WO2017150763A1 - Animal model expressing human cd3 and use thereof - Google Patents

Abstract

The present invention relates to an animal model expressing human CD3 and a use thereof, and more specifically, to: an animal model into which a gene encoding human CD3 has been introduced; a method for producing the animal model; and a method for screening a therapeutic agent for T cell-mediated diseases using the animal model.

Description

Human CD3 Expressing Animal Model and Uses thereof

The present invention relates to a human CD3 expression animal model and its use, and specifically, to an animal model into which a gene encoding human CD3 is introduced, a method for preparing the animal model, and a therapeutic agent for T cell mediated disease using the animal model. It relates to a method of screening.

The human adaptive immune system is a very sophisticated system that can specifically remove not only various infectious diseases but also cancer cells. In particular, in the case of T cells, it plays a role in determining cellular adaptive immunity and recognizes and removes cells when exposed to non-self or abnormal antigens. For T cells, a monoclonal T cell receptor complex (TCR complex) is expressed on the cell membrane to generate a major histocompatibility complex (peptide, pMHC) of an antigen presenting cell (APC). Be aware. For T cells, it expresses 20,000-40,000 TCR molecules per cell and specifically recognizes several antigens (determined by peptide sequence) of 100,000 pMHC molecules of APC to initiate signal transduction. In other words, the TCR molecule must serve as a highly sensitive sensor that must recognize and transmit signals with very sophisticated and subtle antigenic changes.

Such cellular adaptive immunity must be operated with great sophistication to effectively remove infectious diseases and cancer cells of the human body. If the antigen specific adaptive immune system does not work, it poses serious problems with infectious disease response and cancer cell clearance. On the other hand, when cellular adaptive immunity is activated by recognizing human self antigens, autoimmune diseases that destroy self cells and tissues are induced.

Based on the results of TCR complex structure and function studies, it is possible to show a typical structure of TCR on the T cell membrane. The αβTCR complex consists of an αβ heterodimer that specifically recognizes an antigen and CD3 molecules (CD3γε, CD3εδ, CD3ζζ) for signaling. αβ heterodimer has a structure similar to that of an antibody Fab, and CD3ε, γ, and δ each have an Ig domain in an extracellular configuration. Specific antigen recognition of the [alpha] [beta] heterodimer is mediated by the CD3 molecule to signal the cell. This signaling is known to be T-cell activation by intracellular tyrosine kinase (LCK, ZAP70, LAT, PLCγ) phosphorylation, cytoplasmic Ca ⁺⁺ concentration changes and intracellular transcriptional system changes.

Recently, each domain structure was determined by NMR and X-ray structure determination, and structural information on quaternary structure binding on T cell membranes was revealed one by one. In the TCR complex, the specific binding of the extracellular domain is very weak, while the electrical binding of charged amino acids in the cell membrane is important, suggesting that it can be amplified by the environment within the cell membrane. In particular, the important role of human CD3γε for T cell signaling is known, and the major crystal structures of the complexes bound to OKT3 being used as therapeutic antibodies have recently been described (Kjer-Nielsen, PNAS 101, (2004), 7675). -7680).

Signal transduction mechanisms of TCR were found to be mediated by CD3 molecules. Based on this, various CD3 antibodies have been developed for T cell signal transduction research and T cell activation regulation.

Therapies have been developed to regulate T cell immunity by targeting T cell antigen receptor complex signaling. In particular human CD3 monoclonal antibodies (mAbs) are widely used clinically in immunosuppressive therapy. OKT3, a CD3-specific mAb, was the first mAb licensed for use in humans and was used as an immunosuppressive agent in clinical transplantation. Most of the CD3 monoclonal antibodies used to date, such as OKT3, UCHT1 and Leu-4, all have binding capacity to human T cell CD3, but do not have binding capacity to heterologous T cells.

The biggest problem with therapeutic CD3 antibodies is that most CD3 antibodies are species specific. CD3 monoclonal antibodies are generally due to the species specificity of the CD3 molecule, which is the target molecule. For example, OKT3, the most widely used CD3 antibody, reacts with chimpanzee CD3 but not with other primates, macaques monkeys or CD3 in dogs. Most human CD3 antibodies bind only to human CD3 molecules, while CD3 molecules from mice, rats, rabbits, rhesus monkeys, cynomolgus monkeys, or baboon monkeys for preclinical experiments Do not combine.

