Nano antibody, polypeptide containing nano antibody and application of polypeptide
阅读说明：本技术 一种纳米抗体、包含该纳米抗体的多肽及其应用 (Nano antibody, polypeptide containing nano antibody and application of polypeptide ) 是由 贾凌云 任军 王玉凤 于 2020-04-01 设计创作，主要内容包括：本发明涉及纳米抗体、包含该纳米抗体的多肽及其应用,其中,该纳米抗体的氨基酸序列包含特殊结构的互补决定区和框架区。本发明的纳米抗体,是通过噬菌体库筛选发现的具有新的氨基酸序列的抗β2微球蛋白纳米抗体,该纳米抗体及其多肽具有很高的亲和力和活性,能够特异性地识别并结合β2微球蛋白,基于该纳米抗体的吸附剂对β2微球蛋白的吸附能力极强,可应用于血液净化及β2微球蛋白检测领域,有助于促进和改善对透析相关淀粉样变等疾病的诊断和治疗。(The invention relates to a nano antibody, a polypeptide containing the nano antibody and an application thereof, wherein an amino acid sequence of the nano antibody comprises a complementary determining region and a framework region with a special structure, the nano antibody is an anti- β 2 microglobulin nano antibody with a new amino acid sequence, which is found by screening a phage library, the nano antibody and the polypeptide thereof have high affinity and activity, can specifically recognize and combine β 2 microglobulin, and an adsorbent based on the nano antibody has extremely strong adsorption capacity on β 2 microglobulin, can be applied to the fields of blood purification and β 2 microglobulin detection, and is beneficial to promoting and improving the diagnosis and treatment of diseases such as dialysis-related amyloidosis.)
1. A nanobody, characterized in that its amino acid sequence comprises a CDR and a framework FR,
the complementarity determining region CDR includes complementarity determining region CDR1, complementarity determining region CDR2, and complementarity determining region CDR3, wherein,
the complementarity determining region CDR1 is of formula (I):
Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-L ys-Tyr-Cys formula (I) (SEQ ID No:1),
the complementarity determining region CDR2 is of formula (II):
Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23formula (II) (SEQ ID No:2),
the complementarity determining region CDR3 is of formula (III):
Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe formula (III) (SEQ ID No:3),
wherein the content of the first and second substances,
Xaa11independently selected from Ser, Thr, and,Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa12independently selected from Asn, Gln, Asp, Glu and His; and/or the presence of a gas in the gas,
Xaa13independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa21independently selected from Gly, Ala, Ser, Thr and Pro; and/or the presence of a gas in the gas,
Xaa22independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa23independently selected from Thr, Ser, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa31independently selected from Ile, Val, Met, L eu and Cys.
2. The nanobody according to claim 1,
in the formula (I), Xaa11Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
in the formula (I), Xaa12Independently selected from Asn and Gln; and/or the presence of a gas in the gas,
in the formula (I), Xaa13Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
in the formula (II), Xaa21Independently selected from Gly and Ala; and/or the presence of a gas in the gas,
in the formula (II), Xaa22Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
in the formula (II), Xaa23Independently selected from Thr and Ser; and/or the presence of a gas in the gas,
in the formula (III), Xaa31Independently selected from Ile and Val.
3. Nanobody according to claim 1 or 2,
in the formula (I), Xaa11Independently selected from Ser and Thr, Xaa12Independently selected from Asn and Gln, said Xaa13Independently selected from Ser and Thr; and the combination of (a) and (b),
in the formula (II), Xaa21Independently selected from Gly and Ala, Xaa22Independently selected from Ser and Thr, Xaa23Independently selected from Thr and Ser; and the combination of (a) and (b),
in the formula (III), Xaa31Independently selected from Ile and Val.
4. The nanobody according to claim 1 to 3,
in the formula (I), Xaa11Is Ser, said Xaa12Is Asn, said Xaa13Is Ser;
in the formula (II), Xaa21Is Gly, the Xaa22Is Ser, said Xaa23Is Thr;
in the formula (III), Xaa31Is Ile.
5. The nanobody of claims 1 to 4, wherein the framework region FR comprises a framework region FR1, a framework region FR2, a framework region FR3 and a framework region FR4, or wherein the framework region FR comprises an amino acid sequence having 50% or more homology to the framework region FR1, the framework region FR2, the framework region FR3 and the framework region FR4,
alternatively,
the framework region FR1 is of formula (IV):
Gln-Val-Gln-L eu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-L eu-Arg-L eu-Ser-Cys-Ala-Ile-Ala formula (IV) (SEQ ID No:4)
The framework region FR2 is of formula (V):
Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-L ys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly formula (V)
(SEQ ID No:5)
The framework region FR3 is of formula (VI):
Tyr-Tyr-Ala-Asp-Ser-Val-L ys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-L ys-Asn-Thr-L eu-Tyr-L eu-Gln-Met-Asn-Ser-L eu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys formula (VI) (SEQ ID No:6)
The framework region FR4 is of formula (VII):
Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser of formula (VII) (SEQ ID No:7),
alternatively,
the framework region FR1 is of formula (VIII):
Glu-Val-Gln-L eu-Gln-Glu-Ser-Gly-Gly-Gly-L eu-Val-Gln-Pro-Gly-Gly-Ser-L eu-Arg-L eu-Ser-Cys-Ala-Ile-Ala formula (VIII) (SEQ ID No:8)
The framework region FR2 is of formula (IX):
Met-Ser-Trp-Val-Arg-Gln-Ala-Pro-Gly-L ys-Gly-L eu-Glu-Trp-Val-Ser-Arg-Gly formula (IX) (SEQ ID NO:9)
The framework region FR3 is of the following formula (X):
Tyr-Tyr-Ala-Asp-Ser-Val-L ys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-L ys-Asn-Thr-L eu-Tyr-L eu-Gln-Met-Asn-Ser-L eu-Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys formula (X) (SEQ ID No:10)
Said framework region FR4 is of formula (XI):
Trp-Gly-Gln-Gly-Thr-L eu-Val-Thr-Val-Ser-Ser of formula (XI) (SEQ ID No: 11).
