Nanobodies specifically binding cd39 and uses thereof

Abstract

The present disclosure provides a kind of nanobody that specifically binds CD39 and can inhibit soluble CD39 enzyme activity and cell membrane surface CD39 enzyme activity, its preparation method, composition and the like, its protein binding activity is equivalent to patent WO2023186121A1 anti-CD39 nanobody R-Ye-19 (1) -037, R-Ye-19 (1) -046, the maximum enzyme activity inhibition rate at protein level is better than patent WO2023186121A1 anti-CD39 nanobody R-Ye-19 (1) -037, R-Ye-19 (1) -046, the maximum enzyme activity inhibition rate and IC50 of CD39 at cell level are all better than patent WO2023186121A1 anti-CD39 nanobody R-Ye-19 (1) -037, R-Ye-19 (1) -046.

Description

[0001] This application is a divisional application of the invention patent application with the application date of December 27, 2023, the application number of 202311835720.5, and the invention name of Nanobodies specifically binding to CD39 and uses thereof. TECHNICAL FIELD

[0002] The present disclosure relates to the field of antibodies, and more specifically to a nanobody specifically binding to CD39. BACKGROUND

[0003] CD39, also known as ecto-nucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is a full membrane protein that converts ATP or ADP to AMP, which is then dephosphorylated by CD73 to adenosine, an effective immune suppressor, and binds to adenosine receptors (e.g., A2A receptors) on the surface of CD4, CD8 T cells and natural killer (NK) cells, and suppresses T cell and NK cell responses, thereby suppressing the immune system. Adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, inhibiting phagocytosis and antigen presentation, and increasing the secretion of pro-cancer factors (e.g., VEGF, TGFb and IL-6). Elevated adenosine levels mediated by CD39 and CD73 create an immunosuppressive environment, promoting the occurrence and development of cancer. In addition, CD39 also exists as a soluble enzyme in human and mouse blood, 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, etc. In addition to the aforementioned effector T cells and NK cells, CD39 also has regulatory functions on immune system, such as on macrophages, MDSCs, neutrophils, regulatory T cells, antigen-presenting cells, etc. CD39 is associated with immune suppression, immune exhaustion, etc. 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 of CD39 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 immune suppression, thereby inhibiting tumor growth. SUMMARY

[0006] Compared with traditional antibodies, nanobodies have small molecular weight, weak adhesion between monomers, and are easy to be linked in series, which can enrich the design of diabodies and multispecific antibodies. Moreover, nanobody fusion expression has better expression capacity than traditional antibodies. Nanobodies are suitable for prokaryotic expression and various eukaryotic expression systems, and have low cost and short cycle in large-scale production. Moreover, nanobody fusion expression has better expression capacity than traditional antibodies.

[0007] To solve the above problems, the present disclosure provides a nanobody that can specifically bind to CD39 and can inhibit the enzyme activity of soluble and cell membrane surface CD39, a preparation method thereof, a composition, and the like. The benefits provided by the present disclosure are widely applicable to the fields of antibody therapy and diagnosis, and can be used in combination with antibodies that can react with various targets.

[0008] In one aspect, the present disclosure provides a nanobody that specifically binds to CD39, wherein the nanobody comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are as follows:

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

[0010] Further, in some embodiments of the present disclosure, 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 disclosure, the nanobody comprises two or more binding domains, and the two or more binding domains have the same or different amino acid sequences.

[0012] Further, in some embodiments of the present disclosure, an anti-CD39 antibody is provided, and the antibody is a traditional antibody or a functional fragment thereof, and the heavy chain variable region of the antibody is composed of the nanobody against CD39 described above; preferably, the functional fragment is a 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, an immunoconjugate.