This discrimination ability, such as species specificity, is a major obstacle to the development of CD3 monoclonal antibodies for the treatment of human diseases. New drug candidates must undergo rigorous testing in the pre-clinical phase of clinical approval testing. Preclinical studies should be performed in animals and the aim is to confirm the activity and safety of the drug candidate. Possible efficacy as a drug candidate is established and the drug candidate can be approved for human testing by the relevant regulatory authority.

Drug antibody candidates can be tested for safety in animals in two general ways: First, the antibody candidate may utilize a related animal species capable of recognizing orthologous antigens or an antibody candidate surrogate capable of binding to the parallel homologous antigens present in the animal.

A disadvantage in the development of most therapeutic candidate monoclonal antibodies is that candidate monoclonal antibodies should only be tested in the relevant animal species in high-quality primates, particularly chimpanzees. Chimpanzees are an endangered species and therefore the use of these animals for safety testing is very limited.

Recent studies show that TCR signaling can be altered not by CD3 antibody binding capacity or number of binding antibodies, but by the mode of binding of antibodies that bind CD3 (a recognized epitope). These results show that the use of an antibody candidate surrogate that can bind to parallel homologous antigens may differ from the equivalent signaling results.

Transgenic animal models incorporating only the human CD3ε coding gene have been reported to have serious problems with T cell differentiation and production (PNAS 1994 91 (20) 9402-9406). For this reason, transgenic animals which only introduce the human CD3ε coding gene are highly likely to cause problems throughout the immune system, and therefore, they are very limited to be used as experimental animals for immune diseases.

Under this technical background, the inventors of the present application have prepared a genetically modified animal incorporating a human CD3 coding gene, presenting a method for evaluating efficacy in a disease model prepared according to the present invention, In this case, the present invention was completed by confirming that it can be used as a disease model without changing the rodent immune system.

Summary of the Invention

An object of the present invention is to provide a transgenic animal model incorporating human CD3.

An object of the present invention is to provide a method for producing the animal model.

An object of the present invention is to provide a method for screening a therapeutic agent for T cell mediated diseases using the animal model.

In order to achieve the above object, the present invention provides a human CD3 expressing animal model into which a gene encoding human CD3 is introduced.

The present invention also provides a method for producing a human CD3 expressing animal model comprising introducing the gene encoding the human CD3 into an animal.

The present invention also comprises the steps of treating or administering a candidate to the human CD3 expression animal model; And it provides a method for screening a T-cell mediated disease therapeutic agent comprising the step of selecting a candidate for reducing the human CD3 expression compared to the control agent.

Figure 1 shows the results of phenotypic analysis of thymus and spleen cells derived from huCD3γε-TG mouse.

Figures 2a to 2d shows the results of confirming whether the response to the α-human CD3 antibody and / or α-mouse CD3 antibody of huCD3γε-TG mouse.

Figure 3 shows a schematic diagram of the back skin animal model experiment design (A) and ear skin animal model experiment design (B). IMQ = Imiquimod

Figure 4 shows the results of T lymphocyte flow cytometry before and after administration of anti-human CD3 antibody (OKT3) to transgenic mice at 6 hours, 24 hours and 48 hours. (Black = control type (Isotype control), orange = normal mouse)

Figure 5 shows a photograph of the skin symptoms by anti-CD3 antibody (OKT3) treatment and control (Isotype control) in imiquimod induced psoriasis skin disease.

Figure 6 shows the results of PASI by anti-CD3 antibody (OKT3) treatment and control (Isotype control) in imiquimod induced psoriasis skin disease. a-b Other letters on the test day indicate a significant difference. Statistical analysis was performed on the test groups (PBS, OKT3, Isotype control) except for the VAS group. Tukey's post hoc test. VAS = Vaseline, PBS = Phosphate buffered saline.

Figure 7 shows the results of T lymphocyte analysis in blood on the third day of psoriasis induction. ^{a- b} Other characters mean significant differences. Statistical analysis was performed on the test groups (PBS, OKT3, Isotype control) except for the VAS group. Tukey's post hoc test. VAS = Vaseline, PBS = Phosphate buffered saline.

8 shows histopathological findings and evaluation results of skin such as mice. ^{a- b} Other characters mean significant differences. Statistical analysis was performed on the test groups (PBS, OKT3, Isotype control) except for the VAS group. Tukey's post hoc test. VAS = Vaseline, PBS = Phosphate buffered saline.