6. The nanobody according to claim 1 to 5, wherein the amino acid sequence of the nanobody is as follows:
Gln-Val-Gln-Leu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ile-Ala-Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-Lys-Tyr-Cys-Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-Lys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly-Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys-Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe-Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser formula (XII) (SEQ ID No:12),
xaa is11Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa is12Independently selected from Asn and Gln; and/or the presence of a gas in the gas,
xaa is13Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa is21Independently selected from Gly and Ala; and/or the presence of a gas in the gas,
xaa is22Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa is23Independently selected from Thr and Ser; and/or the presence of a gas in the gas,
xaa is31Independently selected from Ile and Val.
7. The nanobody of claim 6,
in the formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Thr, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Ala, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Val; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII)，Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Ser, Xaa31Is Ile.
8. A polypeptide consisting essentially of the nanobody of any one of claims 1 to 7,
optionally, the polypeptide is a multivalent polypeptide;
optionally, the multivalent polypeptide is a bivalent or trivalent polypeptide;
optionally, the divalent or trivalent polypeptide is a specific polypeptide;
alternatively, the amino acid sequence of the bivalent polypeptide is SEQ ID No. 14 or SEQ ID No. 15;
alternatively, the amino acid sequence of the specific polypeptide is SEQ ID No. 16 or SEQ ID No. 17.
9. A nucleic acid encoding the nanobody of any one of claims 1 to 7 and/or the polypeptide of claim 8.
10. A host or host cell capable of expressing a nanobody according to claims 1 to 7 and/or a nucleic acid according to claim 9.
11. An adsorbent comprising a carrier matrix and the nanobody of any one of claims 1 to 7 or the polypeptide of claim 8.
12. Use of the nanobody of any one of claims 1 to 7 and/or the polypeptide of claim 8 for the preparation of β 2 microglobulin adsorbent.
13. Use of the nanobody of claims 1 to 7 and/or the polypeptide of claim 8 for immunodetection, enrichment and/or purification and the like.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nanobody capable of specifically recognizing and binding β 2 microglobulin, and a polypeptide comprising one or more nanobodies, nucleic acid encoding the amino acid sequence and the polypeptide, a host or a host cell expressing or capable of expressing the amino acid sequence and the polypeptide, and application of the amino acid sequence and the polypeptide, particularly application of the amino acid sequence and the polypeptide on β 2 microglobulin adsorbents for prevention, treatment or diagnosis.
Background
β 2 microglobulin (β 2-microrogobulin, β 2M) is the light chain part of human lymphocyte antigen (H L A) present on the surface of nucleated cells, has a molecular weight of 11.8kDa, is separated from the heavy chain during metabolism, and is widely present in blood, urine, cerebrospinal fluid and saliva. β 2 microglobulin serum mass concentration in normal human is relatively stable, generally 1.5-3 mg/L (see non-patent document 1). β 2 microglobulin metabolism is dependent only on kidney, and can reach 20-50 mg/L and even 100 mg/L in the serum of patients with end-stage renal disease.
The pathogenesis of β 2 microglobulin is not clear, but high concentration of β 2 microglobulin is necessary condition for causing amyloidosis, so that the removal of excessive β 2 microglobulin by blood purification is of great significance for preventing diseases such as dialysis-related amyloidosis and the like.
The main two treatment methods for dialysis-related amyloidosis are to remove the pain by cutting damaged tissues through surgical operations such as carpal tunnel laminectomy and cervical vertebra laminectomy, and to directly adsorb and remove β 2 microglobulin in the blood of a patient by adopting a blood purification mode.
The blood purification method for removing β 2 microglobulin commonly used in clinic includes dialysis and whole blood perfusion, the common hemodialysis method has very limited removal amount of β 2 microglobulin, even can cause the level of β 2 microglobulin in a patient to be increased (see non-patent document 2), the blood perfusion is to remove β 2 microglobulin in blood by leading the blood of the patient out of the body and passing the blood through a blood perfusion device filled with an adsorbent, at present, hydrophobic ligands are used for β 2 microglobulin adsorbents which are commercialized (see non-patent document 3), and the hydrophobic ligand adsorbents often cause the loss of other hydrophobic micromolecules in the blood and have certain side effects on the patient.
The affinity adsorbent using the specific antibody as the ligand can avoid the defects, and has good treatment effect and application prospect in the field of blood perfusion. The specific antibody may be IgG (see non-patent document 4), but the production cost of monoclonal antibodies is high and is limited to laboratory studies. Smaller antibody fragments, single chain variable regions (ScFv), have also been used for affinity ligands (see non-patent document 5), however, ScFv have exposed surface hydrophobic groups, have disadvantages of increased non-specific adsorption, susceptibility to coagulation and adhesion, and are in research phase, and no commercial products have been available.
A natural Heavy chain antibody (HCAb) with a deleted light chain exists in a camel body, and the variable region of the Heavy chain antibody is cloned to obtain a minimum antigen binding fragment, namely a Heavy chain variable region (V)HH) Also known as nanobodies. The great advantage of the nanobody is that it is small, has a molecular weight of only 15kDa, about one tenth of that of the conventional antibody (150kDa), and is much smaller than the single-chain antibody (55 kDa). The smaller molecular mass is beneficial to realizing the high-density directional immobilization of the nano antibody on the surface of the matrix and improving the adsorption performance of the blood purification adsorbent.
The university of major graduates applies the immunoadsorbent material based on the nano-antibody to the field of blood purification for the first time, and develops a specific blood purification adsorbent for β 2 microglobulin in renal failure patients (see patent document 1), but the β 2 microglobulin nano-antibody used by the adsorbent has low affinity, so that the adsorption efficiency of β 2 microglobulin is low, and the actual requirements of the patients cannot be met.
Therefore, the β 2 microglobulin adsorbent provided by the invention takes the high-specificity anti-human β 2 microglobulin nano antibody as a ligand, and the application of the nano antibody in the aspect of blood purification treatment of β 2 microglobulin is realized.
Non-patent document 1: timman B, et al, Mass beta 2-microrogobulin progress empirical approach, sensiars in analysis 200922,4,378-380
Non-patent document 2: ameer G, A models for the removal of beta-2-microobulin from hood, Semin Dial 2001.14:103-6.
