Monoclonal antibodies specifically binding to TIM-3 and uses thereof

Abstract

The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3 and uses thereof. The monoclonal antibody hTIM-3_NCC1 of the present invention specifically recognizes human and mouse cells expressing TIM-3 and can be applied to various fields such as the diagnosis and prevention or treatment of diseases mediated by cells expressing TIM-3.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody or an antigen binding fragment thereof that specifically binds to TIM-3 and uses thereof. Background Art

[0002] T cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane glycoprotein composed of a cytoplasmic tail with an immunoglobulin variable domain, a mucin domain, and a tyrosine phosphorylation motif. Initially, TIM-3 was proposed as a T helper type 1 (Th1)-specific protein selectively expressed in terminally differentiated Th1 cells, but further research results showed that TIM-3 is also expressed in Th17 cells, regulatory T (Treg) cells, and even in non-T cells such as dendritic cells (DC), natural killer (NK) cells, monocytes, and macrophages. This suggests that TIM-3 has various immune functions depending on specific cell types and cell states. In particular, TIM-3 appears to play both positive and negative roles depending on the situation. For example, TIM-3 can inhibit the activation of T cells to weaken the response of CD4+ and CD8+ T cells and induce peripheral tolerance. However, under certain circumstances, TIM-3 can participate in the activation of various innate immune cells, including quiescent macrophages, to eliminate pathological stimuli. Recent studies have shown that TIM-3 promotes short-term effector T cells during viral infection. In addition, it has been reported that dysregulation of TIM-3 expression can lead to excessive or inappropriate responses in immune cells.

[0003] Experimental and clinical studies have linked many diseases such as autoimmune diseases, chronic infections and ischemia to TIM-3. Alterations and dysregulation of TIM-3 expression are associated with the onset and severity of pathological conditions in patients with multiple sclerosis (MS) and patients infected with human immunodeficiency virus (HIV) or hepatitis viruses, especially, not only with high levels of expression of IFN-γ and TNF-α in MS patients, but also with distortion of Th1-induced Th2 responses in allergic diseases. Moreover, it has been shown that blockade of TIM-3 affects the pathological severity of experimental allergic encephalomyelitis (EAE) and the pathogenesis of diabetes in obese diabetic (NOD) mice. It is shown that blockade of TIM-3 signaling restores the proliferation of HIV-specific T cells and increases the production of cytokines. In these TIM-3-related diseases, negative regulation of Th1 or Th17 immunity due to TIM-3 may lead to pathological conditions such as T cell dysfunction or exhaustion of T cells, distortion of Th2 responses to Th1 signaling, and pro-inflammatory states of Th1 responses. However, little is known about the detailed characteristics and roles of TIM-3 in specific immune states and distinct diseases, especially in innate immunity-related pathologies.

[0004] TIM-3 has also attracted much attention as a target for immunomodulation in cancer. In recent years, a lot of research has been done on immune checkpoint molecules and immune surveillance in the process of cancer growth and eradication, which has led to the development of many therapeutic strategies, including therapeutic antibodies targeting immune checkpoint molecules. Studies have shown that TIM-3 expression is associated with many cancers and that TIM-3 has a role in regulating tumor growth. For example, it has been shown that TIM-3 is highly expressed in CD4+ and CD8+ tumor-infiltrating T cells in patients with non-small cell lung cancer, and TIM-3 expression in CD4+ T cells is associated with adverse clinical pathological parameters such as nodal metastasis and ongoing cancer, and is characteristically expressed in tumor cells of patients with renal cancer and hepatocellular carcinoma.

[0005] Korean Patent No. 10-2146319 relates to a bispecific antibody specific for PD-1 and TIM-3, which provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, in particular, a bispecific antibody that binds to TIM3 with a lower binding affinity than to PD1.

[0006] However, there is still a need for research on antibodies that bind to TIM-3 with high affinity for use in monitoring TIM-3 itself and diagnosing diseases associated with TIM-3 expression, etc. Summary of the invention

[0007] Technical issues

[0008] In view of this, the present inventors made intensive efforts to provide an antibody that specifically binds to TIM-3 with high affinity, prepared and screened the monoclonal antibody of the present invention, and thus achieved the present invention.

