CN116120450A - Preparation and application of camelidae nanobody targeting human CD38 molecule - Google Patents

Preparation and application of camelidae nanobody targeting human CD38 molecule Download PDF

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CN116120450A
CN116120450A CN202111342220.9A CN202111342220A CN116120450A CN 116120450 A CN116120450 A CN 116120450A CN 202111342220 A CN202111342220 A CN 202111342220A CN 116120450 A CN116120450 A CN 116120450A
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nanobody
antibody
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任华
薛琪
钱旻
何苗壮
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East China Normal University
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Abstract

The invention provides preparation and application of camelid nanobody targeting human CD38 molecules. Specifically, the invention provides 5 camelidae-derived nanobodies targeting human CD38 molecules and applications thereof. In particular to a specific binding nano antibody sequence obtained by screening a camelid natural nano antibody phage display library aiming at the extracellular section of a human CD38 molecule, and a corresponding anti-human CD38 nano antibody protein escherichia coli expression system is constructed. The nano antibody has the advantages of small molecular weight, easy transformation, easy combined construction of bispecific antibody with other antibodies, easy construction of CAR-T, easy preparation of conjugate with other medicines, and the like.

Description

Preparation and application of camelidae nanobody targeting human CD38 molecule
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceuticals, and discloses preparation and application of camelid nanobody targeting human CD38 molecules.
Background
CD38, also known as ADP ribose cyclase or cyclo ADP ribose hydrolase, has a molecular weight of 45kDa, a single transmembrane glycoprotein. It was found in the analysis of human lymphocytes by e.l. reinherez and colleagues in 1980. CD38 is expressed primarily in T cells, dendritic cells, natural Killer (NK) cells and hematopoietic stem cells, playing an important role in the immune process, for example, mediating cytokine production, regulating proliferation of T lymphocytes and protecting mature B lymphocytes and dendritic cells from apoptosis. In addition, CD38 is also expressed on immunosuppressive cells (e.g., regulatory T cells, regulatory B cells, and bone marrow derived suppressor cells). Thus, expression of CD38 can be used to define an inhibitory subpopulation of regulatory T cells that reduces the anti-tumor activity of the immune system. When normal plasma cells deteriorate, their cell surface CD38 expression levels are significantly elevated, and thus CD38 is one of the markers of B lymphocyte tumorigenesis.
Clinical studies have shown that CD38 molecules are highly expressed on more than 90% of malignant plasma cells in Multiple Myeloma (MM) patients, and thus CD38 has become one of the major targets for the development of targeted therapeutic drugs for MM patients. Two therapeutic antibodies specifically targeting CD38 have been marketed by FDA approved by Daratumumab and Isatuximab, and there are several different types of CD38 antibody-related drugs in preclinical and clinical studies, e.g., chimeric antigen receptor T cells targeting CD38 (CAR-T) and CD38 antibody-coupled drugs (ADC), which can exert antitumor activity through multiple effector mechanisms. However, the CD38 antibody in the market and clinical research belongs to the fully human antibody or the humanized antibody, and has the defects of large molecular weight, poor tissue permeability, complex preparation process, high production cost and the like, and limits the therapeutic effect to a certain extent. Therefore, the development of novel antibody molecules targeting CD38 in combination with other therapeutic approaches for CD 38-associated tumor therapy can greatly expand the range of application and therapeutic effect of CD38 antibodies.
The immunoglobulin contained in the serum of mammals is a tetramer composed of two identical heavy and light chains, with a molecular weight of about 150 kDa. In 1993, belgium scientists Hamers-Cazterman et al found a Heavy chain antibody (Heavy-chain Antibodies, hcAbs) with a naturally deleted light chain in camelid serum, which had a single domain variable structure that binds antigen in the HcAbs structure, with a relative molecular mass of about 15kDa, which is only about 1/10 of that of conventional Antibodies, called nanobodies or VHH Antibodies. VHH, consisting of 4 conserved framework regions and 3 hypervariable complementarity determining regions, is the smallest functional antibody structure found in nature today.
Nanobodies exhibit a number of unique advantages over traditional antibody drugs currently on the market:
(1) CDR3 of nanobody and corresponding antibody region of human are in prominent long loop shape, and can bind to hidden epitope, slit-shaped or pocket-shaped epitope which traditional antibody can not bind. For the current mature targeted therapy for tumor, a brand new antibody drug can be developed by utilizing the nano antibody; (2) The sequence of the antibody has high homology with the sequence of the fully human antibody and weak immunogenicity; (3) The molecular weight is small, aggregation is not easy to occur in vivo, and compared with the traditional antibody, the antibody has stronger tissue penetrability; (4) Can maintain good stability in severe environments such as high temperature, strong acid, strong alkali or in the presence of protease. Easy to store and transport, and can be prepared into finished products of medicines with various administration modes; (5) Can be easily connected with other molecules, can be prepared into various antibody drug forms such as multivalent antibodies, bispecific antibodies, targeted immunotoxins, ADC drugs and the like, and expands the application range of the antibody in tumor diagnosis and treatment.
In 2018, EMA approved nanobody drug cablevi was used to treat adult-acquired thrombotic thrombocytopenic purpura (aTTP), cablevi becoming the first nanobody drug on the market worldwide. The nanometer antibody-based therapeutic scheme has wide research prospect in the treatment of cancers.
At present, a plurality of monoclonal antibodies targeting human CD38 on the market belong to fully human antibodies or humanized antibodies, and have the defects of large molecular weight, poor tissue permeability, high production cost, difficulty in preparing bispecific antibodies by combining with other target antibodies, and the like. The camelid nanobody aiming at human CD38 has the advantages of small molecular weight, easy transformation, easy combined construction of bispecific antibody with other antibodies, easy construction of CAR-T, easy preparation of conjugate with other drugs, and the like.
In view of the above, there is a need in the art to develop a camelidae nanobody targeting human CD 38.
Disclosure of Invention
The invention aims at providing a camelidae nanobody targeting human CD 38.
In a first aspect of the invention there is provided an anti-CD 38 nanobody which is capable of specifically binding to CD38 and in which the complementarity determining region CDRs of the VHH chain are selected from one or more of the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19, CDR3 shown in SEQ ID NO. 20;
(4) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 28;
(5) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which retains high affinity binding to CD 38.