Non-patent document 3: furuyoshi S, et al, New absorption column (lixulle) analyte beta-2-microzyme for direct perfusion, Ther Apher.1998.2:13-7
Non-patent document 4: mogi M, et al, Selective removal of beta-2-microlobulin from human plasma by high-performance immunological chromatography, Jchromatography B Biomed Sci appl.1989496:194-
Non-patent document 5: ameer G, A novel immunological apparatus for removingbeta-2-microrogobulin from floor hood, Kidney International,200159:1544-
Patent document 1: CN201410342951A
Disclosure of Invention
The present invention is made in view of solving the existing technical problems, and an object of the present invention is to provide a nanobody having an amino acid sequence with a specific structure, a polypeptide having the nanobody, a nucleic acid encoding the nanobody and/or the polypeptide, and a host or a host cell capable of expressing the nanobody and/or the polypeptide, and an application of the nanobody and/or the polypeptide in the preparation of β 2 microglobulin adsorbent.
The 1 st aspect of the present invention relates to a nanobody having an amino acid sequence comprising a CDR and a framework FR,
the complementarity determining region CDR includes complementarity determining region CDR1, complementarity determining region CDR2 and complementarity determining region CDR3, wherein,
the CDR1 is represented by the following formula (I):
Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-L ys-Tyr-Cys formula (I) (SEQ ID No:1),
the CDR2 is represented by the following formula (II):
Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23formula (II) (SEQ ID No:2),
the CDR3 is represented by the following formula (III):
Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe formula (III) (SEQ ID No:3),
wherein the content of the first and second substances,
Xaa11independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa12independently selected from Asn, Gln, Asp, Glu and His; and/or the presence of a gas in the gas,
Xaa13independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa21independently selected from Gly, Ala, Ser, Thr and Pro; and/or the presence of a gas in the gas,
Xaa22independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa23independently selected from Thr, Ser, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa31independently selected from Ile, Val, Met, L eu and Cys.
In some preferred modes, Xaa is defined above in formula (I)11Independently selected from Ser and Thr; and/or, in the above formula (I), Xaa above12Independently selected from Asn and Gln; and/or, in the above formula (I), Xaa above13Independently selected from Ser and Thr; and/or, in the above formula (II), Xaa above21Independently selected from Gly and Ala; and/or, in the above formula (II), Xaa above22Independently selected from Ser and Thr; and/or, in the above formula (II), Xaa above23Independently selected from Thr and Ser; and/or, in the above formula (III), Xaa above31Independently selected from Ile and Val.
In some preferred modes, Xaa is defined above in formula (I)11Independently selected from Ser and Thr, Xaa as defined above12Independently selected from Asn and Gln, Xaa above13Independently selected from Ser and Thr; and, in the above formula (II), the aboveXaa21Independently selected from Gly and Ala, Xaa above22Independently selected from Ser and Thr, Xaa as defined above23Independently selected from Thr and Ser; and, in the above formula (III), Xaa above31Independently selected from Ile and Val.
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa described above12Asn, Xaa above13Is Ser; in the above formula (II), Xaa is as defined above21Is Gly, Xaa above22Is Ser, Xaa described above23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile.
For some preferred modes, the framework region FR comprises a framework region FR1, a framework region FR2, a framework region FR3 and a framework region FR4, wherein the framework region FR1 is the following formula (IV):
Gln-Val-Gln-L eu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-L eu-Arg-L eu-Ser-Cys-Ala-Ile-Ala formula (IV) (SEQ ID No:4)
The framework region FR2 is represented by the following formula (V):
Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-L ys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly formula (V) (SEQ ID No:5)
The framework region FR3 is represented by the following formula (VI):
Tyr-Tyr-Ala-Asp-Ser-Val-L ys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-L ys-Asn-Thr-L eu-Tyr-L eu-Gln-Met-Asn-Ser-L eu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys formula (VI) (SEQ ID No:6)
The framework region FR4 is represented by the following formula (VII):
Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser of formula (VII) (SEQ ID No:7), or,
the framework region FR1 is represented by the following formula (VIII):
Glu-Val-Gln-L eu-Gln-Glu-Ser-Gly-Gly-Gly-L eu-Val-Gln-Pro-Gly-Gly-Ser-L eu-Arg-L eu-Ser-Cys-Ala-Ile-Ala formula (VIII) (SEQ ID No:8)
The framework region FR2 is represented by the following formula (IX):
Met-Ser-Trp-Val-Arg-Gln-Ala-Pro-Gly-L ys-Gly-L eu-Glu-Trp-Val-Ser-Arg-Gly formula (IX) (SEQ ID No:9)
The framework region FR3 is represented by the following formula (X):
Tyr-Tyr-Ala-Asp-Ser-Val-L ys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-Asp-Asn-Ser-L ys-Asn-Thr-L eu-Tyr-L eu-Gln-Met-Asn-Ser-L eu-Arg-Ala-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys formula (X) (SEQ ID No:10)
The framework region FR4 is represented by the following formula (XI):
Trp-Gly-Gln-Gly-Thr-L eu-Val-Thr-Val-Ser-Ser of formula (XI) (SEQ ID No: 11).
For some preferred modes, the amino acid sequence of the nanobody is as follows:
Gln-Val-Gln-Leu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ile-Ala-Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-Lys-Tyr-Cys-Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-Lys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly-Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys-Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe-Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser formula (XII) (SEQ ID No:12),
xaa is11Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above12Independently selected from Asn and Gln; and/or the presence of a gas in the gas,
xaa as defined above13Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above21Independently selected from Gly and Ala; and/or the presence of a gas in the gas,
xaa as defined above22Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above23Independently selected from Thr and Ser; and/or the presence of a gas in the gas,
xaa as defined above31Independently selected from Ile and Val.
Specific examples of the amino acid sequence include the following sequences:
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Ala, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Val; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Ser, Xaa31Is Ile.
The 2 nd aspect of the present invention provides a polypeptide consisting essentially of the nanobody of any one of the above 1 to 7,
optionally, the polypeptide is a multivalent polypeptide;
optionally, the multivalent polypeptide is a bivalent or trivalent polypeptide;
alternatively, the divalent or trivalent polypeptide is a specific polypeptide;
alternatively, the amino acid sequence of the above divalent polypeptide is SEQ ID No. 14 or SEQ ID No. 15;
alternatively, the amino acid sequence of the specific polypeptide is SEQ ID No. 16 or SEQ ID No. 17;
the 3 rd aspect of the present invention provides a nucleic acid encoding the nanobody of the above or the polypeptide of the above.