[0009] Therefore, an object of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3 and uses thereof.

[0010] Solutions to the problem

[0011] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, comprising: a heavy chain variable region, comprising a heavy chain CDR1 consisting of an amino acid sequence as shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of an amino acid sequence as shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of an amino acid sequence as shown in SEQ ID NO: 3; and a light chain variable region, comprising a light chain CDR1 consisting of an amino acid sequence as shown in SEQ ID NO: 4, a light chain CDR2 consisting of an amino acid sequence as shown in SEQ ID NO: 5, and a light chain CDR3 consisting of an amino acid sequence as shown in SEQ ID NO: 6.

[0012] According to a preferred embodiment of the present invention, the above-mentioned antibody may comprise: a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0013] According to a preferred embodiment of the present invention, the heavy chain variable region and the light chain variable region can be connected via a linker consisting of an amino acid sequence as shown in SEQ ID NO: 9.

[0014] According to a preferred embodiment of the present invention, the above-mentioned antibody may be a humanized antibody or a human antibody.

[0015] The present invention also provides a nucleic acid molecule encoding the heavy chain variable region or the light chain variable region of the above monoclonal antibody or antigen-binding fragment thereof.

[0016] According to a preferred embodiment of the present invention, the above-mentioned nucleic acid molecule may consist of a nucleotide sequence as shown in Sequence 16 or a nucleotide sequence as shown in Sequence 17.

[0017] Furthermore, the present invention may provide a recombinant vector comprising a nucleic acid molecule encoding the heavy chain variable region, a nucleic acid molecule encoding the light chain variable region, or all of the nucleic acid molecules.

[0018] Furthermore, the present invention can provide a host cell comprising the above-mentioned recombinant vector.

[0019] Furthermore, the present invention may provide a method for preparing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, which comprises the step of culturing the above-mentioned host cell.

[0020] Furthermore, the present invention can provide a TIM-3 expression level determination kit, which comprises the above-mentioned monoclonal antibody or its antigen-binding fragment.

[0021] Furthermore, the present invention may provide a method for monitoring the expression level of TIM-3, comprising: step i), contacting the above-mentioned monoclonal antibody or antigen-binding fragment thereof with a biological sample isolated from an individual; and

[0022] Step ii), determining the level of TIM-3 bound to the monoclonal antibody or its antigen-binding fragment in the above biological sample by forming an antigen-antibody complex.

[0023] According to a preferred embodiment of the present invention, the biological sample may be one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, tissue and cells.

[0024] Effects of the Invention

[0025] The monoclonal antibody hTIM-3_NCC1 of the present invention specifically recognizes human and mouse cells expressing TIM-3, and can be applied to various fields such as diagnosis, prevention or treatment of diseases mediated by cells expressing TIM-3. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The ELISA results of antibody candidates specific for TIM-3 are shown. From the left, they correspond to scFv-TIM3#1 to scFv-TIM3#12.

[0027] Figure 2 The amino acid sequences and CDR sequences of the screened hTIM-3_NCC1 antibodies are shown.

[0028] Figure 3 It was shown that the screened hTIM-3_NCC1 antibodies specifically bind to human TIM-3.

[0029] Figure 4 It was shown that the screened hTIM-3_NCC1 antibodies specifically bind to human TIM-3 expressed by Jurkat cells.

[0030] Figure 5 It was shown that the screened hTIM-3_NCC1 antibodies specifically bind to human TIM-3.

[0031] Figure 6It was shown that the screened hTIM-3_NCC1 antibody reacted better and specifically bound to human TIM-3 of human PBMCs than a commercially available anti-human TIM-3 antibody (BV421α-Human TIM-3, BD#565553).

[0032] Figure 7 This indicates that the screened hTIM-3_NCC1 antibody can also bind to mouse TIM-3. hIgG Fc refers to APC-hIgG Fc, and α-hTIM-3 refers to the antibody hTIM-3_NCC1 of the present invention.

[0033] Figure 8 It is shown that the hTIM-3_NCC1 antibody screened can also bind to mouse TIM-3. In the lower end graph, Rat Isotype-PE (eBioscience, cat#12-4321) refers to the isotype antibody of α-mTIM-3-PE antibody (eBioscience, cat#12-5870), and APC-hIgG Fc (Biolegend, cat#409305) refers to the second antibody control group for α-hTIM-3-scFv-hIgG (hTIM-3_NCC1). DETAILED DESCRIPTION

[0034] Hereinafter, the present invention will be described in detail.

