CN116514978A - Preparation method and application of PD-L1 specific nano antibody molecule image probe - Google Patents

Abstract

The invention relates to the technical fields of molecular imaging, nuclear medicine and nanobody for tumor diagnosis and treatment, in particular to a PD-L1 specific nanobody, fusion protein thereof, a PD-L1 specific nanobody molecular imaging probe and a preparation method and application thereof. The PD-L1 specific nano antibody molecule image probe overcomes the defects of long imaging period, large radiation dose and the like of the monoclonal antibody molecule image probe, and has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like.

Description

Preparation method and application of PD-L1 specific nano antibody molecule image probe

Technical Field

The invention relates to the technical fields of molecular imaging, nuclear medicine and nanobody for tumor diagnosis and treatment, in particular to a preparation method and application of a PD-L1 specific nanobody molecular imaging probe.

Background

Programmed death factor ligand 1 (programmed death ligand, PD-L1) inhibits T cell activation and anti-tumor immune responses by interacting with the receptor programmed death factor (programmed cell death protein, PD-1) on the surface of T cells. Immunotherapy based on monoclonal antibody targeting of PD-1/PD-L1 significantly prolongs survival of a variety of tumors. However, there is currently no biomarker in clinical practice that effectively predicts the efficacy of targeted PD-1/PD-L1 immunotherapy. Of particular note, the clinical study data published in the journal of Nature Medicine demonstrate that: immunohistochemical staining revealed that PD-L1 expression levels did not predict therapeutic efficacy of the PD-L1 specific monoclonal antibody, ab-zumab (atezolizumab) (Nat Med.2018; 24:1852-1858.). Meanwhile, another clinical study showed that there was a significant difference in detection efficacy of several immunohistochemical staining methods currently used to detect the expression level of PD-L1, and that the same method had a large difference in staining results between different samples (J Thorac Oncol.2017; 12:208-222.). In addition, immunohistochemical staining also faces defects such as puncture biopsy sampling errors, image judgment errors and the like.

Immune PET (immunoPET) imaging based on radionuclide labeled monoclonal antibody, antibody fragment is a new model of accurate medical time molecular image, and can not only not be usedThe method is used for in-vivo noninvasive visualization of heterogeneous expression of tumor markers, dynamic monitoring of dynamic evolution of the tumor markers before and after treatment, and effective prediction of therapeutic effects of targeted treatment or immunotherapy (Chem Rev.2020;120 (8): 3787-3851.). The applicant has previously prepared long half-life metal nuclides for a number of targeting or immunotherapy related targets such as CD146 (Advanced science.2019Feb27;6 (9): 1801237.), TIM-3 (Advanced therapeutics.2020Jul;3 (7): 2000018.), ICAM-1 (Eur J Nucl Med Mol imaging.2020Nov;47 (12): 2765-2775.), HER2 (Am J Cancer Res.2019Nov1;9 (11): 2413-2427.), tissue factor (Advanced science.2020May 17;7 (13): 1903595.), etc. in the past ⁸⁹ Zr，T1/2＝78.4h； ⁶⁴ Cu, T1/2=12.7h), the above-mentioned probes have achieved good diagnostic efficacy, but monoclonal antibody probes face the defects of long systemic circulation time, long-half-life-needed nuclide labeling, long imaging period up to 1 week, large radiation dose, and great difficulty in clinical transformation application.

In 1993, belgium scientists Hamers et al reported for the first time in Nature journal that an antibody with a naturally deleted light chain was present in alpaca peripheral blood (Nature.1993; 363 (6428): 446-8.), an antibody with a specific domain, namely Heavy chain antibodies (HCAbs). Through molecular biological means, the antigen binding fragment of only the heavy chain variable region can be obtained by cloning the variable region of the heavy chain antibody, namely the nanobody (VHH, variable Domain of Heavy Chain of Heavy Chain Antibody). VHH crystals are 2.5nm wide and 4nm long and have a molecular weight of only 15kDa, and are therefore also known as nanobodies [ (] Ablynx corporation registers trade names). The nano antibody is the currently known minimum antibody unit capable of combining the target antigen, and has the advantages of high affinity, small molecular weight, low preparation cost (not only can be expressed by using escherichia coli, but also can be expressed by using eukaryotic expression systems such as yeast, chinese hamster ovary cells and the like), and easy clinical transformation and popularization and application. Nanobodies are popular targeting vectors for constructing molecular imaging probes in recent years (theranostics.2014; 4 (4): 386-98).

Xing Yan et al prepare ^99m Tc marked PD-L1 specific nano antibody probe ^99m Tc-NM01 (J Nucl Med.2019;60 (9): 1213-1220.). But it is worth noting that the number of the parts, ^99m tc belongs to single photon emission radionuclides, and the prepared probe is used for Single Photon Emission Computed Tomography (SPECT) and is limited by ^99m The physical properties of Tc itself and the low resolution of SPECT imaging devices result in poor clinical diagnostic performance of the probe; in addition, nanobody NM01 has a low affinity of 2.9nM, and may not be able to efficiently detect expression of low abundance PD-L1; finally, free ^99m Tc is often concentrated in normal thyroid and gastric mucosal tissues, resulting in false positive imaging of the imaging. Meanwhile, european scholars prepared the monoclonal antibody Ab-bead monoclonal antibody with specificity of PD-L1 obtained in clinical batch ⁸⁹ Zr-atezolizumab (Nat Med.2018; 24:1852-1858.). However, the cloned antibody probes are not only expensive to prepare, but also have long imaging cycles (up to 1 week). In addition, monoclonal antibody probes have higher uptake in normal tissue organs such as liver, spleen and the like because Fc/FcR fragments mediate uptake of the probes by normal tissue organs, and further cause greater radiation damage to the normal tissue organs. In general, the PD-L1 specific nanobody probe and monoclonal antibody which enter the clinical transformation stage at present have certain defects.

Therefore, a new PD-L1 specific nanobody probe is urgently needed to overcome the defects of low affinity and high off-target effect of the traditional nanobody probe and overcome the defects of long imaging period and large radiation dose of the monoclonal antibody probe.

Disclosure of Invention

The invention aims to construct a PD-L1 specific nano antibody molecule image probe, overcome the defects of long imaging period, large radiation dose and the like of a monoclonal antibody molecule image probe, and realize more convenient layering of patients subjected to targeted PD-L1 treatment and targeted PD-L1 treatment effect monitoring.

PD-L1 specific nanobody

In one aspect, the invention provides a PD-L1-specific nanobody comprising:

(1) CDR1 having the amino acid sequence shown in SEQ ID No.1, CDR2 having the amino acid sequence shown in SEQ ID No.2 and CDR3 having the amino acid sequence shown in SEQ ID No.3,

(2) CDR1 having the amino acid sequence shown in SEQ ID No.8, CDR2 having the amino acid sequence shown in SEQ ID No.9 and CDR3 having the amino acid sequence shown in SEQ ID No.10,

(3) CDR1 having the amino acid sequence shown in SEQ ID No.13, CDR2 having the amino acid sequence shown in SEQ ID No.14 and CDR3 having the amino acid sequence shown in SEQ ID No.15,

(4) CDR1 having the amino acid sequence shown in SEQ ID No.39, CDR2 having the amino acid sequence shown in SEQ ID No.19 and CDR3 having the amino acid sequence shown in SEQ ID No.20,

(5) CDR1 having the amino acid sequence shown in SEQ ID No.23, CDR2 having the amino acid sequence shown in SEQ ID No.24 and CDR3 having the amino acid sequence shown in SEQ ID No.25,

(6) CDR1 having the amino acid sequence shown in SEQ ID No.28, CDR2 having the amino acid sequence shown in SEQ ID No.29, and CDR3 having the amino acid sequence shown in SEQ ID No.30, or

(7) CDR1 having the amino acid sequence shown in SEQ ID No.33, CDR2 having the amino acid sequence shown in SEQ ID No.34, and CDR3 having the amino acid sequence shown in SEQ ID No. 35.

