CN116731169B - Nano antibody with sortilin 1 specificity and application thereof - Google Patents

Nano antibody with sortilin 1 specificity and application thereof Download PDF

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CN116731169B
CN116731169B CN202310995805.3A CN202310995805A CN116731169B CN 116731169 B CN116731169 B CN 116731169B CN 202310995805 A CN202310995805 A CN 202310995805A CN 116731169 B CN116731169 B CN 116731169B
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sortilin
nanobody
antibody
cdr2
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CN116731169A (en
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郝睿
王仲亚
董凤起
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Renoviron Suzhou Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Abstract

The invention discloses a nano antibody with sortilin 1 specificity and application thereof, and belongs to the technical field of biology. The invention provides a plurality of sortilin 1 antibodies which are obviously differentiated from the prior sortilin 1 monoclonal antibody AL001 and have better clinical application prospect. Experimental results show that the anti-sortilin 1 nanobody obtained by the invention can effectively bind sortilin 1 and simultaneously block the interaction with ligand granule protein precursors thereof. More importantly, the plurality of nano antibodies obtained in the invention can obviously improve the level of the particle protein precursor and has the capability of treating various neurodegenerative diseases.

Description

Nano antibody with sortilin 1 specificity and application thereof
Technical Field
The invention relates to a sortilin 1 specific nano antibody and application thereof, and belongs to the technical field of biology.
Background
Sortilin (Sortilin 1) acts as a member of the vacuolar Sortilin 10 (VPS 10P) domain receptor family, acting as a receptor or co-receptor for cells, mediating targeted transport of diverse proteins into cells. The N-terminus of sortilin 1 is located in the extracellular region and the C-terminus is located in the intracellular region, which are linked by a transmembrane structure. Sortilin 1 has a broad distribution on cell membranes but can also be found in the endoplasmic reticulum, golgi apparatus, early endocytic vesicles, etc. inside cells. Sortilin 1 is widely expressed in a variety of tissues and organs, and particularly in the nervous system, sortilin 1 is expressed at high levels. It is expressed in various areas of the brain, including the cortex, hippocampus, striatum, cerebellum, and so on. Sortilin 1 is also expressed in a number of tissues and organs such as the heart, liver, lung, kidney, intestine and immune system. Sortilin 1 plays an important role in intracellular protein transport, neurodevelopment, metabolic regulation and the like. First, sortilin 1 is involved in the localization and transport of intracellular proteins, which can bind a variety of proteins and ligands, such as nerve growth factors, neurotrophins, glycoproteins, etc., and bring them from the cell surface into the endoplasmic reticulum and transport vesicle system by endocytosis, and participate in their localization and transport processes. Second, sortilin 1 plays an important role in the nervous system, and it is involved in the growth and developmental regulation of neurons, associated with neurodegenerative diseases. Sortilin 1 interacts with multiple signaling pathways in neurons, regulating the processes of cell survival, apoptosis, and differentiation. It is also closely related to nerve functions such as synaptic transmission, synaptic plasticity and memory formation. In addition, sortilin 1 plays an important role in metabolic regulation, and it is involved in cholesterol metabolism and insulin release regulation, regulating endocytosis and transport of cholesterol by binding to low density lipoprotein receptor-related proteins (LRPs); sortilin 1 is involved in the maturation and secretion of insulin particles in islet cells.
Programmulin (granulin precursor, PGRN) is an extracellular secreted protein consisting of 588 amino acids. Its structure comprises a plurality of repeating linear building blocks containing serine and tyrosine residues. The granulin precursors are widely distributed in human bodies, are particularly rich in tissues and cells such as central nervous system, immune system, endocrine system, kidney and the like, and play an important role in the aspects of inflammation regulation, neuroprotection, tumor development and the like. During neuroprotection and neurodevelopment, granulin precursors are able to promote neuronal growth, differentiation and survival, and are involved in synapse formation and neurotransmission regulation. The lack of granulin precursors may lead to an increase in inflammatory response, triggering toxic damage to neurons, interfering with the growth of neurons and the formation of synaptic connections, leading to dysfunction of the neural network. The lack of granulin precursors may also lead to abnormal autophagy processes and protein metabolic disorders, further affecting cellular function.
Sortilin 1 is one of the main degradation pathways of granulin precursors: the extracellular domain of sortilin 1 binds to the granulin precursor, which mediated endocytosis brings the granulin precursor from outside the cell into the cell interior, and sortilin 1 promotes the granulin precursor into the degradation pathway by vesicle and lysosome binding, thereby reducing the concentration of granulin precursor outside the cell. Accordingly, inhibiting the function of sortilin 1 may reduce degradation of granulin precursors, thereby increasing intracellular and extracellular granulin precursor levels. Abnormal changes in the levels of granulin precursors can result when abnormal sortilin 1 function occurs or sortilin 1 expression levels are altered. Abnormal degradation of the granulin precursor can be prevented by inhibiting the function of sortilin 1, thereby increasing the available amount of granulin precursor. Studies have shown that in neurodegenerative diseases such as frontotemporal lobar degeneration (Frontotemporal Dementia, FTD), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), and Parkinson's Disease (PD), decreased levels of granulin precursors are associated with the occurrence and progression of the Disease. Thus, by inhibiting the function of sortilin 1 to increase the level of granulin precursors, it may be helpful to restore the physiological function of granulin precursors, thereby having therapeutic potential for FTD, ALS, PD and related diseases.
