Patent Application
20230416360
The present disclosure claims the right of priority of Chinese patent application no. 202011395861.6 titled “ANTI-CD22 NANO ANTIBODY AND USE THEREOF” and filed with the China Patent Office on Dec. 3, 2020, which is incorporated in the present disclosure by reference in its entirety.
The present invention relates to the fields of bioengineering and biomedicine, and mainly relates to an anti-human CD22 nano body or an antigen-binding fragment thereof, and an encoding nucleic acid, an expression vector and an expression cell, a preparation method, a pharmaceutical composition therefor, and their use for treating diseases, such as use for treating tumors and autoimmune diseases.
CD22 is a type I transmembrane glycoprotein, which belongs to the sialic acid-binding immunoglobulin like lectins (Siglec) family. As a B cell differentiation antigen, it is specifically expressed in B cells. The expression of CD22 begins from the pre-B cell (pre-B cell) stage, and stops after B cells differentiate into plasma cells. The broad-spectrum expression of CD22 in B cell development makes it an attractive molecule for targeting B cells.
The extracellular region of CD22 consists of 7 Ig-like domains and 12 predicted N-linked glycosylation sites, and its N-terminal (i.e., distal end of membrane) domain 1 is V Type Ig-like domain, which, as a ligand binding site, can recognize α2,6-coupled sialic acid. The intracellular region of CD22 has immunoreceptor tyrosine-based inhibitory motifs (ITIMs). When the tyrosine on the ITIMs is phosphorylated by the Src family protein kinase, binding sites for molecules containing SH2 (Src homology2) domain would be generated, then SHP-1 (Src homology region 2 domain-containing phosphatase-1) was recruited to inhibit the BCR (B-cell receptor) signaling pathway of normal B cells.
α2,6-coupled sialoglycoprotein exists in hematopoietic cells, some endothelial cells, T cells and B cells, and CD22 protein itself also produces α2,6-coupled sialic acid, so CD22 can form cis-interaction with itself and other sialoglycoproteins on the surface of B cells, and trans-interaction with sialoglycoproteins on the surface of other types of cells. In resting B cells, the cis-interaction between CD22 molecules makes the ligand-binding site of CD22 masked, but once the ligand is presented by an adjacent cell, the masked ligand-binding site of CD22 is exposed and interacts with the ligand of the adjacent cells to form trans-interaction. The cis-interaction between CD22 molecules forms homo-oligomers on the B cell surface and the homo-oligomers can form a dynamic nanocluster and generate an antigen binding signal threshold that must be reached before B cell activation, thereby regulating B cell signaling pathway.
CD22 is expressed in 60% to 90% of B cell malignancies and is not expressed in hematopoietic stem cells. In an early clinical study on acute lymphoblastic leukemia (ALL), CD22 is expressed in 60% to 85% of ALL. In another study, the positive rate of CD22 in B-lineage ALL patients reaches 93%. CD22 is expressed in more than 85% of patients with diffuse large B-cell lymphomas (DLBCLs). There are many clinical trials investigating the effectiveness of drugs that target CD22. Epratuzumab is a CD22 monoclonal antibody that has certain effects in adults and children with B-ALL. CD22 antibody-conjugated drugs have a certain therapeutic effect on B-ALL.
Nano antibody (Nanobody, Nb) is a genetically engineered antibody containing only a single domain. In 1993, Belgian scientist Hamers-Casterman C discovered a natural heavy chain antibody in camel blood that only contained heavy chains but no light chains. Compared with a normal antibody, the heavy chain antibody has no light chains, but still retains the ability to bind to an antigen. After cloning the variable region of the heavy chain antibody in camels, the obtained single domain antibody (sdAb) consisting of only one heavy chain variable region is called nanobody or VHH antibody (variable heavy chain domain of a heavy chain antibody). The nanobody not only has a molecular weight which is only 1/10 of that of a normal antibody, but also has more flexible chemical properties, good stability, high solubility, and high tumor tissue penetration, and is easily expressed and easily coupled to other molecules. Therefore, the nanobody technology has broad prospects of application in development of a therapeutic antibody against CD22.
