WO2020073832A1 - 一种抗人心肌肌钙蛋白i的重组抗体 - Google Patents
一种抗人心肌肌钙蛋白i的重组抗体 Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Definitions
- the present disclosure relates to the technical field of immunology, in particular, to a recombinant antibody against human cardiac troponin I.
- WHO World Health Organization
- myocardial enzyme activity is more sensitive and specific than phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), and lactate dehydrogenation. Enzyme and aspartate aminotransferase and other biomarkers.
- Cardiac troponin I (cTnI) exists only in the myocardium and is a marker of cardiomyocytes.
- cTnI in normal people's blood is generally less than 0.3 ⁇ g / L.
- ELISA enzyme-linked immunosorbent assay
- chemiluminescence chemiluminescence
- colloidal gold etc.
- the present disclosure relates to a novel isolated binding protein containing cardiac troponin I (cTnI) antigen binding domain, and studies on the preparation and application of the binding protein.
- cTnI cardiac troponin I
- the antigen binding domain includes at least one complementarity determining region selected from the following amino acid sequence; or; having a sequence identity of at least 80% with the complementarity determining region of the following amino acid sequence and having K D ⁇ with cardiac troponin I 1.41 ⁇ 10 -9 mol / L affinity;
- the complementarity determining region CDR-VH1 is G-F-N-X1-K-X2-Y-X3-M-H, where,
- X1 is L or I
- X2 is D or E
- X3 is F or Y
- the complementarity determining region CDR-VH2 is R-I-X1-P-E-D-X2-E-T-X3-Y-A-P-E, where,
- X1 is E or D
- X2 is A or G
- X3 is R or K
- the complementarity determining region CDR-VH3 is Y-Y-X1-S-Y-X2-P-F-V-Y, where,
- X1 is T or S, X2 is I, L or V;
- the complementarity determining region CDR-VL1 is Q-S-X1-X2-Y-S-N-X3-H-T-Y, where,
- X1 is I or L
- X2 is I or L
- X3 is R or K
- the complementarity determining region CDR-VL2 is Q-X1-S-X2-R-F-S, where,
- X1 is I, L or V, X2 is N or Q;
- the complementarity determining region CDR-VL3 is S-X1-S-T-H-X2-P-X3-T, where,
- X1 is Q or N
- X2 is L or I
- X3 is F or Y.
- binding protein has strong activity and has a high affinity with human cTnI protein.
- X2 is D
- X1 is D
- X1 is S
- X3 is R
- X2 is N;
- X1 is L in the complementarity determining region CDR-VH1.
- X1 is I in the complementarity determining region CDR-VH1.
- X3 is F in the complementarity determining region CDR-VH1.
- X3 is Y in the complementarity determining region CDR-VH1.
- X2 is A in the complementarity determining region CDR-VH2.
- X2 is G in the complementarity determining region CDR-VH2.
- X3 is R in the complementarity determining region CDR-VH2.
- X3 is K in the complementarity determining region CDR-VH2.
- X2 is I in the complementarity determining region CDR-VH3.
- X2 is L in the complementarity determining region CDR-VH3.
- X2 is V in the complementarity determining region CDR-VH3.
- X1 is I in the complementarity determining region CDR-VL1.
- X1 is L in the complementarity determining region CDR-VL1.
- X2 is I in the complementarity determining region CDR-VL1.
- X2 is L in the complementarity determining region CDR-VL1.
- X1 is I in the complementarity determining region CDR-VL2.
- X1 is L in the complementarity determining region CDR-VL2.
- X1 is V in the complementarity determining region CDR-VL2.
- X2 is L in the complementarity determining region CDR-VL3.
- X2 is 1 in the complementarity determining region CDR-VL3.
- X3 is F in the complementarity determining region CDR-VL3.
- X3 is Y in the complementarity determining region CDR-VL3.
- the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
- the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
- the binding protein is a complete antibody comprising variable and constant regions.
- the binding protein is a "functional fragment" of an antibody, such as Nanobody, F (ab ') 2 , Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit One of them.
- the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and / or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4.
- the binding protein further comprises antibody constant region sequences.
- the constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
- the species source of the constant region is cattle, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
- the constant region is derived from murine
- the sequence of the light chain constant region is shown in SEQ ID NO: 9;
- the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
- the disclosure also relates to an isolated nucleic acid molecule, which is DNA or RNA, which encodes a binding protein as described above.
- the present disclosure also relates to a vector comprising the nucleic acid molecule as described above.
- the present disclosure also relates to a host cell transformed with the vector as described above.
- the present disclosure also relates to a method for producing the binding protein as described above, the method comprising the following steps:
- the host cells as described above are cultured in the culture medium and under appropriate culture conditions, and the binding protein thus produced is recovered from the culture medium or from the cultured host cells.