The use of antibodies to inhibit sortilin 1 function is a potential strategy to achieve increased levels of progranulin. The antibody, as a specific molecular tool, can specifically bind to sortilin 1 and influence the binding of the granulin precursor to sortilin 1, so as to block the degradation path of the granulin precursor, and the strategy is expected to increase the bioactivity and the functional expression of the granulin precursor, so that the antibody has potential benefits for treating related diseases. Various pharmaceutical companies including Lundbeck, sanofi, takeda and GSK have been put into the development of sortilin 1 antibodies worldwide, wherein the monoclonal antibody Latozinemab (AL 001) developed by american biotechnology company Alector Therapeutics has entered clinical stage three for FTD and another sortilin 1 antibody AL01 by Alector company has entered clinical stage one for Alzheimer's Disease (AD), suggesting that sortilin 1 antibodies have therapeutic potential for a variety of neurodegenerative diseases. Nevertheless, no sortilin 1 antibodies are marketed in bulk worldwide, and thus there is still a need to develop new sortilin 1 antibodies that are significantly different and more suitable for clinical use.
The development and application of the conventional IgG antibody have many problems, such as long development period, high production cost, poor stability, high immunogenicity and the like, and limit the application range of the IgG antibody in clinical development. In 1993 Hamers et al reported that there was a heavy chain antibody in the camelid body which naturally lacks the light and heavy chain constant region 1 (CH 1), and cloning the variable region thereof resulted in a single domain antibody consisting of only one heavy chain variable region, called VHH (variable domain of heavy chain of heavy-chain antibodies), now designated as "nanobody" (Nb). The molecular weight of the nano antibody is about 15kD, and the nano antibody is the smallest antigen binding fragment with complete binding function, and the nano antibody has an elliptic structure with the diameter of 2.5nm and the length of 4nm. Nanobodies can more easily cross tissue barriers due to their small size and good permeability, increasing the local concentration of therapeutic drugs compared to common IgG monoclonal antibodies. Because of the small size and structure of the single variable domains, the risk of nanobodies triggering immune responses in humans is relatively low, reducing immune related problems that occur when nanobodies are used by patients, and increasing their acceptability in long-term treatment. The nanobody has good tolerance to environment, stable conformation and easy synthesis. The unique properties enable the nanobody to have unique application value in aspects of disease diagnosis, targeted therapy and the like.
Disclosure of Invention
In order to solve the problems, the invention provides an anti-sortilin 1 nanobody and application thereof, and aims to provide a plurality of sortilin 1 antibodies which are obviously different from the prior sortilin 1 monoclonal antibody AL001 and have better clinical application prospects. The antibody of the invention has wide biological application value and clinical application value, and the application thereof relates to a plurality of fields of diagnosis and treatment of diseases related to sortilin 1, basic medical research, biological research and the like.
A first object of the present invention is to provide a nanobody specific for sortilin 1, the heavy chain variable region of the nanobody comprising complementarity determining regions CDR1, CDR2, CDR3, the complementarity determining regions comprising any one of the following combinations or sequences having homology of not less than 90%:
1) CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.2, CDR3 shown in SEQ ID No. 3;
2) CDR1 shown in SEQ ID No.8, CDR2 shown in SEQ ID No.9, CDR3 shown in SEQ ID No. 10;
3) CDR1 shown in SEQ ID No.15, CDR2 shown in SEQ ID No.16, CDR3 shown in SEQ ID No. 17;
4) CDR1 shown in SEQ ID No.22, CDR2 shown in SEQ ID No.23, CDR3 shown in SEQ ID No. 24;
5) CDR1 shown in SEQ ID No.29, CDR2 shown in SEQ ID No.30, CDR3 shown in SEQ ID No. 31;
6) CDR1 shown in SEQ ID No.36, CDR2 shown in SEQ ID No.37, CDR3 shown in SEQ ID No. 38;
7) CDR1 shown in SEQ ID No.43, CDR2 shown in SEQ ID No.44, CDR3 shown in SEQ ID No. 45;
8) CDR1 shown in SEQ ID No.50, CDR2 shown in SEQ ID No.51, CDR3 shown in SEQ ID No. 52;
9) CDR1 shown in SEQ ID No.57, CDR2 shown in SEQ ID No.58, CDR3 shown in SEQ ID No. 59.
Further, the invention includes those molecules having a CDR-bearing antibody heavy chain variable region, so long as the CDR has 90% or more (preferably 95% or more, most preferably 98% or more, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the above CDR sequences.
Further, a sequence having a homology of not less than 90% is obtained by amino acid substitutions of CDR1, CDR2 and/or CDR3, said amino acid substitutions being selected from one or more of the following:
1) Alanine is replaced by valine, leucine or isoleucine,
2) Arginine is replaced with lysine, glutamine or asparagine,
3) Asparagine is replaced by glutamine, histidine, lysine or arginine,
4) The aspartic acid is replaced by glutamic acid,
5) The substitution of cysteine with serine is performed,
6) The substitution of glutamine with asparagine is performed,
7) The glutamic acid is replaced by aspartic acid,
8) Glycine is replaced by proline or alanine,
9) Histidine is replaced by asparagine, glutamine, lysine or arginine,
10 Isoleucine is replaced with leucine, valine, methionine, alanine or phenylalanine,
11 Leucine is replaced by isoleucine, valine, methionine, alanine or phenylalanine,
12 Lysine is replaced by arginine, glutamine or asparagine,
13 Methionine is replaced by leucine, phenylalanine or isoleucine,
14 Phenylalanine is replaced by leucine, valine, isoleucine, alanine or tyrosine,
15 A) proline is replaced by alanine or glycine,
16 A) replacement of serine with threonine,
17 Threonine is replaced with serine and the amino acid is replaced with serine,
18 Tryptophan is replaced with tyrosine or phenylalanine,
19 Tyrosine is replaced by tryptophan, phenylalanine, threonine or serine,
20 Valine to isoleucine, leucine, methionine, phenylalanine or alanine.
Further, the variable region of the nanobody comprises framework regions FR1, FR2, FR3, FR4, and complementarity determining regions, CDR1, CDR2, CDR3, are disposed between adjacent framework regions, separated by framework regions FR1, FR2, FR3, FR4. Therefore, the nano antibody is sequentially provided with FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
Further, the frame region comprises one or more of the following combinations:
1) FR1 shown in SEQ ID No.4, FR2 shown in SEQ ID No.5, FR3 shown in SEQ ID No.6, FR4 shown in SEQ ID No. 7;
2) FR1 shown in SEQ ID No.11, FR2 shown in SEQ ID No.12, FR3 shown in SEQ ID No.13, FR4 shown in SEQ ID No. 14;
3) FR1 shown in SEQ ID No.18, FR2 shown in SEQ ID No.19, FR3 shown in SEQ ID No.20, FR4 shown in SEQ ID No. 21;
4) FR1 shown in SEQ ID No.25, FR2 shown in SEQ ID No.26, FR3 shown in SEQ ID No.27, FR4 shown in SEQ ID No. 28;
5) FR1 shown in SEQ ID No.32, FR2 shown in SEQ ID No.33, FR3 shown in SEQ ID No.34, FR4 shown in SEQ ID No. 35;
6) FR1 shown in SEQ ID No.39, FR2 shown in SEQ ID No.40, FR3 shown in SEQ ID No.41, FR4 shown in SEQ ID No. 42;
7) FR1 shown in SEQ ID No.46, FR2 shown in SEQ ID No.47, FR3 shown in SEQ ID No.48, FR4 shown in SEQ ID No. 49;
8) FR1 shown in SEQ ID No.53, FR2 shown in SEQ ID No.54, FR3 shown in SEQ ID No.55, FR4 shown in SEQ ID No. 56;
9) FR1 shown in SEQ ID No.60, FR2 shown in SEQ ID No.61, FR3 shown in SEQ ID No.62 and FR4 shown in SEQ ID No. 63.
Further, the nanobody contains a sequence as shown in any one of SEQ ID NO. 64-72.
It is a second object of the present invention to provide polynucleotide molecules encoding the nanobodies described above.
It is a third object of the present invention to provide an expression vector comprising the polynucleotide molecule described above.
Further, the expression vector is selected from the group consisting of DNA, RNA, viral vectors (e.g., lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof), plasmids, transposons, other gene transfer systems, liposome nanoparticles (LNP, DNA or mRNA encoding sortilin 1 nanobody encapsulated therein), or combinations thereof.
It is a fourth object of the present invention to provide host cells containing the nanobodies described above.
Further, the host cell may be a prokaryotic cell or a eukaryotic cell, such as a plant cell, an animal cell, a microorganism, etc.
It is a fifth object of the present invention to provide monovalent, bivalent or multivalent antibodies comprising the above nanobody.
It is a sixth object of the present invention to provide recombinant proteins or immunoconjugates comprising the nanobodies described above.
Further, the recombinant protein contains
(a) An anti-sortilin 1 nanobody, or a bivalent or multivalent antibody as described above;
(b) Tag sequences that facilitate expression and/or purification.
Preferably, the tag sequence comprises an Fc tag, an HA tag, a 6His tag or the like. The fusion protein formed by the fusion protein and the Fc fragment has the structure from the N end to the C end as shown in the formula Ia or Ib:
A-L-B (Ia)，
B-L-A (Ib)，
wherein A is the anti-sortilin 1 nanobody, B is the Fc fragment of IgG, and L is a non-or flexible linker. Preferably, the flexible linker is a peptide linker, more preferably the peptide linker has 1-20 amino acids. The Fc fragment of IgG comprises the Fc fragment of hot IgG, the Fc fragment of IgG being selected from the group consisting of the Fc fragments of IgG1, igG2, igG3, igG4, or a combination thereof.
Further, the immunoconjugate comprises
(a) An anti-sortilin 1 nanobody, or a recombinant protein, bivalent or multivalent antibody as described above;
(b) A coupling moiety selected from one or more of the following: a detectable label, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof.
Preferably, the coupling moiety is selected from: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)) or any form of nanoparticle.
A seventh object of the present invention is to provide the use of the above nanobody, monovalent antibody, bivalent antibody, multivalent antibody, recombinant protein or immunoconjugate for the preparation of a diagnostic, prophylactic or therapeutic product. Forms of the product include, but are not limited to, medicaments, reagents, assay plates, kits, and the like.
Further, the product is used for detecting sortilin 1 molecular levels or modulating granulin precursor levels, in particular for blocking granulin precursor interactions with sortilin 1. Of course, those skilled in the art will appreciate that nanobodies may be delivered in vivo using a variety of pharmaceutically acceptable carriers, such as strategies employing AAV viral vectors or LNP expression of sortilin 1 nanobodies for endogenous expression of sortilin 1 nanobodies in vivo.