9 shows the results of changes in ear thickness according to antibody administration in ear skin models. ^{a- b Any} other character on the test day means a significant difference. Tukey's post hoc test. VAS = Vaseline.

FIG. 10 shows the results of T lymphocyte analysis in blood on day 12 of the ear skin psoriasis induction model by imiquimod. a-b Other characters mean significant differences. Tukey's post hoc test.

Figure 11 shows the results of mouse and human CD3 expressing T lymphocytes in blood on day 12 of the ear skin psoriasis induction model.

Figure 12 shows the results of inflammatory cell analysis in the skin of psoriasis induced ear. ^{a- c} Other characters mean significant differences. Tukey's post hoc test.

Figure 13 shows the histopathological findings and evaluation results of mouse ear skin. ^{a- c} Other characters mean significant differences. Tukey's post hoc test.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a human CD3 expressing animal model into which a gene encoding human CD3 is introduced.

CD3 molecules are the major molecules that mediate the signal transduction of T cell antigen receptor complexes and are the main targets for immunotherapy treatment. CD3 molecules have very high barriers to animal model utilization and validation using human CD3 antibodies due to their interspecificity. The present invention can be used in evaluating the efficacy of the human CD3 molecule as a disease model to overcome the problems of introducing animal models of other species.

In order to overcome serious problems in T cell differentiation and production that may occur in a transgenic animal model in which only a human CD3ε coding gene is introduced, a human CD3 expressing animal model in which a gene expressing both human CD3γ and CD3ε is introduced and a method of manufacturing the same. Was used.

In one embodiment, the gene encoding human CD3 may be transformed with a cloned vector.

As used herein, "vector" refers to a nucleic acid molecule delivered into a cell and may include an "expression vector" for replicating DNA and expressing in a host cell. The expression vector is a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a specific host organism, and if necessary, a promoter, a transcriptional regulatory sequence (e.g., an enhancer sequence). ), Transcription termination factors, and the like.

"Promoter" means a non-readed nucleic acid sequence upstream of a coding region that contains a binding site for polymerase and has a transcription initiation activity to mRNA of a promoter lower gene.

The promoter is operably linked to induce the expression of a gene of interest, a foreign gene, and "operably linked" means that the binding between nucleic acid sequences is functionally related. The case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can employ enzymes commonly known in the art.

In the present invention, the target gene is a human CD3 coding gene, and "CD3" is a molecule that transmits a signal from an αβTCR heterodimer that specifically recognizes an antigen in a TCR complex structure, and includes an Ig domain in an extracellular configuration, and αβ hetero Signaling takes place into cells via CD3 after recognition of the specific antigen of the dimer.

In one embodiment, the human CD3 may be CD3γε, and the gene encoding human CD3 may be a structure in which the CD3γ coding gene of SEQ ID NO: 1 and the CD3ε coding gene of SEQ ID NO: 2 are linked by a self-cleaving peptide coding gene. When the autologous cleavage peptide coding gene is located between the CD3γ coding gene and the CD3ε coding gene, autologous cleavage occurs during translation and divides it into two CD3γ and CD3ε. Using this, it is possible to express two proteins in one plasmid, there is an advantage that makes the expression amount to the same degree.

Specifically, the self-cleaving peptide may be, for example, a 2A peptide. Among the 2A peptides, for example, P2A peptides encoded by the sequence of SEQ ID NO: 3 (SEQ ID NO: 5 is an amino acid sequence) can be used. P2A peptides have superior cleavage efficiency compared to 2A peptides with other sequences.

CD3ε and CD3γ each CD3 gene is linked to the 2A peptide, which is a self-cleaving peptide, thereby simultaneously transcribing from transcription to protein translation to minimize the interference effect on CD3 in transgenic animal models.

The transcription terminator may include any transcription terminator known in the art, for example, may be poly A linked.

The vector may be a linear DNA, a plasmid DNA or a recombinant viral vector, wherein the recombinant viral vector is a retrovirus, adenovirus, herpes simplex virus and lentivirus. ), But is not limited thereto.

"Transformation" may mean changing genetic properties to have new genotypes by recombinant DNA techniques and germ cell engineering methods, rather than by traditional crosses. Transformation may include somatic transformation and germ cell transformation. Somatic transformation may mean that the newly acquired genotype appears in the current generation of animals but is not passed on to the next generation. On the other hand, germ cell transformation refers to a case where a new gene is transferred directly to germ cells or a transformed cell is transferred to germ cells so that a new genotype is transmitted not only to the current generation but also to the next generation. In general, the production of a truly transgenic animal is through germ cell transformation.