[0014] The conventional antibody is structurally composed of two identical heavy chains and two identical light chains, the light chain has a light chain variable region (VL) and a light chain constant region (CL); the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, CH3 and / or CH4). Under the premise that the present application discloses the structure of the nanobody capable of specifically binding to CD39, it is easy for those skilled in the art to conceive that the nanobody of the present application is used to modify the conventional antibody, for example, the CDR region structure of the nanobody of the present application is applied to the conventional antibody, so as to obtain a conventional antibody capable of specifically binding to CD39, and such conventional antibody also belongs to the protection scope of the present application; further, based on the structure of the conventional antibody, part of the structure such as Fab, Fab', (Fab')2, Fv, scFv or sdFv structure also has CD39 binding specificity, which also belongs to the protection scope of the present application.

[0015] In another aspect, the nanobody, the antibody is used in the preparation of a medicament for inhibiting the activity of soluble CD39 enzyme and inhibiting the activity of CD39 enzyme on the surface of cell membrane.

[0016] In another aspect, the present application provides a composition comprising the above-mentioned nanobody, the above-mentioned antibody; and optionally a pharmaceutically acceptable adjuvant.

[0017] In another aspect, the present application provides a polynucleotide encoding the above-mentioned nanobody, the above-mentioned antibody.

[0018] Based on the disclosure of the present application, those skilled in the art can easily obtain the polynucleotide molecule encoding the above-mentioned nanobody and fusion protein by the conventional technology in the art, and based on the degeneracy of codon, the polynucleotide molecule is variable, and there are many possibilities for the specific base sequence, based on this, no matter how the polynucleotide molecule changes, as long as it can encode the nanobody or fusion protein of the present application, it belongs to the protection scope of the present application.

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

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

[0021] In another aspect, the present application provides the use of the above-mentioned CD39 nanobody, the above-mentioned antibody in the preparation of a medicament and / or prolonging the half-life of the medicament, the medicament is used for preventing, treating and / or improving diseases related to CD39 expression.

[0022] In another aspect, the present application provides a use of the above-mentioned CD39 nanobody, which binds an active molecule having a prophylactic or therapeutic effect to the CD39 nanobody through a covalent bond, a non-covalent bond, or a linker molecule. BRIEF DESCRIPTION OF DRAWINGS

[0023] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and are used to explain the present disclosure, but do not limit the present disclosure.

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

[0025] Figures 2A-2D The results of the detection of the protein binding activity of the CD39 nanobody are shown;

[0026] Figures 3A-3C The results of the detection of the protein binding activity of the CD39 nanobody are shown;

[0027] Figure 4 The results of the detection of the protein binding activity of the CD39 nanobody are shown;

[0028] Figure 5 The results of the detection of the protein binding activity of the CD39 nanobody are shown;

[0029] Figure 6 The results of the detection of the protein binding activity of the CD39 nanobody are shown; DETAILED DESCRIPTION

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

[0031] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by a person of ordinary skill in the art. Further, unless otherwise required by context, singular forms "a," "an," and "the" include plural referents. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes," and "included" is not limiting. Also, ranges are used herein to include endpoints and all values between the endpoints.

[0032] Definitions

[0033] To better understand the present application, definitions and explanations of relevant terms are provided as follows.

[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 chain of an antibody can be classified as either kappa or lambda light chain. The heavy chain can be classified as mu, delta, gamma, alpha, or epsilon, which defines the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. In both 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 further comprises a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed 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 complementarity determining regions, CDRs) that are spaced apart by relatively conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from N-terminus to C-terminus. The variable regions of each heavy / light chain pair (VH and VL) form an antigen binding site / portion, respectively. The distribution of amino acids in the various regions or domains follows the numbering definitions of the commonly used systems of Kabat, IMGT, or Chothia, and in the specific embodiments of the present disclosure, the determination of CDR sequences uses the numbering definition in the Kabat system.