- the present disclosure also relates to application.
- the present disclosure also relates to a method of detecting troponin I antigen in a test sample, which includes:
- the immune complex in step a), further includes a second antibody, and the second antibody binds to the binding protein.
- the immune complex further includes a second antibody, and the second antibody binds to the troponin I antigen.
- the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
- the present disclosure also relates to a kit including the binding protein as described above.
- the troponin I antigen is cardiac troponin I antigen.
- the disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with cardiac troponin I.
- the present disclosure also relates to a method of diagnosing diseases associated with cardiac troponin I, including:
- the presence of the immune complex indicates the presence of a disease associated with cardiac troponin I.
- the method is based on fluorescent immunoassay, chemiluminescence, colloidal gold immunoassay, radioimmunoassay, and / or enzyme-linked immunoassay.
- the sample is selected from at least one of whole blood, peripheral blood, serum, plasma, or myocardial tissue.
- the subject is a mammal, such as a primate, such as a human.
- the disease associated with cardiac troponin I is a cardiovascular disease.
- the disease associated with cardiac troponin I is selected from the group consisting of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, myocardial injury, or a combination thereof.
- FIG. 1 is a monoclonal antibody electrophoresis diagram of the recombinant antibody against human cardiac troponin I of the present disclosure.
- Isolated binding protein comprising an antigen binding domain generally refers to any protein / protein fragment comprising a CDR region.
- antibody includes polyclonal antibodies and monoclonal antibodies and antigen compound binding fragments of these antibodies, including Fab, F (ab ') 2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, and these antibodies And fragments of single-chain derivatives.
- the type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
- antibody includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isomeric forms (isoforms).
- antibody is used interchangeably with “immunoglobulin”.
- variable region refers to the amino-terminal domain of the heavy or light chain of the antibody.
- the variable domain of the heavy chain may be referred to as "VH”.
- variable domain of the light chain may be referred to as "VL”. These domains are usually the most variable part of an antibody and contain antigen binding sites.
- the light or heavy chain variable region (VL or VH) is composed of three hypervariable regions called “complementarity determining regions” or "CDRs" and a framework region separating the three complementarity determining regions.
- CDRs complementarity determining regions
- the framework and CDR ranges have been precisely defined, for example, in Kabat (see “Sequences of Proteins of Immunological Interests", E.
- the framework region of the antibody that is, the framework region constituting the combination of the light chain and the heavy chain of the requirements, plays a role of positioning and aligning CDRs, which are mainly responsible for binding to the antigen.
- a "backbone region”, “framework region” or “FR” region means a region of the antibody variable domain that excludes those defined as CDRs.
- the framework region of each antibody variable domain can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.
- variable regions VL / VH of the heavy and light chains can be obtained by connecting the numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the term “purified” or “isolated” in association with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form.
- isolated includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring.
- an isolated polypeptide generally does not contain at least some proteins or other cellular components that it is usually bound to or usually mixed with or in solution.
- Isolated polypeptides include the naturally produced polypeptides contained in cell lysates, the polypeptides in purified or partially purified form, recombinant polypeptides, the polypeptides expressed or secreted by cells, and in heterologous host cells or cultures Of the polypeptide.
- the term isolated or purified indicates that the nucleic acid is not in its natural genomic background (eg, in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).
- bispecific antibody or “bifunctional antibody” refers to an artificial hybrid binding protein with two different heavy / light chain pairs and two different binding sites. Bispecific binding proteins can be produced by a variety of methods, including fusion of hybridomas or attachment of Fab 'fragments.
- sequence identity refers to the similarity between at least two different sequences. This percentage identity can be determined by standard algorithms, such as Basic Local Alignment Search Tool (BLAST); Algorithms such as Needleman, etc .; or algorithms such as Meyers.
- BLAST Basic Local Alignment Search Tool
- Algorithms such as Needleman, etc .
- Meyers or algorithms such as Meyers.
- a set of parameters may be a Blosum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
- the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4: 11-17), which has been incorporated Into the ALIGN program (version 2.0), use the PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4. Percent identity is usually calculated by comparing sequences of similar length.
- affinity refers to the strength of binding of an antigen binding domain of a binding protein or antibody to an antigen or antigen epitope. Affinity can be measured by KD value, the smaller the KD value, the greater the affinity.