[0035] The "antigen" of the present invention refers to a molecule or a part of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. An antigen can have more than one epitope.

[0036] The term "antibody" or "Ab" of the present invention refers to an immunoglobulin molecule that can recognize and bind to a specific target or antigen, such as a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc., through one or more antigen recognition sites located in the variable region of the immunoglobulin molecule. "Antibodies" include: monoclonal antibodies; polyclonal antibodies; "antigen-binding fragments" of antibodies that have the ability to specifically bind to a specific antigen (e.g., TIM-3), such as Fab, Fab', F(ab') 2, Fd, Fv, Fc, etc.; isolated complementary determining regions (CDRs); bispecific antibodies; heteroconjugate antibodies, mutants thereof; fusion proteins with antibodies or antigen-binding fragments thereof (e.g., domain antibodies); single-chain antibodies (ScFv) and single-domain antibodies [e.g., shark and camelid antibodies]; maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFv; humanized antibodies; chimeric antibodies; and all other variants of immunoglobulin molecules containing the required specific antigen recognition site (including antibody glycosylation mutants, antibody amino acid sequence mutants and covalently converted antibodies), but any type of antibody may be included without limitation. Antibodies may be of mouse, rat, human or other origin (including chimeric or humanized antibodies).

[0037] The "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy chain (CDRH1, CDRH2 and CDRH3) and the light chain (CDRL1, CDRL2 and CDRL3) each contain three CDRs, and these CDRs provide the main contact residues for the binding of the antibody to the antigen or epitope.

[0038] An antibody or polypeptide that "specifically binds" to a specific target or antigen (e.g., TIM-3) is a well-known term in the relevant art, and methods for determining such specific binding are also well known in the relevant art. When a particular molecule reacts with a particular cell or substance more frequently, more rapidly, with greater persistence, and / or with greater affinity than it does with other cells or substances, it is considered to exhibit "specific binding." When a particular antibody binds to a specific target or antigen with greater affinity, binding activity, more rapidly, and / or with greater persistence than it binds to other substances, then the specific antibody "specifically binds" to the specific target or antigen.

[0039] The "vector" of the present invention includes nucleic acid molecules that can carry other nucleic acids connected to it. One form of a vector is a "plasmid", which refers to a circular double-stranded DNA loop in which additional DNA fragments can be combined. Another form of a vector is a viral vector, in which additional DNA fragments can be combined with the viral genome. Some vectors can replicate autonomously in the host cells they enter (for example, bacterial vectors with bacterial origin replication and free mammalian vectors). When other vectors (for example, non-free mammalian vectors) enter the host cell, they can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can indicate the expression of genes that are operatively linked to them. The above-mentioned vectors are referred to as "recombinant expression vectors" (or simply, "expression vectors") in this specification. Generally, expression vectors useful in recombinant DNA technology often exist in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include other forms of expression vectors with equivalent functions, for example, viral vectors (for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0040] The term "host cell" of the present invention is used to refer to a cell that is capable of expressing a gene of interest that has been transformed or that has been selected after transformation by a nucleic acid sequence. The above term includes the offspring of a parent cell, whether the offspring is identical to the original parent in morphology or genetic structure, as long as the selected gene is present.

[0041] As described above, the present inventors have made intensive efforts to provide antibodies that can be used for TIM-3 monitoring and disease diagnosis, etc., by strongly binding to TIM-3 specifically and selectively, thereby completing the present invention.

[0042] Since the antibody hTIM-3_NCC1 of the present invention is not only human TIM-3 but also can specifically and selectively bind to mouse TIM-3, it is very suitable for monitoring the expression level of TIM-3. Therefore, when the antibody hTIM-3_NCC1 of the present invention is used, since the expression level of TIM-3 can be very accurately measured, it can be widely used in the field of confirming the expression level of TIM-3, such as the diagnosis of diseases such as cancer, multiple sclerosis or brain diseases or the prediction of their prognosis, and the field of preclinical research using mice, etc.

