Nanometer antibody specifically binding to CD39 and application thereof

Abstract

The invention provides a nano antibody which is specifically bound with CD39 and can inhibit the enzyme activity of soluble CD39 and the enzyme activity of CD39 on the surface of a cell membrane, a preparation method, a composition and the like of the nano antibody, the protein binding activity of the nano antibody is equivalent to that of a nano antibody R-Ye-19 (1)-037 and a nano antibody R-Ye-19 (1)-046 in the patent WO2023186121A1, the maximum enzyme activity inhibition rate of the protein level of the nano antibody is superior to that of the nano antibody R-Ye-19 (1)-037 and the nano antibody R-Ye-19 (1)-046 in the patent WO2023186121A1, and the nano antibody can be used for preparing the nano antibody. The maximum enzyme activity inhibition rate of the CD39 at the cellular level and the IC50 are superior to those of the anti-CD39 nano antibody R-Ye-19 (1)-037 and the anti-CD39 nano antibody R-Ye-19 (1)-046 in the patent WO2023186121A1.

Description

[0001] This application is a divisional application of the invention patent application for nanoantibodies specifically binding to CD39 and their uses, with the application date of December 27, 2023; application number: 202311835720.5; and the invention name: Nanoantibodies specifically binding to CD39 and their uses. Technical Field

[0002] The present disclosure relates to the field of antibodies, and more specifically to a nanobody that specifically binds to CD39. Background Art

[0003] CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is an integral membrane protein that converts ATP or ADP into AMP, which is then dephosphorylated to adenosine by CD73. Adenosine is a potent immunosuppressant and binds to adenosine receptors (e.g., A2A receptors) located on the surface of CD4, CD8 T cells, and natural killer (NK) cells, and inhibits T cell and NK cell responses, thereby suppressing the immune system. Adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, inhibits phagocytosis and antigen presentation, and increases the secretion of tumor-promoting factors (e.g., VEGF, TGFb, and IL-6). Elevated adenosine levels mediated by CD39 and CD73 create an immunosuppressive environment that promotes the initiation and progression of cancer. In addition, CD39 is also constitutively present in human and mouse blood as a soluble enzyme and promotes the metabolism of ADP together with other purinergic enzymes.

[0004] CD39 is widely expressed in various tissues and organs, such as bladder, brain, breast, colon, uterus, stomach, prostate, etc., and is mainly expressed in endothelial cells and immune cells. CD39 is highly expressed in various human tumors, including lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer and testicular cancer. In addition to the effector T cells and NK cells mentioned above, CD39's regulatory function on the immune system is also manifested in macrophages, MDSC, neutrophils, regulatory T cells, antigen-presenting cells and other cells. CD39 is associated with immunosuppression and immune exhaustion in most immune cells, while knocking out CD39 can upregulate the secretion of immune factors, inhibit the activity of regulatory T cells and stimulate the function of immune cells.

[0005] Given the important role CD39 plays in the tumor microenvironment, it has become an emerging target for researchers to develop tumor immunotherapy. In the tumor microenvironment, targeting CD39 and inhibiting CD39 enzyme activity can block adenosine-mediated immunosuppression, thereby inhibiting tumor growth. Summary of the invention

[0006] Compared with traditional antibodies, in the design of bispecific antibodies and multispecific antibodies, nanoantibodies have small molecular weights, weak viscosity between monomers, and are easy to connect in series, which can enrich the design of bispecific antibodies and multispecific antibodies. Moreover, nanoantibody fusion expression has better expression ability than traditional antibodies. Nanoantibodies are suitable for prokaryotic expression and various eukaryotic expression systems, with low large-scale production costs and short cycles. Moreover, nanoantibody fusion expression has better expression ability than traditional antibodies.

[0007] In view of the above problems, the present disclosure provides nanobodies that can specifically bind to CD39 and inhibit soluble and cell membrane surface CD39 enzymatic activity, preparation methods, compositions, etc. The benefits provided by the present disclosure are widely applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies that react with various targets.

[0008] On the one hand, the present invention provides a Nanobody that specifically binds to CD39, comprising CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are:

[0009] CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:24, and CDR3 shown in SEQ ID NO:29.

[0010] Further, in some embodiments of the invention, the Nanobody comprises a sequence having at least 80%, at least 85%, 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% or 100% sequence identity to SEQ ID NO: 5, or consists of SEQ ID NO: 5.