In the 4 th aspect of the present invention, a host or host cell capable of expressing the nanobody described above and/or the polypeptide described above is provided.
In the 5 th aspect of the present invention, there is provided an adsorbent comprising a carrier matrix and the nanobody described above or the polypeptide described above.
In the 6 th aspect of the invention, the invention provides an application of the nanobody and/or the polypeptide in preparing β 2 microglobulin adsorbent.
In the 7 th aspect of the present invention, there is provided an application of the nanobody and/or the polypeptide in immunodetection, enrichment and/or purification, etc.
Advantageous effects
The nano antibody is an anti- β 2 microglobulin nano antibody with a new amino acid sequence discovered by screening of a phage library, so that the nano antibody and polypeptide thereof have high affinity and activity, can specifically recognize and combine β 2 microglobulin, have strong adsorption capacity of an adsorbent to β 2 microglobulin, can be applied to the fields of blood purification and β 2 microglobulin detection, and are helpful for promoting and improving diagnosis and treatment of diseases such as dialysis-related amyloidosis.
Drawings
Fig. 1 is purified nanobody CNb 1;
FIG. 2 is the purified bivalent polypeptides bvCNb1, hbv CNb1, bsCNb1 and bfCNb 1;
fig. 3 is a nanobody CNb1 kinetic sensorgram;
fig. 4 is a kinetic sensorgram of nanobodies (CNb111, CNb121, CNb131, CNb112, CNb122, CNb113, CNb123) of the present invention;
figure 5 is a kinetic sensorgram of humanized nanobody hCNb 1.
Detailed Description
The above and other aspects of the invention will be apparent from the further description below, in which:
(1) unless otherwise indicated or defined, all terms used have the ordinary meaning in the art and are well known to those skilled in the art, see, for example, standard manuals, such as Sambrook Molecular Cloning: A L laboratory Manual, second edition, volumes 1-3, Cold Spring Harbor laboratory Press, 1989, F.Ausubel Current Protocols in Molecular Biology, Green Publishing and Wiley Interscience, 1987, Roitt Immunolology, Immunology, sixth edition, Mosby/Elisevier, 2001, and the general background art cited above.
(2) Unless otherwise indicated, the term "sequence" as used herein (as in analogous to "antibody sequence", "variable region sequence", "V)HHSequence "or" protein sequence "terms) should generally be understood to include related amino acid sequences and nucleic acid sequences or nucleotide sequences encoding the amino acid sequences, unless the context requires a narrower interpretation.
(3) Unless otherwise indicated, all methods, steps, techniques and operations not specifically described are known and well known to those of skill in the art. For example, the reference is still made to the general background cited above and to other references cited therein.
(4) Amino acid residues are shown according to the standard three-or one-letter amino acid code.
(5) The term "specificity" refers to the different types of antigens or antigenic determinants to which a particular antigen-binding molecule (e.g., nanobody or polypeptide of the invention) may bindThe ability to cluster. The specificity of an antigen binding molecule may be determined by its affinity and/or activity. Affinity is expressed as the dissociation equilibrium constant (K) of the antigen and antigen binding moleculeD) Is a measure of the strength of binding between the antigen and the antigen-binding molecule, KDThe smaller the value, the stronger the binding strength between the antigen and the antigen binding molecule, and conversely, KDThe larger the value, the weaker the binding strength between the antigen and the antigen binding molecule. KaDenotes the binding constant, KaLarger, indicating faster binding, KaSmaller indicates slower binding; kdDenotes the dissociation constant, KdThe larger, the faster the dissociation, KdThe smaller, the slower the dissociation; and KD＝Kd/Ka。
(6) Amino acid residues of single domain antibodies are according to Kabat et al "Sequence of proteins of immunological interest" (Sequence of proteins of immunological interest), US Public Health Services (US Public Health service), Publication No.91]"given in relation to VHThe generic numbering of domains is numbering, which is used in the reechmann and muydermans article for V from camelidaeHHA domain. According to this numbering scheme, FR1 of the single domain antibody comprises amino acid residues at positions 1-30, CDR1 of the single domain antibody comprises amino acid residues at positions 31-36, FR2 of the single domain antibody comprises amino acid residues at positions 37-49, CDR2 of the single domain antibody comprises amino acid residues at positions 50-65, FR3 of the single domain antibody comprises amino acid residues at positions 66-94, CDR3 of the single domain antibody comprises amino acid residues at positions 95-102, and FR4 of the single domain antibody comprises amino acid residues at positions 103-113. In this respect, it should be noted that: as in the art with respect to VHDomains and related to VHHAs is well known for domains-the total number of amino acid residues in each CDR can be different and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means, generally according to KabatIn number, the amino acid residues in the actual sequence may be the same or different from the actual numbering. It can also be said that position 1, according to the Kabat numbering, corresponds to the start of FR1, irrespective of the numbering of the amino acid residues of the CDRs, and vice versa; position 36 by Kabat numbering corresponds to the start of FR2, and vice versa, position 66 by Kabat numbering corresponds to the start of FR 3; and vice versa, position 103 according to Kabat numbering corresponds to the start of FR4, and vice versa.
(7) The term "loading" refers to the total amount of ligand coupled per unit volume of affinity media (adsorbent).
The amino acid sequence of the nanobody of the present invention substantially includes complementarity determining regions CDR and framework regions FR.
The CDR includes a CDRs 1, 2 and 3, and the FR includes a framework region FR1, a framework region FR2, a framework region FR3 and a framework region FR 4.
The CDR1 is represented by the following formula (I):
Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-L ys-Tyr-Cys formula (I) (SEQ ID No:1), the complementarity determining region CDR2 is of the following formula (II):
Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23formula (II) (SEQ ID No:2),
the CDR3 is represented by the following formula (III):
Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe formula (III) (SEQ ID No:3),
wherein the content of the first and second substances,
Xaa11independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa12independently selected from Asn, Gln, Asp, Glu and His; and/or the presence of a gas in the gas,
Xaa13independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa21independently selected from Gly, Ala, Ser, Thr and Pro; and/or the presence of a gas in the gas,
Xaa22independently selected from Ser, Thr, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa23independently selected from Thr, Ser, Ala, Pro and Gly; and/or the presence of a gas in the gas,
Xaa31independently selected from Ile, Val, Met, L eu and Cys.