Production of transgenic animals can be carried out using a variety of known methods, such as microinjection, electroporation, particle bombardment, methods using viral vectors, methods using embryonic stem cells, nuclear transfer. Methods, direct muscle injection, insulators, transposons, or methods using sperm. In one embodiment of the present invention, a transgenic animal model was prepared by linearly injecting a vector including a gene to be introduced into a fertilized egg (Example 1). Genetic modification may include the case where the desired gene is introduced randomly and when introduced at a specific site.

The animal model may be, for example, a rodent including a mouse and the like.

In an embodiment of the present invention, a human CD3 expressing transgenic mouse transformed with a vector containing a gene encoding human CD3 was prepared and analyzed. In the transgenic mouse according to the present invention, it was confirmed that human CD3 is normally expressed in T lymphocytes and works functionally.

In another aspect, the present invention relates to a method for producing a human CD3 expressing animal model comprising introducing a gene encoding the human CD3 into an animal.

Human CD3 expression animal model production method also includes a process for introducing a human CD3 coding gene, for example, CD3γ coding gene and CD3ε coding gene by linking with a self-cleaving peptide coding gene and cloning the vector into the animal, the configuration is As it overlaps with the components included in the transgenic animal model mentioned, the description applies equally.

In another aspect, the present invention comprises the steps of treating or administering a candidate to the human CD3 expression animal model; And it relates to a method for screening a T-cell mediated disease therapeutic agent comprising the step of selecting a candidate for reducing the human CD3 expression compared to the control agent.

CD3 molecules have very high barriers to animal model utilization and validation using human CD3 antibodies due to their interspecificity. The present invention overcomes the problem of introducing human CD3 molecules into other animal models, and can utilize a human CD3 expressing animal model to evaluate the efficacy of a drug for treating T cell mediated diseases.

The candidates are suspected of having the potential to inhibit CD3 expression or activity according to conventional selection or randomly selected individual test subjects, eg, compounds, natural products, antisense nucleotides, short interfering RNA ), Short hairpin RNAs, aptamers or antibodies, but are not limited thereto.

In the present invention, the measurement of CD3 expression amount change can be carried out through various methods known in the art. The CD3 expression level change can be measured, for example, changes in gene expression levels, for example reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting Or via a DNA chip. In addition, the CD3 expression level change can be measured, for example, protein expression levels, for example Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunity Staining, immunoprecipitation assays, complement fixation assays, FACS or protein chips.

As used herein, "T cell mediated disease" refers to a condition caused by the proliferation or activity of T cells, and may include, for example, cancer or autoimmune diseases.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Transformation Mouse Construction

To express the human full-length CD3γ epsilon protein on the surface, a construct was constructed in which a full-length CD3γ DNA (SEQ ID NO: 1) and a CD3ε DNA (SEQ ID NO: 2) were linked with a 2A peptide to a pCAGGS vector used for animal cell expression. The 2A peptide is encoded by SEQ ID NO: 3, which is present in the virus and located between the large proteins causes self-cleavage during translation to divide the proteins. This allows the two proteins to be expressed in a single plasmid, with the advantage that the amounts expressed are about the same. The 2A peptide used in this experiment is the P2A peptide of SEQ ID NO: 3 and has a higher cleavage efficiency than the 2A peptide of other sequences. In addition, a separate spacer (SEQ ID NO: 4) was connected to the 5 'direction of the P2A peptide. The amino acid sequence of the construct including the CD3, P2A peptide and the spacer included in the vector used for constructing the transgenic mouse is as described in SEQ ID NO: 6, and the gene sequence encoding it is as described in SEQ ID NO: 7.

The prepared plasmid was cut and linearized by restriction enzymes, and microinjected into fertilized eggs of mice. The procedure was followed according to standard procedures, and individuals transformed with human CD3γε were selected by genomic PCR.