[0011] Further, in some embodiments of the present invention, the Nanobody comprises two or more binding domains, and the two or more binding domains have the same or different amino acid sequences.

[0012] Furthermore, in some embodiments of the present invention, an anti-CD39 antibody is provided, which is a traditional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of the above-mentioned anti-CD39 nanoantibody; preferably, the functional fragment is the Fab, Fab', (Fab')2, Fv, scFv or sdFv structure of the traditional antibody.

[0013] In some embodiments, the CD39 antibody is a heavy chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, or an immunoconjugate.

[0014] Traditional antibodies are structurally composed of two identical heavy chains and two identical light chains, the light chain having a light chain variable region (VL) and a light chain constant region (CL); the heavy chain having a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, CH3 and / or CH4). Under the premise that the present invention discloses the structure of a nano antibody that can specifically bind to CD39, it is easy for a person skilled in the art to think of using the nano antibody of the present invention to transform a traditional antibody, for example, applying the CDR region structure of the nano antibody of the present invention to a traditional antibody to obtain a traditional antibody that can specifically bind to CD39, and such traditional antibodies also fall within the scope of protection of the present invention; further, based on the structure of a traditional antibody, its partial structure, such as Fab, Fab', (Fab')2, Fv, scFv or sdFv structure, etc., also has CD39 binding specificity, which also falls within the scope of protection of the present invention.

[0015] On the other hand, the nanobody and the antibody are used in drugs for inhibiting soluble CD39 enzyme activity and inhibiting CD39 enzyme activity on the cell membrane surface.

[0016] On the other hand, the present invention provides a composition comprising the above-mentioned Nanobody, the above-mentioned antibody; and optional pharmaceutically acceptable excipients.

[0017] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned Nanobody or the above-mentioned antibody.

[0018] Based on the contents disclosed in the present invention, a person skilled in the art can easily obtain polynucleotide molecules encoding the above-mentioned Nanobodies and fusion proteins by conventional techniques in the art, and based on the degeneracy of codons, the polynucleotide molecules are variable, and there are multiple possibilities for their specific base sequences. Based on this, no matter how the polynucleotide molecules change, as long as they can encode the Nanobodies or fusion proteins of the present invention, they belong to the protection scope of the present invention.

[0019] In another aspect, the present invention provides a nucleic acid construct comprising the above-mentioned polynucleotide.

[0020] In another aspect, the present invention provides a host cell comprising the above polynucleotide or the above nucleic acid construct.

[0021] On the other hand, the present invention provides the above-mentioned CD39 nanobody and the use of the above-mentioned antibody in preparing drugs and / or prolonging the half-life of drugs, wherein the drugs are used to prevent, treat and / or improve diseases related to CD39 expression.

[0022] On the other hand, the present invention provides an application of the above-mentioned CD39 nanobody, wherein an active molecule with a preventive or therapeutic effect is combined with the CD39 nanobody through a covalent bond, a non-covalent bond, or a linker molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings are used to provide further understanding of the present disclosure and constitute a part of the specification. They are used to explain the present disclosure together with the embodiments of the present disclosure and do not constitute a limitation of the present disclosure.

[0024] Figure 1A-Figure 1C The results of the CD39 nanobody protein binding activity assay are shown;

[0025] Figure 2A-2D The inhibitory activity of CD39 nanobody on soluble CD39 enzyme activity was shown;

[0026] Figure 3A-3C The CD39 nanobody showed inhibitory activity on cell surface CD39 enzyme activity;

[0027] Figure 4 The results of protein binding activity assays of CD39 nanoantibodies 6-2-9, 7-2-19, DR1-64, DR1-68 and control antibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 are shown;

[0028] Figure 5 The inhibitory activity of CD39 nanobodies 6-2-9, 7-2-19, DR1-64, DR1-68 and control antibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 on soluble CD39 enzyme activity was shown;

[0029] Figure 6 The results show that CD39 nanoantibodies 6-2-9, 7-2-19, DR1-64, DR1-68 and control antibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 have inhibitory activity on cell surface CD39 enzyme activity. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only illustrative embodiments of a part of the present invention, rather than all embodiments. Therefore, the present invention is not limited to the specific embodiments illustrated. In addition, any section titles used herein are not to be construed as limiting the subject matter described.