More specifically, the PD-L1 specific nanobody of the invention has the amino acid sequence shown in SEQ ID No.4, 6, 11, 16, 21, 26, 31 or 36.

In the present invention, for the sake of simplicity, the PD-L1-specific nanobody having the amino acid sequence shown in SEQ ID No.4, 6, 11, 16, 21, 26, 31 or 36 is referred to as WW102, WW101, WW103, WW104, WW105, WW106, WW107 or WW108, respectively.

As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region), typically derived from a variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity. Nanobodies are also known as single-domain antibodies (sdabs) or VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody), which are used interchangeably.

In some embodiments, the invention also encompasses antigen binding fragments of PD-L1 specific nanobodies as described herein.

As used herein, the term "antigen-binding fragment" refers to a polypeptide comprising a fragment of a nanobody that retains the ability to specifically bind to the same antigen to which the nanobody binds, and/or competes with the nanobody for specific binding to an antigen, also referred to as an "antigen-binding portion. Generally, see Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of the present antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the present nanobodies.

Antigen-binding fragments of nanobodies can be obtained from a given nanobody (e.g., a nanobody provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened in the same manner as for whole nanobodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "nanobody" it includes not only whole nanobodies but also antigen-binding fragments of nanobodies.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs are contained in the nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given nanobody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

As used herein, the term "PD-L1 specific" refers to specifically binding to PD-L1.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. A method involves measuringThe rate of antigen binding site/antigen complex formation and dissociation. "binding Rate constant" (k) _a Or k _on ) And "dissociation rate constant" (k) _dis Or k _off ) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). k (k) _dis /k _on Is equal to the dissociation constant K _D (see Davies et al, annual Rev Biochem,1990; 59:439-473). K can be measured by any effective method _D 、k _on And k _dis Values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

In some embodiments, the invention also provides variants of the PD-L1-specific nanobody as described herein that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID nos. 4, 6, 11, 16, 21, 26, 31 or 36, and substantially retain the biological function (e.g., the biological activity of specifically binding PD-L1) of the nanobody from which it is derived.

More specifically, the variants differ from PD-L1-specific nanobodies as described herein only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, up to 5, or up to 1) amino acid residues.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. ApplBiosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (J mobiol. 48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

PD-L1 specific nanobody fusion proteins

In another aspect, the invention provides a PD-L1-specific nanobody fusion protein comprising a nanobody as described herein and a moiety for extending in vivo half-life.

As used herein, the moiety for extending in vivo half-life may include serum albumin or fragments thereof, polyethylene glycol, domains that bind serum albumin (e.g., nanobodies against serum albumin), polyethylene glycol-liposome complexes, and the like.

In the PD-L1 specific nanobody fusion proteins provided herein, the nanobody and the moiety for extending in vivo half-life as described herein may be provided with a linker peptide. The connecting peptide can be a flexible polypeptide chain consisting of alanine (A) and/or serine (S) and/or glycine (G), and the length of the connecting peptide can be 3-30 amino acids, preferably 3-9, 9-12, 12-16 and 16-20.

In a specific embodiment, the invention provides a PD-L1 specific nanobody fusion protein having the amino acid sequence shown in SEQ ID No. 38.

In the present invention, the PD-L1 specific nanobody fusion protein having the amino acid sequence shown in SEQ ID No.38 is referred to as ABDWW102 for the sake of simplicity.

The nanobody fusion protein ABDWW102 of the invention can not only bind with high affinity to human PD-L1, but also bind with high affinity to Human Serum Albumin (HSA) and Murine Serum Albumin (MSA) (FIG. 12). Biacore affinity assay showed that the affinity of ABDWW102 to HSA and MSA was 3.38pM and 52.74pM, respectively (FIG. 13). From the above data, nanobody fusion protein abddww 102 has significantly higher affinity for HSA than MSA.

Polynucleotide

In another aspect, the present invention also provides a polynucleotide encoding the above nanobody or antigen-binding fragment thereof or fusion protein thereof.

More specifically, the polynucleotide has the nucleotide sequence shown as SEQ ID No.5, 7, 12, 17, 22, 27, 32, 37 or 39.

More specifically, the polynucleotide encoding a PD-L1 specific nanobody as described herein has the nucleotide sequence set forth in SEQ ID No.5, 7, 12, 17, 22, 27, 32, or 37. More specifically, the polynucleotide encoding a PD-L1 specific nanobody fusion protein as described herein has the nucleotide sequence set forth in SEQ ID No. 39.

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.

The term "polynucleotide encoding a polypeptide/protein/antibody" may include polynucleotides encoding such polypeptide/protein/antibody, as well as polynucleotides further comprising 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 50%, preferably at least 70%, more preferably at least 80%, most preferably at least 90% identity between the two sequences, and which encode polypeptides/proteins/antibodies having substantially the same function and activity. The 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) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

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.

Carrier body

In another aspect, the invention also provides a vector comprising a polynucleotide encoding the nanobody or antigen-binding fragment thereof or fusion protein thereof described above.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

Host cells

In another aspect, the invention also provides a host cell comprising a vector as described herein.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include single cells or cell populations.

The vector may be introduced into the host cell by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . 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 nanobodies 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. The preferred detectable label is a radionuclide.

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)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.

Binding or coupling of the detectable label or therapeutic agent to the antibody can be performed by conventional methods well known to those skilled in the art. For example, the detectable label may be directly or indirectly bound to the nanobody, e.g., via a cleavable or non-cleavable linker peptide, or incorporated into the nanobody. The detectable label may be bound to the nanobody, in particular by substitution (e.g. by substituting H with I at the tyrosine residue level), by complexation or by chelation. For example, the therapeutic agent may be conjugated to the nanobody via a cleavable linker (e.g., a peptidyl, disulfide, or hydrazone linker).

In a preferred embodiment, the nanobody of the invention is conjugated with a radionuclide for use as a PD-L1 specific molecular imaging probe, as described in more detail below.

PD-L1 specific molecular imaging probe

In another aspect, the invention provides a PD-L1-specific molecular imaging probe comprising a radionuclide-labeled PD-L1-specific nanobody or PD-L1-specific nanobody fusion protein as described herein.

More specifically, a PD-L1 specific nanobody or PD-L1 specific nanobody fusion protein as described herein is labeled with a radionuclide via a bifunctional chelator.

As used herein, a bifunctional chelating agent is a class of chelating agents having both a metal chelating end and a protein anchoring end. The bifunctional chelating agent may be selected from NOTA, MAA-NOTA, p-SCN-Bn-Deferoxamine (DFO), p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA.