[0035] Generally, the antigen-binding properties of an antibody can be described by three specific regions of the variable region of the heavy chain, called the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs), the amino acid sequences of which are relatively conserved and not directly involved in binding. The CDRs form loops or complementarity determining regions connecting the FRs that are held in place in the overall structure by FR secondary interactions. The CDRs on the heavy chain and on the light chain together with their connecting FRs form the three-dimensional antigen binding site of an antibody. It is possible to determine which amino acids form the FRs or the CDRs by comparing the amino acid sequences of antibodies of the same class or subclass.

[0036] The present application includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0037] A "chimeric antibody" is one 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 the variable regions of an antibody of a desired specificity, affinity, and capability from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions correspond to the sequences of another species (usually human) to avoid eliciting an immune response in that species.

[0038] A "nanobody" is generally as defined in WO 2008 / 020079 or WO 2009 / 138519 and in one particular aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH), or generally denotes a sequence optimized VHH (e.g. optimized for chemical stability and / or solubility, maximal overlap with known human framework regions and maximal expression). "Nanobodies" are obtained by genetic engineering methods, and there are mainly three types, the first type is the heavy chain variable region from camelid HCAb, which is a single folding unit, retains the complete antigen binding activity, and is the smallest natural antibody fragment. The second type is the heavy chain variable region from cartilaginous fish IgNAR, denoted as VNAR. The third type is the heavy chain or light chain variable region from human or murine monoclonal antibody, which retains the antigen binding activity, but the affinity and solubility are greatly reduced.

[0039] For 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 to 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 1134231 and WO 02 / 48193 to Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 to Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 to Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 to National Research Council of Canada; WO 03 / 025020 to 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 to Ablynx N.V. and further published patent applications of Ablynx N.V. Reference is also made to 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, can be characterized in that one or more “signature residues” are present in one or more framework sequences.Further description of Nanobodies can be found, for example, in WO 08 / 101985 and WO 08 / 142164, including humanization and / or camelization of Nanobodies, as well as other modifications, portions or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of Nanobodies and their production.

[0040] Thus, in the sense of the present disclosure, the term "antibody single variable domain" or "single variable domain" includes polypeptides derived from non-human sources, preferably from Camelidae, preferably from Camelidae heavy chain antibodies. As described before, they can be humanized. Furthermore, the term includes polypeptides derived from non-Camelidae sources, such as mouse or human, which have been "camelized", for example 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 origin, including mouse, rat, rabbit, donkey, human and Camelidae antibody sequences. It also includes fully human, humanized or chimeric antibody sequences. For example, it includes Camelidae antibody sequences and humanized Camelidae antibody sequences, or camelized antibody single variable domains, such as the camelized dAbs described by Ward et al., 1989 (see, e.g., WO 94 / 04678 as well as Davies and Riechmann 1994, 1995 and 1996) and camelized VHS.

[0042] Likewise, such antibody single variable domains can be derived from any suitable source in any suitable manner, and can be, for example, naturally occurring VHH sequences (i.e., from a suitable Camelidae species) or synthetic or semi-synthetic amino acid sequences, including but not limited to partially or fully "humanized" VHHs, "camelized" antibody sequences (and especially 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 the foregoing.

[0043] The amino acid sequences and structure of antibody single variable domains can be considered - 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", respectively; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively; which are interrupted 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 further references cited therein with respect to antibody single variable domains, it will be clear that the precise number of amino acid residues will also depend on the length of the particular CDRs present in the antibody single variable domain. With respect to CDRs, as is well known in the art, there are multiple definitions and conventions describing the CDRs 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 location of the structural loop regions). Reference is made, for example, to the website http: / / www.bioinf.org.uk / abs / . For the purposes of the present specification and claims, even though the CDRs according to Kabat can also be mentioned, the CDRs are most preferably defined based on the Abm definition (which is based on the Oxford Molecular's AbM antibody modelling software), as this is considered to be the best compromise between the Kabat and Chothia definitions. Reference is again made to the website http: / / www.bioinf.org.uk / abs / ).