[0043] Therefore, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, comprising: a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0044] The above-mentioned “antigen-binding fragment thereof” refers to a fragment having antigen-binding function, including Fab, F(ab'), F(ab') 2 And Fv or single-chain antibody molecules. Among antibody binding fragments, Fab has a structure with variable regions of light and heavy chains, constant regions of light chains, and the first constant region (CH1) of heavy chains, so it has one antigen binding site. The difference between F(ab') and Fab is that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab') 2 When the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv refers to the smallest antibody fragment that has only the heavy chain variable site and the light chain variable site. This antibody fragment can be obtained by proteolytic enzymes, for example, the whole antibody can be restricted by papain to obtain Fab, and by pepsin to obtain F(ab'). 2 The fragments can preferably be prepared by genetic recombination technology.

[0045] According to a preferred embodiment of the present invention, the above-mentioned antibody may comprise: a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0046] According to a preferred embodiment of the present invention, the heavy chain variable region and the light chain variable region can be connected via a linker consisting of an amino acid sequence as shown in SEQ ID NO: 9.

[0047] According to a preferred embodiment of the present invention, the above-mentioned antibody may be a humanized antibody or a human antibody.

[0048] The humanized antibodies have been engineered to contain immunoglobulin domains that are more similar to humans, containing only the complementarity determining regions of animal-derived antibodies. This is achieved by carefully examining the sequences of the hypervariable loops of the variable regions of monoclonal antibodies and adapting the sequences to the structure of human antibody chains.

[0049] A fully human antibody is an antibody molecule in which the entire sequence of the light chain and the heavy chain, including the CDRs, is derived from human genes.

[0050] The present invention may also provide a nucleic acid molecule encoding the heavy chain variable region of the above monoclonal antibody or antigen-binding fragment thereof; or encoding the light chain variable region.

[0051] The above-mentioned "nucleic acid molecule" has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules. The nucleotides as the basic structural units of nucleic acid molecules include not only natural nucleotides, but also analogs (analogues) modified at the sugar or base sites. The sequences of the nucleic acid molecules encoding the heavy chain and light chain variable regions of the present invention can be modified. The above-mentioned modifications include the addition, deletion, non-conservative substitution or conservative substitution of nucleotides. The nucleic acid molecules of the present invention are interpreted as also including nucleotide sequences that show substantial identity with the above-mentioned nucleotide sequences. The above-mentioned substantial identity means that after the above-mentioned nucleotide sequence of the present invention is aligned (aligned) to the maximum extent possible with any other sequence, when the aligned sequences are analyzed by the algorithm commonly used in the art, at least 80% homology is shown, at least 90% homology is shown in a specific example, and at least 95% homology is shown in another specific example.

[0052] According to a preferred embodiment of the present invention, the above-mentioned nucleic acid molecule encoding the heavy chain variable region may consist of the nucleotide sequence shown in Sequence 16, and the above-mentioned nucleic acid molecule encoding the light chain variable region may consist of the nucleotide sequence shown in Sequence 17.

[0053] The present invention can also provide a recombinant vector, which comprises the above-mentioned nucleic acid molecule encoding the heavy chain variable region, the above-mentioned nucleic acid molecule encoding the light chain variable region, or all of the above-mentioned nucleic acid molecules.

[0054] The recombinant vector system of the present invention can be constructed by various methods known in the art. The vector of the present invention can generally be constructed as a vector for cloning or a vector for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells or eukaryotic cells as hosts.

[0055] The present invention can also provide a host cell comprising the above recombinant vector.

[0056] The above-mentioned host cell is a cell transformed by the recombinant vector of the present invention. The host cell that can stably and continuously clone and express the vector of the present invention is known in the art, and therefore any host cell can be utilized, for example, prokaryotic host cells such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus) can be included.

[0057] The present invention may also provide a method for preparing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, which comprises culturing the above-mentioned host cell.

[0058] The host cells used to prepare the above-mentioned antibodies or antigen-binding fragments thereof can be cultured according to appropriate culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use this culture process according to the selected strain. Cell culture can be divided into suspension culture and adherent culture according to the growth mode of cells, and can be divided into batch culture, feed-batch culture, and continuous culture according to the culture method. The culture medium used for culture should appropriately meet the requirements of the specific strain.