Example 2. Transformed Mouse Model Analysis

Analysis of the transgenic mouse model prepared in Example 1 was performed as follows. Wild type mice (B6) and transgenic mice (human CD3γε) of 4-5 weeks old were used. Cells were isolated by homogenizing tissue (thymus, spleen). After centrifugation at 400 g for 5 minutes, the supernatant was removed, and 500 μl of RBC lysis buffer (sigma, # R7757) was added thereto, followed by suspension for 2 minutes to remove red blood cells. 10 ml of medium (RPMI1640, 10% FBS) was added and diluted, and the supernatant was removed by centrifugation at 400 g for 5 minutes. 5 x 105 cells were used per experiment. 0.5 ug of mouse Fc blocker (BD, # 553141) was added and incubated at 4 ° C. for 5 minutes. Anti-mouse CD4-V450 (RM4-5, BD, # 560468), anti-mouse CD8-V500 (53-6.7, BD, # 560776), anti-mouse CD3-A647 (17A2, BD, # 557869), anti Dilute mouse CD3-A488 (UCHT1, BD, # 557694) antibodies with buffer (PBS, 2% FBS, 0.05% sodium azide) in a ratio of 1:50 to make a master mix and add 20ul per experiment The exposure to light was blocked and stained for 20 minutes at 4 ° C. After centrifugation at 400g for 5 minutes, the supernatant was removed and washed twice with 100ul buffer. Finally, the cells were suspended in 200ul buffer and measured by FACSVerse (BD).

The results are shown in FIG. 1, and according to FIG. 1, it was confirmed that human CD3 was specifically expressed in transgenic mice (TG). Thymic analysis revealed that T cell differentiation and development was the same as wild type mice (WT).

Example 3 Confirmation of T Cell Activity by CD3 Antibodies

Dilute purified anti-mouse CD3 (2C11, biolegend, # 100302) and purified anti-human CD3 (UCHT1, eBioscience, # 14-0038) antibodies in DPBS to a concentration of 1 ug / ml, 1 ml each in a 24-well plate. The antibody was coated by incubation at 4 ° C. overnight. Washing was repeated three times with 1 ml DPBS to remove uncoated antibodies. Wild type mice (B6) and transgenic mice (human CD3γε) of 4-5 weeks old were used. Cells were isolated via spleen homogenization. After centrifugation at 400 g for 5 minutes, the supernatant was removed, and 500 μl of RBC lysis buffer (sigma, # R7757) was added and left to stand for 2 minutes to remove red blood cells. 10 ml of medium (RPMI1640, 10% FBS) was added and diluted, and the supernatant was removed by centrifugation at 400 g for 5 minutes. Peripheral blood mononuclear cells (PBMCs) were separated from blood using lymphoprep (stemcell, # 07851) as follows. Blood was mixed with PBS in a ratio of 1: 1. 16 ml of Lymphoprep was placed in a new 50 ml tube and carefully diluted blood was slowly placed on it. Centrifuge at 400g for 30 minutes at room temperature and complete centrifugation without brake. The top plasma was removed and the PBMC layer collected and transferred to tubes. PBS was transferred to a 50ml tube containing PBS, the final volume was adjusted to 50ml, and centrifuged at 400g for 10 minutes. After removal of the supernatant, 30ml of PBS was put and centrifuged at 400g for 10 minutes to wash. After removal of the supernatant, 2 ml of RBC lysis buffer (sigma, # R7757) was added and left to stand for 2 minutes to remove red blood cells. 30 ml of PBS was added and centrifuged at 400 g for 10 minutes. After removal of the supernatant, the suspension was resuspended with 1 ml of medium. The cells were filtered with a cell strainer (352352) to complete PBMC separation. Cells were stimulated by incubating for 1 hour in 1 ml each of wells and uncoated wells coated with an anti-CD3 antibody at a concentration of 1 × 10 ⁶ cells (splenocytes, human PBMC) / ml. The cells were collected by 150ul and centrifuged at 400g for 5 minutes to remove supernatant and washed with buffer (PBS, 2% FBS, 0.05% sodium azide). Four antibody mastermixes were prepared as follows. Anti-mouse CD4-V450 (RM4-5, BD, # 560468), anti-mouse CD8-V500 (53-6.7, BD, # 560776), anti-mouse CD3-A647 (17A2, BD, # 557869), anti Human CD3-A488 (UCHT1, BD, # 557694) antibody was diluted 1:50 with buffer and added to 20 μl of WT, TG splenocytes samples to block exposure to light at 4 ° C. for 20 minutes and stained. Anti-mouse CD4-V450 (RM4-5, BD, # 560468), anti-mouse CD8-V500 (53-6.7, BD, # 560776), anti-mouse CD69-A488 (H1.2F3, biolegend, # 104516) , Anti-mouse CD25-PE (3C7, BD, # 553075) antibody was diluted 1:50 with buffer and added in 20 ul of WT, TG splenocyte samples to block exposure to light and stain for 20 minutes at 4 ° C. It was. Anti-human CD4-V450 (RPA-T4, BD, # 560345), anti-human CD8-V500 (RPA-T8, BD, # 560774), anti-human CD3-A488 (UCHT1, BD, # 557694), anti Human CD69-APC-H7 (FN50, BD, # 560737) antibody was diluted 1:50 with buffer and added to 20 μL of human PBMC samples to block exposure to light at 4 ° C. for 20 minutes and stained. Anti-human CD4-V450 (RPA-T4, BD, # 560345), anti-human CD8-V500 (RPA-T8, BD, # 560774), anti-human CD3-A488 (UCHT1, BD, # 557694), anti -Dilute human CD25-PerCP-Cy5.5 (M-A251, BD, # 560503) antibody with buffer at a ratio of 1:50 and add 20ul to human PBMC samples to block light exposure at 4 ° C for 20 minutes. Stained. After centrifugation at 400g for 5 minutes, the supernatant was removed and washed twice with 100ul buffer. Finally, the cells were suspended in 100ul buffer and measured with FACSVerse (BD).

