CN116655793A

CN116655793A - Preparation method and application of BCMA (bcmA-specific diagnosis and treatment) integrated molecular imaging probe

Info

Publication number: CN116655793A
Application number: CN202310616779.9A
Authority: CN
Inventors: 魏伟军; 潘心冰; 刘建军
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-05-29
Filing date: 2023-05-29
Publication date: 2023-08-29

Abstract

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 BCMA (BCMA specific diagnosis and treatment) integrated molecular imaging probe. The BCMA specific immune PET imaging probe prepared by the invention has the advantages of simple preparation process, low cost, high specificity, high stability, easy clinical transformation and the like. The clinical transformation application is applied, the noninvasive visualization of BCMA heterogeneous expression is hopeful to be realized, and the patients suitable for BCMA specific targeted therapy, immunotherapy or nanobody drug coupling therapy are further screened, so that the clinical significance is remarkable.

Description

Preparation method and application of BCMA (bcmA-specific diagnosis and treatment) integrated molecular imaging 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 BCMA (BCMA specific diagnosis and treatment) integrated molecular imaging probe.

Background

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.;) J nucleic Med.2022Oct;63 (10):1705-1709.). Currently, a variety of short half-life nuclides have been used to label nanobodies and prepare nanobody molecular imaging probes. Nanobody probes labeled with technetium-99 m (99 mtc; t1/2=6.02 h) targeting programmed death ligand 1 (PD-L1) have been successfully transformed into clinic for non-invasive diagnosis of non-small cell lung cancer patients (J nucleic med.2019;60 (9): 1213-1220.); gallium-68 (68 ga; t1/2=1.1h) labeled nanobody probes targeting human epidermal growth factor receptor (HER 2) have also been successfully transformed into clinic for non-invasive diagnosis of breast cancer (J nucleic med.2016;57 (1): 27-33.). The above examples illustrate that the radionuclide-labeled nanobody probe has great clinical transformation application prospect, and can be used for early noninvasive diagnosis of human malignant tumor, visualization of key pathogenic targets, screening of patients treated by monoclonal antibodies (mAbs) and evaluation of curative effects after monoclonal antibody treatment.

B Cell Maturation Antigen (BCMA) also known asTumor necrosis factor receptor superfamily member 17 (TNFRSF 17), a cell surface receptor for tumor necrosis factor receptor superfamily member 13B (TNFSF 13B/BAFF), is mainly expressed in mature B cells, and after binding to BAFF, induces activation of NF-kappaB and MAPK8/JNK signaling pathways, and regulates B cell proliferation, survival and differentiation. Recent evidence suggests that BCMA expression is associated with a variety of hematological malignancies, suggesting that BCMA may serve as a biomarker and an important therapeutic target in a variety of hematological malignancies. In Multiple Myeloma (MM) patients, malignant plasma cell surface BCMA expression is significantly higher than normal plasma cells and other bone marrow cells, and cell surface BCMA high expression is associated with short progression free survival and total survival, suggesting that BCMA has independent prognostic value in multiple myeloma. BCMA expression is also found in partial lymphomas and leukemias such as B lymphoblastic leukemia. The difference in expression of BCMA in normal tissues and tumors makes it a very potential tumor specific marker and can avoid potential side effects. Current common therapeutic modalities for BCMA include bispecific antibodies such as (bispecific T-cell engager) immune tumor therapy, antibody-drug conjugates (ADCs) and chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cell therapies, and have been partially entered into clinical research. Thus, there is an urgent need to develop a diagnostic tool that targets BCMA to enable visualization and monitoring of BCMA expression in hematological malignancies. On the basis of research with diagnostic tools, new therapeutic approaches to BCMA can be further developed.