[0059] The present invention can also provide a TIM-3 expression level determination kit, which comprises a monoclonal antibody or an antigen-binding fragment thereof.

[0060] The above-mentioned expression level determination is determined by a method capable of determining the combination of antigen and antibody, and the above-mentioned method may include any immunological analysis method. This method is well known in the art, for example, it can be Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, immunofluorescence analysis, immunoblotting, Ouchterlony method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, immunochromatography, FACS or protein chip.

[0061] The tools or reagents used for immunological analysis may include a suitable carrier or support, a label capable of generating a detectable signal, a solubilizing agent, a cleaning agent, a stabilizer, etc. As a suitable carrier, although not limited thereto, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity, a suitable buffer solution, a second antibody labeled with a colorimetric enzyme or a fluorescent substance, a colorimetric substrate, and a reaction terminator, etc.

[0062] The above-mentioned markers include all compositions that can be detected by spectral analysis, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical means. Suitable markers include, but are not limited to, magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive markers (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and those commonly used in enzyme-linked immunosorbent assay (ELISA)), and chromogenic markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0063] The antibody comprised by the test kit of the present invention can preferably be fixed on a suitable carrier or support by the whole bag of tricks of recording in the document, as the example of a suitable carrier or support, include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, cross-linked dextran, agarose gel particles, liposome, carboxymethyl cellulose, polyacrylamide, polystyrene, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads or magnetic particles etc. In addition, include cell culture plates, microwell plates, microarrays, ELISA plates, pipes and polymer films as other solid matrices. Above-mentioned support can have any possible form, for example spherical (bead), cylindrical (inner surface of test tube or hole), planar (thin plate, test strip).

[0064] The present invention may also provide a method for monitoring the expression level of TIM-3, comprising: step i), contacting the above-mentioned monoclonal antibody or its antigen-binding fragment with a biological sample isolated from an individual; step ii), determining the level of TIM-3 bound to the monoclonal antibody or its antigen-binding fragment in the above-mentioned biological sample by forming an antigen-antibody complex.

[0065] The individual in step i) above may be a mammal including a human.

[0066] According to a preferred embodiment of the present invention, the biological sample may be one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, tissue and cells.

[0067] Hereinafter, the present invention will be described in detail by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.

[0068] Example 1: Screening and sequence analysis of antibodies specifically binding to TIM-3

[0069] TIM-3 specific antibodies (hTIM-3_NCC1, scFv-TIM3#4) were screened by screening of a human scFv (naive) library using the human TIM-3 antigen and ELISA reaction.

[0070] Specifically, antibodies specific to the Tim-3-His tag protein as a human TIM-3 antigen were screened by a bio-panning method known in the art. First, in order to coat the TIM-3 antigen, the TIM-3-His tag (ACRObiosystems Inc.) was diluted in PBS at 2 μg / ml and injected into the well plate at 100 μl per well. After shaking off the antigen, 100 μl of 5% skim milk in PBS solution was added to each well and reacted at room temperature for 4 hours, and then washed with PBS more than 4 times. On a 96-well plate attached with a TIM-3 protein antigen, the scFv bound to the TIM-3-His tag was collected by bio-panning the antibody library of scFv expressed on the surface of the M13 phage.

[0071] The 12 TIM-3 specific antibody candidates (ScFV-TIM3#1 to ScFV-TIM3#12) obtained by the above human ScFv (naive) library screening were diluted in 1% skim milk in TBST and treated in each well, reacted at room temperature for 1 hour, and then washed 3 times with 1×PBST. After treating the second antibody (anti-humanIgG HRP, 1:5000) and shaking (shaking) at room temperature for one hour to react, it was washed 6 times with 1×PBST, and then for color development, 50ml of TMB solution was treated and reacted at room temperature for 30 minutes. Finally, 100ml of 1N H 2 SO 4After the reaction was terminated by treatment with a stop solution for 10 minutes, the absorbance was measured at 450 nm.

[0072] Results, such as Figure 1 As shown, ScFV-TIM3#2, ScFV-TIM3#4, ScFV-TIM3#7, ScFV-TIM3#8 and ScFV-TIM3#12 antibodies were confirmed from 12 candidates, and ScFV-TIM3#4 (a-hTIM-3-scFv-hIgG) showing the strongest signal was screened out.