[0031] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0032] definition

[0033] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0034] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions, CDRs) separated by relatively conserved regions (called framework regions, FRs). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT or Chothia. In the specific embodiments of the present disclosure, the determination of CDR sequences uses the numbering definitions in the Kabat system.

[0035] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0036] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0037] "Chimeric antibodies" refer to antibodies in which the amino acid sequence of the antibody molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually humans) to avoid eliciting an immune response in that species.

[0038] "Nanoantibodies" are generally defined as in WO 2008 / 020079 or WO 2009 / 138519, and in a specific aspect generally represent VHH, humanized VHH or camelized VH (such as camelized human VH), or generally represent sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). "Nanoantibodies" are obtained by genetic engineering methods, and there are mainly three categories. The first category is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit that retains complete antigen binding activity and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from cartilaginous fish IgNAR such as sharks, represented by VNAR. The third category is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibodies, which retains antigen binding activity, but greatly reduces affinity and solubility.

[0039] For a further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and to the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 98 / 5196, WO 99 / 60816, WO 100 / 11979, WO 110 / 12346 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 to Algonomics NV and Able Links Inc. WO 03 / 050531 to the National Research Council of Canada; WO 01 / 90190 to the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and the further published patent applications of Able Links AG. Reference is also made to the further prior art mentioned in these applications and in particular to the list of references mentioned on pages 41-43 of international application WO 06 / 040153, which list and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) may be characterized in particular by the presence of one or more "Hallmark residues" in one or more framework sequences.Further description of Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of Nanobodies and their preparation can be found for example in WO 08 / 101985 and WO 08 / 142164.

[0040] Thus, in the meaning of the present disclosure, the term "antibody single variable domain" or "single variable domain" includes polypeptides derived from non-human sources, preferably camelids, preferably camelid heavy chain antibodies. As described before, they may be humanized. In addition, the term includes polypeptides derived from non-camelid sources, such as mice or humans, that have been "camelized", such as described in Davies and Riechmann 1994 (FEBS 339: 285-290), 1995 (Biotechnol. 13: 475-479), 1996 (Prot. Eng. 9: 531-537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231: 25-38).

[0041] The term "antibody single variable domain" includes antibody sequences of different origins, including mouse, rat, rabbit, donkey, human and camelid antibody sequences. It also includes fully human, humanized or chimeric antibody sequences. For example, it includes camelid antibody sequences and humanized camelid antibody sequences, or camelized antibody single variable domains, such as the camelized dAb described by Ward et al., 1989 (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.

[0042] Likewise, such antibody single variable domains may be derived from any suitable source in any suitable manner, and may for example be a naturally occurring VHH sequence (i.e. from a suitable Camelid species) or a synthetic or semi-synthetic amino acid sequence, including but not limited to partially or fully "humanized" VHHs, "camelized" antibody sequences (and in particular camelized VHs), as well as Nanobodies and / or VHHs obtained by techniques such as affinity maturation (e.g. starting from synthetic, random or naturally occurring antibody sequences such as VHH sequences), CDR grafting, veneering, combining fragments derived from different antibody sequences, PCR assembly using overlapping primers, and similar techniques known to the skilled person for engineering antibody sequences, or any suitable combination of any of them.

[0043] The amino acid sequence and structure of an antibody single variable domain can be considered, however without limitation, to consist of four framework regions or "FRs", which are referred to in the art and herein as "Framework Region 1" or "FR1"; "Framework Region 2" or "FR2"; "Framework Region 3" or "FR3"; and "Framework Region 4" or "FR4", respectively; which are separated by three complementarity determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region 1" or "CDR1"; "Complementarity Determining Region 2" or "CDR2"; and "Complementarity Determining Region 3" or "CDR3", respectively.

[0044] Based on the examples of antibody single variable domain sequences given herein and in WO 08 / 020079, in WO 06 / 040153 and in the references cited therein for other antibody single variable domains, it will be clear that the exact number of amino acid residues will also depend on the length of the specific CDR present in the antibody single variable domain. With regard to CDR, as is well known in the art, there are multiple definitions and the custom of describing the CDR of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the position of the structural loop region). For example, reference is made to the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, even though it may also be mentioned that the CDR according to Kabat, it is most preferably defined based on the Abm definition (which is based on the AbM antibody modeling software of Oxford Molecular), because it is considered that this is the best compromise between the Kabat and Chothia definitions. Again refer to the website http: / / www.bioinf.org.uk / abs / ).