[0045] The term "phage display library" refers to a "library" of phage expressing a foreign peptide or protein on their surface. The foreign peptide or polypeptide is displayed on the outer surface of the phage coat. The foreign peptide can be displayed as a recombinant fusion protein incorporated as part of the phage coat protein; a recombinant fusion protein that is not normally a phage coat protein but is able to become incorporated into the coat outer surface; or a protein or peptide that becomes covalently or noncovalently attached to the protein. This is accomplished by inserting a foreign nucleic acid sequence into a nucleic acid that can be packaged into a phage particle. The foreign nucleic acid sequence is inserted, for example, into the coding sequence of a phage coat protein gene. If the foreign sequence is cloned in frame, the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody repertoires generated from individual CDRs, can be inserted into phage in this way to create a "phage library." When the peptides and proteins represented by the nucleic acid library are displayed by the phage, a "peptide display library" is created. Although a variety of phage are used in the construction of these libraries, filamentous phage are commonly used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). See, e.g., the following descriptions of phage display libraries.

[0046] "Fc region" or "Fc" refers to a C-terminal region of an antibody heavy chain that contains at least a portion of a hinge region, a CH2 domain and a CH3 domain, which mediates 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 to the first component (e.g., Clq) of the classical complement system, including native-sequence Fc regions and variant Fc regions. Generally, a human IgG heavy chain Fc region is the segment from an amino acid residue at its Cys226 or Pro230 position, or changes can be made to the boundary region. The C-terminal lysine (residue 447, according to EU numbering system) of the Fc region can or can not be present. Fc can also refer to this region isolated, or in the context of a protein polypeptide comprising Fc, e.g., "binding proteins comprising an Fc region," also referred to as "Fc fusion proteins" (e.g., antibodies or immunoadhesins). Native sequence Fc regions in the antibodies of the application include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region in the IgG, IgA, and IgD antibody isotypes comprises the CH2 and CH3 constant domains of each of the heavy chains of the antibody; the IgM and IgE Fc regions comprise the three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

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

[0048] The terms "vector," "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA technologies are often in the form of plasmids. However, other forms of expression vectors are available (e.g., viral vectors such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

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

[0050] A polynucleotide is the basic structural unit of nucleic acids.

[0051] The term "multispecific antibody", an antibody of the present disclosure can be monospecific, bispecific or multispecific. A multispecific antibody can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. An antibody of the present disclosure can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity. Variations of the bispecific antibody formats described above are encompassed 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, e.g., scFv or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-in-hole structures, common light chain (e.g., common light chain with knob-in-hole structure, 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 binding, e.g., where unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. electronic publication: December 4, 2012).

[0052] The term "single domain antibody" is obtained by genetic engineering method, mainly three types, the first type is the heavy chain variable region obtained from camelid HCAb, which is a single folding unit, retains the complete antigen binding activity, and is the smallest natural antibody fragment. The second type is the heavy chain variable region obtained from shark IgNAR, represented by VNAR. The third type is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibody, which retains the 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, a Fab, a Fab', a F(ab')2, a Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (dsdiabody), a single chain Fv (scFv), a scFv dimer (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 bound by the parent antibody or parent antibody fragment (e.g., a parent scFv). In some embodiments, a binding domain can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0054] Both the "on rate constant" (Kaor Kon) and the "off rate constant" (Kdor Koff) for a particular antibody-antigen interaction can be calculated from the concentrations and the actual rates of association and dissociation, and KD, Kaand Kdvalues can be measured using any effective method. In preferred embodiments, the dissociation constant is measured using bioluminometric interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or KinExa.

