CN110596369A

CN110596369A - Kit for detecting human TIM-3 expression level

Info

Publication number: CN110596369A
Application number: CN201910678193.9A
Authority: CN
Inventors: 熊浩
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2019-12-20

Abstract

The invention provides a kit for detecting the expression level of human TIM-3, wherein a humanized monoclonal antibody in the kit can be combined with a TIM-3 antigen in high strength and specificity and has the auxiliary diagnosis effect of efficiently detecting TIM-3 related diseases, Tim-3 is a negative regulatory factor of type 1 immunity, Tim-3 signaling is necessary for inducing antigen specific tolerance, and Tim-3 blocking promotes the generation and development of spontaneous autoimmunity.

Description

Kit for detecting human TIM-3 expression level

Technical Field

The invention relates to the field of in-vitro detection, in particular to a kit for detecting the expression level of human TIM-3.

Background

Tim-3 is a negative regulator of type 1 immunity, Tim-3 signaling is necessary for inducing antigen-specific tolerance, and Tim-3 blockade promotes the development of spontaneous autoimmunity, and C-type lectin Galectin-9 is a Tim-3 ligand, which consolidates the inhibitory function of Tim-3, because triggering Tim-3 by Galectin-9 induces cell death in Tim-3+ Th1 cells and improves EAE.

Another Tim-3 ligand that affects the innate immune response is the high mobility group protein B1(HMGB1), HMGB1 binds to DNA released by cells that are about to die and facilitates the delivery of innate immune cells by binding to advanced glycation end products (RAGE) and Toll-like receptors (TLRs), triggering activation of innate immune cells and the production of proinflammatory cytokines, and the binding of Tim-3 to HMGB1 can interfere with this process, inhibiting the activation of the innate immune response.

TIM-3 was found to be associated with various diseases, TIM-3 positively correlated with CD 4- + T cell expression level and CD 8- + T cell in prostate cancer patients, and the immunohistochemical detection analysis shows that TIM-3 does not stain or stains weakly in prostate hyperplasia epithelial cells, but stains strongly in prostate cancer. Another study showed that TIM-3 expression correlates with the presence or absence of lymph node metastasis, the extent of adenocarcinoma cell differentiation, clinical staging and prognosis (P < 0.05).

Thus, there is a need to develop more sensitive, faster methods for animal models or patient biological samples than existing ELISAs to aid diagnosis and prognosis.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a kit for detecting the expression level of human TIM-3.

The invention is realized by the following technical scheme:

a kit for detecting the expression level of human TIM-3, comprising a TIM-3 antibody coated plate as a solid phase antibody, said TIM-3 antibody comprising:

CDR-L1 comprising the amino acid sequence of SEQ ID NO:01, and/or

CDR-L2 comprising the amino acid sequence of SEQ ID NO 02, and/or

03, and/or CDR-L3 comprising the amino acid sequence of SEQ ID NO

04, and/or CDR-H1 comprising the amino acid sequence of SEQ ID NO:04

CDR-H2 comprising the amino acid sequence of SEQ ID NO. 05, and/or

CDR-H3 comprising the amino acid sequence of SEQ ID NO: 06.

Further, methods of using the kit include:

sequentially adding a sample into antibody-coated micropores, adding an HRP-labeled enzyme labeling solution to form an antibody-antigen-enzyme-labeled compound, completely washing, and adding TMB for color development;

step two, TMB is blue under the catalysis of HRP enzyme, and is yellow after the termination solution is added, and the color depth is positively correlated with the content of TIM-3 antigen;

and step three, determining the OD450nm of the solution, and quantitatively calculating the TIM-3 protein content according to a standard curve.

Further, the antibody is selected from a monoclonal antibody, an antibody fragment specifically binding to human TIM-3, a fusion protein comprising an antibody fragment specifically binding to human TIM-3, a murine antibody, a humanized antibody, a murine monoclonal antibody, preferably, the antibody is a humanized monoclonal antibody.

Further, the antibody comprises an antibody heavy chain variable region amino acid sequence comprising a VH sequence set forth as SEQ ID NO. 7 or having at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 10.

Further, the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence as set forth in SEQ ID NO 8 or having at least 99% sequence identity to the amino acid sequence of SEQ ID NO 12.