2A to 2D, it was shown that T cells derived from transgenic mice are activated by UCHT1, a human CD3 specific antibody.

Example 4 Psoriasis Model Experiment

1. Preparation for experiment

Mice and Test Substance Administration

Female or male transgenic mice (huCD3γε ^+/− ) and normal mice of 8-9 weeks of age were supplied from macrogen and used in the experiment. Two normal mice and seven transgenic mice were used in experiments to confirm expression of human CD3 introduced into transgenic mice and responses to anti-human CD3 antibodies (OKT3). Orbital vein bleeding was performed 6 hours, 24 hours and 48 days after administration of the antibody, and the fluorescently labeled antibody was used to measure the expression of human CD3 and the number of T lymphocytes by flow cytometry.

Next, two types of psoriasis models (back and ear skin models) were tested. Twenty-one female transgenic mice were used for the dorsal skin model, and sixteen male transgenic mice were used for the ear skin model. To induce psoriasis, the mouse abdominal cavity was anesthetized by injection anesthesia solution (ketamine + xylazine mixture), and the back hairs were removed using a hair removal machine and the remaining hairs were completely removed using a hair removal cream (VEET). The back skin model is applied with 62.5 mg aldara cream (5% imiquimod Imiquimod) for 4 days every day using spatula to induce psoriasis on the back (van der Fits et al. 2009), and the ear skin model is psoriasis on both ears. 12.5 mg aldara cream was applied to the ear for 5 days every day, followed by 3 additional doses every 2 days (Hemmerle et al. 2014). Mice in the group that did not cause psoriasis were treated with petrolatum cream. Isotype control and test substance were administered in 1 mg / mL and 0.2 mL intraperitoneally three times on 0 and 2 days before psoriasis induction in skin model such as (OKT3), and psoriasis in ear skin model. Three doses were administered at 0.5 mg / mL, 0.1 mL intraperitoneally on days 7, 9 and 11 post-induction (FIG. 3). Mice were bred in a conventional breeding environment with free water and feed at 23 ± 3 ° C and 40-70%. The animal experiment was performed with approval of the Meditox Animal Experiment Ethics Committee (A-2014-014).

Psoriasis Skin Symptom Assessment

To evaluate the degree of psoriasis induced on the back skin, the clinically applied Psoriasis Area and Severity Index (PASI) assessment was applied (van der Fits et al. 2009). Erythema, scaling, and skin thickening were evaluated. Each item was evaluated by 0-4 points (0: normal, 1: weak, 2: moderate, 3: severe, 4: very severe). Back and skin thicknesses were measured using a vernier caliper, and psoriasis lesions on the back skin were photographed using a digital camera and stereomicroscope (SMZ18, Nikon). Ear skin model measured the thickness of both ears using a vernier caliper and used the average of both ears. Psoriasis lesions were evaluated by two evaluators, and the score of each assessment item was added to evaluate the extent of psoriasis with a total score of 0-12.