- the present disclosure relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the following amino acid sequence; or; the complementarity determining region with the following amino acid sequence has at least 80% sequence identity and affinity with cardiac troponin I K D ⁇ 1.41 ⁇ 10 -9 mol / L;
- the complementarity determining region CDR-VH1 is G-F-N-X1-K-X2-Y-X3-M-H, where,
- X1 is L or I
- X2 is D or E
- X3 is F or Y
- the complementarity determining region CDR-VH2 is R-I-X1-P-E-D-X2-E-T-X3-Y-A-P-E, where,
- X1 is E or D
- X2 is A or G
- X3 is R or K
- the complementarity determining region CDR-VH3 is Y-Y-X1-S-Y-X2-P-F-V-Y, where,
- X1 is T or S, X2 is I, L or V;
- the complementarity determining region CDR-VL1 is Q-S-X1-X2-Y-S-N-X3-H-T-Y, where,
- X1 is I or L
- X2 is I or L
- X3 is R or K
- the complementarity determining region CDR-VL2 is Q-X1-S-X2-R-F-S, where,
- X1 is I, L or V, X2 is N or Q;
- the complementarity determining region CDR-VL3 is S-X1-S-T-H-X2-P-X3-T, where,
- X1 is Q or N
- X2 is L or I
- X3 is F or Y.
- the complementarity determining region of the antigen binding domain and the following amino acid sequence has at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, Or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, Or at least 97%, or at least 98%, or at least 99% sequence identity and having K D ⁇ 1.41 ⁇ 10 -9 mol / L with cardiac troponin I, such as 1 ⁇ 10 -10 mol / L, 2 ⁇ 10 -10 mol / L, 3 ⁇ 10 -10 mol / L, 4 ⁇ 10 -10 mol / L, 4.5 ⁇ 10 -10 mol / L, 5 ⁇ 10 -10 mol / L, 6 ⁇ 10 -10 mol / L L, 7 ⁇ 10 -10 mol / L
- KD is less than or equal to 1 ⁇ 10 -10 mol / L, 2 ⁇ 10 -10 mol / L, 3 ⁇ 10 -10 mol / L, 4 ⁇ 10 -10 mol / L, 4.5 ⁇ 10 -10 mol / L, 5 ⁇ 10 -10 mol / L, 6 ⁇ 10 -10 mol / L, 7 ⁇ 10 -10 mol / L, 8 ⁇ 10 -10 mol / L, 9 ⁇ 10 -10 mol / L, 1 ⁇ 10 -11 mol / L, 3 ⁇ 10 -11 mol / L, 5 ⁇ 10 -11 mol / L, 5 ⁇ 10 -11 mol / L, 7 ⁇ 10 -11 mol / L, or 9 ⁇ 10 -11 mol / L.
- the affinity is measured according to the method in the present specification.
- X2 is D
- X1 is D
- X1 is S
- X3 is R
- X2 is N;
- X1 is L in the complementarity determining region CDR-VH1.
- X1 is I in the complementarity determining region CDR-VH1.
- X3 is F in the complementarity determining region CDR-VH1.
- X3 is Y in the complementarity determining region CDR-VH1.
- X2 is A in the complementarity determining region CDR-VH2.
- X2 is G in the complementarity determining region CDR-VH2.
- X3 is R in the complementarity determining region CDR-VH2.
- X3 is K in the complementarity determining region CDR-VH2.
- X2 is I in the complementarity determining region CDR-VH3.
- X2 is L in the complementarity determining region CDR-VH3.
- X2 is V in the complementarity determining region CDR-VH3.
- X1 is I in the complementarity determining region CDR-VL1.
- X1 is L in the complementarity determining region CDR-VL1.
- X2 is I in the complementarity determining region CDR-VL1.
- X2 is L in the complementarity determining region CDR-VL1.
- X1 is I in the complementarity determining region CDR-VL2.
- X1 is L in the complementarity determining region CDR-VL2.
- X1 is V in the complementarity determining region CDR-VL2.
- X2 is L in the complementarity determining region CDR-VL3.
- X2 is 1 in the complementarity determining region CDR-VL3.
- X3 is F in the complementarity determining region CDR-VL3.
- X3 is Y in the complementarity determining region CDR-VL3.
- the mutation site of each complementarity determining region is selected from any one of the following mutation combinations:
- X1 appearing in the six CDR regions of the binding protein described in the present disclosure each independently of each other represents the amino acid defined in the present disclosure; appearing in the six of the binding protein described in the present disclosure X2 in each CDR region independently represents the amino acids defined in the present disclosure; X3 appearing in the six CDR regions of the binding protein described in the present disclosure each independently represents the amino acids defined in the present disclosure.
- the binding protein includes at least 3 CDRs; alternatively, the binding protein includes at least 6 CDRs.
- the binding protein is a complete antibody comprising variable and constant regions.
- the binding protein is a "functional fragment" of an antibody, such as Nanobody, F (ab ') 2 , Fab', Fab, Fv, scFv, bispecific antibody, and antibody minimum recognition unit One of them.
- the "functional fragment” described in the present disclosure particularly refers to an antibody fragment having the same specificity as cTnI as the parent antibody. In addition to the above functional fragments, it also includes any fragments whose half-life has been increased.