[0055] The terms "antibody drug conjugate," "immunoconjugate," consist of an antibody, a linker, and a drug. The linker is either a cleavable linker combination or a non-cleavable linker. The antibody is a globular protein with a number of amino acid sites available for conjugation of the drug-linker. Due to its tertiary and quaternary structure, only solvent accessible amino acids are available for conjugation. In fact, high yield conjugation typically occurs at the epsilon-amino groups of lysine residues or the thiol groups of cysteine residues. The large number of lysine side chains on the surface of the antibody protein results in a large number of sites available for drug conjugation, resulting in a mixture of antibody drug conjugates with different drug conjugation numbers (drug / antibody ratio, DAR) and conjugation sites. The conjugation product provided by the present disclosure, although still a mixture, has a very narrow range of DAR distribution compared to antibody drug conjugates obtained by traditional methods. Its average DAR value is close to 4, close to the optimal range of average DAR value (2-4) of antibody drug conjugates. In addition, the conjugation product contains very few naked antibodies (DAR=0), which are not effective in killing cells. At the same time, the conjugation product also contains very few heavily conjugated products (DAR=8), which are quickly cleared in vivo relative to the 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 of the present disclosure preferably retain their antigen binding ability as in their original wild-type state. Thus, the antibodies of the present disclosure are capable, and preferably specifically, bind to an antigen. In an effort to develop effective cell level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells, while being expressed little or not at all on the surface of one or more non-cancerous cells. Typically, such tumor-associated polypeptides are over-expressed on the surface of cancer cells relative to the surface of non-cancerous cells. Identification of such tumor-associated factors can greatly enhance the specific targeting properties of antibody-based therapies for cancer.

[0057] The terms "conjugate", "link", "couple" refer to the association of two or more molecules. The linkage can also be genetic (i.e., recombinant fusion). In a particular context, the terms include reference to the linkage of a ligand (e.g., an antibody moiety) to an effector molecule. Such linkage can be achieved using a variety of art-recognized techniques, e.g., by chemical or recombinant means. By "chemical means" is meant a reaction between the antibody moiety and the effector molecule to form a covalent bond between the two molecules to form one molecule.

[0058] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Host cells can be heterologous or homologous to the vector(s) with which they are being used.

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

[0060] Examples

[0061] The application described generally 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 present application. Additionally, the experimental methods described in the following examples are routine methods unless otherwise specified. The starting materials, reagents, materials, etc. used in the following examples are commercially available unless otherwise specified.

[0062] Example 1: Immunization of Llama

[0063] Alpacas were immunized with Human CD39 Protein (purchased from ACRO BIOSYSTEMS, catalog number CD9-H52H4) antigen. For the initial immunization, a 1:1 mixture of complete Freund's adjuvant and Human CD39 Protein antigen (0.4 mg) was emulsified and administered subcutaneously at multiple sites. Booster immunizations were performed every two weeks using a 1:1 mixture of incomplete Freund's adjuvant and Human CD39 Protein antigen (0.4 mg). A total of seven immunizations were administered. Blood was collected on day 7 after each immunization, and serum was separated. Serum titers were determined using an immunogenic protein-coated ELISA plate.

[0064] Example 2: Construction of Phage Display Library

[0065] Peripheral blood was collected from alpacas, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from the PBMCs using the HiPure Total RNA Mini Kit (Magen, catalog number R4111-02), and then... III. The First-Strand Synthesis System for RT-PCR (Invitrogen, catalog number 18080-051) kit was used for reverse transcription. The alpaca VHH gene fragment was amplified from cDNA and inserted into the phage display vector pComb3XSS (purchased from HonorGene, catalog number HG-VSW0922). This vector was then electroporated into ER2738 cells (BIOSEARCH, catalog number 60522-2) to construct a nanobody library. The bacterial library was expanded to the logarithmic growth phase, and then superinfected with helper phage M13KO7 (purchased from NEB, catalog number N0315S, which amplifies spontaneously after infecting the host) to package and amplify the nanobody phage display library. The nanobody library obtained in this example has a volume greater than 1.0 × 10⁻⁶. 9 The obtained nanobody phage display library titer is greater than 1.0 × 10⁻⁶. 12 cfu / mL.