Preferably, the bifunctional chelating agent is selected from p-SCN-Bn-NOTA or p-SCN-Bn-deferoxamine.

As used herein, the NOTA is 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;

the MAA-NOTA is (2, 2' - (7- (2- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -2-oxoethyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;

The p-SCN-Bn-NOTA is 2-S- (4-isothiocyanatophenyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid;

the p-SCN-Bn-Deferoxamine (DFO) is 1- (4-isothiocyanatophenyl) -3- [6, 17-dihydroxy-7,10,18,21-tetraoxo-27- (N-acetylhydroxyamino) -6,11,17,22-tetraazaheptyldisaccharide ] thiourea;

the p-SCN-NODA is 1,4, 7-triazacyclooctane-1, 4-diacetic acid-7-p-isothiocyanatobenzyl;

the MAA-GA-NODA is 2,2' - (7- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxobutyl) -1,4, 7-triazacyclononane-1, 4-diyl) diacetic acid;

the MAA-DOTA is 2,2' - (10- (1-carboxy-4- ((2- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) ethyl) amino) -4-oxybutyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid ];

the DOTA-NHS is 2,2' - (10- (2- ((2, 5-dioxopyrrolidin-1-yl) oxy) -2-oxoethyl) -1,4,7, 10-triazacyclododecane-1, 4, 7-triyl) triacetic acid;

the iEDTA is 1- (4-isothiocyanatobenzyl) ethylenediamine-N, N, N ', N' -tetraacetic acid;

the p-SCN-Bn-DTPA is 2- (4-isothiocyanatobenzyl) -diethylenetriamine pentaacetic acid.

More specifically, PD-L1 specific nanobodies as described herein are labeled with a radionuclide via p-SCN-Bn-NOTA. More specifically, PD-L1 specific nanobody fusion proteins as described herein are labeled with a radionuclide via p-SCN-Bn-NOTA. More specifically, PD-L1 specific nanobody fusion proteins as described herein are labeled with a radionuclide via p-SCN-Bn-deferoxamine.

More specifically, the radionuclide is selected from Tc-99m, ga-68, F-18, I-123, I-125, I-131, I-124, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, Y-86, mn-52, sc-44, lu-177, Y-90, ac-225, at-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra-223, ru-106, na-24, sr-89, tb-149, th-227, xe-133, yb-169 or Yb-177.

More specifically, the radionuclide is selected from Ga-68.

More specifically, PD-L1 specific nanobodies as described herein are labeled with Ga-68 (examples of one such PD-L1 specific molecular imaging probe are described in the examples ⁶⁸ Ga]Ga-NOTA-WW 102). More specifically, PD-L1 specific nanobody fusion proteins as described herein are labeled with Ga-68 (examples of one such PD-L1 specific molecular imaging probe are described in the examples ⁶⁸ Ga]Ga-NOTA-aBDWW 102). More specifically, PD-L1 specific nanobody fusion proteins as described herein are Cu-64 labeled (examples of one such PD-L1 specific molecular imaging probe are described in the examples ⁶⁴ Cu]Cu-NOTA-ABDWW102)。

In another aspect, the invention also provides a method for preparing a PD-L1 specific molecular imaging probe, comprising modifying a PD-L1 specific nanobody by a bifunctional chelating agent to obtain a radionuclide-labeled precursor; and labeling the radionuclide labeling precursor with a radionuclide to obtain the PD-L1 specific molecular imaging probe.

Composition and method for producing the same

In another aspect, the invention provides a composition comprising a PD-L1 specific nanobody, a PD-L1 specific nanobody fusion protein, a polynucleotide, a vector, a host cell, or a molecular imaging probe as described herein. The composition can be used for detecting the expression level of PD-L1, diagnosing PD-L1 related tumors, predicting the therapeutic effect of PD-L1 related tumors or treating PD-L1 related tumors.

In some embodiments, the composition may be a pharmaceutical composition.

In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

In some embodiments, in the pharmaceutical composition, the PD-L1 specific nanobody, PD-L1 specific nanobody fusion protein, polynucleotide, vector, host cell, or molecular imaging probe as described herein and the additional pharmaceutically active agent may be provided as separate components or as a mixed component. Thus, a PD-L1 specific nanobody, PD-L1 specific nanobody fusion protein, polynucleotide, vector, host cell, or molecular imaging probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

In some embodiments, the pharmaceutically acceptable carrier and/or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a PD-L1 specific nanobody, PD-L1 specific nanobody fusion protein, polynucleotide, vector, host cell, or molecular imaging probe as described herein. "prophylactically effective amount" means an amount sufficient to prevent, arrest or delay the onset of a disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a PD-L1 specific nanobody, a PD-L1 specific nanobody fusion protein, a polynucleotide, a vector, a host cell, or a molecular imaging probe as described herein.

The kit can be used for detecting the expression level of PD-L1, diagnosing PD-L1 related tumors, predicting the treatment effect of PD-L1 related tumors or treating PD-L1 related tumors.

The kit may further comprise further containers, instructions for use, and other reagents and buffers required for the actual application, such as lysis media for lysing the sample, various buffers, detection labels, detection substrates, etc.

Diagnostic and therapeutic applications

The PD-L1 specific nano antibody has extremely high affinity to PD-L1, so that the nano antibody can be used for detecting the expression level of PD-L1, diagnosing PD-L1 related tumors, predicting the treatment effect of PD-L1 related tumors or treating PD-L1 related tumors.

Particularly, the PD-L1 specific molecular image probe prepared from the PD-L1 specific nano antibody has the characteristics of obviously improved affinity, obviously reduced non-specific uptake of normal tissue uptake and obviously improved image quality, and can be used for noninvasively, accurately and efficiently detecting the expression of human PD-L1, so that the PD-L1 specific molecular image probe is particularly suitable for diagnosing PD-L1 related tumors and predicting the treatment effect of PD-L1 related tumors. After proper radionuclides are selected for coupling, the method can also be used for accurately treating PD-L1 related tumors.

Thus, in another aspect, the invention also relates to the use of a PD-L1-specific nanobody, a PD-L1-specific nanobody fusion protein, a polynucleotide, a vector, a host cell, or a molecular imaging probe as described herein in the preparation of a kit or a medicament for detecting the expression level of PD-L1, diagnosing a PD-L1-related tumor, predicting the therapeutic effect of a PD-L1-related tumor, or treating a PD-L1-related tumor.

As used herein, PD-L1-associated tumors may include various tumors or cancers well known in the art. For example, PD-L1-associated tumors may include gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymphatic cancer, adrenal tumor, or bladder tumor.

The beneficial effects of the invention are that

The invention reports a high affinity and high specificity nano antibody probe ⁶⁸ Ga]Ga-NOTA-WW102, wherein the affinity of the PD-L1 specific nanobody WW102 is 15.29pM. 189.6 times of PD-L1 specific nanobody NM01 is reported in the literature.

The invention reports another molecular imaging probe based on nanobody WW102 fusion protein ABDWW102 [ ⁶⁸ Ga]Ga NOTA-aBDWW102, wherein PD-L1 specific nano antibody fusion protein ABDWW102 can be combined with human PD-L1 with high affinity (the affinity is 3.714 pM) and can also be combined with human serum albumin (human serum albumin, HSA) and mouse serum albumin (murine serum albumin, MSA) with high affinity, wherein the affinities are 3.382pM and 52.74pM respectively.