Further, diseases that the medicament may prevent or treat include, but are not limited to: frontotemporal lobar degeneration (Frontotemporal Dementia, FTD), amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS), alzheimer's Disease (AD) and Parkinson's Disease (PD), depression, neurological disorders (neuropsyciatric disorder), vascular dementia, seizures (seizuries), retinal dystrophies (retinal dystrophy), senile maculopathy (age related macular degeneration), glaucoma, traumatic brain injury, aging, wound healing, stroke, arthritis (arthoritis), atherosclerosis, and the like.
Further, the detection includes flow detection, cellular immunofluorescence detection, enzyme-linked immunosorbent assay (ELISA) detection, etc.
The invention has the beneficial effects that:
through extensive and intensive studies, the inventors succeeded in obtaining a plurality of nanobodies against human sortilin 1 through a large number of screens. Specifically, the invention utilizes the humanized sortilin 1 extracellular antigen protein to immunize camels, and obtains a high-quality immune nanobody gene library. And coupling sortilin 1 protein molecules on an ELISA plate, and screening immune nanobody gene libraries (camel heavy chain antibody phage display gene libraries) by using phage display technology on antigens in the form, so as to obtain sortilin 1 specific nanobody genes. The antibody can block the interaction between the cell surface sortilin 1 and the granulin precursor, and has high affinity and strong specificity; can effectively bind sortilin 1 protein, and has similar binding activity with the control antibody AL 001. In addition, the related experimental results show that the plurality of nano antibodies obtained in the invention can obviously improve the level of the granulin precursor in the U251 cell culture medium, and the capability of improving the level of the extracellular granulin precursor is close to or better than that of a control antibody AL001, so that the nano antibodies have the potential capability of treating various neurodegenerative diseases.
Drawings
FIG. 1 shows the SDS-PAGE detection of the recombinant protein of example 1.
FIG. 2 shows the identification of human sortilin 1 binding nanobody in bacterial periplasmic space extract by ELISA after amplification and induced expression of 576 randomly selected monoclonal antibodies in a three-round sortilin 1 positive library.
Figure 3 is a graph showing the results of an ELISA assay for the competitive inhibition of binding of human sortilin 1 to its ligand granule protein precursor by 27 anti-human sortilin 1 nanobodies.
FIGS. 4-8 are results of flow cytometry testing the binding capacity of different antibodies to cell surface sortilin 1.
FIGS. 9-11 show the results of affinity assay experiments for Gator detection of nanobody binding to recombinant human sortilin 1 protein.
FIG. 12 shows the results of an Epitope grouping (Epitope binding) experiment in which a gate detects the binding of a plurality of nanobodies to recombinant human sortilin 1 protein.
FIG. 13 shows ELISA results for the detection of the levels of granulin precursors in the cell culture medium after nanobody treatment of U251 cells.
FIG. 14 shows the results of particle protein precursor level measurements in cell culture after treatment of U251 cells with different concentrations of sortilin 1 antibodies, including nanobodies Nb171, nb184 and AL 001.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The term interpretation referred to in the following examples:
in the present invention, the terms "antibody of the present invention", "anti-sortilin 1 nanobody", "sortilin 1 nanobody" and "sortilin 1 nanobody" have the same meaning, and are used interchangeably, to refer to an antibody that specifically recognizes and binds sortilin 1 (including human sortilin 1).
In the present invention, the term "antibody" or "immunoglobulin" is an iso-tetralin of about 150000 daltons, which consists of two identical light chains (L) and two identical heavy chains (H), having the same structural characteristics. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
In the present invention, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody" (single domain antibody, sdAb, or nanobody) have the same meaning and are used interchangeably, referring to cloning the variable region of the antibody heavy chain, constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed.
In the present invention, "multivalent" refers to a fusion protein comprising a plurality of repeated anti-sortilin 1 nanobody VHH chains, anti-sortilin 1 nanobodies, or anti-sortilin 1 nanobodies.
In the present invention, the term "variable" means that some portion of the variable region in an antibody differs in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions known as complementarity determining regions (complementarity determiningregion, CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are substantially in a beta-folded configuration, joined by three CDRs forming a linker loop, which in some cases may form part of a beta-folded structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Pub1.No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
In the present invention, immunoconjugates and fusion expression products include: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the anti-TIGIT nanobodies or fragments thereof.
In the present invention, the term "heavy chain variable region" is used interchangeably with "VH".
In the present invention, the term "variable region" is used interchangeably with "complementarity determining region".
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to a polypeptide that specifically binds sortilin 1, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragments, derivatives or analogues of the invention may be (i) a polypeptide having one or more, preferably conservative amino acid residues, substituted or non-conservative amino acid residues, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or a polypeptide, such as a polypeptide that extends the half-life, for example a nanobody to an antisera albumin, or an engineered antibody Fc domain, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a pro-protein sequence, or a fusion protein formed with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having sortilin 1 binding activity, comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising an antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions as described above.
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 90%, preferably at least 95%, more preferably at least 98% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃, or (2) hybridization with denaturing agents, e.g., 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaC12 method using procedures well known in the art. Another approach is to use MgC12. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.