[0066] Example 3: Screening of Nanobody Phage Display Library

[0067] SuperBlock PBS Blocking Buffer (Thermo, catalog number 37580) was used to block 1.0 × 10⁻⁶ PBS samples. 12After phage library of cfu and streptavidin conjugated magnetic beads Beaver Beads Mag Streptavidin (purchased from Beaver, Cat. No. 22305-1) were added Biotinylated Human CD39 Protein (purchased from ACROBIOSYSTEMS, Cat. No. CD9-H82E5) for incubation, after PBST (self-made, 0.05% Tween 20 in 1x PBS) rinsing, the magnetic beads-antigen were added to the blocked phage library for incubation. After PBST rinsing, the magnetic beads were separated by magnetic stand, and 1 mg / mL Trypsin (trypsin, derived from bovine pancreas, purchased from Sigma-Aldrich, Cat. No. T8802-50MG) was used to elute the phage and infect the host ER2738 for the next round of panning. After 1-3 rounds of selection, single clones were picked, cultured in a deep well plate, and then infected with M13KO7 helper phage (purchased from NEB, Cat. No. N0315S, and then amplified by oneself) to package the single clone recombinant phage. The supernatant was collected and screened for positive clones that could bind to the immunogen by ELISA method and gene sequencing.

[0068] Example 3: Nanobody production

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

[0070] Example 4: Detection of nanobody protein binding activity

[0071] Human CD39 Protein was diluted with PBS to 1 pg / mL, and 100 pL / well was used to coat the enzyme-labeled plate. After PBST washing, 2% BSA was blocked for 1 h. The nanobody was diluted by 3-fold gradient starting from 70 nM using PBST solution containing 1% BSA (purchased from Sigma Aldrich, Cat. No. B2064-50g), and was added to the 96-well plate, 100 pL / well, incubated at room temperature for 1 h. After washing, rabbit anti-human IgG H&L (HRP) (abcam Cat. No. ab6759) was incubated, and the OD450 value was detected. The nanobody with better binding activity was screened, and the number was 7-2-19; it contained CDR1 as shown in SEQ ID NO: 18, CDR2 as shown in SEQ ID NO: 24, and CDR3 as 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 parts are CDRs. The activity detection results thereof are shown in Figure 1A , Figure 1B and Figure 1C .

[0072] Example 5: Detection of Nanobody Protease Activity Inhibition Activity

[0073] Dilute Human CD39 Protein to 0.6 μg / mL with reaction solution (25 mM Tris, 5 mM MgCl2, pH 7.5), add to 96-well plates (Greiner, item number 655209), and add 30 μL of reaction solution to each well of the Control wells without adding protein; dilute the nanobodies to be tested with antibody diluent, starting at 30 μg / mL, and dilute by 4-fold gradient, to prepare 7 concentration gradients. Add the nanobodies to be tested diluted by gradient to the 96-well plates, 30 μL per well. Incubate at 37°C for 10 min; dilute 10 mM ATP (Sigma, item number: A7699-1G) to 150 μM with reaction solution, and add 30 μL per well to the 96-well plates, and incubate at 37°C for 30 min without adding ATP to the NC (negative control) wells; add 50 μL of CTG detection reagent (Promega, item number G7572) per 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 results are shown in Figure 2A , Figure 2B , Figure 2C and Figure 2D . The 14 nanobodies all have good protease activity inhibition activity.

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

[0075] Take SK-MEL-28 cells (purchased from ATCC, item number SK-MEL-28-HTB-72|ATCC) in the logarithmic growth phase, wash with PBS, and treat with Accutase (Invitrogen, item number: 00-4555-56). Centrifuge the cell suspension at 300g for 5 min, resuspend with antibody diluent, and adjust the density to 2.0 x 10 5The antibody diluent was used to dilute the nanobodies to be tested, starting at 30 pg / ml, 4-fold gradient dilution, a total of 7 gradients, and ATP was diluted to 150 mM with reaction solution (25 mM Tris, 5 mM CaCl2, pH 7.5). After mixing the cell suspension, 50 mΐ was added to each well of a 96-well U-shaped plate, 50 mΐ of the diluted nanobodies was added to each well, and incubated at 37°C for 30 min; 50 mΐ of ATP was added to each well and mixed, and incubated at 37°C for 60 min; after centrifugation at 300 g for 5 min, 50 mΐ of supernatant was taken to a 96-well plate, 50 mΐ of CTG detection reagent (Promega, item number G7572) was added to each well, and incubated at room temperature for 5 min, then the luminescence value was detected by a microplate reader.