Early series of basic and clinical studies by the applicant showed that by subtly fusing the extraordinary targeting specificity of antibodies with the superior sensitivity and resolution of Positron Emission Tomography (PET), immunopet can better show the distribution and abundance of targets of interest in vivo, particularly heterogeneous expression, and better predict response to targeted or immunotherapy (Chem rev.2020;120 (8): 3787-3851.) compared to Immunohistochemical staining (IHC) or other conventional predictive markers. For example, the value of immune PET imaging probes targeting HER 2 in breast cancer has been clinically demonstrated. Based on the evidence above and our previous findings, we hypothesize that BCMA-targeted immune PET imaging probes can non-invasively display intratumoral BCMA expression and provide a better approach for diagnosis and monitoring of BCMA positive hematologic tumors. Furthermore, there has been evidence that Radioimmunotherapy (RIT) and pretargeted radioimmunotherapy (pr it) may help tumor patients alleviate the condition for a long period of time, even eradicating multiple cancer types.

At present, no BCMA specific molecular imaging probes or nuclide labeled diagnosis and treatment integrated probes are reported in clinical practice and literature report. The use of radiolabeled monoclonal antibodies is severely hampered by the high cost, necessity of using long half-life radionuclides, cumbersome imaging procedures within a week, and associated radiation exposure. In order to improve the clinical application of antibody diagnostics, the molecular imaging field is actively exploring pretargeting imaging strategies or using smaller molecular weight antibody derivatives to achieve the current day molecular imaging (same-day imaging). In the small antibody format, the nanobody or single domain antibody from the family camelidae is the smallest antigen binding portion with a molecular weight of about 15 kDa. The small size, high affinity and ease of engineering makes nanobodies an excellent alternative to molecular imaging (J nucleic Med.2022Oct;63 (10): 1705-1709.). In recent years, we have focused on the development and clinical transformation of nanobody-derived tracers to exert their superior molecular imaging properties. Although radiolabeled monovalent nanobodies are ideal companion diagnostic tools, the half-life in vivo is too short and the renal uptake is high, leaving room for further improvement. To develop an integrated diagnostic therapeutic platform, the introduction of Albumin Binding Domains (ABD) targeting human/murine albumin into monovalent nanobodies significantly prolongs the half-life of monovalent nanobody derivatives in vivo, further optimizing the pharmacokinetics and pharmacodynamics of molecular imaging probes. Research shows that the bispecific nanometer antibody derivative targeting tumor antigen and albumin simultaneously improves in vivo biodistribution and can be used as a carrier for developing a therapeutic and diagnostic kit.

At present, no BCMA specific molecular imaging probes or nuclide labeled diagnosis and treatment integrated probes are reported in clinical practice and literature report. However, the clinical transformation application of the monoclonal antibody immune PET imaging probe is severely limited by the reasons of high preparation cost, large molecular weight, long in vivo circulation time, long imaging period, high radiation dose, large toxic and side effects and the like.

Therefore, those skilled in the art are working to develop a nanobody immune PET imaging probe which has low preparation cost, small molecular weight, short in vivo circulation time, short imaging period, low radiation dose and easy clinical transformation application.

Disclosure of Invention

To fill the gap in this field, we describe herein the construction of nanobody-derived BCMA targeting diagnostic pairs and characterize their diagnostic and therapeutic value in cell-derived xenograft (CDX) models. The invention aims to construct a BCMA specificity diagnosis and treatment integrated molecular image probe, noninvasively displays the expression of BCMA in tumors, and provides a better method for diagnosing and monitoring BCMA positive solid tumors. On this basis, new therapeutic approaches targeting BCMA are to be further developed. The invention realizes noninvasive visualization of human BCMA molecular expression, and further realizes noninvasive diagnosis of blood tumor, and the probe 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.

BCMA specific nanobodies

In one aspect, the present invention provides a BCMA 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.6, CDR2 having the amino acid sequence shown in SEQ ID No.7 and CDR3 having the amino acid sequence shown in SEQ ID No.8,

(3) CDR1 having the amino acid sequence shown in SEQ ID No.11, CDR2 having the amino acid sequence shown in SEQ ID No.12 and CDR3 having the amino acid sequence shown in SEQ ID No.13,

(4) CDR1 having the amino acid sequence shown in SEQ ID No.16, CDR2 having the amino acid sequence shown in SEQ ID No.17 and CDR3 having the amino acid sequence shown in SEQ ID No.18,

(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 BCMA specific nanobody of the present invention has the amino acid sequence shown in SEQ ID No.4, 9, 14, 19, 21, 26, 31 or 36.