[0073] The selected ScFV-TIM3#4 clone was named hTIM-3_NCC1, and its base sequence and amino acid sequence were analyzed. The sequence information of the antibody CDR site, i.e., the heavy chain variable site and the light chain variable site, was confirmed based on the sequence analysis results (Table 1, Figure 2 ).

[0074] Table 1

[0075]

[0076]

[0077]

[0078] Example 2: Confirmation of the specific binding of the screened antibodies to human TIM-3

[0079] In order to confirm the specificity of the antibody hTIM-3_NCC1 screened in Example 1 above for TIM-3, ELISA analysis was performed.

[0080] Specifically, in order to coat the TIM-3 antigen, the TIM-3-His tag (ACRObiosystems Inc.) was diluted in PBS at 2 μg / ml and 100 μl was injected into each well. After shaking off the antigen, 100 μl of 5% skim milk in PBS solution was added to each well and reacted at room temperature for 4 hours, and then washed with PBS more than 4 times. After hTIM-3_NCC1 was diluted in 1% skim milk in TBST and treated in each well, after reacting at room temperature for 1 hour, it was washed 3 times with 1×PBST. After treating the second antibody (anti-human IgG HRP, 1:5000) and shaking (shaking) at room temperature for one hour to react, it was washed 6 times with 1×PBST, and then for color development, 50 ml of TMB solution was treated and reacted at room temperature for 30 minutes. Finally, 100 ml of 1N H 2 SO 4 After treating with a stop solution for 10 minutes to stop the reaction, the absorbance was measured at 450 nm. The ELISA experimental conditions are shown in Table 2 below.

[0081] Table 2

[0082] Antigen TIM-3-His-tag, 200 ng / well Primary Ab Anti-TIM-3-hscFv-hIgG1 Secondary Ab-HRP 1：3000 TMB substrate incubation 10 minutes Measurement filter 450nm Plate reader Infinite F50

[0083] Results, such as Figure 3 As shown, hTIM-3_NCC1 was confirmed to bind specifically to TIM-3. The result of ELISA QC showed that 1:128000 could be detected. In this case, the amount of antigen coated on the immunoplate was about 0.2 μg per well (200 ng / well).

[0084] Example 3: Confirmation of the Screened Antibodies' Binding to Human TIM-3

[0085] <3-1> Confirmation of the reactivity of human T cell lymphoma cells to human TIM-3

[0086] In order to confirm the specificity of the antibody hTIM-3_NCC1 screened in Example 1 above for TIM-3, FACS analysis was performed.

[0087] Specifically, 3×10 5Jurkat immortalized human T lymphocyte cells (T cell leukemia) expressing TIM-3, or the cells were reacted with 1 μg of antibody hTIM-3_NCC1 for 30 minutes, and then the surface was stained with a secondary antibody (APC-Human IgG Fc or PE-Human IgG Fc) for 10 minutes and measured by flow cytometer.

[0088] Results, such as Figure 4 As shown, it was confirmed that the antibody hTIM-3_NCC1 specifically binds to cells expressing TIM-3.

[0089] <3-2> Confirmation of reactivity to mouse NIH3T3 cells overexpressing human TIM-3

[0090] In order to confirm the specificity of the antibody hTIM-3_NCC1 screened in Example 1 above for TIM-3, FACS analysis was performed.

[0091] Specifically, the method for overexpressing human TIM-3 in mouse NIH / 3T3 cells using Lenti particles encoding TIM3 cDNA prepared using Lenti-Human TIM-3-GFP plasmid DNA (Origene, cat#RC209440L4) and Lenti-control particles-GFP (Origene, cat#PS100093V) is as follows: ① As a control group, Lenti-control particles-GFP and 10 MOI of Lenti particles were treated with polybrene for 5×10 4 NIH3T3 cells were transfected with puromycin, and only transduced cells (NIH3T3-GFP) were selectively selected. ② As an experimental group, 293FT cells were transfected with lenti-Human TIM-3-GFP plasmid DNA and packing DNA for 3 days to obtain lenti-Human TIM-3-GFP particles. NIH / 3T3 was treated with polybrene together with the culture medium containing lenti-Human TIM-3-GFP particles. Puromycin was used to selectively select only cells overexpressing human TIM-3 (NIH3T3-GFP-Human TIM-3).