Sortilin 1 is a cell membrane receptor protein, belongs to the VPS10P superfamily, and plays an important role in intracellular protein transport, neurodevelopment, metabolic regulation and the like. Sortilin 1 can bind a variety of proteins and ligands, such as nerve growth factors, neurotrophins, glycoproteins, etc., and take them from the cell surface into the endoplasmic reticulum and transport vesicle system by endocytosis, participating in their localization and transport processes. Sortilin 1 interacts with multiple signaling pathways in neurons, which are involved in neuronal growth and developmental regulation, and are also closely related to neural functions such as synaptic transmission, synaptic plasticity and memory formation. Sortilin 1 plays an important role in the nervous system, and studies have shown that sortilin 1 function is associated with neurodegenerative diseases. In addition, sortilin 1 is overexpressed in cancers such as prostate, ovarian, triple negative breast, skin, lung, colorectal and pancreatic cancers and aids in the adsorption and invasion of cancer cells during breast metastasis.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 100 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Typically, the anti-sortilin 1 nanobody may comprise at least two VHH chains, and the VHH chains are linked by a linker.
In the present invention, the linker is selected from the following sequences: (GaSb) x- (GmSn) y, wherein a, b, m, n, x, y=0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably a=4 and b=1, m=3 and n=1), i.e. the linker is selected from the group consisting of: GGGGSGGGS.
Labeled antibodies
In the present invention, the antibody is provided with a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels.
Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the invention, the antibodies to sortilin 1 are labeled with colloidal gold to obtain colloidal gold-labeled antibodies.
The anti-sortilin 1 nanobody of the invention can effectively bind sortilin 1 proteins on the cell surface.
Detection method
The invention also relates to a method for detecting sortilin 1 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of sortilin 1 protein in the solubilized sample.
In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, and the like.
The invention also provides a detection kit for detecting the sortilin 1 level, which comprises an antibody for identifying sortilin 1 protein, a lysis medium for dissolving a sample, and a general reagent and a buffer solution required for detection, such as various buffers, detection markers, detection substrates and the like. The detection kit may be an in vitro diagnostic device.
The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in (Sambrook and Russel1 et al, molecular cloning: A laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press), or by the manufacturer's recommendations. Percentages and parts are by weight unless otherwise indicated.
Example 1 expression of human sortilin 1 extracellular domain fusion proteins
Expression purification of human Sortilin-Fc fusion protein: transient expression of human sortilin 1 extracellular fragment protein using mammalian cells, expi-293F: mixing pcDNA3.1-H-SORT1-hFc recombinant plasmid cloned with human sortilin 1 extracellular segment gene with a transfection reagent Polyethylenimine (PEI), and transfecting to an Expi-293F cell; 37 ℃,5% CO 2 Culturing in a shaking incubator for 5 days; cell supernatants were then collected and bound to Protein a beads for 1h at room temperature; after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M glycine solution pH 3.0; the eluted proteins were then ultrafiltered into phosphate buffer, and samples were taken for SDS-PAGE detection after yield determination. The detection result is shown in the A of figure 1, the purity of the expressed and purified H-SORT1-hFc protein is more than 90%, and the protein can be used for camel immunity and antibody screening (wherein M represents Marker, and lane 1 is human sortilin 1 protein with Fc domain).
Expression and purification of human Sortilin-HisTag fusion protein: transient expression of human sortilin 1 extracellular fragment protein using mammalian cells, expi-293F: mixing pcDNA3.1-H-SORT1-Hisx6 recombinant plasmid cloned with human sortilin 1 extracellular segment gene with a transfection reagent PEI, and then transfecting the mixture into an Expi-293F cell; 37 ℃,5% CO 2 Culturing in a shaking incubator for 5 days; the cell supernatant was then collected and bound to Ni affinity chromatography beads, and the protein was eluted with 300mM imidazole, 500mM imidazole solution; the eluted proteins were then ultrafiltered into PBS solution, and samples were taken for SDS-PAGE detection after yield determination. Inspection and detectionAs shown in the B of FIG. 1, the purity of the expressed and purified H-SORT1-Hisx6 protein is more than 90%, and the protein can be used for camel immunity and antibody screening (wherein M represents Marker, and lane 2 is human Sortilin protein with Hisx6 tag).
Example 2 construction of camel immune library and screening and expression purification of anti-human sortilin 1 nanobody
Purified human Sortilin-Hisx6 fusion protein was combined with freund's adjuvant 1:1, four-wheel immunization is carried out on a healthy alpaca by adopting a multipoint subcutaneous injection method, a small amount of blood is extracted during the period to separate serum, and the serum titer is detected. 100mL of peripheral blood was collected 7-10 days after the end of the last immunization, and isolation of mononuclear cells (PBMC) was performed using Ficoll lymphocyte isolate (GE Healthcare 17-1440-03 FICOLL PAQUE PLUS).
Total mRNA from PBMC was extracted with phenol-chloroform and RNA reverse-converted to cDNA using SuperScript ™ IV First-Strand cDNA Synthesis Reaction kit (Thermo, 18091200). The heavy chain variable region fragment (Q5 High-Fidelity 2X Master Mix, NEB, M0492L) was amplified by nested PCR using cDNA as template. The primers used for the two rounds of PCR are shown in Table 1 below:
TABLE 1 primers for nested PCR amplification of VHH genes
The antibody fragment recovered from the gel of the second round of PCR and phage vector pADL were digested with Not1 and Bstx1 restriction enzymes, respectively, and the digested products were subjected to agarose electrophoresis and purified, followed by an enzymatic ligation reaction. The enzyme-linked product was electrotransformed into TG1 competent cells (indigenous organisms, DE 1055M) to construct a library of e.coli containing nanobody fragments. Clone counts indicated that the library was at a stock of 5E8; 40 monoclonal antibodies are randomly selected for sequencing, and the sequencing result shows that the insertion rate of the nanobody genes is about 95%.