In the present invention, BCMA-specific nanobodies having the amino acid sequence shown in SEQ ID nos. 4, 9, 14, 19, 21, 26, 31, or 36 are referred to as MMBC1, MMBC2, MMBC3, MMBC6, MMBC7, MMBC8, MMBC9, or MMBC10, respectively, for simplicity.

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 BCMA 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 "BCMA specific" refers to specifically binding BCMA.

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. One method involves measuring the 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). Can be any ofEfficient method for measuring K _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 BCMA-specific nanobodies 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 shown in SEQ ID nos. 4, 9, 14, 19, 21, 26, 31 or 36 and substantially retain the biological function of the nanobody from which they are derived (e.g., the biological activity of specifically binding BCMA).

More specifically, the variants differ from BCMA-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).

BCMA-specific nanobody fusion proteins

In another aspect, the present invention provides a BCMA-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 BCMA-specific nanobody fusion protein provided by the present invention, the nanobody and the moiety for extending the 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.

Specifically, the connecting peptide is (G4S) _n ，n＝1,2,3,4,5,6,7,8,9,10。

In a specific embodiment, the present invention provides a BCMA specific nanobody fusion protein comprising a nanobody as described herein and an albumin binding domain.

More specifically, the albumin binding domain is a serum protein binding domain ABD035, having the amino acid sequence shown in SEQ ID No. 38.

In a specific embodiment, the present invention provides a BCMA specific nanobody fusion protein having an amino acid sequence shown as SEQ ID No.39, SEQ ID No.41, SEQ ID No.43, SEQ ID No.45, SEQ ID No.47, SEQ ID No.49, SEQ ID No.51, or SEQ ID No. 53.

In the present invention, BCMA-specific nanobody fusion proteins having the aforementioned amino acid sequences are respectively referred to as ABDMMBC1, ABDMMBC2, ABDMMBC3, ABDMMBC6, ABDMMBC7, ABDMMBC8, ABDMMBC9, or ABDMMBC10 in order for simplicity.

In a specific embodiment, the invention also provides a BCMA specific nanobody fusion protein having the amino acid sequence shown in SEQ id No. 55.

In the present invention, for the sake of simplicity, BCMA-specific nanobody fusion proteins having the amino acid sequences shown above are referred to as ABDMMBC2-cys.

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 set forth in SEQ ID No.5, 10, 15, 20, 22, 27, 32, 37, 40, 42, 44, 46, 48, 50, 52, 54 or 56.

More specifically, the polynucleotide encoding a BCMA specific nanobody as described herein has the nucleotide sequence shown in SEQ ID No.5, 10, 15, 20, 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 nos. 40, 42, 44, 46, 48, 50, 52, 54, or 56.

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 BCMA specific molecular imaging probe, as described in more detail below.

BCMA specific molecular imaging probe

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

More specifically, BCMA-specific nanobodies or BCMA-specific nanobody fusion proteins as described herein are 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 the group consisting of p-SCN-Bn-NOTA.

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

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, BCMA-specific nanobodies as described herein are labeled with Ga-68 (examples of one such BCMA-specific molecular imaging probe are described in the examples ⁶⁸ Ga]Ga-NOTA-MMBC2)。

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

BCMA-specific nanobody drug conjugates

In another aspect, the present invention provides a BCMA-specific nanobody drug conjugate comprising a BCMA-specific nanobody fusion protein and a cytotoxic drug.

As used herein, cytotoxic drugs are a class of drugs that are effective in killing immune cells and inhibiting their proliferation. Such as MMAF, MMAE, SN-38, irinotecan, etc.

Preferably, the cytotoxic drug is selected from SN38, MMAE.

As used herein, the MMAF is monomethyl auristatin F;

the SN38 is 7-ethyl-10-hydroxycamptothecin;

the MMAE is monomethyl auristatin E.