In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
In another preferred embodiment, the VHH chain further comprises a framework region FR, which is one or more selected from the group consisting of:
(1) FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 2E);
(2) FR1 shown in SEQ ID NO. 13, FR2 shown in SEQ ID NO. 14, FR3 shown in SEQ ID NO. 15 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 7G);
(3) FR1 shown in SEQ ID NO. 21, FR2 shown in SEQ ID NO. 22, FR3 shown in SEQ ID NO. 23 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 1E);
(4) FR1 shown in SEQ ID NO. 29, FR2 shown in SEQ ID NO. 30, FR3 shown in SEQ ID NO. 31 and FR4 shown in SEQ ID NO. 32 (corresponding to the FR of nanobody 5G);
(5) FR1 shown in SEQ ID NO. 29, FR2 shown in SEQ ID NO. 30, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 8A).
In another preferred embodiment, the VHH chain of the anti-CD 38 nanobody is selected from one or more of SEQ ID NO. 8, SEQ ID NO. 16, SEQ ID NO. 24, SEQ ID NO. 33 or SEQ ID NO. 37.
In a second aspect of the invention there is provided an anti-CD 38 antibody comprising one or more anti-CD 38 nanobodies according to the first aspect of the invention.
In another preferred embodiment, the anti-CD 38 antibody comprises one or more VHH chains having the amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 16, SEQ ID NO. 24, SEQ ID NO. 33 or SEQ ID NO. 37.
In another preferred example, the antibody may be a monomer, a bivalent antibody, and/or a multivalent antibody.
In a third aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: nanobody according to the first aspect of the invention, or anti-CD 38 antibody according to the second aspect of the invention.
In another preferred embodiment, the nucleotide sequence of the polynucleotide comprises one or more of SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 25, SEQ ID NO. 34 or SEQ ID NO. 38.
In another preferred embodiment, the polynucleotide is RNA, DNA or cDNA.
In a fourth aspect of the invention, there is provided an expression vector expressing a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
In a fifth aspect of the invention there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having integrated into its genome a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli and yeast cells.
In a sixth aspect of the invention, there is provided a method of producing anti-CD 38 nanobodies comprising the steps of:
(a) Culturing the host cell of the fifth aspect of the invention under conditions suitable for nanobody production, thereby obtaining a culture comprising said anti-CD 38 nanobody; and
(b) Isolating or recovering said anti-CD 38 nanobody from said culture; optionally, a plurality of metal sheets
(c) Purifying and/or modifying the CD38 nanobody obtained in step (b).
In another preferred embodiment, the anti-CD 38 nanobody has an amino acid sequence as shown in SEQ ID NO. 8, SEQ ID NO. 16, SEQ ID NO. 24, SEQ ID NO. 33 or SEQ ID NO. 37.
In a seventh aspect of the invention, there is provided an immunoconjugate comprising:
(a) Nanobodies according to the first aspect of the invention, or anti-CD 38 antibodies according to the second aspect of the invention; and operatively connected to
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or a combination thereof.
In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-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, ra223, ru-106, na24, sr89, tb-149, th-227, xe-133 Yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the coupling moiety is a drug or a toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxybenzodiazepines), vinca alkaloids (vinca alkaloids), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-chain of jezosin, α -octacocin, gelonin, mitogellin, restrictocin (retproctrocin), phenol, enomycin, curcin, crotonin, calicheamicin, saporin (Sapaonaria officinalis), glucocorticoids, or combinations thereof.
In another preferred embodiment, the coupling moiety is a detectable label.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
In another preferred embodiment, the coupling moiety is a microtubule inhibitor DM1.
In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g., bivalent) anti-CD 38 nanobodies as described above, or anti-CD 38 antibodies as described above.
In another preferred embodiment, the multivalent means that a plurality of repeats of the anti-CD 38 nanobody as described above, or the anti-CD 38 antibody as described above, are comprised in the amino acid sequence of the immunoconjugate.
In another preferred embodiment, the immunoconjugate is used for the diagnosis or prognosis of cancer, in particular for CD38 expressing tumors (i.e. CD38 positive tumors).
In another preferred embodiment, the immunoconjugate is used for diagnosing and/or treating a tumor that expresses CD38 protein.
In an eighth aspect of the invention there is provided the use of a nanobody as described in the first aspect of the invention, or an anti-CD 38 antibody as described in the second aspect of the invention, for the preparation of (a) a reagent for detecting a CD38 molecule; (b) a medicament for the treatment of tumors.
In another preferred embodiment, the assay is an in vivo assay or an in vitro assay.
In another preferred embodiment, the detection comprises flow detection, cellular immunofluorescence detection.
In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) Nanobodies according to the first aspect of the invention, or anti-CD 38 antibodies according to the second aspect of the invention, and/or immunoconjugates according to the seventh aspect of the invention;
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of an injection.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.
In another preferred embodiment, the pharmaceutical composition is used for the treatment of tumors expressing the CD38 protein (i.e. CD38 positive).
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor selected from the group consisting of: myeloma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymph cancer, adrenal tumor, or bladder tumor.
In another preferred embodiment, the myeloma is Multiple Myeloma (MM).
In a tenth aspect of the invention there is provided the use of a nanobody as described in the first aspect of the invention, or an anti-CD 38 antibody as described in the second aspect of the invention, an immunoconjugate as described in the seventh aspect of the invention, or one or more of a pharmaceutical composition as described in the ninth aspect of the invention:
(a) For detecting CD38 molecules;
(b) For streaming detection;
(c) The method is used for cell immunofluorescence detection;
(d) For treating tumors;
(e) Is used for tumor diagnosis.
(f) For the preparation of cell therapy products that specifically bind to human CD 38;
(g) Antibody drugs for treating tumors, immunotoxins targeting human CD38, antibody drug conjugate products;
(h) Is used for preparing cell therapy products for treating tumors.
In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
In an eleventh aspect of the present invention, there is provided a recombinant protein having:
(i) Nanobodies according to the first aspect of the invention or anti-CD 38 antibodies according to the second aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag and an HA tag.