Preferably, the amino acid sequence of the antibody heavy chain constant region is shown as SEQ ID NO. 7, and the amino acid sequence of the antibody light chain constant region is shown as SEQ ID NO. 8.

Further, the antibody production step comprises culturing a host cell containing a nucleotide sequence expressing the above antibody in a medium and under suitable culture conditions, and recovering and purifying the antibody and/or antigen-binding fragment from the medium and/or the host cell by a conventional method.

Furthermore, the kit also comprises an enzyme-labeled coated plate, a standard buffer, an enzyme-labeled solution, a sample diluent, a TMB color developing agent, a washing solution and a stop solution.

Preferably, the detection kit assists in diagnosing the types of diseases with high TIM-3 protein expression in lung cancer pancreatic cancer, non-small cell lung cancer, diabetes, nephropathy and other diseases.

The humanized monoclonal antibody in the kit provided by the invention can be well and specifically combined with the TIM-3 antigen, and has an auxiliary diagnosis effect of efficiently detecting TIM-3 related diseases.

Detailed Description

Definition of

The term "antibody" is used in the broadest sense of the present invention to include a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific antibodies, and antibody fragments, so long as they exhibit the specified antigen binding activity, and the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds, and antibody fragments including, but not limited to, Fv, Fab', bispecific antibodies, linear antibodies, single chain antibody molecules (e.g., scFv), and/or multispecific antibodies formed from antibody fragments.

The class of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main types of antibodies: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "IgG" of the present invention is an abbreviation for immunoglobulin G (immunoglobulin G). The term "humanized" of the present invention refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

The binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established and well-known techniques of the art. It is well known in the art that an antigen binding domain refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity of action, and that sequences recognizing one target molecule are generally unable to recognize other molecular sequences.

The term "binding" of the present invention refers to a non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, the term "binding force" as used herein refers to the intrinsic binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can be generally expressed by the dissociation constant (KD), and can be measured by common methods known in the art.

The terms "identity", "similarity", and "similarity" of the present invention are the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art.

Studies have shown that TIM-3 is a key factor in tumor formation. TIM-3 concentration has high correlation with diseases such as diabetes, nephropathy, tumor and the like. Numerous studies have been performed in the day ahead to show a strong correlation between circulating TIM-3 levels and tumor burden, and suggest that TIM-3 levels are a potential prognostic marker, and accurate measurement of TIM-3 is of potential importance in understanding its role in many biological processes. The ability to measure endogenous TIM-3 levels depends on the availability of sensitive and specific assays. Enzyme-linked immunosorbent assays (ELISAs) based on colorimetric, chemiluminescent, and fluorescent assays have been reported for TIM-3.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.

Example 1:

female BALB/c mice (purchased from Jersey laboratory animals Co., Ltd., Shanghai) of 6-8 weeks old were used as experimental animals for the mouse immunization test. For the primary immunization, 50 μ g of human TIM-3 protein (purchased from Beijing Hokkaiyuan Biotechnology Co., Ltd.) was mixed with complete Freund's adjuvant to form an emulsion, the emulsion was injected into the abdominal cavity of a mouse at an injection rate of 0.5 ml/injection amount, 25 μ g of human TIM-3 protein was sufficiently mixed with incomplete Freund's adjuvant every 2 weeks to form an emulsion for boosting, the boosting was performed three times, after 1 week of the final immunization, venous blood and serum of the mouse were collected and separated, and the titer of the antibody was measured by ELISA method and mouse cells having high titer were selected to prepare a hybridoma to prepare a monoplasmatic cell suspension.

Collecting logarithmic growth myeloma cells (SP2/0) to prepare immune spleen cell suspension, mixing myeloma cells and spleen cells at a ratio of 1:5, washing with incomplete culture solution, centrifuging for 8 minutes, discarding supernatant, preheating cell precipitate in 40 deg.C water bath, adding 1ml of PEG-4000 solution preheated to 40 deg.C to the cell precipitate, and adding 25ml of incomplete culture medium preheated to 40 deg.C to reaction solution within 1min after the occurrence of particles to terminate the reaction. Standing, adding 2ml HAT culture medium, slightly blowing the precipitated cells to make them suspended and uniformly mixed, then supplementing HAT culture medium until the concentration of splenocytes in the centrifugal tube reaches 1.5X 10⁷And/ml, subpackaging the cell suspension into a 96-well plate for culture and observing, and sucking out a supernatant sample for antibody detection when the cell surface area reaches above 1/2 of the plate.