Among them, amino acid substitutions may be generally described as amino acid substitutions in which an amino acid residue may be substituted with an amino acid having a similar chemical structure, or with an amino acid having a dissimilar chemical structure, so long as there is little or no effect on the function, activity, or other biological properties of the polypeptide. Preferably, the amino acid residue may be substituted with an amino acid having a similar chemical structure.
As the above substitution pattern, for example, the cases disclosed in documents WO04/037999, GB2357768A, WO 98/49185, WO 00/46383 and WO 01/09300 can be cited, but not limited thereto, and further, selection of the (preferred) type and/or combination of the substitution based on the relevant information of other references cited in WO04/037999 and WO 98/49185 can be cited.
The amino acid substitution of the present invention includes, but is not limited to, substitution in which one amino acid in the following groups (a) to (e) is substituted with another amino acid in the same group, (a) Ala, Ser, Thr, Pro and Gly, (b) Asp, Asn, Glu and Gln, (c) His, L ys and Arg, (d) Met, L eu, Ile, Val and Cys, and (e) Phe, Tyr and Trp.
Preferred amino acid substitutions are exemplified by, but not limited to, Ala substituted with Gly or Ser, Arg substituted with L ys, Asn substituted with Gln or His, Asp substituted with Glu, Cys substituted with Ser or Thr, Gln substituted with Asn, Glu substituted with Asp, Gly substituted with Ala or Pro, His substituted with Asn or Gln, Ile substituted with L eu or Val, L eu substituted with Ile or Val, L ys substituted with Arg, Glu or Gln, Met substituted with L eu, Tyr or Ile, Phe substituted with Met, Tyr or L eu, Ser substituted with Thr, Thr substituted with Ser, Tyr substituted with Trp, Trp substituted with Tyr.
In some preferred modes, Xaa is defined above in formula (I)11Independently selected from Ser and Thr; and/or, in the above formula (I), Xaa above12Independently selected from Asn and Gln; and/or, in the above formula (I), Xaa above13Independently selected from Ser and Thr; and/or, in the above formula (II), Xaa above21Independently selected from Gly and Ala; and/or, in the above formula (II), Xaa above22Independently selected from Ser and Thr; and/or, in the above formula (II), Xaa above23Independently selected from Thr and Ser; and/or, in the formula (III), Xaa above31Independently selected from Ile and Val.
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa12Is Asn, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Gly, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 1")
In some preferred modes, Xaa is defined above in formula (I)11Is Thr, Xaa12Is Asn, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Gly, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 111")
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa12Is Asn, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Ala, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 121")
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa12Is Asn, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Gly, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Val. (denoted as "CNb 131")
In some preferred modes, Xaa is defined above in formula (I)11Is Thr, Xaa12Is Gln, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Gly, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 112")
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa12Is Asn, Xaa13Is Thr; in the above formula (II), Xaa is as defined above21Is Ala, Xaa22Is Thr, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 122")
In some preferred modes, Xaa is defined above in formula (I)11Is Thr, Xaa12Is Gln, Xaa13Is Thr; in the above formula (II), Xaa is as defined above21Is Gly, Xaa22Is Ser, Xaa23Is Thr; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 113")
In some preferred modes, Xaa is defined above in formula (I)11Is Ser, Xaa12Is Asn, Xaa13Is Ser; in the above formula (II), Xaa is as defined above21Is Ala, Xaa22Is Thr, Xaa23Is Ser; in the above formula (III), Xaa is as defined above31Is Ile. (denoted as "CNb 123")
Furthermore, in some preferred embodiments, Xaa is defined as11Is Thr, Xaa12Is Asp, Xaa13Is Thr; in the above formula (II), Xaa is as defined above21Is Ala, Xaa22Is Thr, Xaa23Is Ala; in the above formula (III), Xaa is as defined above31Is Met.
Furthermore, in some preferred embodiments, Xaa is defined as11Is Ala, Xaa12Is Asp, Xaa13Is Pro; in the above formula (II), Xaa is as defined above21Is Ala, Xaa22Is Thr, Xaa23Is Ala; in the above formula (III), Xaa is as defined above31Is Met.
Furthermore, in some preferred embodiments, Xaa is defined as11Is Ala, Xaa12Is Asp, Xaa13Is Pro; in the above formula (II), the aboveXaa21Is Ala, Xaa22Is Pro, Xaa23Is Gly; in the above formula (III), Xaa is as defined above31Is Met.
Furthermore, in some preferred embodiments, Xaa is defined as11Is Ser, Xaa12Is Asn, Xaa13Is Pro; in the above formula (II), Xaa is as defined above21Is Pro, Xaa22Is Gly, Xaa23Is Gly; in the above formula (III), Xaa is as defined above31Cys is used.
Furthermore, in some preferred embodiments, Xaa is defined as11Is Ser, Xaa12Is Asn, Xaa13Is Pro; in the above formula (II), Xaa is as defined above21Is Pro, Xaa22Is Gly, Xaa23Is Gly; in the above formula (III), Xaa is as defined above31Cys is used.
Furthermore, in some preferred embodiments, Xaa is defined as11Is Gly, Xaa12Is Asp, Xaa13Is Pro; in the above formula (II), Xaa is as defined above21Is Thr, Xaa22Is Ser, Xaa23Is Ser; in the above formula (III), Xaa is as defined above31Is L eu.
The amino acid sequence and structure of the nanobody include a framework region in addition to 3 complementarity determining regions (CDR1 to CDR 3).
For the framework regions, they are more conserved than the complementarity determining regions. Those skilled in the art can reasonably screen the sequence structure of the framework region according to the actual use and function of the nanobody. The amino acid sequence of the framework region is preferably an amino acid sequence having a homology of 50% or more, more preferably an amino acid sequence having a homology of 70% or more, and even more preferably an amino acid sequence having a homology of 95% or more.
As the framework region, there may be exemplified framework region FR1, framework region FR2, framework region FR3 and framework region FR4, but it is considered that it is not limited thereto.