After amplifying the obtained sortilin 1 immune library, adding VCSM13 helper phage in the logarithmic growth phase, and infecting bacteria for 1 hour at 37 ℃; after centrifugation of the bacterial culture, the supernatant was discarded and the bacteria were resuspended in 2XYT medium with antibiotics and 0.5% glucose and the phages were packaged overnight at a constant temperature shaker at 30℃and 225 rpm. The phage particles were precipitated with a second daily polyethylene glycol/sodium chloride. Library screening is performed by utilizing phage display technology, and the nanobody phage population which expresses and binds to the H-Sort1-Hisx6 fusion protein is enriched through 3 rounds of screening processes of adsorption-washing-enrichment. After 576 randomly selected monoclonal antibodies from the enriched phages were amplified and IPTG induced to express, bacterial outer walls were swelled with hypotonic solution PPB (phosphate peptone buffer) to obtain Periplasmic Protein Extract (PPE), and binding of PPE to human sortilin 1 (ECD) protein was identified by ELISA. The results showed that 117 of 576 random clones bound to sortilin (figure 2, clones with elisa readings 5-fold above background were defined as positive clones).
To identify whether these clones were specific for sortilin 1 binding, the selected 117 clones were interacted with insulin-coated ELISA plates, and the results showed that 108 of the 117 clones did not interact with insulin, suggesting that these clones were specific for sortilin 1 binding.
All the obtained 108 clones specifically combined with sortilin 1 are subjected to sequencing identification, 25 non-repeated VHH coding sequences obtained through sequencing are cloned to pcDNA3.1-VHH-hFc plasmid with human Fc domain coding genes, and the VHH-hFc fusion Protein is expressed by transfected Expi-293F cells, and the purified nanobody-Fc fusion Protein is obtained through the steps of Protein-A magnetic bead affinity combination, glycine solution elution and the like.
Example 3 ELISA experiments in which anti-Sort 1 nanobodies compete for binding of granulin precursors to sortilin 1
Coating an ELISA plate with 50 mu L4 mu g/mL H-Sort1-His antigen at 4 ℃ overnight; washing 3 times with 0.1% pbst (phosphate tween buffer), adding 5% bsa (bovine serum albumin) 150 μl, and standing for 1 hour at room temperature; washing 3 times with 0.1% PBST, adding 6ug/mL Bio-PGRN (biotin-modified human granulin precursor) mixed with 4ug/mL VHH-Sort1, 6ug/mL Bio-PGRN alone as control 1,6ug/mL Bio-PGRN and 4ug/mL Hyhel-VHH (non-specific control nanobody) as control 2, and 1 hour at room temperature; wash 3 times with 0.1% pbst, add 50 μl horseradish peroxidase labeled streptavidin (1:5000) (diluted with 5% BSA), room temperature for 1 hour; washing 3 times with 0.1% PBST, adding 50 mu LTMB (3, 3', 5' -tetramethyl benzidine) for color development for 8-10 minutes, and stopping the reaction with 50 mu L of stop solution; the absorbance of each well was read at a wavelength of 450 nm. If nanobodies compete for binding of sortilin 1 to the granulin precursor, which results in a decrease in Bio-PGRN binding to sortilin 1, the corresponding sample will be read lower than the sample without nanobody.
The effect of 25 candidate antibodies on sortilin 1 binding to its ligand granulin precursor is shown in figure 3. The results showed that of the 25 candidate antibodies, 24 were able to inhibit the interaction of the Sortilin-His fusion protein with the granulin precursor in an ELISA experiment, we selected to perform further characterization analysis on these 25 antibodies.
Example 4 detection of the Activity of anti-sortilin 1 nanobody binding to cell surface antigen
The pcDNA3.1-HSort1 recombinant plasmid cloned with the full length of the human sortilin 1 protein gene is mixed with a transfection reagent PEI to transfect 293T cells. After 48 hours of transfection, the cells were collected by pipetting, centrifuging to remove the medium and washing the cells twice with pre-chilled PBS, suspending the cells in pre-chilled PBS solution containing 1% BSA, transferring the cells to a 96-well plate with a U-shaped bottom according to 10 ten thousand cells/well after cell counting, adding purified anti-sortilin 1 nanoantibody VHH-Fc to an antibody concentration of 50 ng/ml, and incubating on ice for 30 minutes; cells were washed 2 times with pre-chilled PBS, secondary anti-human Fc domain with APC fluorescent label was added, incubated on ice for 30 min, cells were washed 2 times with chilled PBS, suspended in pre-chilled PBS solution containing 1% BSA, and analyzed for antibody binding to cell surface antigen with flow cytometry.
In a parallel experiment of the control group, untransfected 293T cells were treated with the same nanobody and the flow cytometer detected the background binding of the antibody to cell surface non-specific proteins.
The results are shown in FIGS. 4-8 and Table 2. The result shows that 9 nano antibodies in 25 candidate nano antibodies can be efficiently combined with the human sortilin 1 protein which is highly expressed on the surface of 293T cells, and the combination activity of the human sortilin 1 protein is similar to that of a control antibody AL 001. Thus, 9 preferred antibodies, nb107, nb110, nb118, nb128, nb145, nb151, nb171, nb184, nb252 were obtained, and the sequence information is shown in tables 3-4.