In another preferred embodiment, the recombinant protein specifically binds to CD38 protein.
In a twelfth aspect of the invention there is provided the use of a nanobody according to the first aspect of the invention or an anti-CD 38 antibody according to the second aspect of the invention, an immunoconjugate according to the seventh aspect of the invention, or a pharmaceutical composition according to the ninth aspect of the invention, for the preparation of a medicament, reagent, assay plate or kit;
Wherein the reagent, assay plate or kit is for: detecting CD38 protein in the sample;
wherein the agent is for the treatment or prevention of a CD38 expressing tumor.
In another preferred embodiment, the tumor comprises: myeloma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymph cancer, adrenal tumor, or bladder tumor.
In a thirteenth aspect of the invention there is provided a kit comprising a nanobody according to the first aspect of the invention or an anti-CD 38 antibody according to the second aspect of the invention, an immunoconjugate according to the seventh aspect of the invention, or a pharmaceutical composition according to the ninth aspect of the invention.
In a fourteenth aspect of the present invention, there is provided a method of detecting CD38 protein in a sample, the method comprising the steps of:
(1) Contacting a sample with a nanobody according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of CD38 protein in the sample.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method is a non-therapeutic non-diagnostic method.
In a fifteenth aspect of the present invention there is provided a method of treating a CD 38-associated disease, the method comprising administering to a subject in need thereof a nanobody as described in the first aspect of the invention or an anti-CD 38 antibody as described in the second aspect of the invention, an immunoconjugate as described in the seventh aspect of the invention, or a pharmaceutical composition as described in the ninth aspect of the invention.
In a sixteenth aspect of the invention there is provided a CAR-T cell expressing a chimeric antigen receptor CAR, the antigen binding domain of the CAR having a nanobody as described in the first aspect of the invention.
In a seventeenth aspect of the invention, there is provided a formulation comprising a CAR-T cell according to the sixteenth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, the dosage form of the formulation comprises an injection.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
Fig. 1: flow cytometry analysis of binding of targeted human CD38 nanobody to cd38+ -CHO and CD 38-CHO cells. a is the binding analysis of the anti-human CD38 nanobody and MM1S cells; b is the combination analysis of the anti-human CD38 nano antibody and CD 38-CHO cells; c is the binding analysis of the anti-human CD38 nanobody and CD38+ -CHO cells; PC is an anti-human CD38 positive human ScFv antibody control.
Fig. 2: SDS-PAGE electrophoresis of anti-human CD38 nanobody purification.
Fig. 3: flow cytometry analysis of anti-human CD38 nanobody affinity.
Fig. 4: three-dimensional structure simulation of anti-human CD38 nanobody interactions with CD 38.
Fig. 5: flow cytometry evaluated endocytosis of anti-human CD38 nanobodies.
Fig. 6: in vitro killing activity of anti-human CD38 nanobody (FA-2E) -DM1 conjugate on cells was examined.
Detailed Description
The inventor of the present invention has studied extensively and intensively, and succeeded in obtaining a group of anti-CD 38 nanobodies through a large number of screening. Specifically, the candidate nanobody targeting human CD38 is obtained by a solid phase screening method by using a high-purity human CD38 extracellular protein with biological activity and utilizing a camelidae natural nanobody phage display technology. After protein level and cell level are detected and analyzed by enzyme-linked immunosorbent assay (ELISA) and Flow Cytometry (FCM), respectively, 5 nanobodies which specifically bind to human CD38 extracellular domain proteins and can bind to CD38+ cells are finally obtained. The invention has wide application prospect in the aspect of researching and developing therapeutic antibody medicaments targeting human CD38 aiming at the human CD38 specific nano antibody. The present invention has been completed on the basis of this finding.
In addition, the invention also provides 5 camel natural nanobodies aiming at the extracellular segment of human CD38, nucleotide and amino acid sequences of the nanobodies, and a vector and a host cell containing the coding sequences.
Terminology
As used herein, the term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the terms "nanobody" (single domain antibody, sdAb, or VHH) "," nanobody "(nanobody) have the same meaning, referring to the variable region of a cloned antibody heavy chain, to construct a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Typically, the naturally deleted light and heavy chain constant region 1 (CH 1) antibodies are obtained first, and then the variable region of the heavy chain of the antibody is cloned to construct nanobodies (VHHs) consisting of only one heavy chain variable region.
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the terms "single domain antibody (VHH)", "nanobody" have the same meaning, referring to cloning the variable region of the heavy chain of an antibody, constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the anti-CD 38 protein antibodies or fragments thereof.
As used herein, the terms "heavy chain variable region" and "V H "interchangeably used.
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to CD38 protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.
The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of which 4 FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibodies of the invention refer to polypeptides having CD38 protein binding activity that include the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the nanobodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
Figure BDA0003352581800000121
Figure BDA0003352581800000131
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) 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. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
The VHH chain of the nanobody provided by the invention comprises CDRl, CDR2 and CDR3 selected from the following combinations:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3 (corresponding to the CDR of nanobody 2E);
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12 (corresponding to the CDR of nanobody 7G);
(3) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19, CDR3 shown in SEQ ID NO. 20 (corresponding to the CDR of nanobody 1E);
(4) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 28 (corresponding to the CDR of nanobody 5G);
(5) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36 (corresponding to the CDR of nanobody 8A).
In another preferred example, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 8, SEQ ID NO. 16, SEQ ID NO. 24, SEQ ID NO. 33 or SEQ ID NO. 37; the nucleotide sequences are shown as SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 25, SEQ ID NO. 34 or SEQ ID NO. 38 respectively. Further, nanobodies of the invention also include or have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or more to the above sequences.
In another preferred embodiment, the nucleotide sequence shown as SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 25, SEQ ID NO. 34 or SEQ ID NO. 38 may be added, substituted, deleted or inserted with one or several nucleotide sequences to obtain a derivative sequence which retains the ability to bind with high affinity to CD 38.