Example 2:

hybridoma culture supernatants were screened for anti-human TIM-3 antibodies. Specifically, human TIM-3 (purchased from Hitachi bioscience, Inc., Beijing) was coated with a buffer solution on a 96-well high-adsorption enzyme-labeled plate in an amount of 100. mu.L per well, and then washed with the buffer solution 3 times; blocking with a buffer solution containing 1% BSA and incubating at 25 ℃ for 1h, wherein the blocking amount is 280. mu.L/well, after the incubation is completed, washing with the buffer solution for 3 times, respectively adding 75. mu.L of a supernatant sample (S1-S85) and positive serum (control, CK1-5) into wells 1-90, incubating at 25 ℃ for 1 hour, and washing with the buffer solution for 5 times; adding 100 μ L of anti-mouse IgG antibody diluted in 1/10000 in 1% BSA buffer solution to each well, said anti-mouse IgG antibody being labeled with horseradish peroxidase, incubating for 1 hour at 25 ℃ and washing 5 times with the buffer solution; adding 100 mu L of colorimetric substrate 3,3',5,5' -Tetramethylbenzidine (TMB) into each well, developing for 10min at 30 ℃, stopping the color development reaction, reading the absorbance at 450nm on an enzyme-linked immunosorbent assay, and selecting a positive clone capable of secreting human TIM-3 binding antibody according to the strength of OD450 nm.

Example 3:

the clones obtained by screening and having both the antigen-binding activity and the antigen-neutralizing activity were subjected to the measurement of the antibody DNA sequence. Cellular mRNA was first extracted using the RNA prep Pure kit (Tiangen) according to the instructions. First strand cDNA was then synthesized using the Quant Script RT kit (Tiangen). The first chain of cDNA generated by reverse transcription is used for subsequent PCR reaction, a target band obtained by PCR amplification is cloned into a pGEM-T vector, and single clone is selected to complete sequencing by Nanjing Jinsry Biotech Co.

Obtaining an antibody light chain variable region and an antibody heavy chain variable region through PCR amplification, and obtaining a complementary determining region sequence after eliminating a framework region sequence; wherein the three complementarity determining regions CDR-L1 amino acid sequences of the light chain are shown in SEQ ID NO 1; the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 3; the amino acid sequences of three complementarity determining regions CDR-H1 of the heavy chain are shown as SEQ ID NO. 4, CDR-H2 is shown as SEQ ID NO. 5, and CDR-H3 is shown as SEQ ID NO. 6; the antibody light chain constant region amino acid sequence is derived from murine IgVH4-21 x 07, antibody heavy chain constant region sequence murine IgVH2-09 x 01, and the light chain full-length sequence is obtained by connecting the antibody light chain variable region with the light chain constant region; the heavy chain full-length sequence is obtained by connecting an antibody heavy chain variable region with a heavy chain constant region, and the variable region sequence and the constant region sequence are respectively cloned into a eukaryotic cell expression vector TL10-11 (a vector framework pEGFP-N1 purchased from Chimana Shanghai). Antibody light chain and antibody heavy chain expression vectors were transfected into 293F cell line (purchased from shanghai jingning biotechnology). Cells were seeded the day before transfection, harvested by centrifugation the day of transfection, resuspended in fresh expression medium at a cell density of 1.5X 10⁷cells/mL. Plasmid was added to a final concentration of 39.1. mu.g/mL and linear polyethyleneimine was added to a final concentration of 45. mu.g/mL, according to the transfection volume. And (3) putting the mixture into a cell culture box for culturing at 37 ℃ for 1 hour, then adding a fresh culture medium into the culture solution until the final volume is 20 times of the transfection volume, continuing culturing for 5-6 days, and collecting the supernatant.