ABDWW102 can be combined with serum protein with high efficiency, so that the in vivo circulation time of the ABDWW is obviously longer than that of monovalent nanobody WW102, and the positron nuclide ⁶⁸ The Ga half-life is only 1.1h, and is more difficult to match with the ABDWW102 in the systemic circulation time. Thus, the present invention further discloses a positron nuclide ⁶⁴ Cu(T _1/2 =12.7h) labeled PD-L1 specific nanobody fusion protein probes [ ⁶⁴ Cu]Cu-NOTA-ABDWW102。

The invention discloses a PD-L1 specific nano antibody probe [ ⁶⁸ Ga]Ga-NOTA-WW102, and two nanobody fusion protein probes [ ⁶⁸ Ga]Ga-NOTA-aBDWW102 and [ ⁶⁴ Cu]Cu-NOTA-aBDWW102 is used for noninvasively, accurately and efficiently detecting the expression of human PD-L1.

Compared with the prior art ^99m Tc-labeled nanobody probes (e.g ^99m Tc-NM01, J Nucl Med.2019;60 (9) 1213-1220.) PD-L1 nanobody probes reported in the present invention [ ⁶⁸ Ga]Ga-NOTA-WW102 has the characteristics of obviously improved affinity, obviously reduced non-specific uptake of normal tissue uptake and obviously improved image quality; compared with the prior art ⁸⁹ Zr-labeled monoclonal antibodies (e.g ⁸⁹ Zr-atezolizumab, nat med.2018; 24:1852-1858.), the PD-L1 nanobody fusion protein probes reported in the invention [ [ i.e. ⁶⁸ Ga]Ga-NOTA-aBDWW102 and [ ⁶⁴ Cu]Cu-NOTA-aBDWW102 also has the advantage of obviously improving the affinity, and more particularly, the radiation dose of the probe to normal tissues and organs is obviously lower, because the half-life period of the used positron nuclide is shorter [ ] ⁸⁹ Zr、 ⁶⁴ Cu and ⁶⁸ ga half-life is 78.4h, 12.7h and 1.1h, respectively), and the targeting vector used is shorter in systemic circulation (monoclonal antibody circulation time>Nanobody fusion protein circulation time>Nanobody circulation time).

The probe disclosed by the invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easiness in clinical transformation and the like.

At present, the clinical detection of the PD-L1 expression level mainly depends on immunohistochemical staining of surgical excision specimens or puncture biopsy tissues, but the immunohistochemical staining has defects of sampling errors, incapability of evaluating the overall appearance of the PD-L1 expression level of the heterogeneous tumor and incapability of evaluating the PD-L1 expression level of the metastatic tumor. More particularly, a recent clinical study showed that several immunohistochemical staining methods currently used to detect PD-L1 expression levels have significant differences in detection efficacy, and that the same method has a large difference in staining results between different samples (J Thorac Oncol.2017; 12:208-222.). Thus, in the age of accurate medicine, immunotherapy, there is a clinical need for new methods and strategies for noninvasive visualization of PD-L1 heterogeneity expression. The research of the applicant team and the same line shows that the immune PET imaging based on the antibody and nano antibody molecular imaging probe is a key target of tumor and a sharp tool for noninvasive visualization of immune checkpoints, and is an ideal choice for accompanying diagnosis in the accurate medical era (Chem Rev.2020;120 (8): 3787-3851).

The invention further develops three positron nuclide labeled nanobodies and nanobody fusion protein molecular imaging probes on the basis of developing the ultra-high affinity PD-L1 specific nanobody WW 102. Wherein [ among others ] ⁶⁸ Ga]Ga-NOTA-WW102 is characterized in that the same day imaging can be realized, namely, the imaging process of a patient can be realized within 1-2 hours after the probe is injected, the clinical transformation application is convenient, and the potential of clinical popularization and application is higher. However, almost all of the radiationThe radionuclide marked monovalent nano antibody molecular imaging probe has higher enrichment in the kidney and has the possibility of causing potential nephrotoxicity.

Successful development of monovalent nanobody molecular imaging probes ⁶⁸ Ga]On the basis of Ga-NOTA-WW102, the invention further reports that the nano antibody fusion protein ABDWW102 with better pharmacokinetics and higher affinity has double specificity at a specific point, can be combined with human/mouse serum protein and also can be combined with human PD-L1 protein, and the affinity of the nano antibody fusion protein ABDWW102 with better pharmacokinetics and higher affinity (4.12 times) with the human PD-L1 protein is obviously higher than that of WW102 with the human PD-L1. Furthermore, the invention provides two novel probes by using different positron nuclides to label the nanometer antibody fusion protein ABDWW102 ⁶⁸ Ga]Ga-NOTA-aBDWW102 and [ ⁶⁴ Cu]Cu-NOTA-aBDWW102. The above-mentioned probe kidney enrichment is significantly lower than monovalent nanobody probes [ ⁶⁸ Ga]Ga-NOTA-WW102, thus leading to a significantly reduced potential for nephrotoxicity. Comparison [ ⁶⁸ Ga]Ga-NOTA-ABDWW102，[ ⁶⁴ Cu]The Cu-NOTA-aBDWW102 has better imaging performance and better image quality, and is more beneficial to the accurate visualization of PD-L1.

Drawings

FIG. 1 is a schematic diagram of an illustrative example of a radionuclide-labeled molecular imaging probe;

FIG. 2 is a diagram of experimental results of SDS-PAGE of the anti-PD-L1 specific high affinity nanobody WW102 disclosed in the invention for determining the expression status;

FIG. 3 shows the affinity measurement results of the nanobody WW102 disclosed by the invention and the human PD-L1 protein;

FIG. 4 is a graph showing the affinity assay results of the nanobody WW102 disclosed in the present invention with human PD-L1 protein after coupling with NOTA;

FIG. 5 is a use of ⁶⁸ Probe constructed by Ga-marked nano antibody WW102 ⁶⁸ Ga]Quality control diagram of Ga-NOTA-WW 102;

FIG. 6 shows a probe according to the present invention ⁶⁸ Ga]PET/CT imaging of Ga-NOTA-WW102 in RKO colon cancer tumor model;

FIG. 7 shows a probe according to the present invention ⁶⁸ Ga]Ga-NOTA-WW102 in RKOROI and in vitro biological distribution map in colon cancer tumor model;

FIG. 8 is a diagram showing experimental results of SDS-PAGE of anti-PD-L1 specific high affinity nanobody fusion protein ABDWW 102;

FIG. 9 is a graph showing the measurement results of affinity between the nanobody fusion protein ABDWW102 disclosed in the invention and the human PD-L1 protein;

FIG. 10 is a graph showing the results of affinity measurement of nanobody fusion protein ABDWW102 disclosed in the present invention with human PD-L1 protein after coupling with NOTA;

FIG. 11 is a graph showing the results of affinity measurement of the nanobody fusion protein ABDWW102 disclosed in the present invention with human PD-L1 protein after coupling with DFO;