Amino acid sequence engineering can be performed on the nanobody sequences of the invention using affinity maturation and computer simulation techniques to obtain new nanobody sequences.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The invention provides an expression system for expressing the human CD38 nanobody, and a host cell comprises the expression vector. The host cell is preferably E.coli.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which 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 . If neededAlternatively, transformation may be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The invention provides a preparation method of a nanometer antibody aiming at human CD38, which comprises the following steps: the method specifically comprises the following steps: firstly, synthesizing human CD38 extracellular protein and CD38 high expression cell strain, coupling the CD38 protein on an ELISA plate, displaying human CD38 spatial conformation, screening nanobody by using camelid natural nanobody phage display library technology, thereby obtaining nanobody specifically combined with human CD38, transferring candidate nanobody gene into escherichia coli, and establishing an expression system capable of efficiently expressing nanobody in escherichia coli.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. drug-activated enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticle, etc.
Immunoconjugates
The invention also provides an immunoconjugate comprising:
(a) A VHH chain of an anti-CD 38 nanobody according to the first aspect of the invention, or an anti-CD 38 nanobody according to the second aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: radionuclides, enzyme antibodies, cells, or combinations thereof.
The invention provides a conjugate of a nanometer antibody targeting human CD38 and DM1, wherein the nanometer antibody resisting CD38 is conjugated with a microtubule inhibitor DM1 to obtain a drug conjugate based on the nanometer antibody resisting human CD38, and the cytotoxicity of the drug conjugate on CD38+ cells is detected by CCK-8.
The conjugate of the nanometer antibody targeting human CD38 and DM1 provided by the invention has obvious in vitro killing effect on CD38+ myeloma cells.
The immunoconjugate of the invention can be used for noninvasively detecting CD38 expression of an object to be detected, has small size and high specificity, is suitable for whole body detection of primary and metastatic tumors in a targeting mode, and has high accuracy and small radiation dose.
Cytotoxic agents
The conjugation moieties comprising the antibody conjugates of the invention include: toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to: auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin A-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin A chain, bozosin A chain, alpha-octacocin, gelonin, mitogellin, restrictocin (retsfactocin), phenomycin, enomycin, curcin (curcin), crotonin, calicheamicin, saporin (Sapaonaria officinalis) and other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, re186, re188, sm153, bi212 or 213, P32 and radioisotopes of Lu including Lu 177. The antibodies may also be conjugated to an anticancer prodrug-activating enzyme capable of converting the prodrug into its active form.
Preferred small molecule drugs are highly cytotoxic compounds, preferably monomethyl auristatin (monomethyl auristatin), calicheamicin, maytansinoids, or combinations thereof; more preferably selected from: monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or combinations thereof.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein or immunoconjugate thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention can be used directly to bind CD38 protein molecules and thus can be used to treat tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the nanobody (or conjugate thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 nanograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day, more preferably from 50 nanograms per kilogram of body weight to about 1 milligrams per kilogram of body weight or from 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight.
In addition, the polypeptides of the invention or conjugates thereof may be used with other therapeutic agents (e.g., antineoplastic agents or immunomodulators).
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 nanograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 50 nanograms per kilogram of body weight to about 1 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Labeled nanobodies
In a preferred embodiment of the invention, the nanobody is provided with a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels.
Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-CD 38 nanobody is labeled with colloidal gold to obtain a colloidal gold-labeled nanobody.
The anti-CD 38 nano antibody has good specificity and high potency.
CAR-T cells
As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to CAR-T cells according to the nineteenth aspect of the invention.
As used herein, a chimeric immune antigen receptor (Chimeric antigen receptor, CAR) includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding member (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory molecule and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.
As used herein, "antigen binding domain" and "single chain antibody fragment" refer to Fab fragments, fab 'fragments, F (ab') 2 Fragments, or single Fv fragments. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the scFv comprises a VHH chain according to the first aspect of the invention or a nanobody according to the second aspect of the invention.
For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.
The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
When CAR is expressed in T cells, the extracellular segment recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN- γ, etc., affecting the tumor cells, causing the tumor cells to not grow, to be caused to die or otherwise be affected, and causing the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain.
Detection method
The invention also relates to methods of detecting CD38 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of CD38 protein in the solubilized sample.
In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, and the like.
The invention also provides a detection kit for detecting the level of CD38, which comprises an antibody for recognizing the CD38 protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The detection kit may be an in vitro diagnostic device.
The invention also provides a kit comprising an immunoconjugate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, an isotope tracer, and one or more reagents selected from the group consisting of: contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nanometer magnetic particle and imaging agent.
Preferably, the kit of the invention is an in vivo diagnostic kit for non-invasively detecting CD38 expression in a subject.
Application of
As described above, the nanobody of the present invention has a wide range of biological and clinical applications, and its application relates to various fields such as diagnosis and treatment of CD 38-associated diseases, basic medical research, biological research, etc. One preferred application is for clinical diagnosis and targeted therapy against CD 38.
The invention also provides application of the conjugate of the nanometer antibody targeting human CD38 and DM1 in preparing tumor medicaments.
The main advantages of the invention include
(1) The nanometer antibody of the invention for resisting human CD38 has small molecular weight, is easy to reconstruct and combine with other antibodies to construct bispecific antibody, is easy to construct CAR-T, and is easy to prepare into conjugates with other medicines.
(2) The nano antibody can be expressed by adopting a prokaryotic expression system, so that the production cost is reduced, the production is simple and convenient, and the application range of the targeted human CD38 therapeutic antibody is expanded.
(3) The anti-CD 38 nanobody drug conjugate of the invention has smaller molecular weight than the conventional full-size antibody conjugate drug, and theoretically has better tumor tissue penetrability, thus having possibly unique advantages in the aspect of solid tumor treatment. Nanobodies have significant advantages in terms of production, transport and administration.
The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Antibody sequences according to the invention
TABLE 1 nanobodies and sequences thereof according to the invention
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Example 1 screening of nanobodies against human CD38
(1) Screening was performed using solid phase screening, human CD38 protein was diluted to 10. Mu.g/mL with PBS, 100. Mu.L was coated in 96-well ELISA plates, and coated overnight at 4 ℃.
(2) The next day, the protein solution was discarded and 10% mbps was blocked for 2h at room temperature.
(3) After 2h, 100. Mu.L of a natural camelidae nanobody phage library was added and combined with shaking at room temperature for 2h.
(4) After 2h, the phage were washed 10 times with PBST and 10 times with PBS to wash away unbound phage.