[0092] Make the above screened 1×10 5 After reacting each cell with 0.1 μg of antibody hTIM-3_NCC1 for 30 minutes, the cells were stained with secondary antibodies (PE-Human IgG Fc, APC-Human IgG Fc) for 20 minutes and then confirmed by flow cytometry. The expression of human TIM-3 was confirmed by GFP expression, and the expression difference between the transduction groups was confirmed by Lenti-contorl particles and Lenti-Human TIM-3particles in cells expressing GFP.

[0093] Results, such as Figure 5 As shown, it can be confirmed that the screened antibody hTIM-3_NCC1 specifically reacts with human TIM-3.

[0094] <3-3> Confirmation of reactivity to human PBMC

[0095] FACS analysis was performed to compare the reactivity of hTIM-3_NCC1 (α-hTIM-3-scFv-hIgG) screened in Example 1 and currently commercially available anti-human TIM-3 antibodies to TIM-3.

[0096] Specifically, human blood was collected and only peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE healthcare, cat#17-5442-02). 6 PBMC and 0.5 μg of the screened antibody, 1 μg of PE-Cy7-conjugated anti-CD3 antibody (BD, cat#557851, USA), 0.25 μg of FITC-conjugated anti-CD11b antibody (ebioscience, cat#11-0112, USA) were reacted for 30 minutes, and then the surface was stained with a secondary antibody (PE-Human IgG Fc) and measured by flow cytometry. Commercially available BV421-conjugated anti-TIM-3 antibody (BV421-Human TIM-3; BD, cat#565553, USA) was used as a positive control group. In CD3 high CD11b - 、CD3 high CD11b mid 、CD3 - CD11b midand CD3 - CD11b high The level of TIM-3 was measured in the cells.

[0097] Results, such as Figure 6 As shown, it was confirmed that hTIM-3_NCC1 reacts by recognizing TIM-3 expressed by a cell population expressing CD3 and CD11b of human PBMC, and has a higher reactivity than the commercially available antibody used as a positive control group.

[0098] Example 4: Confirmation of the Binding of Screened Antibodies to Mouse TIM-3 and Applicability for Preclinical Studies

[0099] FACS analysis was performed to determine whether the screened antibody hTIM-3_NCC1 has the ability to specifically bind to human TIM-3 and mouse TIM-3.

[0100] Specifically, primary glial cells of mice were reacted with 0.5 μg of antibody hTIM-3_NCC1 for 30 minutes, and then stained with a secondary antibody (APC-humanIgG Fc; Biolegend, cat#409305). In addition, mouse CD11b overexpressing mouse TIM-3 was distinguished by BV421-conjugated anti-CD11b antibody (a-BV421-conjugated a-mCD11b; BD, cat#562605). + Cells (3×10 5 After that, 0.5 μg of hTIM-3_NCC1 antibody was reacted with PE-conjugated anti-mouse TIM-3 antibody for 30 minutes. These cells were surface stained with a secondary antibody (APC-Human IgGFc) and then measured by flow cytometry.