FIG. 12 is a schematic diagram showing the binding of the nanobody fusion protein ABDWW102 disclosed by the invention to human and mouse serum albumin in vivo;

FIG. 13 is a graph showing the results of affinity assay of binding of the nanobody fusion protein ABDWW102 disclosed in the present invention to human and murine serum albumin;

FIG. 14 is a use ⁶⁸ Probe constructed by Ga marked nano antibody fusion protein ABDWW102 ⁶⁸ Ga]Quality control diagram of Ga-NOTA-aBDWW 102;

FIG. 15 is a diagram showing a probe according to the present invention ⁶⁸ Ga]Multiple time point PET/CT images of Ga-NOTA-ABDWW102 in RKO colon cancer tumor model;

FIG. 16 is a diagram showing a probe according to the present invention ⁶⁸ Ga]ROI and in vitro biological distribution map of Ga-NOTA-aBDWW102 in RKO colon cancer tumor model;

FIG. 17 is a diagram showing a probe according to the present invention ⁶⁴ Cu]Multiple time point PET/CT images of Cu-NOTA-ABDWW102 in RKO colon cancer tumor model;

FIG. 18 shows a probe according to the present invention ⁶⁴ Cu]In vitro biological profile of Cu-NOTA-aBDWW102 in RKO colon cancer tumor model.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. It is noted that the invention is not limited to the particular methods, protocols, cell lines, constructs, and reagents described herein, and may vary as well. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1: preparation of anti-PD-L1 specific high-affinity nanobody

The anti-PD-L1 specific high-affinity nanobody WW101-108 is obtained by immunizing healthy alpaca (Vicugnapacos), phage screening and enzyme-linked immunosorbent assay (ELISA) with the extracellular domain of PD-L1 protein for a plurality of times. Nanobodies WW102, WW101, WW103, WW104, WW105, WW106, WW107 and WW108 have amino acid sequences as shown in SEQ ID nos. 4, 6, 11, 16, 21, 26, 31 and 36 in the sequence listing and gene sequences as shown in SEQ ID nos. 5, 7, 12, 17, 22, 27, 32 and 37 in the sequence listing, respectively.

Then, the monovalent nanobody WW101-108 is expressed in HEK293 cells in a recombination mode, and the specific steps are as follows: plasmid extraction, HEK293 transient expression, antibody purification and antibody basic quality control.

In the following, WW102 is taken as an example, and experimental materials and experimental steps used for the plasmid extraction are as follows:

experimental materials:

1) A plurality of extraction kits;

2) 70% ethanol: adding 375mL of absolute ethyl alcohol into 125mL of sterilized deionized water, uniformly mixing, and preserving at 4 ℃;

3) Isopropyl alcohol;

4) Buffer P1: mu.L of RNase A (20 mg/mL) was added to 120mL of the P1 solution in advance to give a final concentration of RNase A of 100. Mu.g/mL.

The experimental steps are as follows:

1) 50mL of the cultured (15 hours) bacterial liquid is taken and added into a 50mL centrifuge tube which is well marked, whether the serial numbers are consistent or not is checked, and the supernatant is poured out after centrifugation at 5000rpm at 4 ℃.

2) To this centrifuge tube 5mL of Rnase a-added suspension buffer P1 was added and bacterial pellet was thoroughly suspended with a vortex mixer.

3) To the centrifuge tube, 5mL of buffer P2 (preheated for 20 minutes at 37℃in winter) was added, and the mixture was immediately and gently inverted and mixed, and left at room temperature for 4 minutes. At this time, the bacterial liquid is changed from turbid to clear viscous liquid (if the bacterial liquid is not clear viscous liquid, the bacterial liquid is presented with turbid or whitish and timely reported, and the table is marked).

4) To the centrifuge tube, 5mL of buffer P3 was added, and the mixture was immediately and gently inverted, at which point a white flocculent precipitate appeared.

5) Centrifuge at 4℃at 9000rpm for 10 minutes, filter the supernatant with filter paper into a clean 50mL centrifuge tube with labeling, and check whether the numbers are consistent.

6) To the collected filtrate, 2mL of ER buffer was added, and the mixture was thoroughly mixed upside down, and ice-cooled for 30 minutes.

7) Column balance: in the process of collecting bacterial liquid and centrifuging, placing the empty column on a centrifuge tube rack, using ddH ₂ And filling the empty column with O, cleaning the packing, and filling the empty column with eluent to balance the packing.

8) Loading: the ER buffer treated supernatant was passed under gravity through the equilibrated column.

9) The column was gravity washed once with Wash buffer filled.

10 Place the column into a clean 50mL centrifuge tube with a label made, check if the numbers are consistent. The plasmid was eluted under gravity with 5mL elution buffer.

11 5mL ice-cold isopropanol was added to the collected filtrate, mixed upside down, centrifuged at 12000rpm for 18min, the supernatant was carefully decanted, and inverted on absorbent paper to observe the position of the precipitate at any time, preventing the precipitate from being decanted.

12 10mL of 70% ice-cold ethanol was added to thoroughly rinse the precipitate, and the precipitate was centrifuged at 12000rpm for 10 minutes. And (3) gently pouring out the supernatant, inverting the supernatant on absorbent paper, observing the position of the sediment at any time, preventing the sediment from being poured out, and finally marking the position of the sediment.

13 Placing the centrifuge tube in an open place in an ultra-clean workbench for about 10-20 minutes to fully volatilize the ethanol. 500. Mu.L of ultrapure water was added to the centrifuge tube, and the pellet was sufficiently dissolved by a pipette.

14 Transferring the dissolved solution into a clean 1.5mL centrifuge tube with marked, shaking and uniformly mixing, and sampling to detect the concentration and endotoxin level.

The experimental materials and experimental steps used for HEK293 transient expression are as follows:

experimental materials: shaking table, centrifuge, water bath, expi293 culture solution, transfection reagent, pipettes of various specifications, shake flasks of various specifications.

Reagent(s)	Manufacturer(s)	Numbering device
			293 culture solution	Baiying organism	BY20220701
293 transfection reagent	Baiying organism	BY20220701

The experimental steps are as follows:

1) And (5) culturing the cells.

2) Transient transfection and expression: 30 mL.

Solution 1: diluting 60 mug of plasmid with 1mL of culture solution, and uniformly mixing;

solution 2: diluting 15 mu L of transfection reagent with 1mL of culture solution, and uniformly mixing;

adding the solution 2 into the solution 1, uniformly mixing, incubating for 15 minutes at 37 ℃, dropwise adding the mixed transfection solution into the cell liquid, shaking while adding, placing into a shaking table for culturing, expressing for one week, collecting the supernatant, and centrifuging at 8000rpm for 5 minutes.

The experimental materials and experimental steps used for the antibody purification are as follows:

Experimental materials: stirrer, 1xPBS, imidazole, ni-Smart pre-packed column, centrifuge tubes of various specifications:

reagent(s)	Manufacturer(s)	Numbering device
			1xPBS	Baiying organism	BY20220807
150mM imidazole pH8.0	Baiying organism	BY20220801
			5mM imidazole pH8.0	Baiying organism	BY20220801

The experimental steps are as follows: ni-Smart affinity chromatography column purification

1) Equilibrium chromatographic column 1xPBS, flow rate 1mL/min,20mL

2) Loading: flow rate 1mL/min

3) Washing impurities with 1xPBS, flow rate of 1mL/min,20mL;5mM imidazole, flow 1mL/min

4) Eluting: 150mM imidazole, 1mL/min, collected in separate tubes, about 500uL per tube. A total of 10 tubes were collected and absorbance values at 280nm were read using a NanoDrop instrument.