(5) 200. Mu.L of 0.1M glycine HCl (0.1M Gly-HCl, pH 3.0) was added, followed by shaking for 20min, followed by neutralization with 60. Mu.L of 1M Tris-HCl, and gently pipetting to obtain 520. Mu.L of eluate.
(6) Phage bound to CD38 collected by elution was transferred to E.coli TG1 cells in the logarithmic growth phase, cultured with shaking at 37℃for 1 hour, plated on 2YT (1.6% (W/V) tryptone, 1% (W/V) yeast extract, 0.5% (W/V) sodium chloride) plates containing ampicillin (Amp) resistance at a final concentration of 100. Mu.g/mL, and cultured overnight at 37 ℃.
(7) The following day, 20. Mu.L of the overnight grown bacteria liquid obtained by scraping plates is added into 10mL of 2YT-Amp culture liquid, shake-cultured at 37 ℃ to logarithmic phase, a proper amount of auxiliary phage M13KO7 is added, and shake-cultured at 37 ℃ for 1h after infection.
(8) The next day, phage supernatants were collected by centrifugation for the next round of solid phase screening. The same screening was repeated 3 more times to obtain enrichment step by step.
EXAMPLE 2 screening of specific monoclonal antibodies Using phage enzyme-Linked immunosorbent assay (ELISA)
(1) Monoclonal phage ELISA detection: from among the phage-containing TG1 cell colonies obtained after the above 4 th round of screening, single colonies were randomly selected and inoculated into 100. Mu.L of 2YT-Amp culture solution in a 96-well plate, shake-cultured at 37℃to logarithmic growth phase, and after 1h of infection, a proper amount of helper phage M13KO7 was added, and shake-cultured at 37℃overnight. The following day, phage supernatants were collected by centrifugation, blocked with MPBST at room temperature for 2h, transferred to antigen-coated ELISA plates (blocked with MPBST at room temperature for 2 h) at 4℃overnight, and reacted at room temperature for 2h. PBST was washed to remove unbound phage, HRP-anti-M13 antibody was added, and the reaction was carried out at room temperature for 1h. PBST is again washed to remove unbound antibody, TMB color development solution is added, reaction is carried out for 10min at room temperature, and 2M H is carried out 2 SO 4 The reaction was terminated. The absorbance was read at 450nm wavelength on an microplate reader. A positive clone was identified when phage monoclonal bound to CD38-Fc antigen with an OD450 reading greater than 1.2 and bound to IgG4-Fc tag protein with an OD450 reading less than 0.5. And (3) sending the colony with positive detection result to a sequencing company for sequencing, comparing the sequencing result by DNAMAN analysis software, and combining the candidate nanobodies with the same sequence.
(2) Crude extraction of antibody periplasmic protein: the nanobody TG1 infection strain with positive result obtained by the screening is cultivated to logarithmic phase at 37 ℃ in a shaking way, IPTG with final concentration of 1mM is added, and the culture is carried out at 30 ℃ in a shaking way for overnight. The following day, the expressed cells were collected by centrifugation. The pellet was resuspended in 1mg/mL lysozyme solution and digested on ice for 20 to 30min. The antibody expression supernatant periplasmic protein crude sample was collected by high-speed freeze centrifugation.
(3) Periplasmic protein cell binding assay: cell samples to be tested were collected by post-incubation digestion, and 2% FPBS was resuspended and washed 2 times. 200. Mu.L of different periplasmic protein samples were added and incubated at 4℃for 40min.2% FPBS was washed 2 times. Incubating a secondary antibody: 100 mu L of anti-Flag-APC secondary antibody is added into each tube, and incubated for 20-30 min at 4 ℃ in a dark place. After washing the cells with 2% FPBS, 400. Mu.L of 2% FPBS was resuspended, and filtered for transfer into a flow tube for FCM analysis. Screening to obtain CD38 according to the flow detection result + Candidate nanobodies bound by Daudi cells (human B lymphomatoid cells).
Example 3 expression and purification of candidate targeting human CD38 nanobodies in E.coli hosts
(1) Plasmids carrying the different nanobody expression sequences are transformed into BL21/DE3 escherichia coli competent cells, and the plasmids are coated in a 2YT-Amp culture plate and cultured overnight at 37 ℃.
(2) The following day, the monoclonal colonies on the plates were picked up and added to 2YT broth (1.6% (W/V) tryptone, 1% (W/V) yeast extract, 0.5% (W/V) sodium chloride), and OD was cultured by shaking at 37 ℃ 600 The value is 0.6-0.8, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mM is added, and the expression is induced overnight (about 16 h) at 30 ℃.
(3) And centrifugally collecting thalli, preparing lysozyme solution with the final concentration of 1mg/mL by using PBS buffer solution, suspending centrifugal thalli sediment again, and placing the thalli sediment on ice for incubation for 20-30 min.
(4) The supernatant after the bacterial disruption treatment was collected by centrifugation at 4℃for subsequent purification.
(5) Nanobody purification using his-nickel column affinity chromatography: the supernatant was subjected to sample column at a flow rate of about 1mL/min, 5 column volumes NP-10 (10 mM imidazole, 50mM NaH) 2 PO 4 300mM NaCl) to wash the Ni-NTA column; the Ni-NTA column was then washed 10-fold with NP-20 (20 nM imidazole). The target protein was eluted with NP-250 (250 nM imidazole), and the eluted protein solution was collected and subjected to SDS-PAGE to determine the protein purity.
(6) The nanobody buffer system was replaced by centrifugal ultrafiltration and the protein sample concentration was concentrated. Transferring the nano antibody sample eluted by the affinity chromatography into a ultrafiltration tube, centrifuging at a low speed at 4 ℃, adding precooled PBS with the same volume when the volume of the sample is concentrated to about 1mL, and concentrating to 1mL of sample volume. Repeated 3 times.
(7) And collecting the nano antibody sample after concentrating the displacement buffer solution, and storing at-80 ℃.
Example 4 specific detection of candidate targeting human CD38 nanobodies
4.1. Enzyme-linked immunosorbent assay (ELISA) detection:
(1) Human CD38, BSA protein was diluted to 1. Mu.g/ml in PBS, 100. Mu.L/Kong Baobei in 96-well ELISA plates and incubated overnight at 4 ℃.