[0101] Results, such as Figure 7 As shown in , it can be confirmed that hTIM-3_NCC1 also reacts with TIM-3 expressed in mouse primary glial cells. Figure 8 As shown, it can be confirmed that the mouse hTIM-3_NCC1 antibody also has reactivity with TIM-3 of mouse cells. This result shows that the novel antibody of the present invention can also be used for preclinical studies such as monitoring mice expressing TIM-3. SEQUENCE LISTING <110> NATIONAL CANCER CENTER <120> Monoclonal antibodies specifically binding to TIM-3 and uses thereof <130> POPC212113k02PCT <150> KR 10-2020-0023273 <151> 2020-02-25 <150> PCT / KR2021 / 002369 <151> 2021-02-25 <160> 18 <170> PatentIn version 3.2 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1_amino acid <400> 1 Ser Ile Phe Ser Asn Gly Tyr Met Thr 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2_amino acid <400> 2 Ser Ile Asn Ser Arg Gly Val Thr Asn 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR3_amino acid <400> 3 Ala Ala Leu Leu Pro His Glu Ile Arg Lys Tyr 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1_amino acid <400> 4 Lys Ser Ser Gln Ser Val Phe Tyr Asn Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Gly <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2_amino acid <400> 5 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR3_amino acid <400> 6 Cys Gln Gln Tyr Tyr Ser Thr Pro Arg Thr 1 5 10 <210> 7 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region_amino acid <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Asn Gly 20 25 30 Tyr Met Thr Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ser Ser Ile Asn Ser Arg Gly Val Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Leu Leu Pro His Glu Ile Arg Lys Tyr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 8 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region_amino acid <400> 8 Val Asp Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser 1 5 10 15 Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Phe 20 25 30 Tyr Asn Ser Asn Asn Lys Asn Tyr Leu Gly Trp Tyr Gln Lys Lys Pro 35 40 45 Gly Gln Pro Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg Glu Ser 50 55 60 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 80 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 85 90 95 Gln Gln Tyr Tyr Ser Thr Pro Arg Thr Leu Gly Gly Gly Thr Lys Val 100 105 110 Asp Ile Lys Arg 115 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Linker_Amino Acid <400> 9 Gly Gly Gly Ser His Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR1_nucleotide <400> 10 tcactgcgtc tgagctgcgc ggccagc 27 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR2_nucleotide <400> 11 gcgcccggga aaggtctgga attggtg 27 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR3_nucleotide <400> 12 gccgccctgc ttcctcatga gattcggaag tat 33 <210> 13 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR1_nucleotide <400> 13 aagtccagcc agagtgtttt ttacaactcc aacaataaga actacttagg t 51 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR2_nucleotide <400> 14 tgggcatcta cccgggaatc c 21 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR3_nucleotide <400> 15 tgtcagcaat attatagcac tcctcgcact 30 <210> 16 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region_nucleotide <400> 16 gaagtgcagc tggtggaatc gggtggtggt ctggttcaac cgggcggctc actgcgtctg 60 agctgcgcgg ccagcggtag tatctttagt aatggttata tgacttggta tcgtcaggcg 120 cccgggaaag gtctggaatt ggtgtcgtcg attaattcga ggggagtgac caattatgca 180 gactctgtta aaggccgttt taccatcagc cgcgataact ctaaaaatac gctgtatctg 240 caaatgaact ctctgcgcgc ggaagatacg gcagtgtatt actgcgccgc cctgcttcct 300 catgagattc ggaagtattg gggtcagggt acccaagtga cggttagctc t 351 <210> 17 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region_nucleotide <400> 17 gtcgacgaca tccagatgac ccagtctcca gactccctgg ctgtgtctct gggcgagagg 60 gccaccatca actgcaagtc cagccagagt gttttttaca actccaacaa taagaactac 120 ttaggttggt accagaagaa accaggacag cctcctaagc tgctcatttt ctgggcatct 180 acccgggaat ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 240 ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 300 agcactcctc gcactctcgg cggagggacc aaagtggata tcaaacgt 348 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Linker_Nucleotide <400> 18 ggtggcggtt cacatggtgg cggcagcgga ggtggatcc 39

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, characterized in that: Include: A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; as well as The light chain variable region comprises a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

6.

2. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3 according to claim 1, characterized in that: The above antibodies include: A heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and The light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3 according to claim 2, characterized in that: The heavy chain variable region and the light chain variable region are connected via a linker consisting of the amino acid sequence shown in SEQ ID NO:

9.

4. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to TIM-3 according to claim 1, characterized in that: The above antibodies are humanized antibodies or human antibodies.

5. A nucleic acid molecule, characterized in that Encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The above-mentioned nucleic acid molecule comprises the nucleic acid molecule encoding the heavy chain variable region composed of the nucleotide sequence shown in Sequence 16 and the nucleic acid molecule encoding the light chain variable region composed of the nucleotide sequence shown in Sequence 17.

7. A recombinant vector, characterized in that: The method comprises a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. A host cell, characterized in that The method comprises a recombinant vector comprising a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3 according to any one of claims 1 to 4.

9. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TIM-3, characterized in that: Comprising the step of culturing the host cell of claim 8.

10. A kit for determining TIM-3 expression level, characterized in that: Comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1.

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