- antibody fragments of the present disclosure can obtain the above-mentioned functions by methods such as enzymatic digestion (including pepsin or papain) and / or methods of chemically reducing the cleavage of disulfide bonds according to the contents described in the description of the present disclosure. Fragment.
- Antibody fragments can also be obtained by peptide synthesis by recombinant genetics techniques that are also known to those skilled in the art or by, for example, automatic peptide synthesizers such as those sold by Applied BioSystems.
- the binding protein includes light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 whose sequence is as shown in SEQ ID NO: 1-4, and / or Or, the sequence is as shown in SEQ ID NO: 5-8 heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4.
- the species source of the skeleton region may be human to constitute a humanized antibody.
- the binding protein further comprises antibody constant region sequences.
- the constant region sequence is selected from any one of the constant region sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
- the species source of the constant region is cattle, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink , Chicken, duck, goose, turkey, cockfight or human.
- the constant region is derived from murine
- the sequence of the light chain constant region is shown in SEQ ID NO: 9;
- the sequence of the heavy chain constant region is shown in SEQ ID NO: 10.
- the disclosure also relates to an isolated nucleic acid molecule, which is DNA or RNA, which encodes a binding protein as described above.
- the present disclosure also relates to a vector comprising the nucleic acid molecule as described above.
- the present disclosure further includes at least one nuclear construct encoding the nucleic acid molecule as described above, such as a plasmid, and further an expression plasmid, and a method for constructing the vector will be introduced in an embodiment of the present application.
- the present disclosure also relates to a host cell transformed with the vector as described above.
- the host cell may be a eukaryotic cell, such as a mammalian cell.
- the host cell is a CHO cell.
- the present disclosure also relates to a method for producing the binding protein as described above, the method comprising the following steps:
- the host cells as described above are cultured in the culture medium and under appropriate culture conditions, and the binding protein thus produced is recovered from the culture medium or from the cultured host cells.
- the present disclosure also relates to application.
- the present disclosure also relates to a method of detecting troponin I antigen in a test sample, which includes:
- the binding protein may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
- the immune complex further includes a second antibody, and the second antibody binds to the binding protein
- the binding protein forms a paired antibody with the second antibody in the form of a first antibody for binding to different epitopes of cTnI;
- the second antibody may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
- the immune complex further includes a second antibody that binds to the troponin I antigen
- the binding protein serves as an antigen of the second antibody
- the second antibody may be labeled with an indicator that shows signal intensity, so that the complex is easily detected.
- the indicator showing the signal intensity includes fluorescent substances, quantum dots, digoxin-labeled probes, biotin, radioisotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electrons Any one of dense substance, chemiluminescent marker, ultrasound contrast agent, photosensitizer, colloidal gold or enzyme.
- the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY -FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, 5-carboxy-2 ′, 4 ′ , 5 ′, 7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,
- the radioisotope includes 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y , 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re , 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb and 83 Sr.
- the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
- the fluorescent microspheres are: polystyrene fluorescent microspheres, with rare earth fluorescent ion europium wrapped inside.
- the present disclosure also relates to a kit including the binding protein as described above.
- the troponin I antigen is cardiac troponin I antigen.
- the disclosure also relates to the use of the binding proteins described herein in the diagnosis of diseases associated with cardiac troponin I.
- cardiac troponin I-related disease refers to a disease that uses cardiac troponin I, including its protein or encoding nucleic acid, as a marker.
- a disease associated with cardiac troponin I may refer to a disease characterized by an increase in the level of cardiac troponin I in blood.
- a cardiac troponin I-related disease may refer to a disease characterized by a decrease in cardiac troponin I levels in cardiac muscle tissue or cardiomyocytes.
- the present disclosure also relates to a method of diagnosing diseases associated with cardiac troponin I, including:
- the presence of the immune complex indicates the presence of a disease associated with cardiac troponin I.
- the method is based on fluorescent immunoassay, chemiluminescence, colloidal gold immunoassay, radioimmunoassay, and / or enzyme-linked immunoassay.
- the sample is selected from at least one of whole blood, peripheral blood, serum, plasma, or myocardial tissue.
- the subject is a mammal, such as a primate, such as a human.
- the disease associated with cardiac troponin I is a cardiovascular disease.
- the disease associated with cardiac troponin I is selected from the group consisting of acute myocardial infarction, acute coronary syndrome, pulmonary infarction, unstable angina, myocardial injury, or a combination thereof.
- This embodiment provides an exemplary method for preparing a recombinant antibody against human cardiac troponin I.
- restriction enzyme and Prime DNA polymerase were purchased from Takara;
- BD SMART TM RACE cDNA Amplification Kit was purchased from Takara;
- the pMD-18T vector was purchased from Takara;
- the plasmid extraction kit was purchased from Tiangen Company;
- the anti-cTnI 5G8 monoclonal antibody was secreted as an existing hybridoma cell line, and was recovered for use.