(2) The next day, the coating solution was discarded. 300 uL/well PBST was washed 3 times and blocked with 5% MPBST at room temperature for 1h. While 5% mpbst blocked nanobody samples for 1h.
(3) The blocking solution was discarded and washed 3 times at 300. Mu.L/well PBST. Adding the purified nano antibody sample, and incubating for 1h at room temperature.
(4) The nanobody sample solution was discarded and washed 3 times at 300. Mu.L/well PBST. anti-Flag-HRP secondary antibody was added and incubated at room temperature for 1h in the dark.
(5) The secondary antibody was discarded and washed 6 times at 300. Mu.L/well PBST. TMB color development liquid is added, and OD450nm reading value is detected after 10 min. According to the analysis of the read result, the nanobody which is combined with the CD38-Fc protein and is not combined with the protein label IgG1-Fc is judged as a positive candidate antibody.
4.2. Flow Cytometry (FCM) detection and human CD38 + Candidate nanobodies that cell-specific bind:
(1) The positive nanobodies obtained by the screening of example 4.1 were further subjected to cell binding detection. The wall-attached cultured myeloma cell line MM1S (CD 38) + ) CHO from self construction (CD 38) + ) And CHO (CD 38) - ) Cells were collected by centrifugation after digestion with pancreatin, respectively, and 2% fpbs washed the cells 2 times.
(2) 100. Mu.L of the candidate positive nanobody obtained in example 4 was added to each tube, and incubated at room temperature for 1h. Cells were collected by centrifugation, antibody incubations were discarded, and cells were washed 3 times with 2% fpbs. Meanwhile, the ScFv region of the fully human anti-CD 38 antibody with the patent number of US9951144B2 is used as a positive control (marked as PC in figure 1, recombinant plasmid is constructed through the synthesis of corresponding nucleotide sequences of the antibody, and is transferred into a prokaryotic expression system of escherichia coli BL21 (DE 3), and is purified by a his-nickel column affinity chromatography method for standby after IPTG induction expression.
(3) 100 mu L of anti-Flag-APC secondary antibody of the targeting candidate nano antibody and a positive control recombinant antibody Flag label is added into each tube, and incubated for 30min in a dark place.
(4) The supernatant was discarded by centrifugation, resuspended in 2% FPBS, washed 3 times, and cells were collected by centrifugation.
(5) 400 μL of 2% FPBS was resuspended, filtered and transferred into a flow tube for FCM analysis.
Based on the streaming detection (FIG. 1), it will be associated with CD38 + MM1S and CD38 of (c) + CHO cells binding to and not to CD38 - Candidate nanobodies bound to CHO were judged to be capable of binding to CD38 + Positive nanobodies bound by cells. Finally screening to obtain 5 specific CD38 targeting nanometer antibody sequences,
numbered 2E, 7G, 1E, 5G and 8A. The nucleotide sequences are SEQ ID NO 9,SEQ ID NO:17,SEQ ID NO:25,SEQ ID NO:34 and SEQ ID NO 38; the corresponding amino acid sequences are SEQ ID NO. 8,SEQ ID NO:16,SEQ ID NO:24,SEQ ID NO:33 and SEQ ID NO. 37.
The purification chart of 5 nano antibodies aiming at CD38 is shown in figure 2, and the result shows that the purity of the nano antibodies can reach more than 90% after affinity purification.
Example 5 nanobody affinity evaluation
Detection of nanobodies at different concentrations with myeloma cell line MM1S (CD 38) by flow cytometry FCM + ) The average fluorescence intensity of binding reflects the affinity of nanobody binding to CD38 protein, while comparing with the affinity of positive control PC (PC control is ScFv sequence segment of anti-CD 38 antibody sequence in CD38 CAR-T project of Sorrento company, plasmid expression of the ScFv segment was constructed as PC positive control).
(1) The positive nanobody obtained by the above screening of example 5 was diluted three-fold from 100. Mu.g/. Mu.l, and 100. Mu.L of the antibody sample was added to the myeloma cell MM1S cells resuspended in 2% FPBS. Incubate for 1h at room temperature.
(2) Cells were collected by centrifugation, antibody incubations were discarded, and cells were washed 3 times with 2% fpbs.
(3) To each tube, 100. Mu.L of anti-Flag-APC secondary antibody was added and incubated for 30min in the dark.
(4) The supernatant was discarded by centrifugation, resuspended in 2% FPBS, washed 3 times, and cells were collected by centrifugation.
(5) 400. Mu.g/. Mu.l 2% FPBS was resuspended, filtered and transferred into a flow tube for FCM analysis.
(6) The collected data were analyzed using flowjo7.6 and Graphpad Prism 5 analysis software to calculate nanobody affinity values.
The results show (see fig. 3) that nanobody 2E has an affinity superior to the positive control antibody and that 7G exhibits an affinity level comparable to the control antibody.
EXAMPLE 6 analysis of nanobody interaction with CD38
(1) The protein structure database PDB was searched for complex structures of known CD38 antibodies binding to CD38 (CD 38-antibody complex numbered 4cmh in PDB database was used as an interaction structure analysis template) to analyze CD38 binding to antibodies. Wherein, FIG. 4A is a diagram of the interaction space structure analysis of CD38 and antibody (surface model) in the PBD database 4cmh, and the red structure region is the structure part of CD38 interacting with 4 cmh.
(2) The three-dimensional protein structures of the candidate nanobody are predicted by comprehensively utilizing a plurality of protein three-dimensional structure modeling software I-TASSER, FR-t5-M, FALCON and the like.
(3) And (3) performing dock prediction of the CD38 and the candidate nanobody by utilizing ZDOCK3.0.2 and combining the template interaction structure obtained in the step (1) and the nanobody space structure predicted in the step (2). And in addition, according to the evaluation of tools such as MEFTop and QIPI on the locking conformation, the optimal locking model is found out through comparison and selection.