Specific examples of the above-mentioned framework regions include the following:
the framework region FR1 is represented by the following formula (IV):
Gln-Val-Gln-L eu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-L eu-Arg-L eu-Ser-Cys-Ala-Ile-Ala formula (IV) (SEQ ID No:4)
The framework region FR2 is represented by the following formula (V):
Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-L ys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly formula (V) (SEQ ID No:5)
The framework region FR3 is represented by the following formula (VI):
Tyr-Tyr-Ala-Asp-Ser-Val-L ys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-L ys-Asn-Thr-L eu-Tyr-L eu-Gln-Met-Asn-Ser-L eu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys formula (VI) (SEQ ID No:6)
The framework region FR4 is represented by the following formula (VII):
Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser formula (VII) (SEQ ID No:7)
The framework region may be composed of other sequences in addition to the above-mentioned examples of framework regions, and may be, for example, an amino acid sequence having a homology of 50% or more, preferably 70% or more, and more preferably 95% or more, with the above-mentioned FR1 to FR4 sequences.
The amino acid sequence of the nanobody includes the following:
Gln-Val-Gln-Leu-Gln-Glu-Ser-Gly-Gly-Gly-Ser-Val-Gln-Ala-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ile-Ala-Ser-Gly-Xaa11-Gly-Phe-Ser-Xaa12-Xaa13-Lys-Tyr-Cys-Met-Ser-Trp-Phe-Arg-Gln-Ala-Pro-Gly-Lys-Glu-Arg-Glu-Trp-Val-Ser-Arg-Gly-Ile-Asn-Gly-Xaa21-Xaa22-Lys-Asp-Ile-Xaa23-Tyr-Tyr-Ala-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Phe-Ser-Gln-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-Ser-Leu-Glu-Pro-Glu-Asp-Thr-Ala-Thr-Tyr-Tyr-Cys-Ala-Thr-Asn-Xaa31-Pro-Val-Arg-Cys-Arg-Asp-Ile-Val-Ala-L ys-Gly-Ser-Gly-Ser-Asp-Gly-Tyr-Arg-Phe-Trp-Gly-Gln-Gly-Thr-Gln-Val-Thr-Val-Ser-Ser formula (XII) (SEQ ID No:8),
xaa as defined above11Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above12Independently selected from Asn and Gln;and/or the presence of a gas in the gas,
xaa as defined above13Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above21Independently selected from Gly and Ala; and/or the presence of a gas in the gas,
xaa as defined above22Independently selected from Ser and Thr; and/or the presence of a gas in the gas,
xaa as defined above23Independently selected from Thr and Ser; and/or the presence of a gas in the gas,
xaa as defined above31Independently selected from Ile and Val.
Specific examples of the amino acid sequence of the nanobody are as follows:
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile (SEQ ID No: 18); alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Ala, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Val; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Ser, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Gln, Xaa13Is Thr, Xaa21Is Gly, Xaa22Is Ser, Xaa23Is Thr, Xaa31Is Ile; alternatively, the first and second electrodes may be,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Ser, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Ser, Xaa31Is the compound of Ile, or,
in the above formula (XII), Xaa11Is Thr, Xaa12Is Asp or Xaa13Is Thr, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Ala, Xaa31Is a Met, or alternatively,
in the above formula (XII), Xaa11Is Ala, Xaa12Is Asp or Xaa13Is Pro, Xaa21Is Ala, Xaa22Is Thr, Xaa23Is Ala, Xaa31Is a Met, or alternatively,
in the above formula (XII), Xaa11Is Ala, Xaa12Is Asp or Xaa13Is Pro, Xaa21Is Ala, Xaa22Is Pro, Xaa23Is Gly, Xaa31Is a Met, or alternatively,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Pro, Xaa21Is Pro, Xaa22Is Gly, Xaa23Is Gly, Xaa31Is a Cys, or alternatively,
in the above formula (XII), Xaa11Is Ser, Xaa12Is Asn, Xaa13Is Pro, Xaa21Is Pro, Xaa22Is Gly, Xaa23Is Gly, Xaa31Is a Cys, or alternatively,
in the above formula (XII), Xaa11Is Gly, Xaa12Is Asp or Xaa13Is Pro, Xaa21Is Thr, Xaa22Is Ser, Xaa23Is Ser, Xaa31L eu, etc., but are not limited to the examples given above, as long as there is little or substantially no effect on the function, activity or other biological properties of the polypeptide.
In addition, the total number of residues of the nanobody may be in the range of 110-120, preferably 112-115, and most preferably 113. However, the portions, fragments or analogs of nanobodies are not particularly limited in their length and/or size, as long as such portions, fragments or analogs meet the further requirements set forth below and are also suitable for the purposes described herein.
The method for producing the "nanobody" is not limited to a specific biological resource or a specific production method in its broadest sense. For example, nanobodies of the present invention may be obtained by: (1) by isolating the V of naturally occurring heavy chain antibodiesHHA domain; (2) encoding naturally occurring V by expressionHHA nucleotide sequence of a domain; (3) by converting naturally occurring VHHDomains are "humanized" (as described below) or encode such humanized V by expressionHHA nucleic acid of a domain; (4) the use of synthetic or semi-synthetic techniques to prepare protein, polypeptide or other amino acid sequences; (5) preparing a nucleic acid encoding a nanobody by applying a nucleic acid synthesis technique, and then expressing the thus-obtained nucleic acid; and/or (6) by any combination of the foregoing.
In addition, a variant based on the nanobody of the present invention also includes a nanobody with naturally occurring VHHNanobodies with amino acid sequences corresponding to the domains but which have been humanized. Humanization i.e.V of conventional 4-chain antibodies from humansHSubstitution of said naturally occurring V by one or more amino acid residues present at corresponding positions in the domainHHOne or more amino acid residues of the domain sequence.
Specific examples of the humanized amino acid sequence nanobody include, but are not limited to, SEQ ID No. 13 (designated as "hCNb 1").
Furthermore, the invention relates to a composition comprising at least one VHHA domain or at least one protein or polypeptide based thereon.