TABLE 2 flow cytometer detection results
TABLE 3 list of nanobody sequences
Table 3 (subsequent)
TABLE 4 list of nanobody sequences
Example 5 determination of affinity of nanobody binding to sortilin 1 Using biological Membrane interferometry
Biological layer interferometry assays were performed using the gate Prime system (gate Bio) v2.7.3.0728 (https:// www.gatorbio.com /), to evaluate the binding kinetics of nanobodies to sortilin 1 fusion proteins.
The glass hFC probe (gate Bio) was first immersed in Q buffer (gate Bio) for about 30 seconds to obtain a baseline signal. Then, probes were loaded with 3-5 μg/ml nanobody-Fc fusion protein (VHH-Fc) diluted in Q buffer for 180 seconds, followed by a Q buffer wash step of 30 seconds. Next, VHH-Fc-binding probes were conjugated to 300-1.2 nM human Sortilin-Hisx6 fusion protein, including Sortilin-free 1 protein control (0 nM), for about 180 seconds, followed by a 180 second dissociation step. The association and dissociation curves were plotted and calculated by GatorPrime (Gator Bio) software to yield the binding kinetics values (dissociation rate constant koff, association rate constant Kon, and dissociation constant KD). The results are shown in FIGS. 9-11.
The binding kinetics of the control antibody Latozinemab to human sortilin 1 fusion protein were determined in the same manner and the binding kinetics values (koff, kon and KD) obtained under the same conditions were used for parallel comparison with VHH-Fc affinity. The results are shown in Table 5.
TABLE 5 Gator determination of affinity data for human Sortilin-Hisx6 fusion proteins for each nanobody (VHH-Fc)
Example 6 determination of epitope grouping of nanobodies recognizing sortilin 1 Using a duplex epitope partitioning assay
The epitope binding of each nanobody to sortilin 1 fusion protein was assessed using the gate Prime system (gate Bio) v2.7.3.0728 (https:// www.gatorbio.com /) using a biological layer interferometry assay.
The glass His-tag probe (Gator Bio) was first immersed in Q buffer (Gator Bio) for about 30 seconds to obtain a baseline signal. Then, the probes were loaded with 100nM human Sortilin-Hisx6 fusion protein diluted in Q buffer for 180 seconds; then a 30 second Q buffer wash step was performed. Next, the Sortilin-Hisx 6-binding probe was bound to the 300nM control antibody AL001 diluted in Q buffer for about 180 seconds; the Sortilin-AL 001-bound probe was then immersed in a 200nM nanobody-Fc fusion protein (VHH-Fc) solution diluted in Q buffer for 180 seconds of duplex binding. The duplex binding curves were plotted and calculated by GatorPrime (Gator Bio) software to obtain groupings of different antibody anti-binding epitopes.
The results are shown in FIG. 12. Indicating that the epitope bound by the selected nanobody is different from the epitope bound by the control antibody AL 001.
EXAMPLE 7 determination of the levels of granulin precursor in cell culture Medium after nanobody treatment of U251 cells
Spreading U251 cells in logarithmic growth phase into 96-well cell culture plate, 1E4 cells per well, placing in culture medium containing 5% CO 2 Is a cell incubator at 37 ℃. After 24 hours of plating, VHH-Fc nanobody at a working concentration of 40nM or control antibody AL001 at the same molar concentration was added, and after 72 hours the cell culture supernatant was taken and the concentration of the granulin precursor was detected by granulin precursor ELISA KIT (EAGLE BIOSCIENCES Cat #: E103). The detection method comprises the following steps: taking cell supernatant, and diluting the cell supernatant by 15 times by using a dilution buffer solution provided by a kit; to each ELISA well of the kit was added 50uL of the antibody conjugate provided in the kit and 50ul of diluted sample (including antibody-treated cell culture supernatant, granulin precursor standard solution and positive control), and incubated for 1 hour (.gtoreq.350 rpm) on a shaker at room temperature; discarding the supernatant, and washing with a washing solution for 5 times; adding the enzyme conjugate provided by the 100 uL kit, and incubating for 30 minutes (more than or equal to 350 rpm) at room temperature; discarding the supernatant, and washing with a washing solution for 5 times; adding a substrate solution provided by a 100 uL kit, and incubating for 30 minutes at room temperature in a dark place; 100 uL stop solution was added to each well and the absorption of 450 nm was detected within 30 minutes. In the data analysis, the concentration of the particle protein precursor in the cell culture supernatant is converted according to the absorbance curve of the standard sample.
The results indicate that the selected nanobodies all increased the level of granulin precursors in the U251 cell culture medium, wherein antibodies such as Nb128, nb171, nb184, nb252 had an effect on increasing granulin precursors to or above that of the control antibody AL001 (fig. 13).
To further determine the relationship between antibody concentration and the level of secretion of the granulin precursor by U251 cells, U251 cells were treated with sortilin 1 nanobody VHH-Fc at different concentration gradients, respectively, ELISA was performed after 72 hours of treatment to determine the level of granulin precursor in the cell culture medium, and the effect of the antibodies was calculated by comparison with the granulin precursor concentration in the U251 cell culture medium without antibodies. The results showed that in the test range of 0.004 ug/mL-4.0 ug/mL, the effects of Nb171 and Nb184 on the induction of the elevation of granulin precursors were approximately positively correlated with the antibody concentration, and the elevation of granulin precursors was more pronounced by high concentrations of antibody. It is worth mentioning that both Nb171 and Nb184 induced an increase in granulin precursor at the same antibody concentration, which was superior to AL001 (fig. 14).