More specifically, BCMA-specific nanobody drug conjugates as described herein are conjugates of nanobody fusion protein ABDMMBC2 with MMAE, referred to as ABDMMBC2-MMAE; or a conjugate of the nanobody fusion protein ABDMMBC2-cys and SN38, which is called ABDMMBC2-cys-SN38.

Composition and method for producing the same

In another aspect, the invention provides a composition comprising a BCMA specific nanobody, BCMA specific nanobody fusion protein, polynucleotide, vector, host cell, or molecular imaging probe as described herein. The compositions are useful for detecting expression levels of BCMA, diagnosing BCMA-related tumors, predicting the therapeutic effect of BCMA-related tumors, or treating BCMA-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, a BCMA specific nanobody, BCMA 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 BCMA specific nanobody, BCMA 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 BCMA specific nanobody, BCMA 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 BCMA specific nanobody, BCMA specific nanobody fusion protein, polynucleotide, vector, host cell, or molecular imaging probe as described herein.

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

Particularly, the BCMA specific molecular image probe prepared by the BCMA 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 BCMA, so that the BCMA specific molecular image probe is particularly suitable for diagnosing BCMA-related tumors and predicting the therapeutic effect of BCMA-related tumors. After proper radionuclides are selected for coupling, the method can also be used for accurately treating BCMA related tumors.

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

As used herein, BCMA-related tumors can include various tumors or cancers well known in the art. For example, BCMA-related tumors may include hematological tumors, such as leukemia, bone cancer, lymphoma, and the like.

The beneficial effects of the invention are that

BCMA-specific immunoPET imaging probes prepared according to the invention are described as follows ⁶⁸ Ga]Ga-NOTA-MMBC2 has the advantages of simple preparation process, low cost, high specificity, high stability, easy clinical transformation and the like. The clinical transformation application is applied, the noninvasive visualization of BCMA heterogeneous expression is hopeful to be realized, and the patients suitable for BCMA specific targeted therapy, immunotherapy or nanobody drug coupling therapy are further screened, so that the clinical significance is remarkable.

Drawings

FIG. 1 shows the results of SDS-PAGE determination of nanobody MMBC1, MMBC2 and MMBC3 expression;

FIG. 2 shows the result of SDS-PAGE determination of nanobody fusion protein ABDMMBC2 expression;

FIG. 3 shows immunohistochemical staining results of MM1S tumor model BCMA;

FIG. 4 shows the results of affinity assays of nanobodies MMBC1, MMBC2 and MMBC3 and nanobody fusion protein ABDMMBC2 with human BCMA;

FIG. 5 shows the results of an affinity assay of nanobody fusion protein ABDMMBC2 with human serum albumin and mouse serum albumin;

FIG. 6 shows BCMA-specific molecular imaging probes of the invention [ ⁶⁸ Ga]Radiochemical purity measurement results of Ga-NOTA-MMBC 2;

FIG. 7 shows[ ⁶⁸ Ga]PET and CT experimental results of Ga-NOTA-MMBC2 immune PET imaging diagnosis of multiple myeloma;

FIG. 8 shows [ sic ] ⁶⁸ Ga]ROI analysis chart of Ga-NOTA-MMBC2 immune PET imaging diagnosis multiple myeloma;

FIG. 9 shows immunohistochemical staining results for MM.1S tumor sections;

FIG. 10 shows the results of the determination of ABDMMBC2-MMAE concentration and UV absorbance spectra using NanoDrop.