[0045] The term "phage display library" refers to a "library" of phages on which exogenous peptides or proteins are expressed on their surfaces. Foreign peptides or polypeptides are displayed on the outer surface of the phage capsid. Foreign peptides can be displayed as recombinant fusion proteins incorporated as part of a phage coat protein; recombinant fusion proteins that are usually not phage coat proteins, but can become incorporated into the outer surface of the capsid; or proteins or peptides that become covalently or non-covalently linked to the protein. This is achieved by inserting an exogenous nucleic acid sequence into a nucleic acid that can be packaged into a phage particle. The exogenous nucleic acid sequence is, for example, inserted into the coding sequence of a phage coat protein gene. If the foreign sequence is an in-frame clone, the protein it encodes will be expressed as part of the coat protein. Therefore, a library of nucleic acid sequences, such as a library of antibody repertoires made from gene segments encoding the entire B cell repertoire of one or more individuals, can be inserted into a phage to produce a "phage library". When those peptides and proteins representing peptides and proteins encoded by a nucleic acid library are displayed by a phage, a "peptide display library" is produced. Although a variety of phages are used in the construction of such libraries, filamentous phages are often used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). See, for example, the following description of phage display libraries.

[0046] "Fc region" or "Fc" refers to the C-terminal region of the antibody heavy chain, which contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the binding of the antibody to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component of the classical complement system (e.g., C1q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is a segment from the amino acid residue at the Cys226 or Pro230 position to the carboxyl terminus, but its boundaries may vary. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may or may not be present. Fc may also refer to this region in isolation, or in the case of a protein polypeptide comprising Fc, such as a "binding protein comprising an Fc region", also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). The native sequence Fc region in the antibodies of the present invention includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the antibody's two heavy chains; IgM and IgFc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0047] "Specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of the immunobinding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, where the smaller the KD value, the higher the affinity. The immunobinding properties between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of antigen binding sites / antigen complexes.

[0048] The term "vector", "nucleic acid construct" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, a bacterial vector with a bacterial replication origin and an episomal mammalian vector). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can instruct the expression of the gene to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply referred to as "expression vector"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play equivalent functions.

[0049] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0050] Polynucleotides are the basic structural units of nucleic acids.

[0051] The term "multispecific antibody", the antibodies of the present disclosure may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide or may contain antigen binding domains that are specific for more than one target polypeptide. See, for example, Tutt et al., 1991, Journal of Immunology 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The antibodies of the present disclosure may be linked to another functional molecule (e.g., another peptide or protein) or co-expressed with it. For example, an antibody or a fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to prepare a bispecific or multispecific antibody with a second binding specificity. Variations on the bispecific antibody format described above are within the scope of the present disclosure. Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or bifunctional antibody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, knobs-in-holes, common light chains (e.g., common light chains with knobs-in-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes of defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. Epub: Dec. 4, 2012).

[0052] The term "single domain antibody" is obtained by genetic engineering methods, and there are mainly three categories. The first category is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit, retains complete antigen binding activity, and is the smallest natural antibody fragment. The second category is the heavy chain variable region obtained from cartilaginous fish IgNAR such as sharks, represented by VNAR. The third category is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibodies, which retains antigen binding activity, but the affinity and solubility are greatly reduced.

[0053] A binding domain refers to an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain Fv (scFv), an scFv dimer (a bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelid single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. A binding domain is capable of binding to the same antigen that a parent antibody or parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, a binding domain may comprise one or more CDRs from a specific human antibody grafted to a framework region from one or more different human antibodies.

[0054] The "association rate constant" (Ka or Kon) and "dissociation rate constant" (Kd or Koff) for a particular antibody-antigen interaction can both be calculated from the concentration and the actual rates of association and dissociation, and the KD, Ka and Kd values ​​can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured by bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured by surface plasmon resonance technology (e.g., Biacore) or KinExa.