Example 4:

the kinetic constants of the binding of the anti-human TIM-3 murine monoclonal antibody (hereinafter abbreviated as OM-anti-TIM-3) obtained in example 1 to the antigen were determined. The instrument optical surface plasma resonance technology is used for detecting the combination and dissociation between the molecules coupled and coated on the biochip and the molecules to be detected. Briefly, OM-anti-TIM-3 was dissolved in sodium acetate buffer (pH 5.0) and coupled to CM chips, blocked with 1M ethanolamine. Different concentrations of OM-anti-TIM-3 were injected at a rate of 27. mu.L/min for 2.5min during the binding phase and 27. mu.L/min for 8min during the dissociation phase, and the binding kinetic constants and dissociation kinetic constants were calculated by analysis using Biacore 3000 software. OM-anti-TIM-3 has a binding kinetic constant of 4.77E +04(1/Ms), a dissociation kinetic constant of 1.64E-05(1/s), and a dissociation equilibrium constant of 0.01 (nM).

Example 5:

humanized versions of anti-human TIM-3 antibodies were prepared by reference to the preparation of molecular Immunol, selecting the humanized template best matched to OM-anti-TIM-3 non-CDR region in the Germinle database, wherein the template for heavy chain variable region was human IgVH 4-28A 03, the template for light chain variable region was human IGKV 1-16A 02, grafting the murine antibody CDR region onto the selected humanized template, and substituting to obtain the humanized antibody heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO. 7, and the humanized antibody light chain variable region, whose amino acid sequence is shown in SEQ ID NO. 8. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) obtained by selecting suitable sites for back mutation by sequence alignment are shown in table 1.

TABLE 1 amino acid sequences obtained by back-mutation

VH	SEQ ID NO
		ORI	SEQ ID NO:7
MeH-037	SEQ ID NO:9
		MeH-012	SEQ ID NO:10
VL	SEQ ID NO
		ORI	SEQ ID NO:8
MeL-005	SEQ ID NO:11
		MeL-058	SEQ ID NO:12

The heavy chain variable region (SEQ ID NO: 7-10) of the humanized anti-human TIM-3 monoclonal antibody was ligated to the heavy chain constant region (SEQ ID NO:13) of human antibody IgG1 to obtain the corresponding heavy chain full-length sequences, respectively. The light chain variable region (SEQ ID NO: 11-12) of the humanized anti-human TIM-3 monoclonal antibody is connected with the constant region (SEQ ID NO:14) of the human antibody Kappa light chain to respectively obtain corresponding light chain full-length sequences, all the heavy chain full-length sequences and the light chain full-length sequences are combined to obtain the humanized antibody full-length sequence, and the humanized antibody full-length sequence is connected into a TL10-11 (carrier framework pEGFP-N1) carrier through enzyme digestion.

Example 6:

the kinetic constants of the obtained humanized TIM-3 monoclonal antibodies (ORI: SEQ ID NO:7 and SEQ ID NO:8, MeH-037: SEQ ID NO:9 and SEQ ID NO:11, MeH-012: SEQ ID NO:10 and SEQ ID NO:12) combined with the antigen TIM-3 are detected, and the combination and dissociation between the molecules coupled and coated on the biochip and the molecules to be detected are detected by using an instrument optical surface plasma resonance technology. The method is the same as example 4, and the binding kinetic constant, dissociation kinetic constant and dissociation equilibrium constant of the humanized TIM-3 monoclonal antibody are shown in Table 2, and the results show that the humanized TIM-3 monoclonal antibody has strong binding ability to the antigen.

TABLE 2 kinetic constants for binding of humanized TIM-3 monoclonal antibody to its antigen

Example 7:

in order to determine the thermal stability of ORI, MeH-037 and MeH-012 monoclonal antibodies, samples of ORI, MeH-037 and MeH-012 were subjected to high temperature conditions of 40 ℃ and samples were taken at weeks 2 and 3, respectively, for SE-HPLC to observe thermal stability, and the assay was performed by chromatography using phosphate buffer (0.1mol/L), sodium sulfate buffer (0.1mol/L, pH6.7) as mobile phases; the flow rate is 0.6 mL/min; the temperature of the chromatographic column is 27 ℃; the temperature of the sample cell is 4 ℃; the detection wavelength is 280 nm; the samples were diluted to 2mg/mL loading in 20. mu.L volume with buffer, and the data were processed by calculating the ratio of the main peaks using area normalization, and the results of the thermal stability test for humanized TIM-3 monoclonal antibody are shown in Table 3.

TABLE 3 humanized TIM-3 monoclonal antibody thermal stability test results

The thermal stability tests showed that ORI, MeH-037 and MeH-012 monoclonal antibodies all showed good and comparable stability.