The present invention provides a nanobody or an antigen-binding fragment that specifically binds to CD22, a nucleic acid encoding the antibody and the antigen-binding fragment, a pharmaceutical composition and a kit comprising the antibody and the antigen-binding fragment, and they can be used in the preparation of drugs for treating tumors, autoimmune diseases, etc.
In some embodiments, the nanobody or the antigen-binding fragment that specifically binds CD22, the nanobody or the antigen-binding fragment comprises a combination of CDRs, the combination of CDRs comprises: CDR1, CDR2, and CDR3; the CDR1, CDR2 and CDR3 have any sequence combination selected from the following, or a sequence combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:
In particular, for example, the nanobody or the antigen-binding fragment of the invention, wherein:
In another specific embodiment, the invention provides an antibody or an antigen-binding fragment thereof comprising:
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the present invention binds to human CD22 with a dissociation constant (KD) of no more than 50 nM.
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the present invention comprises a sequence of the constant region of any one of human or murine antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, comprises a sequence of the constant region of human or murine antibody IgG1, IgG2, IgG3 or IgG4.
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the invention further comprises a heavy chain constant region sequence without a CH1 fragment.
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region sequence with CH2 and CH3 fragments.
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the invention is chimeric or humanized or fully human; preferably, the antibody or the antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (for example, a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab′, F(ab′)2, Fd, Fv, scFv, a diabody or a single domain antibody.
In a preferred embodiment, the antibody or the antigen-binding fragment thereof of the invention is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescence label, a ultrasound contrast agent or a photosensitizer.
In a preferred embodiment, the present invention also provides a multispecific antigen-binding molecule; preferably, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprises the antibody or the antigen-binding fragment described in any one of the above, the second antigen-binding module specifically binds to other antigens than CD22 or binds to a different CD22 epitope than the first antigen-binding module;
In a preferred embodiment, the present invention provides a chimeric antigen receptor (CAR); preferably, the chimeric antigen receptor at least comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen-binding domain comprises any of the CD22 antibody or the antigen-binding fragment described above.
In a preferred embodiment, the present invention provides an immune effector cell; preferably, the immune effector cell comprises the chimeric antigen receptor described above or a nucleic acid fragment encoding the chimeric antigen receptor described above;
In a preferred embodiment, the present invention provides an isolated nucleic acid molecule encoding the nanobody, the antigen-binding fragment, or any combination thereof according to any one of the above aspects of the present invention, the multispecific antigen-binding molecule described above or the chimeric antigen receptor described above.
In some embodiments, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the present invention described above.
In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule or the expression vector of the present invention described above.
In a preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell is selected from HEK293E or Chinese hamster ovary (CHO) cell.
In some embodiments, the present invention provides a method for preparing an antibody or an antigen-binding fragment or a multispecific antigen-binding molecule, the method comprises culturing the host cell of the present invention described above under appropriate conditions, and isolating the antibody or the antigen-binding fragment or the multispecific antigen-binding molecule.
In some embodiments, the present invention provides a method for preparing an immune effector cell, wherein the CAR nucleic acid fragment described above is introduced into the immune effector cell, preferably, the method further comprises enabling the immune effector cell to express the CAR described above.
In some embodiments, the present invention provides a pharmaceutical composition comprising the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, or a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, and a pharmaceutically acceptable carrier.
In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional antineoplastic agent.