- 5’RACE upstream primers for amplification of heavy and light chains 5’RACE upstream primers for amplification of heavy and light chains:
- NUP Nested Universal Primer A
- RNA from the hybridoma cell line secreting Anti-cTnI 5G8 monoclonal antibody and use the SMARTERTM RACE cDNA Amplification Kit kit and the SMARTER II oligonucleotide and 5'-CDS primers in the kit to perform first-strand cDNA After synthesis, the obtained first-strand cDNA product is used as a template for PCR amplification.
- the light chain genes were amplified with universal primer A mixture (UPM), nested universal primer A (NUP) and mIg-kR primers, and the heavy chain genes were amplified with universal primer A mixture (UPM), nested universal primer A (NUP) and The mIg-HR primer is amplified.
- the primer pair of the light chain amplifies the target band of about 0.72KB
- the primer pair of the heavy chain amplifies the target band of about 1.4KB.
- VL gene sequence is 321bp, which belongs to the VkII gene family, with a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 357bp, which belongs to the VH1 gene family, which has 57bp Leader sequence.
- pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced into polyclonal restriction sites such as HindIII, BamHI, EcoRI, etc., and is named pcDNA3.4A expression vector, subsequently referred to as 3.4A expression vector; according to pMD-18T Based on the sequencing results of the variable region of the medium antibody, anti-cTnI 5G8 antibody VL and VH gene-specific primers were designed with HindIII and EcoRI cleavage sites and protective bases at both ends. The primers are as follows:
- cTnI-5G8-HF 5 ’> CCCAAGCTTATGGAATGCAGCTGTGTCATGCTCTTCTTC ⁇ 3’;
- cTnI-5G8-LF 5 ’> CCCAAGCTTATGAAGTTGCCTGTTAGGCTGTTGG ⁇ 3’;
- cTnI-5G8-LR 5 ’> CCCGAATTCCTAACACTCATTCCTGTTGAAGCTCTTGACAA ⁇ 3’;
- the 0.72KB light chain gene fragment and 1.4kb heavy chain gene fragment were amplified by PCR amplification.
- the heavy chain and light chain gene fragments were digested with HindIII / EcoRI double digestion, and the 3.4A vector was digested with HindIII / EcoRI double digestion. After the fragments and vector were purified and recovered, the heavy chain gene and light chain gene were connected to the 3.4A expression vector, respectively. Recombinant expression plasmids for heavy and light chains were obtained.
- the complementary determination region (WT) of the heavy chain After analysis, the complementary determination region (WT) of the heavy chain:
- CDR-VH1 is G-F-N-L (X1) -K-E (X2) -Y-F (X3) -M-H
- CDR-VH2 is R-I-E (X1) -P-E-D-A (X2) -E-T-R (X3) -Y-A-P-E;
- CDR-VH3 is Y-Y-T (X1) -S-Y-I (X2) -P-F-V-Y;
- CDR-VL1 is Q-S-I (X1) -I (X2) -Y-S-N-K (X3) -H-T-Y;
- CDR-VL2 is Q-I (X1) -S-Q (X2) -R-F-S;
- CDR-VL3 is S-N (X1) -S-T-H-L (X2) -P-F (X3) -T;
- X1, X2, and X3 are all sites to be mutated.
- the inventors made the above mutations in the CDR sites in WT to obtain antibodies with better activity.
- the purified antibody is diluted with PBST to 10ug / ml, CTNI quality control product recombinant protein (PK2-CTNI-1, 170120, produced by the company)
- PBST gradient dilution 400nmol / ml, 200nmol / ml, 100nmol / ml, 50nmol / ml, 25nmol / ml, 12.5nmol / ml, 6.25nmol / ml, 0nmol / ml;
- Operation process equilibrate in buffer 1 (PBST) for 60s, solidify antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH1.69GLY solution
- buffer 3 performs sensor regeneration and outputs data.
- mutation 1 is used as the backbone sequence to screen for mutation sites with good titers (ensure that the activity of the antibody obtained by screening is similar to mutation 1, and the antibody activity is ⁇ 10%), some results are as follows.
- the purified antibody was diluted with PBST to 10ug / ml, and the CTNI quality control product recombinant protein (PK2-CTNI-1, 170120, produced by the company) was gradient diluted with PBST: 400nmol / ml, 200nmol / ml, 100nmol / ml, 50nmol / ml, 25nmol / ml, 12.5nmol / ml, 6.25nmol / ml, 0nmol / ml;
- the present disclosure provides isolated binding proteins that include an antigen binding domain that binds to cardiac troponin I, including specific heavy chain CDRs and light chain CDRs.