Example 8:

the 2 amino acid sequences (ORI, MeH-012) can be respectively used for preparing a detection kit, and the specific method comprises the steps of preparing an antibody coating plate into a solid-phase antibody, sequentially adding samples into micropores of the coated antibody, adding HRP-labeled enzyme labeling liquid to form an antibody-antigen-enzyme labeled antibody compound, completely washing, adding a substrate TMB for developing color, converting the TMB into blue under the catalysis of HRP enzyme, converting the TMB into yellow under the action of a stop solution, positively correlating the color depth and the TIM-3 content, measuring OD (optical density) under the wavelength of 450nm by using an enzyme labeling instrument, and calculating the TIM-3 content by labeling, wherein the kit adopts a novel humanized TIM-3 monoclonal antibody which has stronger binding capacity with human TIM-3 and higher binding rate, can be used for quickly and accurately detecting the TIM-3 content and provides more sensitive TIM-3 related diseases for auxiliary diagnosis, Accurate data, as shown in table 4, the kit specifically included the ingredients described in the table below, stored at 4 ℃.

TABLE 4 high sensitivity detection kit for human TIM-3

Kit composition	96 wells
		Enzyme-labeled coated plate	1X 96 Standard
TMB color-developing agent	5mL X2 tube
		Cleaning solution	10mL X2 tube
Stopping liquid	5mL X2 tube
		Standard article (500pg/mL)	1mL X2 tube
Sample buffering agent	10mL X1 tube
		Enzyme labeling liquid	1mL X2 tube
Sample diluent	10mL X1 tube

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

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85 90 95

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105 110

Claims

1. A kit for detecting the expression level of human TIM-3, wherein a TIM-3 antibody is coated onto a plate to form a solid phase antibody, said TIM-3 antibody comprising:

CDR-L1 comprising the amino acid sequence of SEQ ID NO:01, and/or

CDR-L2 comprising the amino acid sequence of SEQ ID NO 02, and/or

03, and/or CDR-L3 comprising the amino acid sequence of SEQ ID NO

04, and/or CDR-H1 comprising the amino acid sequence of SEQ ID NO:04

CDR-H2 comprising the amino acid sequence of SEQ ID NO. 05, and/or

CDR-H3 comprising the amino acid sequence of SEQ ID NO: 06.

2. The kit of claim 1, wherein the method of using the kit comprises:

step three, determining the OD of the solution_450nmAnd quantitatively calculating the content of the TIM-3 protein according to a standard curve.

3. Kit according to claim 1, characterized in that said antibody is selected from the group consisting of monoclonal antibodies, antibody fragments specifically binding to human TIM-3, fusion proteins comprising antibody fragments specifically binding to human TIM-3, murine antibodies, humanized antibodies, murine monoclonal antibodies, preferably said antibodies are humanized monoclonal antibodies.

4. The kit of claim 1, wherein the antibody comprises an antibody heavy chain variable region amino acid sequence comprising a VH sequence as set forth in SEQ ID No. 7 or having at least 99% sequence identity to the amino acid sequence of SEQ ID No. 10.

5. The kit of claim 1, wherein the antibody comprises an antibody light chain variable region amino acid sequence comprising a VL sequence as set forth in SEQ ID No. 8 or having at least 99% sequence identity to the amino acid sequence of SEQ ID No. 12.

6. The kit of claim 1, wherein the antibody heavy chain constant region amino acid sequence is set forth in SEQ ID NO. 7 and the antibody light chain constant region amino acid sequence is set forth in SEQ ID NO. 8.

7. The kit of any one of claims 1 to 6, wherein the antibody production step comprises culturing a host cell comprising a nucleotide sequence that expresses the antibody of claims 1 to 6 in a culture medium under suitable culture conditions, and recovering and purifying the antibody and/or antigen-binding fragment from the culture medium and/or the host cell by conventional methods.

8. The kit of claim 7, further comprising an enzyme-labeled coating plate, a standard buffer, an enzyme labeling solution, a sample diluent, a TMB color developing agent, a washing solution and a stop solution.

9. The kit according to claims 1-8, wherein the detection kit is used for assisting in diagnosing the types of diseases with high TIM-3 protein expression in lung cancer pancreatic cancer, non-small cell lung cancer, diabetes, nephropathy and other diseases.