5) And (3) dialysis: the high concentration protein was sucked into a dialysis bag and dialyzed in a beaker of 1 XPBS.

The experimental materials and experimental steps used for antibody basic quality control are as follows:

1) And (5) detecting concentration.

2) Purity detection (SDS-PAGE), the detection results are shown in FIG. 2.

3) Endotoxin detection:

experimental materials: vortex oscillator, electrothermal constant temperature incubator, endotoxin working standard, limulus reagent and endotoxin test water.

The experimental steps are as follows:

1) Sample positive control solution preparation: 2 times the concentration of the test solution and endotoxin standard (1.00 EU/ml) were mixed 1:1.

2) Preparing a test solution:

sample dilution factor: mvd=c·l/λ

* And (3) injection: MVD: maximum effective dilution of the test sample; l: test bacterial endotoxin limit (1 EU/mg)

C, the concentration of a test sample; lambda: the limulus reagent labeling sensitivity; 110uL sample fluid = C/MVD 110uL.

Limulus reagent (0.25 EU/mL)	Water for endotoxin test	Sample of
			Negative control tube	200uL	Without any means for
Positive control tube	100uL	100uL endotoxin standard (0.50 EU/mL)
			Sample positive control tube	100uL	100uL sample positive control solution
Sample tube	100uL	100uL test solution

Closing the tube orifice, shaking gently, vertically placing into a 37 ℃ constant temperature incubator, and incubating for 60min

Experimental results: the limulus reagent state is clear, transparent and non-clotting.

Conclusion of experiment: by endotoxin detection, the result is<1EU/mgMeets the requirements.

From the above results, it was found that the anti-PD-L1 specific high affinity nanobody WW102 of the present example was well expressed in HEK293 cells with a soluble expression level of 29.4-39.5mg/L. SDS-PAGE showed that WW102 was more than 95% pure (FIG. 2). The affinity of WW102 with human recombinant PD-L1 protein (PD 1-H5229, ACRO Biosystems Group) was very high, 15.29pM (fig. 3), significantly higher than the affinity of the same class of nanobody as determined by Biacore.

Likewise, it was also confirmed that other anti-PD-L1 specific affinity nanobodies WW101 and WW103-108 of the invention also have high affinity for human recombinant PD-L1 protein.

Example 2: ⁶⁸ ga-marked PD-L1 specific nano antibody probe ⁶⁸ Ga]Preparation of Ga-NOTA-WW102 (schematic diagram shown in FIG. 1)

1mg of WW102 was dissolved in 1mL of Phosphate Buffer (PBS) and treated with 0.1mL of 0.1M sodium carbonate (Na ₂ CO ₃ Ph=11.4) buffer solution the nanobody solution PH was adjusted to 9.0-10, the reaction system volume was 1.1mL. P-SCN-Bn-NOTA (CAS Number:147597-66-8; macromolecules) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the nanobody solution at a molar ratio of p-SCN-Bn-NOTA/WW102 of 10:1. The reaction system is placed at room temperature for reaction for 2 hours, PBS is used as a mobile phase, a pre-balanced PD-10 desalting column (GE Healthcare) is used for purifying the nano antibody modified by p-SCN-Bn-NOTA, and NOTA-WW102 is collected; concentrating with ultrafiltration tube (Merck Millipore) with cutoff value of 10kDa, measuring NOTA-WW102 concentration with NanoDrop, and packaging at-20deg.C. After random coupling with the bifunctional chelating agent p-SCN-Bn-NOTA (B-605, 147597-66-8; macrocycle) by Biacore assay affinity, WW102 had an affinity of 47.23pM for PD-L1 (FIG. 4).

Germanium gallium generator (Eckert) was rinsed with 4mL of 0.05M hydrochloric acid solution (HCl)&Ziegler Radiopharma Inc), and collecting equivalent volume activity of about 370-555MBq ⁶⁸ Ga leaches; middle section with highest activity ⁶⁸ Ga leacheate 2mL, added with 0.1mL1M sodium acetate solution (NaoAc) to regulate ⁶⁸ The pH value of the Ga leacheate is 4.0-4.5; adding the coupled NOTA-WW102 100-200 μg to the mixture ⁶⁸ Ga leacheate, reaction system volume<2.5mL; placing the reaction system in a constant temperature oscillator to react for 5-10 minutes at room temperature; after the labeling reaction, PBS was used as a mobile phase to allowSeparation of free with pre-equilibrated PD-10 desalting column ⁶⁸ Ga. Purifying the final product; the unattenuated corrected radiochemical yield (Radiochemical yield, RCY) was obtained according to the procedure described above>50％。

[ ⁶⁸ Ga]The Ga-NOTA-WW102 quality control steps are as follows: suction 10 mu L [ ⁶⁸ Ga]Ga-NOTA-WW102 was spotted on a silica gel plate using a 0.1M sodium citrate solution (pH=5) as a mobile phase, and was purified by a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) determination of the radiochemical purity of the probes (Radiochemical purity, RCP), probes prepared according to the invention [ ⁶⁸ Ga]The radiochemical purity of Ga-NOTA-WW102 was greater than 99% (FIG. 5).

Example 3: [ ⁶⁸ Ga]Ga-NOTA-WW102 immune PET imaging through noninvasive visualization PD-L1 expression diagnosis colorectal cancer related to the research ⁶⁸ The PET/CT imaging acquisition of the Ga-labeled probe was all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Each subcutaneous RKO tumor Balb/c nude mice was successfully prepared by tail vein injection of 3.7-7.4MBq [ ⁶⁸ Ga]Ga-NOTA-WW102, 1 hour after injection, anesthetize mice with isoflurane mixed with oxygen (concentration of 2%), place the mice into deep anesthesia state on PET/CT scanning bed in supine position, continuously acquire PET and CT images, complete image reconstruction with IRIS system self-contained software, as shown in FIG. 6 [ ⁶⁸ Ga]Ga-NOTA-WW102 probe is mainly excreted through kidney, liver has small amount of uptake, and the tumor site is obviously enriched by the probe. As shown in the left half of fig. 7, the region of interest (Region of interest, ROI) of tumor and major tissue organs (heart, liver, lung, kidney, muscle) was delineated on the reconstructed PET image using an OsiriX Lite image processing workstation (Pixmeo SARL), and the radioactive uptake value was calculated in% ID/g (percent of injected dose per gram) unit to quantitatively analyze the pharmacokinetics of the probe in vivo, and it can be seen that uptake of tumor sites was only lower than that of kidney and liver. After PET/CT imaging is finished, the mice are killed by adopting a cervical dislocation method, tumors and other tissue organs of the mice are collected, a gamma counter is used for carrying out radioactive counting, in-vitro biodistribution experiments are carried out, and the results and PET/CT imaging results are obtained And consistent ROI results, there was more pronounced uptake at the tumor site, as shown in the right half of fig. 7. The above results indicate that [ ⁶⁸ Ga]Ga-NOTA-WW102 probe can noninvasively visualize the PD-L1 expression condition inside tumor.