[0055] The terms "antibody drug conjugate" and "immunoconjugate" are composed of an antibody, a linker and a drug, wherein the linker is a cleavable linker combination or a non-cleavable linker. Antibodies are globular proteins containing a series of amino acid sites that can be used to conjugate drug-linkers. Due to their tertiary and quaternary structures, only solvent-accessible amino acids are available for conjugation. In fact, high-yield conjugation usually occurs on the ε-amino group of lysine residues or the sulfhydryl group of cysteine ​​residues. The large number of lysine side chains on the surface of antibody proteins leads to a large number of sites available for drug conjugation, resulting in the generated antibody drug conjugate being a mixture containing different drug conjugation numbers (drug / antibody ratio, DAR) and conjugation sites. The conjugated product provided by the present disclosure, although still a mixture, has a very narrow DAR distribution range compared to the antibody drug conjugate obtained by conventional conjugation. Its average DAR value is close to 4, close to the average DAR value (2-4) range of the best antibody drug conjugate. In addition, the conjugated product rarely contains naked antibodies (DAR=0), and this component has no effect on cell toxicity. At the same time, the conjugated product does not contain heavy conjugated products (DAR=8), which are cleared very quickly in the body compared to low DAR components. Therefore, the heterogeneity of the antibody drug conjugate product provided by the present disclosure is greatly improved.

[0056] The antibodies that make up the antibody drug conjugates in the present disclosure preferably retain their original antigen binding ability in the wild state. Therefore, the antibodies in the present disclosure are able to, preferably specifically, bind to the antigen. In order to develop effective cellular level targets that can be used for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, and rarely or not expressed on the surface of one or more non-cancerous cells. Generally, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancerous cells. Confirming such tumor-associated factors can greatly improve the specific targeting properties of antibody-based cancer treatment.

[0057] The terms "conjugate", "link", "couple" refer to the association of two or more molecules. The link can also be genetic (i.e., recombinant fusion). In a specific context, the terms include reference to linking a ligand (e.g., an antibody portion) to an effector molecule. The link can be achieved using a variety of art-recognized techniques, such as chemical or recombinant. "Chemical" refers to the reaction between the antibody portion and the effector molecule, resulting in the formation of a covalent bond between the two molecules to form one molecule.

[0058] "Vector" refers to a nucleic acid molecule that can transport another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which other DNA segments can be connected. Another type of vector is a viral vector, in which an extra DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, a bacterial vector with a bacterial replication origin and an episomal mammalian vector). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after importing the host cell, and thus replicated with the host genome. In addition, some vectors can instruct the expression of the gene to which they are effectively connected.

[0059] "Nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0060] Example

[0061] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0062] Example 1: Alpaca Immunization

[0063] Human CD39 Protein (purchased from ACRO BIOSYSTEMS, catalog number CD9-H52H4) was used to immunize alpacas. For the first immunization, complete Freund's adjuvant was mixed with Human CD39 Protein antigen protein (0.4 mg) at a ratio of 1:1 and emulsified, and injected subcutaneously at multiple points. Every 2 weeks, Freund's incomplete adjuvant was emulsified with Human CD39 Protein antigen protein (0.4 mg) at a ratio of 1:1, and then boosted for a total of 7 immunizations. Blood was collected on the 7th day after each immunization to separate serum, and the immunogenic protein was used to coat the ELISA plate, and the serum titer was determined by ELISA.

[0064] Example 2: Phage display library construction

[0065] Peripheral blood was collected from alpacas and peripheral blood mononuclear cells (PBMC) were isolated. PBMC Total RNA was extracted using HiPure Total RNA Mini Kit (Magen, Cat. No. R4111-02) and used III First-Strand Synthesis System for RT-PCR (Invitrogen, Catalog No. 18080-051) kit for reverse transcription. Alpaca VHH gene fragments were amplified from cDNA, inserted into the phage display pComb3XSS (purchased from HonorGene, Catalog No. HG-VSW0922) vector, and electroporated ER2738 cells (BIOSEARCH, Catalog No. 60522-2) to construct a nanoantibody library. The bacterial library was expanded and cultured to the logarithmic growth phase, and the helper phage M13KO7 (M13KO7 Helper Phage purchased from NEB, Catalog No. N0315S, amplified after infecting the host) was added for superinfection, and the nanoantibody phage display library was packaged and amplified. The nanoantibody library obtained in this example has a storage capacity of more than 1.0×10 9 The titer of the obtained nanobody phage display library is greater than 1.0×10 12 cfu / mL.