FIG. 4B shows that the protein interaction regions (red regions) between CD38 and the three nanobodies of FA-2E, FB-1E and FB-5G are far apart. The interaction region (red region) of FA-7G and FB-8A with CD38 is similar to the structural region responsible for CD 38-antibody complex docking in the known 4cmh model, and the binding region of the two nanobodies with CD38 is clearly different from that of FA-2E, FB-1E and FB-5G.
Example 7 preparation of anti-human CD38 antibody-DM 1 conjugate
7.1. Coupling of nanobody and maytansine DM1, and analytical detection:
(1) According to the antibodies: the molar ratio of SMCC-DM1 is 1: 18. Mu.L of nanobody solution (10 mg/mL) was mixed with SMCC-DM1 (5 mM/L) in the corresponding ratio, and reacted at room temperature for 2 to 4 hours under stirring.
(2) Subsequently, the reaction mixture was transferred to a 0.5mL centrifugal filter (10 kDa filter membrane), and supplemented with 300. Mu.L PBS, centrifuged at 5000g for 15min 2 to 3 times, and excess SMCC-DM1 contained in the reaction system was removed.
(3) Collecting the coupled product, subpackaging, and storing at-80deg.C.
(4) The nano antibody-DM 1 conjugate prepared by mass spectrometry is: the nanobody solution and the corresponding conjugated conjugate solution were diluted to 100pM/L with PBS. 1 mu L of diluted antibody solution and ADC solution are respectively taken, 1 mu L of 2, 5-dihydroxybenzoic acid (DHB) is added and uniformly mixed, and then the mixture is respectively spotted on a sample target plate and naturally dried. The sample target plate is placed in a MALDI-TOF target plate well. Parameters are set, the laser intensity is adjusted, and data are collected.
7.2. Flow cytometry detection of anti-human CD38 nanobody endocytosis:
(1) The FA-2E and the FA-7G with highest affinity in the candidate anti-human CD38 nano-antibodies are respectively incubated with the target cell MM1S for 30min, and unbound antibodies are washed away.
(2) Control was placed at 4℃and experimental was placed at 37℃to promote endocytosis of the cells. Samples were taken at 1h, 2h, 3h, and 4h, respectively, and then anti-Flag-APC secondary antibodies were incubated, and the average fluorescence intensity (Median Fluorescence Intensity, MFI) of APCs was detected on-line. The endocytosis rate of the antibody was calculated as follows: endocytosis rate of antibody= (amount of surface binding at 4 ℃ c-amount of surface residue at different time points of 37 ℃ c-amount of dissociation of each group from surface)/amount of surface binding at 4 ℃ c×100%.
The fewer the number of antibodies bound to the endocytic cell surface of the cell, the weaker the secondary anti-fluorescence bound thereto.
Panel a of FIG. 5 shows that the MFI of the experimental groups FA-2E and FA-7G gradually decreased over time as compared to the control group, and that the surface fluorescent-labeled antibodies were gradually endocytosed into the target cells. Figure 5 b shows that the ratio of target cells endocytosing both FA-2E and FA-7G nanobodies increases with time, wherein the endocytic effect of FA-2E nanobodies is more pronounced, thus subsequently prepared as DM1 conjugates.
7.3. Cytotoxicity detection of anti-human CD38 antibody-DM 1 conjugate:
(1) The myeloma cell MM1S was diluted to 2X 10 in culture 5 Each of the cells was inoculated at 50. Mu.l/well into a 96-well plate.
(2) FA-2E and FA-2E-DM were diluted 1-fold to gradient concentrations, added to 96-well plates which had been seeded with MM1S cells, each concentration was repeated 3 times, control wells without naked antibody or conjugate samples were set up, and after incubation at 37℃for 72h, detection was performed using CCK-8.
FIG. 6 shows the results of the administration of naked antibody FA-2E to MM1S cells (CD 38 + ) Has no killing effect, and the FA-2E-DM1 administration group shows the cell killing activity with concentration gradient dependence. Experiments show that the anti-human CD38 antibody-DM 1 conjugate (FA-2E-DM 1) has the effect of killing target tumor cells. The killing effect of FA-2E-DM1 is based on targeting and endocytosis of FA-2E nanobodies targeting cell surface CD38, thereby allowing DM1 to enter cells to exert antitumor activity.
Discussion of the invention
At present, clinical researches aiming at CD38 targets comprise the fields of monoclonal antibodies, antibody coupling drugs, bispecific antibodies, chimeric antigen receptor T cells and the like. 11 months 2015, the FDA approved Daratumumab as a single drug therapy, becoming the first CD 38-targeting humanized full-length monoclonal antibody in the world to treat Multiple Myeloma (MM). The FDA approved the isatuximab antibody developed by Sanofi corporation for use in combination with IMiD in the treatment of R/R MM patients, month 3 2020. In addition, several CD38 antibodies are in preclinical research stages (e.g., TAK-169, dara-DM4, etc.), and antibody drug development against CD38 is not only monoclonal antibody drug, but also CAR-T, ADC drug, etc. Such as the CD38 CAR-T project of Sorrento corporation; CD38 ADC project at state university, ohio. However, no effective cases of constructing CART cell therapy and ADC drug therapy by using the CD38 nano antibody are found at home.