According to a non-limiting embodiment of the invention, the above-mentioned polypeptide consists essentially of nanobodies. "consisting essentially of … …" means that the amino acid sequence of the polypeptide of the invention is identical to or corresponds to the amino acid sequence of a nanobody, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino terminus (N-terminus) and/or the carboxy terminus (C-terminus) of said nanobody or polypeptide.
The amino acid residues described above may not alter the biological properties of the nanobody, and other functionalities may be added to the nanobody. For example, the amino acid residues may:
a tag formation, i.e. an amino acid sequence or residue that facilitates purification of the nanobody, e.g. using affinity techniques for said sequence or residue. Some preferred but non-limiting examples of such residues are poly-histidine residues (His-tag) and glutathione residues;
b is an N-terminal Met residue, e.g., whereby expression in a heterologous host cell or host organism is possible;
c is a C-terminal Cys residue, e.g., whereby it can react with-SH on a ligand or with an Au surface;
d is one or more amino acid residues which may be provided with functional groups and/or have been functionalized in a known manner, e.g. amino acid residues such as lysine or cysteine allow attachment of PEG groups as is known in the art.
The polypeptides of the invention may also comprise 2 or more of said nanobodies, also referred to as multivalent polypeptides.
The bivalent polypeptide of the invention comprises 2 nanobodies, optionally linked by one hinge sequence, and the trivalent polypeptide of the invention comprises 3 nanobodies, optionally linked by two hinge sequences.
In the multivalent polypeptide of the present invention, the 2 or more nanobodies may be the same or different. For example, 2 or more nanobodies in a multivalent polypeptide of the invention: may be directed against the same antigen, i.e. against the same epitope of said antigen or against 2 or more different epitopes of said antigen; may be directed against different antigens; or a combination thereof.
For example, a bivalent polypeptide of the invention may comprise 2 identical nanobodies; a first nanobody directed to an epitope of a first antigen and a second nanobody directed to the same epitope or different epitopes of the antigen may be included; a first nanobody directed to a first antigen and a second nanobody directed to a second antigen different from the first antigen may be included. Specific examples of the amino acid sequence of the bivalent polypeptide include, but are not limited to, SEQ ID No. 14 (denoted as "bv CNb 1"). Further, as a specific example of the humanized divalent polypeptide, SEQ ID No. 15 (denoted as "hbvCNb 1") can be cited, but the humanized divalent polypeptide is not limited thereto.
For example, a trivalent polypeptide of the invention may comprise 3 identical nanobodies; can include the same or different nanobodies against the same antigen; 2 identical or different nanobodies that may include identical or different epitopes for a first antigen and a third nanobody that is directed to a second antigen different from the first antigen; a first nanobody directed to a first antigen, a second nanobody directed to a second antigen different from the first antigen, and a third nanobody directed to a third antigen different from the first and second antigens may be included.
The polypeptide of the invention comprises at least 2 nanobodies, of which at least 1 is directed against a first antigen and at least 1 is directed against a second nanobody different from said first antigen (or against a different epitope of the first antigen), also called "multispecific" antibody. Thus, a bispecific antibody is a monoclonal antibody comprising at least 1 nanobody against a first antigen and at least 1 other nanobody against a second antigen, while a trispecific antibody is a monoclonal antibody comprising at least 1 nanobody against a first antigen, at least 1 other nanobody against a second antigen, and at least 1 other nanobody against a third antigen; and so on.
As specific examples of the amino acid sequence of the specific polypeptide, there can be cited bispecific polypeptides composed of a single domain antibody having the amino acid sequence of SEQ ID NO. 18 and another single domain antibody against a different epitope of β 2 microglobulin (see CN104098694), which have a higher affinity for β 2 microglobulin than the single domain antibodies, such as SEQ ID No. 16 (denoted as "bs CNb 1"), SEQ ID No. 17 (denoted as "bf CNb 1"), but not limited thereto.
With respect to the inclusion of one or more VHHMultivalent and multispecific polypeptides of domains and their preparation can be referred to the description in EP 0822985.
Hinges for multivalent and multispecific polypeptides should be well known to those skilled in the art, e.g., include Gly-Ser, as described in WO 99/42077 (Gly)4Ser)3Or (Gly)3Ser2)3(ii) a Or a naturally occurring heavy chain antibody hinge region or a partial region thereof. For other suitable hinges, reference is also made to the general background cited above.
Furthermore, in addition to the 1 or more nanobodies, the polypeptide of the present invention may comprise functional groups, moieties or residues, such as therapeutically active substances, and/or labels, such as fluorescein labels, isotope labels, biotin labels, enzyme-catalyzed labels, and the like.
In addition, dissociation equilibrium constant (K) of the binding of the nanobody or polypeptide of the present invention to β 2 microglobulinD) Is 10-5～10-12Mol/liter (M), preferably 10-7～10-12Mol/liter (M), more preferably 10-7～10-10Mol/liter (M), more preferably 10-8～10-10Moles/liter (M).
Specific binding between the above antigen and the antigen binding molecule may be determined by any suitable method known, including Scatchard Analysis (Scatchard Analysis) and/or competitive binding assays such as Radioimmunoassay (RIA) and enzyme-linked immunoassay (E L ISA), as well as other novel methods known in the art, such as plasmon resonance (SPR) and/or biofilm interference (B L I) techniques, and the like.
Nanobodies, polypeptides, and nucleic acids encoding the same of the present invention may be prepared in known ways, as will be apparent to those skilled in the art from the further description herein. One particularly useful method for making such nanobodies, polypeptides and nucleic acids generally comprises the steps of:
(1) expressing a nucleic acid encoding said nanobody or polypeptide of the invention, optionally followed by expression, in a suitable host cell or host organism or in another suitable expression system;
(2) isolating and/or purifying the nanobody or polypeptide of the present invention thus obtained.
Alternatively, other methods may be used including the following steps:
(3) culturing and/or maintaining a host of the invention under conditions such that the host of the invention expresses and/or produces a nanobody and/or polypeptide of the invention; optionally followed by;
(4) isolating and/or purifying the nanobody or polypeptide of the present invention thus obtained.
The nucleic acids of the invention may be in the form of single-or double-stranded DNA or RNA, and preferably are in the form of double-stranded DNA. For example, the nucleic acid sequence of the invention may be genomic DNA, cDNA or synthetic DNA (e.g., DNA having a codon usage particularly suited for expression in the host cell or host organism in which it is to be used, i.e., codon optimized).