In some embodiments, the present invention provides a method for preventing and/or treating a B cell disease, the method comprises administering the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above to a patient in need thereof. The B cell disease is preferably a tumor or an autoimmune disease;
In some embodiments, the present invention provides the use of the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above in the preparation of a drug for preventing and/or treating a B cell disease, and the B cell disease is preferably a tumor or an autoimmune disease;
In some embodiments, the present invention provides the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above for preventing and/or treating a B cell disease; The B cell disease is preferably a tumor or an autoimmune disease;
In some embodiments, the present invention provides a kit comprising the antibody or the antigen-binding fragment of the present invention described above, the multispecific antigen-binding molecule of the present invention described above, the chimeric antigen receptor of the present invention described above, the immune effector cell of the present invention described above, the isolated nucleic acid molecule of the present invention described above, the expression vector of the present invention described above, the cell of the present invention described above, or a product (e.g., an antibody and an antigen-binding fragment) prepared by the method of the invention described above, or the pharmaceutical composition of the invention described above, and instructions for use.
Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For a term that is explicitly defined herein, the meaning of the term is to be determined based on the definition.
As used herein, the term “antibody” (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest and includes polyclonal, monoclonal, genetically engineered, and other modified forms of an antibody (including but not limited to a chimeric antibody, a humanized antibody, a fully human antibody, a heteroconjugate antibody (e.g. a bispecific, trispecific and tetraspecific antibody, a diabody, a triabody and a tetrabody, a antibody conjugate)) and an antigen-binding fragment of an antibody (including, for example, Fab′, F(ab′)2, Fab, Fv, rIgG and scFv fragment). Furthermore, unless otherwise indicated, the term “monoclonal antibody” (mAb) is intended to include both an intact antibody molecule and an incomplete antibody fragment (such as Fab and F(ab′)2 fragment, which lacks the Fc fragment of the intact antibody (cleared more quickly from circulation in animals), thus lacking Fc-mediated effector function) capable of specifically binding to a target protein (see Wahl et al., J. Nucl. Med. 24:316, 1983; the content of which is incorporated herein by reference).
An “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanaco, alpaca, sheep, rabbits, mice, rats or cartilaginous fishes (such as sharks).
The term “natural antibody” herein refers to an antibody produced and paired by the immune system of a multicellular organism. The term “engineered antibody” herein refers to a non-natural antibody obtained through genetic engineering, antibody engineering, etc. For example, “engineered antibody” includes a humanized antibody, a small molecule antibody (such as scFv, etc.), a bispecific antibody, etc.
The term “monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
The term “multispecific” herein refers to having at least two antigen binding sites, each of which binds a different epitope of the same antigen or a different epitope of a different antigen. Thus, terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/an antigen-binding molecule can bind.
The term “valence” herein refers to the presence of a defined number of binding sites in an antibody/an antigen-binding molecule. Thus, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to the presence of one binding site, two binding sites, four binding site and six binding sites in an antibody/an antigen-binding molecule, respectively.
“Full-length antibody”, “complete antibody” and “intact antibody” are used interchangeably herein to mean that they have a structure substantially similar to that of a natural antibody.
As used herein, the term “antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The antibody fragment can be Fab, F(ab′)2, scFv, SMIP, a diabody, a triabody, an affibody, a nanobody, an aptamer or a domain antibody. Examples of a binding fragment encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) an Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (V) dAb comprising VH and VL domains; (vi) dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) dAb consisting of VH or VL domains; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, the CDRs may optionally be linked by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using a recombinant method through a linker that enables forming a single protein chain (referred to as a single chain Fv (scFv); see e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988) in which the VL and VH regions pair to form a monovalent molecule. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as an intact antibody. The antigen-binding fragment can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of an intact immunoglobulin, or in some embodiments by chemical peptide synthesis procedures known in the art.
As used herein, the term “CD22” refers to Siglec-2, a molecule belonging to the SIGLEC lectin family, which is present on the surface of mature B cells and to a lesser extent on certain immature B cells. The term “CD22” includes CD22 proteins of any human and non-human animal species, and specifically includes human CD22 as well as CD22 of non-human mammals.
As used herein, the term “bispecific antibody” refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected against an antigen epitope of CD22, and the other can be detected against another antigen epitope of CD22 or any other antigen except CD22, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterium-derived protein, or a bacterial surface protein.