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 BCMA specific nano antibody and fusion protein thereof

According to the method previously published by the inventors (refer to patent No. ZL202011131233.7 entitled "molecular imaging probe for diagnosing multiple myeloma," which is incorporated herein by reference in its entirety), novel BCDA-specific monovalent nanobodies MMBC1 (SEQ ID No. 4), MMBC2 (SEQ ID No. 9), MMBC3 (SEQ ID No. 14), MMBC6 (SEQ ID No. 19), MMBC7 (SEQ ID No. 21), MMBC8 (SEQ ID No. 26), MMBC9 (SEQ ID No. 31) and MMBC10 (SEQ ID No. 36), nanobody fusion proteins ABDMMBC1 (SEQ ID NO. 39), ABDMMBC2 (SEQ ID NO. 41), ABDMMBC3 (SEQ ID NO. 43), ABDMMBC6 (SEQ ID NO. 45), ABDMMBC7 (SEQ ID NO. 47), ABDMMBC8 (SEQ ID NO. 49), ABDMMBC9 (SEQ ID NO. 51), ABDMMBC10 (SEQ ID NO. 53), ABDMMBC2-cys (SEQ ID NO. 55) were cloned into pET-30a (+) expression vectors and expressed recombinantly in E.coli (E.coli) respectively (SEQ ID NO.5, 10, 15, 20, 22, 27, 32, 37, 40, 42, 44, 46, 48, 50, 52, 54, 56).

SDS-PAGE determination of nanobody MMBC1, MMBC2 and MMBC3 expression is shown in FIG. 1; SDS-PAGE shows the expression of the nanobody fusion protein ABDMMBC2 as shown in FIG. 2.

Example 2: establishment of BCMA expression positive tumor-bearing mouse model

The MM1S tumor model BCMA expression was found positive by immunohistochemical staining with anti-human BCMA monoclonal antibody (No. 88183, cell Signaling Technology) as primary antibody, as shown in fig. 3. Will be 0.5X10 ⁶ The MM.1S tumor cells were inoculated into NCG (NOD-Prkdcem 26Cd52IL2rgem26Cd 22/Nju) mice via tail vein to establish MM.1S tumor implantation model.

Example 3: affinity assay for BCMA-specific nanobodies and fusion proteins thereof

The detection instrument is a Biacore T200 instrument (Cytiva). Human serum albumin, murine serum albumin and human BCMA were immobilized on CM5 chip surfaces by amine coupling at a flow rate of 10 μl per minute under 10mM sodium acetate buffer and different ph conditions (4.0 for human serum albumin, 5.0 for murine serum albumin, 5.5 for BCMA), and 50mM N-hydroxysuccinimide (NHS) and 200mM 1-methyl-3- (-dimethylaminopropyl) carbodiimide (EDC) mixture was injected for 7 minutes to activate the sensor surfaces. Then 10ug/ml of human serum albumin and murine serum albumin were injected to 500RU and the same concentration of human BCMA to 300RU. The sensor surface was blocked with 1M ethanolamine and ph adjusted to 8.5. A series of concentrations of MMBC1, MMBC2 and abdmbc 2 were injected into the flow system and analyzed for 120 seconds with dissociation set at 1200 seconds. After each concentration injection analysis, human serum albumin and murine serum albumin were regenerated with glycine-hydrogen chloride solution having a ph of 2.0 for 30 seconds and MMBC2 was regenerated with glycine-hydrogen chloride solution having a ph of 2.5 for 20 seconds. All binding assays were performed in Phosphate Buffered Saline (PBS) with a pH of 0.05% (v/v) Tween-20 of 7.4 and 25 ℃. Prior to analysis, the bulk refractive index variation, injection noise, and data drift were eliminated using a double reference subtraction. Binding affinity was determined by the Langmuir 1:1 binding model in Biacore Evaluation software (cytova, sweden). Nanobody Affinity assay of MMBC1, MMBC2 and MMBC3 with human BCMA as shown in FIG. 4, K _D Values of 1.460nM, 181.1pM and 289.5pM, respectively; affinity assay of nanobody fusion protein ABDMMBC2 and human BCMA as shown in FIG. 4, K _D A value of 88.17pM; the affinity measurement of the nanobody fusion protein ABDMMBC2 and human serum albumin and mouse serum albumin is shown in figure 5, K _D The values were 7.797pM and 112.6pM, respectively.