Flow cytometry

Back skin model was performed on day 3 and ear skin model was orbital vein collected from 3 to 4 mice per test group on day 12. In the ear skin model experiment, after euthanasia on day 13, the left ear was removed and 400 U / mL collagenase IV (17104-019, Gibco) and 200 U / mL hyaluronidase (H3506, Sigma) were placed in PBS for flow cytometry. Incubated for 1 hour at 36 ℃, 5% CO ₂ conditions to separate into single cells. The blood samples and single cells of the ear skin were reacted with the antibody diluted at a ratio of 1: 100 at room temperature for 30 minutes, centrifuged at 400xg for 5 minutes, and washed twice with 100 μL of buffer. 400-500 μL of buffer was then added and measured using a flow cytometer (FACSVerse ™, BD). Antibodies used in flow cytometry are as follows. Anti-mouse CD3-A647 (17A2, BD, # 557869), anti-human CD3-PE (UCHT1, BD, 555333), anti-mouse CD4-V450 (RM4-5, BD, # 560468), anti-mouse CD8a -V500 (53-6.7, BD, # 560776), anti-mouse CD69-A488 (H1.2F3, Biolegend, # 104516), anti-mouse CD11b-A647 (M1 / 70, Biolegend, # 101218), anti-mouse Gr-1-A488 (RB6-8C5, Biolegend, # 108417), anti-mouse F4 / 80-PE (BM8, Biolegend, # 123110), anti-CD16 / CD32 (2.4G2, BD, # 553142). Neutrophils in skin tissue were classified into Gr-1 + CD11b + F4 / 80- and macrophages to F4 / 80 +.

Histopathological Evaluation

Psoriasis-induced back or ear skin tissues were fixed in 10% neutral formalin at room temperature for 3 to 4 days, followed by paraffin embedding (Microm STP-120, Thermo scientific), and then cut into 5 μm size (Microm HM 340E, Thermo scientific). ) To make two or more slides per sample. The dissected tissue was subjected to H & E staining and histopathological evaluation and imaging under a microscope (FSX100, Olympus). 0-4 points (0: normal, 1: weak) for epidermal edema / hyperplasia and immune cell influx in at least 5 zones randomly selected by two evaluators for all tissue samples. , 2: moderate, 3: severe, 4: very severe).

Statistical analysis

The experimental results were expressed as mean ± standard error, and statistical analysis was verified by the one-way ANOVA and Tukey's post hoc test using SPSS 17.0 software (Statistical Package for the Social Sciences, SPSS Inc.). P <was considered statistically significant, when the 0.05.

2. Experimental results of back skin psoriasis model

(1) Human CD3 expressing T lymphocytes and concentration dependence test results of transgenic mice

When anti-human CD3 antibody (OKT3) and control antibody (Isotype control) antibodies were administered to transgenic mice, human CD3 expression was reduced in mouse T lymphocytes in proportion to the amount of antibody administered (FIG. 4).

In addition, it can be seen that as the amount of OKT3 administered 6 hours after administration increases the ratio of CD69 + T lymphocytes in mouse CD4 + T lymphocytes, mouse T lymphocytes are activated by the administered OKT3.

(2) back skin psoriasis model

By imiquimod, psoriasis similar to human psoriasis, keratin, and skin thickening were observed in the skin of mice and the like (FIG. 5). Skin lesions increased rapidly after 3 days of induction, and erythema and skin thickening were significantly reduced by OKT3 administration (FIG. 6).

(3) Flow cytometry results

Induction of psoriasis by imiquimod significantly reduced the number of mouse CD4 + and CD8 + T lymphocytes in the blood on the 3rd day compared to the petrolatum group, and in particular, the OKT3 administration group significantly reduced the number of T lymphocytes than the PBS administration group (FIG. 7). ).

(4) histopathological evaluation results

Imiquimod showed psoriasis abnormal keratinization, epidermal thickening, hyperplasia of keratinocytes and infiltration of inflammatory cells. However, in the mouse group administered OKT3, the thickness of the epidermis was reduced compared to the group administered with PBS or control (Isotype control), and the infiltration and abnormal keratinization of inflammatory cells were also reduced (FIG. 8). When scored, the OKT3 group scored significantly lower than PBS and Isotype control.

3. Ear skin psoriasis model experiment results

(1) Ear thickness measurement result

Psoriasis skin thickening occurred by the application of imiquimod to both ears, and skin thickness was significantly reduced by three OKT3 administrations from day 7 (FIG. 9).

(2) flow cytometry results

It was confirmed that the number of CD4 + and CD8 + T cells in blood was significantly reduced by the administration of OKT3 (FIG. 10).

In addition, analysis of mouse CD3 and human CD3 expressing T lymphocytes in blood CD4 + T lymphocytes on the 12th day of psoriasis induced expression of mouse and human CD3 in all groups, and the expression of human CD3 was reduced by OKT3 administration compared to the other two groups. (Move left on graph) (FIG. 11).