Thus, this example demonstrates the invention ⁶⁸ Ga-marked PD-L1 specific nano antibody probe ⁶⁸ Ga]Ga-NOTA-WW102 can noninvasively and accurately detect the heterogeneous expression of PD-L1 in an RKO model (PD-L1 expression positive).

Likewise, other anti-PD-L1 specific affinity nanobodies of the invention have been prepared using the methods described above ⁶⁸ Ga-labeled probes, i.e. [ ⁶⁸ Ga]Ga-NOTA-WW101 and [ ⁶⁸ Ga]Ga-NOTA-WW103-108, and confirm that the heterogeneous expression of PD-L1 can be detected noninvasively and accurately.

Example 4: preparation of fusion protein ABDWW102 of PD-L1 specific nano antibody

The preparation method of the fusion protein ABDWW102 of the PD-L1 specific nanobody is the same as the preparation method of WW102 described in example 1, but the expression system is 200mL. The expression results were as follows:

1) And (5) detecting concentration.

2) Purity detection (SDS-PAGE), the detection results are shown in FIG. 8.

3) Endotoxin detection results were as follows:

closing a pipe orifice, shaking gently, vertically placing into a constant temperature incubator at 37 ℃ for incubation for 60min;

Experimental results: the limulus reagent state is clear, transparent and non-clotting.

Conclusion of experiment: the endotoxin detection result is <1EU/mg, which is satisfactory.

From the above results, the nanobody fusion protein ABDWW102 of the example was well expressed in HEK293 cells with a soluble expression level of 75.9-76.6mg/L. SDS-PAGE showed that WW102 was more than 95% pure (FIG. 8). By Biacore assay affinity, abddww 102 was very high with human recombinant PD-L1 protein (PDL 1-H5229, ACRO Biosystems Group), 3.71pM (fig. 9).

Further, nanobody fusion protein abdw 102 was able to bind not only to human PD-L1 with high affinity, but also to Human Serum Albumin (HSA) and Murine Serum Albumin (MSA) with high affinity (fig. 12). Biacore affinity assay showed that the affinity of ABDWW102 to HSA and MSA was 3.38pM and 52.74pM, respectively (FIG. 13). From the above data, nanobody fusion protein abddww 102 has significantly higher affinity for HSA than MSA.

Likewise, other fusion proteins of anti-PD-L1 specific affinity nanobodies of the invention, namely ABDWW101 and ABDWW103-108, were also prepared using the methods described above and demonstrated similar effects as ABDWW 102.

Example 5: ⁶⁸ ga-marked PD-L1 specific nano antibody fusion protein probe ⁶⁸ Ga]Preparation of Ga-NOTAN-aBDWW 102

1mg of ABDWW102 was dissolved in 1mL of Phosphate Buffer (PBS) and treated with 0.1mL of 0.1M sodium carbonate (Na ₂ CO ₃ Ph=11.4) buffer solution the nanobody solution PH was adjusted to 9.0-10, the reaction system volume was 1.1mL. The p-SCN-Bn-NOTA (CAS Number:147597-66-8; macromolecules) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the nanobody fusion protein solution at a molar ratio of p-SCN-Bn-NOTA/ABDWW102 of 10:1. The reaction system is placed at room temperature for reaction for 2 hours, PBS is used as a mobile phase, a pre-balanced PD-10 desalting column (GE Healthcare) is used for purifying the nano antibody modified by p-SCN-Bn-NOTA, and NOTA-aBDWW102 is collected; concentrating with ultrafiltration tube (Merck Millipore) with cutoff value of 10kDa, measuring NOTA-aBDWW102 concentration with NanoDrop, and packaging at-20deg.C. Affinity was determined by Biacore, in combination with the double columnAfter random coupling of the functional chelator p-SCN-Bn-NOTA (B-605, 147597-66-8; microcycles), the affinity of ABDWW102 for PD-L1 was 79.51pM (FIG. 10), and after random coupling with the bifunctional chelator p-SCN-Bn-deferoxamine (B-705,1222468-90-7; microcycles), the affinity was 105.6pM (FIG. 11).

[ ⁶⁸ Ga]The preparation method and quality control method of Ga-NOTAN-aBDWW 102 probe are as described in example 2 ⁶⁸ Ga]The Ga-NOTA-WW102 probe method is consistent. FIG. 14 shows the produced [ ⁶⁸ Ga]The in vitro radiochemical purity of the Ga-NOTA-aBDWW102 probe is as high as 99%.

Example 6: [ ⁶⁸ Ga]Noninvasive visualization of Ga-NOTA-aBDWW102 through immune PET imaging for PD-L1 expression diagnosis of colorectal cancer

Successful preparation of 3.7-7.4MBq [ ⁶⁸ Ga]Ga-NOTA-aBDWW102 probe is injected into a subcutaneous RKO tumor Balb/c nude mouse through tail vein, PET/CT imaging is carried out at 0.5 hour, 2 hours, 4 hours and 6 hours after injection, and the image acquisition mode is consistent with the above. As shown in fig. 15, with increasing time, uptake of the probe at the tumor site gradually increased and probe enrichment of the heart and kidneys gradually decreased. The analysis of the ROI data in the left half of FIG. 16 confirms the results observed with PET/CT imaging, with a significant difference in uptake values of tumor site probes between 0.5 and 6 hours by statistical analysis (P<0.05). In vitro biodistribution experiments were performed after 6 hours of imaging, and the results showed higher uptake at the tumor sites.

Therefore, this example demonstrates [ ⁶⁸ Ga]Ga-NOTA-aBDWW102 can effectively visualize the expression level of RKO tumor PD-L1.

Likewise, other anti-PD-L1 specific affinity nanobody fusion proteins of the invention were also prepared using the methods described above ⁶⁸ Ga-labeled probes, i.e. [ ⁶⁸ Ga]Ga-NOTA-aBDWW101 and [ ⁶⁸ Ga]Ga-NOTA-aBDWW103-108 and confirm that it can also effectively visualize the expression level of RKO tumor PD-L1.

However, from the ROI and biodistribution data, it can be seen that at the 6 hour time point, there is still more probe in the circulation, indicating that at 6 hours the probe is not in vivoReach equilibrium, visible positron nuclides ⁶⁸ Ga cannot be completely matched with the nanobody fusion protein ABDWW102 in the systemic circulation time, so that the systemic circulation time and targeting performance of the nanobody fusion protein cannot be effectively evaluated. To this end, the invention further prepares a long half-life positive electron nucleus ⁶⁴ Cu]Cu-NOTA-ABDWW102。

Example 7: ⁶⁴ cu marked PD-L1 specific nano antibody fusion protein probe ⁶⁴ Cu]Preparation of Cu-NOTA-aBDWW102

The precursors used for this probe were as described in example 5, ⁶⁴ the Cu marking steps are as follows: production using solid targets ⁶⁴ Cu, then 259-296MBq was collected ⁶⁴ Cu nuclide, adding 300 mu L of 250mmol/L ammonium acetate, regulating the pH to about 5.5, then adding about 500 mu g of precursor, namely NOTA-aBDWW102, and placing the reaction system in a constant temperature oscillator to react for 30 minutes at 42 ℃; after the labeling reaction, PBS was used as a mobile phase, and a pre-equilibrated PD-10 desalting column was used to separate the free phase ⁶⁴ Cu, purifying the final product.