The nucleic acids of the invention may be prepared or obtained in a manner known per se, based on the information given herein on the amino acid sequence of the nanobody or polypeptide of the invention, and/or may be isolated from a suitable natural source. For example, for naturally occurring VHHThe nucleic acid sequence of the domain is subjected to gene site-directed mutagenesis to provide a nucleic acid of the invention encoding the analog.
The nucleic acids of the invention may also be in a form present in and/or part of a genetic construct, as is well known to those skilled in the art. Such genetic constructs typically comprise at least one nucleic acid of the invention, and may be in the form of a vector, such as a plasmid, YAC, viral vector or transposon. In particular, the vector may be an expression vector, i.e., a vector that can provide for expression in vitro and in vivo (e.g., in a suitable host cell, host organism, and/or expression system).
The nucleic acids of the invention and/or the genetic constructs of the invention may be used for transforming a host cell or a host organism, i.e. for expressing and/or producing the nanobody or polypeptide of the invention. Suitable hosts or host cells are well known to those skilled in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or organelle or organism, such as: bacterial strains including, but not limited to, the genera Escherichia coli and Bacillus subtilis; fungal cells including, but not limited to, Trichoderma (Trichoderma), Aspergillus (Aspergillus), or other filamentous fungi; yeast cells, including but not limited to Saccharomyces (Saccharomyces) and Pichia (Pichia); amphibian cells or cell lines, such as Xenopus oocytes (Xenopus oocytes); insect-derived cells or cell lines, such as Spodoptera exigua (Spodoptera) Sf9 and Sf21 cells or Drosophila (Drosophila) cell lines Schneider and Kc cells; plants or plant cells, such as tobacco (tobaco) plants; mammalian cells or cell lines, such as those derived from humans, and/or other mammals, including but not limited to CHO-cells, BHK-cells, HeLa cells, CHS cells, and the like; as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments, including but not limited to single domain antibodies and ScFv fragments, are well known to those skilled in the art.
For production, the nanobodies and polypeptides of the present invention may be produced in the milk of a transgenic mammal, such as a rabbit, cow, goat or sheep, or in a plant or part of a plant, including but not limited to their leaves, flowers, fruits, roots or seeds.
As mentioned above, one advantage of the use of nanobodies is that the polypeptides based thereon may be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, host cells, etc. are well known to those skilled in the art, as in the references cited above. It should be noted, however, that the present invention in its broadest sense is not limited to expression in bacterial systems.
Preferably, in the present invention, the nanobody or polypeptide is produced in a bacterial cell, in particular a bacterial cell suitable for large scale drug production, as described above.
When the nanobody or polypeptide of the present invention is expressed for production in a cell, the nanobody or polypeptide of the present invention may be produced intracellularly (e.g., in the cytoplasmic or periplasmic space) and then isolated from the host cell and optionally further purified; or may be produced extracellularly (i.e., secreted expression) and then isolated from the culture medium, and optionally further purified.
Some preferred but non-limiting vectors for use with these host cells include the vectors for expression in mammalian cells-pMANneo (Clonetech), pUCTtag (ATCC37460) and pMClneo (Stratagene); vectors for expression in bacterial cells-pET vectors (Novagen) and pQE vectors (Qiagen); expression vectors for use in yeast or other fungal cells-pYES 2(Invitrogen) and Pichia vector (Pichaxpression vector) (Invitrogen); expression vectors for use in insect cells-pBlueBac II (Invitrogen) and other baculovirus vectors; and so on.
Corresponding techniques for transforming the host or host cell of the invention are well known to those skilled in the art.
After transformation, one can test and select those hosts that have successfully transformed the nucleotide sequence/genetic construct of the present invention. Transformed host cells (which may be in the form of stable cell lines) or host organisms (which may be in the form of stable mutant lines or strains) form a further aspect of the invention.
Preferably, these host cells or host organisms are such that they express or are capable of expressing (e.g., under suitable conditions) the amino acid sequence of the invention (if in the case of a host organism, in at least one cell, part, tissue or organ thereof). The invention also includes other generations, progeny and/or progeny of the host cells or host organisms of the invention, which may be obtained, for example, by cell division or by sexual or apomictic reproduction.
The amino acid sequence of the invention can then be isolated from the host cell/host organism and/or from the medium in which the host cell or host organism is cultured, by protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoretic techniques, differential precipitation techniques, affinity techniques (e.g. using a specific/cleavable amino acid sequence fused to the amino acid sequence of the invention) and/or preparative immunological techniques (i.e. using antibodies directed against the amino acid sequence to be isolated).
The nanobodies or polypeptides of the invention may specifically bind to the antigen β 2 microglobulin, and thus one preferred but non-limiting application of the invention is β 2 microglobulin adsorbent, which comprises a carrier matrix and the nanobodies or polypeptides.
The carrier matrix may be a porous material, for example, agarose gel microspheres, cellulose spheres, magnetic beads, silica gel microspheres, activated carbon or resin microspheres, or the like.
The carrier for the foregoing adsorbent can be obtained commercially, and as specific examples, there are agarose gel Sepharose C L-6B (GE Healthcare, US), resin microsphere Nanomicro series (Suzhou nano micro technology Co., Ltd.), but not limited thereto.
When the above-mentioned carrier is used, preferably, the above-mentioned carrier may be activated. The activation method can be, for example, but not limited to, the following method: first epoxy activation, second diaminepropylimine (DADPA) activation, and finally iodoacetic acid activation, etc.
The aforementioned adsorbent is obtained by coupling nanobody or polypeptide to activated carrier, and the specific method is not particularly limited, and for example, the purified antibody is reduced to obtain nanobody or polypeptide solution, and then the nanobody or polypeptide is mixed with carrier, and separated by centrifugation, and finally the gel is washed/filtered to obtain the final adsorbent.
The adsorbent of the present invention can be used to specifically recognize β 2 microglobulin.
The nano antibody or polypeptide or adsorbent can be used for preparing β 2 microglobulin removal reagents and can also be used for preparing medical instruments for removing or detecting β 2 microglobulin.
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