[0066] Example 3: Nanobody phage display library screening

[0067] 1.0×10 12After adding the phage library of cfu and the streptavidin-coupled magnetic beads BeaverBeads Mag Streptavidin (purchased from Beaver Bio, item number 22305-1), add Biotinylated Human CD39 Protein (purchased from ACROBIOSYSTEMS, item number CD9-H82E5) to the magnetic beads for incubation, rinse with PBST (homemade, 1xPBS containing 0.05% Tween 20), and then add the blocked phage library to the magnetic beads-antigen for incubation. After rinsing with PBST, separate the magnetic beads with a magnetic stand, use 1mg / mL Trypsin (trypsin, derived from bovine pancreas, purchased from Sigma-Aldrich, item number T8802-50MG) to elute the phage and infect the host ER2738 for the next round of panning. After 1 to 3 rounds of selection, single clones were picked, cultured in deep-well plates, infected with M13KO7 helper phage (purchased from NEB, catalog number N0315S, and then amplified by ourselves), and monoclonal recombinant phages were packaged. The supernatant was collected and the positive clones that could bind to the immunogen were screened by ELISA and gene sequencing was performed.

[0068] Example 3: Nanobody Production

[0069] The DNA encoding the nanobody is synthesized and subcloned into an expression vector containing the human IgG constant region gene in advance. The vector is transfected into mammalian cells for recombinant protein expression, and the cell culture supernatant is collected and the expressed fusion nanobody is purified using a Protein A affinity chromatography column.

[0070] Example 4: Nanobody protein binding activity detection

[0071] Human CD39 Protein was diluted to 1μg / mL with PBS, and 100μL / well was coated on the ELISA plate. After washing with PBST, 2% BSA was blocked for 1h. The nanobody was diluted 3 times from 70nM using a PBST solution containing 1% BSA (purchased from Sigma Aldrich, catalog number B2064-50g), added to a 96-well plate, 100μL / well, and incubated at room temperature for 1h. After washing, incubate with rabbit anti-human IgGH&L (HRP) (abcam catalog number ab6759) and detect the OD450 value. The nanobody with good binding activity was screened out and numbered: 7-2-19; ​​it contains CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 24, and CDR3 shown in SEQ ID NO: 29. The 7-2-19 antibody sequence is as follows: QVQLVDSGGGLVQPGGSLRLSCEASPSISS LAYMG W HRQAPGKRRELVA AITAGGRTNYLDSVRGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCYA MED GTWFGNESDY WGQGTLVTVSS. The underlined part is CDR. The activity test results are as follows Figure 1A , Figure 1B and Figure 1C shown.

[0072] Example 5: Nanobody protease inhibitory activity detection

[0073] Dilute Human CD39 Protein to 0.6μg / mL with reaction solution (25mM Tris, 5mM MgCl2, pH 7.5) and add to a 96-well plate (Greiner, Cat. No. 655209). The control wells do not add protein, 30μL per well, and add 30μL of reaction solution; dilute the nanoantibody to be tested with antibody diluent, starting at 30μg / ml, 4-fold gradient dilution, and prepare a total of 7 concentration gradients. Add the gradient diluted nanoantibody to be tested to a 96-well plate, 30μl per well. Incubate at 37°C for 10 min; dilute 10 mM ATP (Sigma, Catalog No.: A7699-1G) to 150 μM with the reaction solution, add 30 μl to each well of the 96-well plate, and do not add ATP to the NC (negative control) wells, incubate at 37°C for 30 min; add 50 μL CTG detection reagent (Promega, Catalog No. G7572) to each well, and detect the chemiluminescence value after incubation for 5 min. Calculate the inhibition rate, inhibition rate = (RLU-RLU NC ) / (RLU Control -RLU NC )*100%. The result is Figure 2A , Figure 2B , Figure 2C and Figure 2D As shown, all 14 nanoantibodies have good protease inhibitory activity.