[ ⁶⁴ Cu]The Cu-NOTA-aBDWW102 quality control steps are as follows: suction 10 mu L [ ⁶⁴ Cu]Cu-NOTAN-aBDWW 102 was spotted on a silica gel plate using 50mmol/L EDTA (pH=5) as a mobile phase, and a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) determination of the radiochemical purity of the probes (Radiochemical purity, RCP), probes prepared according to the invention [ ⁶⁴ Cu]The radiochemical purity of Cu-NOTA-aBDWW102 meets the requirement of in vivo imaging.

Example 8: [ ⁶⁴ Cu]Cu-NOTA-aBDWW102 immune PET imaging noninvasive visual PD-L1 expression diagnosis colorectal cancer

Successful preparation of 3.7-7.4MBq [ ⁶⁴ Cu]The Cu-NOTA-aBDWW102 probe was injected into subcutaneous RKO tumor Balb/c nude mice via tail vein and PET/CT visualizations were performed at 1 hour, 4 hours, 24 hours and 48 hours, respectively, after injection. As shown in fig. 17, with increasing time, the uptake of the probe at the tumor site gradually increased, and the probe enrichment of the heart and kidney gradually decreased, and at the time point of 48 hours, it can be seen that the uptake value at the tumor site was significantly higher than that at the heart and kidney, etc. FIG. 18Biodistribution data confirm the results observed with PET/CT imaging. Using ⁶⁴ The Cu-marked nanobody fusion protein ABDWW102 has definite complete pharmacokinetics in vivo, and fully proves the obviously prolonged in-vivo circulation time and the visualization capability for tumor PD-L1 expression. The significantly prolonged circulation time in the nanometer antibody fusion protein ABDWW102 body indicates that the nanometer antibody fusion protein ABDWW102 can be used as a treatment carrier to realize the antibody coupling drug treatment or the radioimmunotherapy targeting PD-L1.

Likewise, other anti-PD-L1 specific affinity nanobody fusion proteins of the invention were also prepared using the methods described above ⁶⁴ Cu-labeled probes, i.e. [ ⁶⁴ Cu]Ga-NOTA-aBDWW101 and [ ⁶⁴ Cu]Ga-NOTAN-aBDWW 103-108 and confirmed to have the same meaning as [ ⁶⁴ Cu]Cu-NOTA-aBDWW 102.

Thus, the present invention has confirmed that the prepared PD-L1 specific nanobody probe [ ⁶⁸ Ga]Ga-NOTA-WW101-108, and two nanobody fusion protein probes ⁶⁸ Ga]Ga-NOTAN-aBDWW 101-108 and [ ⁶⁴ Cu]Cu-NOTA-aBDWW101-108 can be used for noninvasively, accurately and efficiently detecting the expression of human PD-L1.

It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are not to be construed as additional limitations of the invention, but are provided for a more thorough understanding of the present invention. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.

Claims (10)

1. A PD-L1-specific nanobody, comprising:

(1) CDR1 having the amino acid sequence shown in SEQ ID No.1, CDR2 having the amino acid sequence shown in SEQ ID No.2 and CDR3 having the amino acid sequence shown in SEQ ID No.3,

(2) CDR1 having the amino acid sequence shown in SEQ ID No.8, CDR2 having the amino acid sequence shown in SEQ ID No.9 and CDR3 having the amino acid sequence shown in SEQ ID No.10,

(3) CDR1 having the amino acid sequence shown in SEQ ID No.13, CDR2 having the amino acid sequence shown in SEQ ID No.14 and CDR3 having the amino acid sequence shown in SEQ ID No.15,

(4) CDR1 having the amino acid sequence shown in SEQ ID No.39, CDR2 having the amino acid sequence shown in SEQ ID No.19 and CDR3 having the amino acid sequence shown in SEQ ID No.20,

(5) CDR1 having the amino acid sequence shown in SEQ ID No.23, CDR2 having the amino acid sequence shown in SEQ ID No.24 and CDR3 having the amino acid sequence shown in SEQ ID No.25,

(6) CDR1 having the amino acid sequence shown in SEQ ID No.28, CDR2 having the amino acid sequence shown in SEQ ID No.29, and CDR3 having the amino acid sequence shown in SEQ ID No.30, or

(7) CDR1 having the amino acid sequence shown in SEQ ID No.33, CDR2 having the amino acid sequence shown in SEQ ID No.34 and CDR3 having the amino acid sequence shown in SEQ ID No.35,

Preferably, the PD-L1 specific nanobody has the amino acid sequence shown in SEQ ID No.4, 6, 11, 16, 21, 26, 31 or 36.

2. A PD-L1 specific nanobody fusion protein comprising the nanobody according to claim 1 and a moiety for extending the half-life in vivo,

preferably, the moiety for extending the in vivo half-life comprises serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin or a polyethylene glycol-liposome complex,

preferably, the PD-L1 specific nanobody fusion protein has an amino acid sequence shown in SEQ ID No. 38.

3. A polynucleotide encoding the PD-L1-specific nanobody according to claim 1 or the PD-L1-specific nanobody fusion protein according to claim 2,

preferably, the polynucleotide has the nucleotide sequence shown as SEQ ID No.5, 7, 12, 17, 22, 27, 32, 37 or 39.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4.

6. A PD-L1 specific molecular imaging probe comprising a radionuclide-labeled PD-L1 specific nanobody according to claim 1 or a PD-L1 specific nanobody fusion protein according to claim 2.

7. The molecular imaging probe of claim 6, wherein the PD-L1 specific nanobody according to claim 1 or the PD-L1 specific nanobody fusion protein according to claim 2 is labeled with a radionuclide via a bifunctional chelator,

preferably, the bifunctional chelating agent is selected from p-SCN-Bn-NOTA or p-SCN-Bn-deferoxamine.

8. The molecular imaging probe of claim 6 or 7, wherein the radionuclide is selected from Tc-99m, ga-68, F-18, I-123, I-125, I-131, I-124, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, Y-86, mn-52, sc-44, lu-177, Y-90, ac-225, at-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra-188, ru-106, na-24, sr-89, tb-149, th-227, yb-133, yb-169, or Yb-177,

preferably, the radionuclide is selected from Ga-68,

preferably, the PD-L1-specific nanobody fusion protein according to claim 2 is Cu-64 labeled.

9. A kit or composition for detecting the expression level of PD-L1, diagnosing a PD-L1-associated tumor, predicting the therapeutic effect of a PD-L1-associated tumor, or treating a PD-L1-associated tumor, comprising a PD-L1-specific nanobody according to claim 1, a PD-L1-specific nanobody fusion protein according to claim 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5, or a molecular imaging probe according to any one of claims 6-8.

10. Use of a PD-L1-specific nanobody according to claim 1, a PD-L1-specific nanobody fusion protein according to claim 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5 or a molecular imaging probe according to any one of claims 6-8 in the preparation of a kit or medicament for detecting the expression level of PD-L1, diagnosing a PD-L1-related tumor, predicting the therapeutic effect of a PD-L1-related tumor or treating a PD-L1-related tumor.