To further verify the effect of anti-sortilin 1 nanobody in vivo, we injected purified nanobody-Fc fusion protein into cynomolgus monkeys intravenously at a dose of 100mg/kg, and collected monkey peripheral blood before and 24-72 hours of dosing, and measured the concentration of the granulin precursor in serum using granulin precursor ELISA KIT (ams.e 09p 0138). The results show that the level of granulin precursors in monkey serum is significantly increased after intravenous injection of anti-sortilin 1 nanobody, indicating that the nanobody can increase the level of functional granulin precursors in vivo by preventing sortilin 1-mediated endocytosis and degradation of granulin precursors.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (9)

1. A nanobody specific for sortilin 1, wherein the heavy chain variable region of said nanobody comprises complementarity determining regions CDR1, CDR2, CDR3, the complementarity determining regions being any combination of:
1) CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.2, CDR3 shown in SEQ ID No. 3;
2) CDR1 shown in SEQ ID No.8, CDR2 shown in SEQ ID No.9, CDR3 shown in SEQ ID No. 10;
3) CDR1 shown in SEQ ID No.15, CDR2 shown in SEQ ID No.16, CDR3 shown in SEQ ID No. 17;
4) CDR1 shown in SEQ ID No.22, CDR2 shown in SEQ ID No.23, CDR3 shown in SEQ ID No. 24;
5) CDR1 shown in SEQ ID No.29, CDR2 shown in SEQ ID No.30, CDR3 shown in SEQ ID No. 31;
6) CDR1 shown in SEQ ID No.36, CDR2 shown in SEQ ID No.37, CDR3 shown in SEQ ID No. 38;
7) CDR1 shown in SEQ ID No.43, CDR2 shown in SEQ ID No.44, CDR3 shown in SEQ ID No. 45;
8) CDR1 shown in SEQ ID No.50, CDR2 shown in SEQ ID No.51, CDR3 shown in SEQ ID No. 52;
9) CDR1 shown in SEQ ID No.57, CDR2 shown in SEQ ID No.58, CDR3 shown in SEQ ID No. 59.
2. The nanobody of claim 1, wherein: the variable region of the nanobody includes framework regions FR1, FR2, FR3, FR4.
3. Nanobody according to claim 2, wherein the framework regions of nanobody complementarity determining region combinations 1) -9) correspond as follows:
1) FR1 shown in SEQ ID No.4, FR2 shown in SEQ ID No.5, FR3 shown in SEQ ID No.6, FR4 shown in SEQ ID No. 7;
2) FR1 shown in SEQ ID No.11, FR2 shown in SEQ ID No.12, FR3 shown in SEQ ID No.13, FR4 shown in SEQ ID No. 14;
3) FR1 shown in SEQ ID No.18, FR2 shown in SEQ ID No.19, FR3 shown in SEQ ID No.20, FR4 shown in SEQ ID No. 21;
4) FR1 shown in SEQ ID No.25, FR2 shown in SEQ ID No.26, FR3 shown in SEQ ID No.27, FR4 shown in SEQ ID No. 28;
5) FR1 shown in SEQ ID No.32, FR2 shown in SEQ ID No.33, FR3 shown in SEQ ID No.34, FR4 shown in SEQ ID No. 35;
6) FR1 shown in SEQ ID No.39, FR2 shown in SEQ ID No.40, FR3 shown in SEQ ID No.41, FR4 shown in SEQ ID No. 42;
7) FR1 shown in SEQ ID No.46, FR2 shown in SEQ ID No.47, FR3 shown in SEQ ID No.48, FR4 shown in SEQ ID No. 49;
8) FR1 shown in SEQ ID No.53, FR2 shown in SEQ ID No.54, FR3 shown in SEQ ID No.55, FR4 shown in SEQ ID No. 56;
9) FR1 shown in SEQ ID No.60, FR2 shown in SEQ ID No.61, FR3 shown in SEQ ID No.62 and FR4 shown in SEQ ID No. 63.
4. The nanobody of claim 1, wherein: the sequence of the nano antibody is any one of the sequences shown in SEQ ID NO. 64-72.
5. A polynucleotide molecule encoding the nanobody of any of claims 1-4.
6. An expression vector comprising the polynucleotide molecule of claim 5.
7. A host cell comprising the nanobody of any of claims 1-4.
8. A monovalent antibody, a bivalent antibody, a multivalent antibody, a recombinant protein or an immunoconjugate comprising the nanobody of any one of claims 1-4.
9. Use of a nanobody according to any one of claims 1-4, a polynucleotide molecule according to claim 5, an expression vector according to claim 6, a host cell according to claim 7 or a monovalent antibody, a bivalent antibody, a multivalent antibody, a recombinant protein or an immunoconjugate according to claim 8 for the preparation of a product for detecting sortilin 1 molecular levels or increasing levels of progranulin.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1344724A (en) * 2000-09-29 2002-04-17 上海博德基因开发有限公司 New polypeptide mitochondrion sorting protein 1-39.71 and polynucleotides encoding this polypeptide
CN111372655A (en) * 2018-07-13 2020-07-03 艾利妥 Anti-sortilin antibodies and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1344724A (en) * 2000-09-29 2002-04-17 上海博德基因开发有限公司 New polypeptide mitochondrion sorting protein 1-39.71 and polynucleotides encoding this polypeptide
CN111372655A (en) * 2018-07-13 2020-07-03 艾利妥 Anti-sortilin antibodies and methods of use thereof

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