- the binding protein can specifically recognize and bind cardiac troponin I protein, and has high sensitivity and specificity.
- the binding protein has high affinity with human cTnI protein.
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Abstract
Description
样品浓度ng/ml | WT | 突变1 | 突变2 | 突变3 | 突变4 | 突变5 |
1000 | 2.039 | 2.401 | 2.378 | 2.349 | 2.219 | 2.197 |
200 | 1.901 | 2.395 | 2.417 | 2.370 | 2.148 | 2.204 |
40 | 1.762 | 2.348 | 2.300 | 2.249 | 1.976 | 1.943 |
8 | 1.598 | 2.066 | 1.798 | 1.857 | 1.753 | 1.694 |
1.6 | 0.534 | 0.973 | 0.724 | 0.831 | 0.689 | 0.721 |
0.32 | 0.310 | 0.341 | 0.233 | 0.301 | 0.314 | 0.189 |
0 | 0.027 | 0.032 | 0.040 | 0.028 | 0.030 | 0.042 |
K D(M) | K on(1/Ms) | K off(1/S) | |
突变1 | 7.51E-10 | 2.06E+05 | 1.55E-04 |
突变1-1 | 8.65E-10 | 3.59E+05 | 3.11E-04 |
突变1-2 | 6.23E-10 | 4.30E+05 | 2.68E-04 |
突变1-3 | 6.76E-10 | 1.81E+05 | 1.22E-04 |
突变1-4 | 9.63E-10 | 2.39E+05 | 2.30E-04 |
突变1-5 | 6.23E-10 | 1.61E+05 | 1.00E-04 |
突变1-6 | 7.34E-10 | 3.62E+05 | 2.66E-04 |
突变1-7 | 8.04E-10 | 6.97E+05 | 5.60E-04 |
突变1-8 | 3.03E-10 | 8.51E+04 | 2.58E-05 |
突变1-9 | 8.66E-10 | 1.06E+05 | 9.18E-05 |
突变1-10 | 9.22E-10 | 4.48E+05 | 4.13E-04 |
突变1-11 | 6.06E-10 | 7.31E+05 | 4.43E-04 |
突变1-12 | 8.34E-10 | 3.09E+05 | 2.58E-04 |
突变1-13 | 8.06E-10 | 3.11E+05 | 2.51E-04 |
突变1-14 | 4.15E-10 | 6.73E+05 | 2.79E-04 |
突变1-15 | 9.17E-10 | 2.67E+05 | 2.45E-04 |
突变1-16 | 6.62E-10 | 1.52E+05 | 1.01E-04 |
突变1-17 | 7.22E-10 | 7.82E+05 | 5.65E-04 |
突变1-18 | 6.80E-10 | 4.15E+05 | 2.82E-04 |
突变1-19 | 4.87E-10 | 3.93E+05 | 1.91E-04 |
突变1-20 | 9.45E-10 | 1.67E+05 | 1.58E-04 |
突变1-21 | 6.76E-10 | 7.05E+05 | 4.77E-04 |
突变1-22 | 7.25E-10 | 2.97E+05 | 2.15E-04 |
突变1-23 | 9.68E-10 | 6.95E+05 | 6.73E-04 |
突变1-24 | 4.08E-10 | 6.35E+05 | 2.59E-04 |
突变1-25 | 9.70E-10 | 6.79E+05 | 6.59E-04 |
突变1-26 | 6.92E-10 | 3.33E+05 | 2.30E-04 |
突变1-27 | 8.50E-10 | 9.93E+05 | 8.44E-04 |
突变1-28 | 1.89E-10 | 5.36E+05 | 1.01E-04 |
突变1-29 | 6.98E-10 | 1.65E+05 | 1.15E-04 |
突变1-30 | 6.69E-10 | 5.41E+05 | 3.62E-04 |
突变1-31 | 7.95E-10 | 3.78E+05 | 3.01E-04 |
突变1-32 | 5.09E-10 | 1.65E+05 | 8.40E-05 |
突变1-33 | 9.86E-10 | 8.94E+05 | 8.81E-04 |
突变1-34 | 1.41E-09 | 4.24E+05 | 5.98E-04 |
突变1-35 | 5.74E-10 | 4.80E+05 | 2.76E-04 |
突变1-36 | 8.09E-10 | 5.59E+05 | 4.52E-04 |
突变1-37 | 6.67E-10 | 4.00E+05 | 2.67E-04 |
突变1-38 | 2.40E-10 | 4.31E+05 | 1.03E-04 |