Example 4: preparation of BCMA specific molecular image probe

P-SCN-Bn-NOTA modified MMBC2 to prepare an intermediate NOTA-MMBC2. The method comprises the following specific steps: 1mg of MMBC2 was dissolved in 1mL of Phosphate Buffer (PBS), 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:170597-66-8; macromolecules) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the nanobody solution in a molar ratio of p-SCN-Bn-NOTA to nanobody=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 nanometer antibody modified by NOTA, and NOTA-MMBC2 is collected; concentrating with ultrafiltration tube (Merck Millipore) with cutoff value of 10kDa, measuring NOTA-MMBC2 concentration with NanoDrop, and packaging at-20deg.C.

⁶⁸ Ga-labeled NOTA-MMBC2 preparation ⁶⁸ Ga]Ga-NOTA-MMBC2. The method comprises the following specific steps: 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 0.1mL 1M sodium acetate solution (NaoAc) to regulate ⁶⁸ The pH value of the Ga leacheate is 4.0-4.5; 200 μg of NOTA-MMBC2 is added to the solution after coupling 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, and the pre-equilibrated PD-10 desalting column was used again to separate the free phase 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]Ga-NOTA-MMBC2 quality control. Suction 10 mu L [ ⁶⁸ Ga]Ga-NOTAMBC 2 is spotted on a silica gel plate using a 0.1M sodium citrate solution (pH=5) as the mobile phase, and is purified by a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) the radiochemical purity of the probe was determined (Radiochemical purity, RCP). Freshly prepared [ as shown in FIG. 6 ] ⁶⁸ Ga]Ga-NOTA-MMBC2 RCP is greater than 99%.

Example 5: [ ⁶⁸ Ga]Ga-NOTA-MMBC2 immune PET imaging diagnosis multiple myeloma

The PET/CT imaging acquisitions of the animals involved in this study were all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Each mouse was injected via the tail vein with 3.7-7.4MBq [ ⁶⁸ Ga]Ga-NOTA-MMBC2 (a group of 3), 1 hour after injection, the mice were anesthetized with isoflurane mixed with oxygen (concentration of 2%), and the mice put into deep anesthesia were placed in a supine position on a PET/CT scanning bed, PET and CT images were continuously acquired, and image reconstruction was accomplished with the IRIS system self-contained software, as shown in FIG. 7. The region of interest (Region of interest, ROI) of heart and major tissue organs (liver, lung, kidney, muscle) were delineated on the reconstructed PET image using an OsiriX Lite image processing workstation (Pixmeo SARL) (fig. 8). BCMA-specific nanobody probes can be seen [ ⁶⁸ Ga]Ga-NOTA-MMBC2 has higher uptake in tumor tissues and higher nonspecific uptake in major excretory (kidney) and metabolic (liver) tissues. Analysis by delineating ROI [ ⁶⁸ Ga]Distribution of Ga-NOTA-MMBC2 in vivo. The above results indicate that [ ⁶⁸ Ga]Ga-NOTA-MMBC2 probes can non-invasively visualize BCMA expression. Furthermore, immunohistochemical staining of tumors with BCMA-specific antibodies (No. 88183, cell Signaling Technology) confirmed the expression of BCMA inside the tumors, as shown in fig. 9.

Example 6: targeting of nano antibody fusion protein ABDMMBC2-cys group and cytotoxic drug SN38 site-directed labeling

2mg of SN38 was dissolved in 50uL of DMSO, and 100mg of TCEP was dissolved in 1ml of double distilled water to prepare a TECP preservative solution. 4mg of ABDMMBC2-cys was dissolved in 1mL of phosphate buffer [. Sup.PBS), molar ratio TECP nanobody (abdmbc 2-cys) =2: 1, TECP preservative solution was added to the ABDMMBC2-cys system, 0.1mL, 0.1M sodium carbonate (Na ₂ CO ₃ Ph=11.4) buffer the PH of the resulting nanobody solution was adjusted to 7.4 and the reaction was performed on a shaker at room temperature for 2 hours. SN38 freshly dissolved in dimethyl sulfoxide (DMSO) was added to the nanobody solution after the above reaction in a molar ratio SN-38:nanobody=10:1 ratio and reacted for 30 minutes in a shaker at room temperature. The reaction product (ABDMMBC 2-cys-SN 38) was purified by washing 4 times with PBS-AsA (4000 g centrifugation at 4 ℃) through an ultrafiltration tube (Merck Millipore) with a cut-off value of 10kDa, concentrated to a volume of less than 500uL, assayed using nanodrop, and sub-packaged for use at 4 ℃.