In addition, inflammatory cell analysis of psoriasis-induced ear skin confirmed that both CD4 + and CD8 + T lymphocytes, neutrophils, and macrophages increased in ear skin due to psoriasis induced by imiquimod. However, administration of OKT3 significantly reduced the infiltration of ear skin of CD4 + T lymphocytes and macrophages (FIG. 12).

(3) histopathological evaluation results

As a result of histopathological evaluation, the thickening and hyperproliferation of mouse ear epidermis and the infiltration of inflammatory cells can be confirmed by imiquimod. However, OKT3 administration showed a decrease in epidermal thickness and inflammatory cell infiltration, and when scored, the OKT3 administration group was significantly lower than the control (Isotype control) (FIG. 13).

As a result of 46 transgenic and normal mice experiments, it was confirmed that the human CD3 injected into the mouse is normally expressed in mouse T lymphocytes, and the concentration of anti-human CD3 antibody (OKT3) was administered. Within 6 hours, mouse T lymphocytes were activated and expression of human CD3 decreased with concentration. Therefore, it was confirmed that human CD3 is normally expressed and functionally operated in T lymphocytes of the transformed mouse according to the present invention.

Psoriasis was induced in the back and ear skin using imiquimod using human CD3 expressing transgenic mice and then tested with anti-human CD3 antibody (OKT3). Mouse T lymphocyte activation and depletion reduced the number of T lymphocytes and inflammatory cells in blood and psoriasis skin tissue. In addition, administration of anti-human CD3 antibody significantly reduced psoriasis skin symptoms and histopathology.

Therefore, it was confirmed that anti-human CD3 antibody may have a therapeutic effect in psoriasis. In addition, it was confirmed from the results that T lymphocytes play an important role in psoriasis induction process, and it can be confirmed that human CD3 of the manufactured transgenic mouse functionally works in mouse T lymphocytes. Human CD3 expressing transgenic mice are likely to be used in various experiments, such as antibody screening of human CD3 targets and confirmation of antibody therapeutic efficacy in autoimmune diseases such as psoriasis.

The transgenic human CD3 expressing animal model into which the gene encoding human CD3 according to the present invention is introduced can be used as a disease model without changing the immune system of the animal model. Through this, a transgenic animal model expressing human CD3 can be applied for drug evaluation or screening for T cell mediated disease therapeutics.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic document attached.

Claims (15)

1. A human CD3 expressing animal model into which a gene encoding human CD3 is introduced.
2. The animal model of claim 1, wherein the gene encoding human CD3 is transformed with the cloned vector.
3. The animal model of claim 1, wherein the gene encoding human CD3 has a structure in which a CD3γ coding gene of SEQ ID NO: 1 and a CD3ε coding gene of SEQ ID NO: 2 are linked to a self-cleaving peptide coding gene.
4. The animal model of claim 3, wherein the self-cleaving peptide is a 2A peptide.
5. The animal model of claim 4, wherein the 2A peptide is encoded by a gene sequence of SEQ ID NO: 3. 6.
6. The animal model according to claim 1, which is a mouse.
7. The animal model of claim 6, wherein the human CD3 is expressed in T lymphocytes of a mouse.
8. A method for producing a human CD3 expressing animal model comprising introducing a gene encoding human CD3 into an animal.
9. The production method according to claim 8, wherein the gene encoding human CD3 is transformed into a cloned vector and a gene encoding human CD3 is introduced into the animal.
10. The method of claim 8, wherein the gene encoding human CD3 has a structure in which a CD3γ coding gene of SEQ ID NO: 1 and a CD3ε coding gene of SEQ ID NO: 2 are linked to a self-cleaving peptide coding gene.
11. The method of claim 10, wherein the self-cleaving peptide is a 2A peptide.
12. 12. The method of claim 11, wherein the 2A peptide is encoded by the gene sequence of SEQ ID NO: 3.
13. The production method according to claim 8, which is a transgenic mouse.
14. Treating or administering a candidate to a human CD3 expressing animal model according to any one of claims 1 to 7; And

    A method for screening a T cell mediated disease therapeutic agent comprising the step of selecting a candidate for reducing human CD3 expression as a therapeutic agent as compared to the control group.
15. The method of claim 14,

    The T cell mediated disease is a screening method, characterized in that the cancer or autoimmune disease.

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