突变1-39 | 7.56E-10 | 6.20E+05 | 4.69E-04 |
突变1-40 | 8.18E-10 | 2.00E+05 | 1.64E-04 |
突变1-41 | 7.23E-10 | 5.44E+05 | 3.93E-04 |
突变1-42 | 3.47E-10 | 5.20E+05 | 1.80E-04 |
突变1-43 | 6.51E-10 | 1.19E+05 | 7.75E-05 |
突变1-44 | 8.65E-10 | 5.37E+05 | 4.64E-04 |
突变1-45 | 1.68E-10 | 1.93E+05 | 3.24E-05 |
突变1-46 | 7.23E-10 | 3.73E+05 | 2.70E-04 |
突变1-47 | 8.10E-10 | 6.26E+05 | 5.07E-04 |
突变1-48 | 9.56E-10 | 6.80E+05 | 6.50E-04 |
突变1-49 | 5.38E-10 | 5.53E+05 | 2.98E-04 |
突变1-50 | 3.86E-10 | 1.42E+05 | 5.48E-05 |
突变1-51 | 7.65E-10 | 9.86E+04 | 7.54E-05 |
突变1-52 | 9.41E-10 | 2.43E+05 | 2.29E-04 |
突变1-53 | 7.21E-10 | 3.37E+05 | 2.43E-04 |
K D(M) | K on(1/Ms) | K off(1/S) | |
WT | 6.70E-10 | 5.61E+05 | 3.76E-04 |
WT 1-3 | 5.49E-10 | 6.33E+05 | 3.48E-04 |
WT 1-14 | 7.49E-10 | 5.32E+05 | 3.98E-04 |
WT 1-29 | 1.23E-09 | 8.96E+04 | 1.10E-04 |
WT 1-50 | 7.02E-10 | 6.96E+05 | 4.89E-04 |
Claims (18)
- 一种包含抗原结合结构域的分离的结合蛋白,其特征在于,其中所述抗原结合结构域包括选自下述氨基酸序列的至少一个互补决定区:或;与下述氨基酸序列的互补决定区具有至少80%的序列同一性且与心肌肌钙蛋白I具有K D≤1.41×10 -9mol/L的亲和力;互补决定区CDR-VH1为G-F-N-X1-K-X2-Y-X3-M-H,其中,X1是L或I,X2是D或E,X3是F或Y;互补决定区CDR-VH2为R-I-X1-P-E-D-X2-E-T-X3-Y-A-P-E,其中,X1是E或D,X2是A或G,X3是R或K;互补决定区CDR-VH3为Y-Y-X1-S-Y-X2-P-F-V-Y,其中,X1是T或S,X2是I、L或V;互补决定区CDR-VL1为Q-S-X1-X2-Y-S-N-X3-H-T-Y,其中,X1是I或L,X2是I或L,X3是R或K;互补决定区CDR-VL2为Q-X1-S-X2-R-F-S,其中,X1是I、L或V,X2是N或Q;互补决定区CDR-VL3为S-X1-S-T-H-X2-P-X3-T,其中,X1是Q或N,X2是L或I,X3是F或Y;优选的:所述互补决定区CDR-VH1中,X2是D;所述互补决定区CDR-VH2中,X1是D;所述互补决定区CDR-VH3中,X1是S;所述互补决定区CDR-VL1中,X3是R;所述互补决定区CDR-VL2中,X2是N;所述互补决定区CDR-VL3中,X1是Q;优选的,所述互补决定区CDR-VH1中,X1是L;优选的,所述互补决定区CDR-VH1中,X1是I;优选的,所述互补决定区CDR-VH1中,X3是F;优选的,所述互补决定区CDR-VH1中,X3是Y;优选的,所述互补决定区CDR-VH2中,X2是A;优选的,所述互补决定区CDR-VH2中,X2是G;优选的,所述互补决定区CDR-VH2中,X3是R;优选的,所述互补决定区CDR-VH2中,X3是K;优选的,所述互补决定区CDR-VH3中,X2是I;优选的,所述互补决定区CDR-VH3中,X2是L;优选的,所述互补决定区CDR-VH3中,X2是V;优选的,所述互补决定区CDR-VL1中,X1是I;优选的,所述互补决定区CDR-VL1中,X1是L;优选的,所述互补决定区CDR-VL1中,X2是I;优选的,所述互补决定区CDR-VL1中,X2是L;优选的,所述互补决定区CDR-VL2中,X1是I;优选的,所述互补决定区CDR-VL2中,X1是L;优选的,所述互补决定区CDR-VL2中,X1是V;优选的,所述互补决定区CDR-VL3中,X2是L;优选的,所述互补决定区CDR-VL3中,X2是I;优选的,所述互补决定区CDR-VL3中,X3是F;优选的,所述互补决定区CDR-VL3中,X3是Y;优选的,各互补决定区的突变位点选自下述突变组合中的任一种:
- 如权利要求1所述包含抗原结合结构域的分离的结合蛋白,其特征在于,所述结合蛋白中包括至少3个CDRs;或者,所述结合蛋白包括至少6个CDRs;优选的,所述结合蛋白为纳米抗体、F(ab’) 2、Fab’、Fab、Fv、scFv、双特异抗体和抗体最小识别单位中的一种;优选的,所述结合蛋白包括序列依次如SEQ ID NO:1-4所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4,和/或,序列依次如SEQ ID NO:5-8所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4。