Example 7: random labelling of cytotoxic drug MMAE by targeting nanobody fusion protein ABDMMBC2 groups

ABDMMBC2 buffer was replaced with PBS and 1ml volume, 0.1ml 0.1m sodium carbonate (Na ₂ CO ₃ Ph=11.4) buffer the nanobody solution (solution of ABDMMBC2 with PBS) was PH adjusted to 9-10. MMAE freshly dissolved in dimethyl sulfoxide (DMSO) was added to the above described ABDMMBC2 solution in a molar ratio MMAE: nanobody=5:1, and reacted for 2 hours at room temperature with shaking. Then PBS is taken as a mobile phase, a pre-balanced PD-10 desalting column (GE Healthcare) is used for purifying the nano antibody fusion protein ABDMMBC2 coupled with the MMAE, the ABDMMBC2-MMAE is collected, the concentration of the ABDMMBC2-MMAE and the ultraviolet absorbable spectrum are measured by using a Nanodrop, and the result is shown in figure 10, and the split charging is carried out at 4 ℃ for standby.

Likewise, other anti-BCMA specific affinity nanobodies of the invention were also prepared using the methods described above ⁶⁸ Ga-labeled probes, e.g. [ ⁶⁸ Ga]Ga-NOTA-MMBC3、[ ⁶⁸ Ga]Ga-NOTA-MMBC6, etc., and confirm that it may also be non-invasively visualizable for BCMA expression.

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

1. A BCMA specific nanobody comprising:

(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 BCMA specific nanobody has the amino acid sequence shown in SEQ ID No.4, 9, 14, 19, 21, 26, 31 or 36.

2. A BCMA specific nanobody fusion protein comprising a nanobody according to claim 1 and an albumin binding domain,

preferably, the albumin binding domain has the amino acid sequence shown in SEQ ID No. 38;

preferably, the BCMA specific nanobody fusion protein has the amino acid sequence shown in SEQ ID No.39, 41, 43, 45, 47, 49, 51, 53, or 55.

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

preferably, the polynucleotide has the nucleotide sequence set forth in SEQ ID No.5, 10, 15, 20, 22, 27, 32, 37, 40, 42, 44, 46, 48, 50, 52, 54 or 56.

4. A vector comprising the polynucleotide of claim 3.

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

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

7. The molecular imaging probe of claim 6, wherein the BCMA-specific nanobody according to claim 1 or the BCMA-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 the group consisting of p-SCN-Bn-NOTA,

preferably, 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,

preferably, the radionuclide is selected from Ga-68.

8. A BCMA specific nano-antibody drug conjugate, which comprises the BCMA specific nano-antibody fusion protein and a cytotoxic drug according to claim 2,

preferably, the cytotoxic drug is selected from SN38, MMAE.

9. A kit or composition for visualizing BCMA expression, diagnosing BCMA-related tumors, predicting progression and prognosis of BCMA-related tumors, predicting therapeutic effect of BCMA-related tumors, or treating BCMA-related tumors, comprising a BCMA-specific nanobody according to claim 1, a BCMA-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, a molecular imaging probe according to any one of claims 6-7, or a BCMA-specific nanobody drug conjugate according to claim 8.

10. Use of a BCMA specific nanobody according to claim 1, a BCMA 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, a molecular imaging probe according to any one of claims 6-7 or a BCMA specific nanobody drug conjugate according to claim 8 in the preparation of a kit or composition for visualizing BCMA expression, diagnosing BCMA-related tumors, predicting progression and prognosis of BCMA-related tumors, predicting the therapeutic effect of BCMA-related tumors or treating BCMA-related tumors.