In summary, the targeted human CD38 antibodies used in current immunotherapy are all fully human antibodies or humanized antibodies, and have been limited to the field of hematological tumor treatment. The existing antibody has the problems of large molecular weight, relatively poor tissue permeability, difficult construction and expression of bispecific antibody combined with other targets, and the like, and limits the application of CD38 antibody medicaments and the treatment range of indications. Therefore, searching for novel antibodies of a smaller molecular weight, which are easy to prepare into various types including bispecific antibodies, antibody-conjugated drugs, antibody immunotoxins, etc., will promote the application range and therapeutic effect of targeted CD38 therapeutic antibodies.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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Preparation and application of <120> camelidae nanobody targeting human CD38 molecule
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tccgactccg tgaagggccg attcaccatc tcctcagaca acgccaagaa tacggtctat 240
ctccaaatga acgacctgaa gcctgaggac acggccatgt attactgcgc gcgaaaggat 300
ttgacgttcg gatgtccgac gccactgagc gaatatacgt actggggtca ggggacccag 360
gtcaccgtct cctcaagt 378
<210> 18
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Gly Ala Thr Phe Ser Tyr Asn Ser Met Ala
1 5 10
<210> 19
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Tyr Ile Tyr Ile Leu Asp Gly Thr Thr Ser Tyr Thr
1 5 10
<210> 20
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Ala Ala Ala Ser Val Thr Gly Ser Gly Met Trp Arg Pro Gly Tyr Asn
1 5 10 15
Tyr
<210> 21
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 22
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val Ala
1 5 10
<210> 23
<211> 35
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn
1 5 10 15
Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met
20 25 30
Tyr Tyr Cys
35
<210> 24
<211> 125
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ala Thr Phe Ser Tyr Asn
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Tyr Ile Tyr Ile Leu Asp Gly Thr Thr Ser Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Met Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Val Thr Gly Ser Gly Met Trp Arg Pro Gly Tyr Asn
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser
115 120 125
<210> 25
<211> 375
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag cctctggagc caccttcagt tacaactcca tggcctggtt ccgccaggct 120
ctagggaagg agcgcgaggg ggtcgcatat atttatattc ttgatggtac cacaagctat 180
accgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa tatggtgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc agccgctagt 300
gtaacgggga gtggtatgtg gcgtccgggg tataactact ggggccaggg gacccaggtc 360
accgtctcct caagt 375
<210> 26
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly
1 5 10
<210> 27
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Ala Ile Asn Ser Gly Gly Gly Asp Thr Tyr Tyr Ala
1 5 10
<210> 28
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Ala Ala Arg Arg Gly Tyr Gly Asn Ser Cys Thr Gly Pro Ser Leu
1 5 10 15
<210> 29
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 30
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10
<210> 31
<211> 35
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn
1 5 10 15
Thr Val Tyr Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Thr
20 25 30
Tyr Tyr Cys
35
<210> 32
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ser
1 5 10
<210> 33
<211> 123
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Asn Ser Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Ala Arg Arg Gly Tyr Gly Asn Ser Cys Thr Gly Pro Ser Leu Trp
100 105 110
Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ser
115 120
<210> 34
<211> 369
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctacagt agctactgca tgggttggtt ccgccaggct 120
ccagggaagg agcgtgaggg ggtcgcagct attaatagtg gtggtggtga cacatactac 180
gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgca acctgaggac acggccacgt attactgtgc ggctcgtcga 300
gggtacggta atagctgcac ggggccttca ctatggagcc aggggaccca ggtcaccgtc 360
tcctcaagt 369
<210> 35
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala
1 5 10
<210> 36
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Ala Ala Gly Arg Ser Tyr Gly Ser Tyr Cys Ser Ala Asn Lys Tyr
1 5 10 15
<210> 37
<211> 123
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Gly Arg Ser Tyr Gly Ser Tyr Cys Ser Ala Asn Lys Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser
115 120
<210> 38
<211> 369
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctatagt agctactgca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgaggg ggtcgcagct attaatagtg gtggtggtag cacatactac 180
gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa tacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc agcaggccgg 300
tcgtatggta gttactgtag tgcgaataag tactggggcc aggggaccca ggtcaccgtc 360
tcctcaagt 369

Claims (10)

1. An anti-CD 38 nanobody, wherein said nanobody is capable of specifically binding to CD38 and the complementarity determining region CDRs of the VHH chain of said nanobody are selected from one or more of the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19, CDR3 shown in SEQ ID NO. 20;
(4) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 28;
(5) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36.
2. The nanobody of claim 1, wherein the VHH chain further comprises a framework region FR, said framework region FR being one or more selected from the group consisting of:
(1) FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 2E);
(2) FR1 shown in SEQ ID NO. 13, FR2 shown in SEQ ID NO. 14, FR3 shown in SEQ ID NO. 15 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 7G);
(3) FR1 shown in SEQ ID NO. 21, FR2 shown in SEQ ID NO. 22, FR3 shown in SEQ ID NO. 23 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 1E);
(4) FR1 shown in SEQ ID NO. 29, FR2 shown in SEQ ID NO. 30, FR3 shown in SEQ ID NO. 31 and FR4 shown in SEQ ID NO. 32 (corresponding to the FR of nanobody 5G);
(5) FR1 shown in SEQ ID NO. 29, FR2 shown in SEQ ID NO. 30, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7 (corresponding to the FR of nanobody 8A).
3. The nanobody of claim 1, wherein the VHH chain of the anti-CD 38 nanobody is selected from one or more of SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 33, or SEQ ID No. 37.
4. An anti-CD 38 antibody comprising one or more anti-CD 38 nanobodies of claim 1.
5. A polynucleotide encoding a protein selected from the group consisting of: the nanobody of claim 1, or the anti-CD 38 antibody of claim 4.
6. An expression vector expressing the polynucleotide of claim 5.
7. A host cell comprising the expression vector of claim 6, or having integrated into its genome the polynucleotide of claim 5.
8. A method of producing anti-CD 38 nanobodies comprising the steps of:
(a) Culturing the host cell of claim 7 under conditions suitable for nanobody production, thereby obtaining a culture comprising said anti-CD 38 nanobody; and
(b) Isolating or recovering said anti-CD 38 nanobody from said culture; optionally, a plurality of metal sheets
(c) Purifying and/or modifying the CD38 nanobody obtained in step (b).
9. An immunoconjugate, characterized in that the immunoconjugate comprises:
(a) The nanobody of claim 1, or the anti-CD 38 antibody of claim 4; and operatively connected to
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or a combination thereof.
10. The nanobody of claim 1, or the use of an anti-CD 38 antibody of claim 4, for the preparation of (a) a reagent for detecting a CD38 molecule; (b) a medicament for the treatment of tumors.
CN202111342220.9A 2021-11-12 2021-11-12 Preparation and application of camelidae nanobody targeting human CD38 molecule Pending CN116120450A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111342220.9A CN116120450A (en) 2021-11-12 2021-11-12 Preparation and application of camelidae nanobody targeting human CD38 molecule

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111342220.9A CN116120450A (en) 2021-11-12 2021-11-12 Preparation and application of camelidae nanobody targeting human CD38 molecule

Publications (1)

Publication Number Publication Date
CN116120450A true CN116120450A (en) 2023-05-16

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN116120450A (en)

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