- 如权利要求1或2所述包含抗原结合结构域的分离的结合蛋白,所述结合蛋白还包含抗体恒定区序列;优选的,所述恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列;优选的,所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人;优选的,所述恒定区来源于小鼠;轻链恒定区序列如SEQ ID NO:9所示;重链恒定区序列如SEQ ID NO:10所示。
- 一种分离的核酸分子,其特征在于,所述核酸分子为DNA或RNA,其编码权利要求1~3任一项所述的结合蛋白。
- 一种载体,其包含权利要求4所述的核酸分子。
- 一种宿主细胞,其被权利要求5所述的载体转化。
- 一种生产权利要求1~3任一项所述的结合蛋白的方法,其特征在于,包括如下步骤:在培养基中和合适的培养条件下培养权利要求6所述的宿主细胞,从培养基中或从所培养的宿主细胞中回收如此产生的结合蛋白。
- 权利要求1~3任一项所述的结合蛋白在制备用于诊断急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤的诊断剂或试剂盒中的应用。
- 一种检测测试样品中的肌钙蛋白I抗原的方法,其包括:a)在足以发生抗体/抗原结合反应的条件下,使所述测试样品中的肌钙蛋白I抗原与权利要求3所述的结合蛋白接触以形成免疫复合物;和b)检测所述免疫复合物的存在,所述复合物的存在指示所述测试样品中所述肌钙蛋白I抗原的存在;优选的,所述免疫复合物中还包括第二抗体,所述第二抗体与所述结合蛋白结合;优选的,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与所述肌钙蛋白I抗原结合。
- 如权利要求9所述的方法,其中所述肌钙蛋白I抗原为心肌肌钙蛋白I抗原。
- 一种试剂盒,其包括权利要求1~3任一项所述的结合蛋白。
- 如权利要求1~3任一项所述的结合蛋白,在诊断与心肌肌钙蛋白I相关的疾病的应用。
- 一种诊断与心肌肌钙蛋白I相关的疾病的方法,包括:A)在足以发生结合反应的条件下,使来自受试者的样品与权利要求1~3中任一项所述的结合蛋白接触以进行结合反应;以及B)检测结合反应产生的免疫复合物,其中,所述免疫复合物的存在指示与心肌肌钙蛋白I相关的疾病的存在。
- 根据权利要求13所述的方法,其中所述方法基于荧光免疫技术、化学发光技术、胶体金免疫技术、放射免疫分析和/或酶联免疫技术。
- 根据权利要求13或14所述的方法,其中所述样品选自全血,外周血,血清,血浆或心肌组织中的至少一种。
- 根据权利要求13~15中任一项所述的方法,其中所述受试者为哺乳动物,优选地为灵长类 动物,更优选地为人类。
- 根据权利要求12所述的应用或根据权利要求13~16中任一项所述的方法,其中所述与心肌肌钙蛋白I相关的疾病为心血管疾病。
- 根据权利要求12所述的应用或根据权利要求13~16中任一项所述的方法,其中所述与心肌肌钙蛋白I相关的疾病选自由急性心肌梗死、急性冠状动脉综合征、肺梗、不稳定性心绞痛、心肌损伤或其组合组成的组。
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US17/284,234 US20210340231A1 (en) | 2018-10-10 | 2019-09-27 | Recombinant antibody against human cardiac troponin i |
EP19870959.4A EP3865509A4 (en) | 2018-10-10 | 2019-09-27 | RECOMBINANT ANTIBODIES AGAINST HUMAN CARDIAC TROPONIN I |
KR1020217012109A KR20210068067A (ko) | 2018-10-10 | 2019-09-27 | 인간 심장 트로포닌 i에 대한 재조합 항체 |
JP2021515092A JP7299309B2 (ja) | 2018-10-10 | 2019-09-27 | 抗ヒト心筋型トロポニンi組換え抗体 |
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EP4345108A1 (en) * | 2022-09-30 | 2024-04-03 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Cardiac troponin i specific antibody, kit and uses thereof |
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JP2022511323A (ja) | 2022-01-31 |
EP3865509A1 (en) | 2021-08-18 |
US20210340231A1 (en) | 2021-11-04 |
CN111018974A (zh) | 2020-04-17 |
JP7299309B2 (ja) | 2023-06-27 |
KR20210068067A (ko) | 2021-06-08 |
CN111018974B (zh) | 2022-04-01 |
EP3865509A4 (en) | 2022-07-13 |
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