[0074] Example 6: Nanobody Cell Enzyme Activity Inhibition Detection

[0075] SK-MEL-28 cells in the logarithmic growth phase (purchased from ATCC, catalog number SK-MEL-28-HTB-72|ATCC) were taken, washed with PBS and treated with Accutase (Invitrogen, catalog number: 00-4555-56). The cell suspension was centrifuged at 300 g for 5 min and resuspended in antibody diluent to adjust the density to 2.0×10 5 / ml. Dilute the nanoantibody to be tested with antibody diluent, starting at 30μg / ml, 4-fold gradient dilution, a total of 7 gradients, and dilute ATP to 150μM with reaction solution (25mM Tris, 5mM CaCl2, pH 7.5). After mixing the cell suspension, add 50μl / well to a 96-well U-shaped plate, add 50μl of diluted nanoantibody to each well, and incubate at 37°C for 30min; add 50μl ATP to each well and mix, incubate at 37 degrees for 60min; centrifuge at 300g for 5min, take 50μL of supernatant to a 96-well plate, add 50μL CTG detection reagent (Promega, catalog number G7572) to each well, incubate at room temperature in the dark for 5min, and then detect the luminescence value with an enzyme reader.

[0076] Calculate the inhibition rate, inhibition rate = (RLU-RLU NC ) / (RLU Control -RLU NC )*100%. The result is Figure 3A , Figure 3B and Figure 3C As shown, all 14 nanoantibodies have good cell enzyme inhibitory activity.

[0077] Example 7: Comparison of Nanobody Activity

[0078] The obtained nanobodies, 6-2-9, 7-2-19, DR1-64, and DR1-68, which have better inhibitory effects on enzyme activity at the protein and cell levels, were compared with the CD39 nanobodies R-Ye-19(1)-037 and R-Ye-19(1)-046 of patent WO2023186121A1 (Pumis Biotechnology Co., Ltd.). The gene sequences of nanobodies R-Ye-19(1)-037 (SEQ ID NO: 32) and R-Ye-19(1)-046 (SEQ ID NO: 33) in patent WO2023186121A1 were fused with the human IgG1Fc region sequence to synthesize the gene, constructed into a eukaryotic expression vector, and the vector was transfected into mammalian cells for recombinant protein expression, and the expressed fusion nanobodies were purified using a Protein A affinity chromatography column (refer to the method used in the embodiment of patent WO2023186121A1). The CD39 protein binding activity was compared by referring to the method of Example 4, and the nanobody was diluted 4-fold starting from 80 nM, with a total of 12 gradients. The CD39 protease activity inhibition was compared by referring to the method of Example 5, and the nanobody was diluted 2-fold starting from 10 nM, with a total of 11 gradients. The cell-level CD39 enzyme activity inhibition was compared by referring to the method of Example 6, and the nanobody was diluted 3-fold starting from 100 nM, with a total of 11 gradients.

[0079] The results are as follows Figure 4, Figure 5 , Figure 6 As shown, the CD39 protein binding activity of 6-2-9, 7-2-19, DR1-64, and DR1-68 is comparable to that of the anti-CD39 nanoantibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 of patent WO2023186121A1, and the maximum enzyme activity inhibition rate at the protein level is better than that of the anti-CD39 nanoantibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 of patent WO2023186121A1. The maximum enzyme activity inhibition rate and IC50 of CD39 at the cellular level are better than those of the anti-CD39 nanoantibodies R-Ye-19(1)-037 and R-Ye-19(1)-046 of patent WO2023186121A1, indicating that they have better enzyme activity inhibition functional activity against soluble CD39 and cell membrane surface CD39.

[0080] Incorporated by Reference

[0081] Each patent document and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0082] Equivalence

[0083] The present invention may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and it is intended that all changes within the equivalent meaning and scope of the claims are included therein.

Claims

1. A nanobody that specifically binds to CD39, comprising CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are: CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:24, and CDR3 shown in SEQ ID NO:

29.

2. The Nanobody according to claim 1, which is shown in SEQ ID NO:

5.

3. Use of the nanobody according to any one of claims 1 to 2 in the preparation of an agent for inhibiting soluble CD39 enzyme activity or inhibiting cell membrane surface CD39 enzyme activity.

4. A composition comprising the Nanobody according to any one of claims 1-2, and optionally a pharmaceutically acceptable excipient.

5. A polynucleotide encoding the Nanobody according to any one of claims 1-2. A nucleic acid construct comprising the polynucleotide according to claim 5.

7. A host cell comprising the polynucleotide of claim 5 or the nucleic acid construct of claim 6.

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