[0076] The inhibition rate was calculated, inhibition rate = (RLU-RLU NC ) / (RLU Control -RLU NC )*100%. The results are shown in Figure 3A 、 Figure 3B and Figure 3C , and 14 strains of nanobodies all have good cell enzyme activity inhibition activity.

[0077] Example 7: Comparison of nanobody activity

[0078] The 6-2-9, 7-2-19, DR1-64, and DR1-68 nanobodies obtained had better protein and cell level enzyme activity inhibition effect, and the CD39 nanobodies R-Ye-19(1)-037 and R-Ye-19(1)-046 in the patent WO2023186121A1 (Pumis Biotechnology Co., Ltd.) were compared for activity. The gene sequences of the nanobodies R-Ye-19(1)-037 (SEQ ID NO: 32) and R-Ye-19(1)-046 (SEQ ID NO: 33) in the patent WO2023186121A1 were fused with the human IgG1 Fc region sequence and synthesized into a gene, which was constructed in a eukaryotic expression vector, 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 adopted in the patent WO2023186121A1 example). The CD39 protein binding activity comparison was carried out according to the method of Example 4, the nanobodies were diluted by 4-fold gradient from 80 nM, a total of 12 gradients. The CD39 protein enzyme activity inhibition effect comparison was carried out according to the method of Example 5, the nanobodies were diluted by 2-fold gradient from 10 nM, a total of 11 gradients. The comparison of cell level CD39 enzyme activity inhibition effect was carried out according to the method of Example 6, the nanobodies were diluted by 3-fold gradient from 100 nM, a total of 11 gradients.

[0079] The results are shown in Figure 4、 Figure 5 、 Figure 6 Definitions As shown in Table 1, the 6-2-9, 7-2-19, DR1-64, DR1-68 CD39 protein binding activity is equivalent to the patent WO2023186121A1 anti-CD39 nanobody R-Ye-19(1)-037, R-Ye-19(1)-046, the maximum enzyme activity inhibition rate at the protein level is better than the patent WO2023186121A1 anti-CD39 nanobody R-Ye-19(1)-037, R-Ye-19(1)-046, the maximum enzyme activity inhibition rate and IC50 at the cell level are better than the patent WO2023186121A1 anti-CD39 nanobody R-Ye-19(1)-037, R-Ye-19(1)-046, which shows that it has better enzyme activity inhibition function activity for soluble CD39 and cell membrane surface CD39.

[0080] incorporated by reference

[0081] The entire contents of each patent and scientific document referred to herein is incorporated by reference herein for all purposes.

[0082] equivalents

[0083] The application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are merely illustrative, not restrictive. The scope of the application should be determined by the appended claims and their legal equivalents rather than by the description above, and all changes which come within the meaning and range of equivalency of the claims are to be embraced therein.

Claims

1. A Nanobody that specifically binds to CD39, wherein comprises CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 are: CDR1 as set forth in SEQ ID NO: 18, CDR2 as set forth in SEQ ID NO: 24, and CDR3 as set forth in SEQ ID NO:

29.

2. The Nanobody of claim 1, as set forth in SEQ ID NO:

5.

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

4. A polynucleotide encoding the Nanobody of any one of claims 1-2.

5. A nucleic acid construct comprising the polynucleotide of claim 4.

6. A host cell comprising the polynucleotide of claim 4 or the nucleic acid construct of claim 5. ​

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