptide linker. The canine CD3ε-specific antibody or antigen-binding fragment thereof according to claim 4, wherein the peptide linker uses the sequence of SEQ ID NO: 13 (SSRSSSGGGSSGGGGS) or SEQ ID NO: 14 (GGGGSGGGGSGGGGS). A polynucleotide encoding the canine CD3ε-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. The polynucleotide of claim 6, wherein the polynucleotide comprises a nucleic acid sequence represented by SEQ ID NO: 11. An expression vector comprising the polynucleotide of claim 6. A transformant other than a human, into which the expression vector of claim 8 is introduced. A pharmaceutical composition for the treatment of canine cancer comprising the canine CD3ε-specific antibody or antigen-binding fragment thereof according to claim 1. A composition for activating canine immunity, comprising the canine CD3ε-specific antibody or antigen-binding fragment thereof according to claim 1 . A pharmaceutical composition for improving or treating autoimmune diseases in canines comprising the canine CD3ε-specific antibody or antigen-binding fragment thereof according to claim 1 . According to claim 12, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, lupus, autoimmune hemolytic anemia, ulcerative colitis, Crohn's disease, and Addison's disease, for the improvement or treatment of autoimmune diseases in canine animals. composition.

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