As used herein, the term “chimeric” antibody refers to an antibody that has a variable sequence derived from an immunoglobulin of one organism (such as a rat or mouse) and a constant region derived from an immunoglobulin of a different organism, such as human. Methods for producing the chimeric antibody are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; The above documents are incorporated herein by reference.
As used herein, the term “heavy chain antibody” refers to an antibody that lacks the light chains of an conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
As used herein, the term “nanobody” refers to a natural heavy chain antibody without light chains in camel and cloning its variable region can obtain a single domain antibody, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which only consists of a heavy chain variable region, and is the smallest functional antigen-binding fragment. For a further description of VHH and nanobody, 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 the Free University of Brussels; WO 94/25591, WO 99/3768, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 of the National Research Council of Canada; WO 03/025020 (=EP 1433793) of 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 of Ablynx N.V., and further published patent applications of Ablynx N.V. Reference is also made to the additional prior art mentioned in these applications, in particular 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, nanobody (in particular VHH sequences and partially humanized nanobody) may inter alia be characterized by the presence of one or more “signature residues” in one or more framework sequences. Further descriptions of nanobody can be found in, for example, WO 08/101985 and WO 08/142164, including humanization and/or camelization of nanobody, as well as other modifications, parts or fragments, derivatives or “nanobody fusion”, multivalent constructs (including some non-limiting examples of linker sequences) and various modifications that increase the half-life of a nanobody and a formulation thereof. For a further general description of nanobody, reference is made to the prior art cited herein, for example WO 08/020079 (page 16).
As used herein, the term “complementarity determining region” (CDR) refers to hypervariable regions found in both light and heavy chain variable domains. The more conserved portions in variable domains are called the framework regions (FR). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered to be within the hypervariable regions under one set of criteria (such as IMGT or KABAT) but outside the hypervariable regions under a different set of criteria (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions. The invention includes an antibody comprising modifications in these hybrid hypervariable positions. The variable domains of the native heavy and light chains respectively comprise four framework regions that largely adopt a sheet configuration and connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets, and in some cases form part of the sheet structure. The CDRs in each chain are held tightly together by the FR regions in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, herein, CDR1-VH, CDR2-VH, and CDR3-VH refer to the first CDR, the second CDR, and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL, and CDR3-VL refer to the first CDR, the second CDR, and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).
As used herein, the term “monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody is produced.
As used herein, the term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv or Fab. The term “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
The term “heavy chain constant region” herein refers to the carboxy-terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, and has a more conserved amino acid sequence relative to the antibody's variable domain. A “heavy chain constant region” comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof “Heavy chain constant region” includes “full-length heavy chain constant region” and “heavy chain constant region fragment”, the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a part of the full-length heavy chain constant region”. For example, a typical “full-length antibody heavy chain constant region” consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical “heavy chain constant region fragment” can be selected from CH1, Fc or CH3 domains.
The term “light chain constant region” herein refers to the carboxy-terminal portion of the light chain of an antibody, which is not directly involved in the binding of the antibody to an antigen. The light chain constant region may be selected from a constant κ domain or a constant λ domain.
The term “Fc” herein refers to the carboxy-terminal portion of an antibody obtained by papain hydrolysis of the intact antibody, which typically includes the CH3 and CH2 domains of the antibody. Fc region includes, for example, a native sequence Fc region, a recombinant Fc region and a variant Fc region. Although the boundary of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region may comprise or may not comprise Lys447.
The term “humanized antibody” herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Generally speaking, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (for example, variable region FR and/or constant region) are derived from human Immunoglobulin (recipient antibody). Humanized antibody usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
The term “fully human antibody” herein refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibody herein may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, a “fully human antibody” herein is not intended to include an antibody in which CDR sequences derived from the germline of another mammalian species (for example, mouse) have been grafted onto human framework sequences.
The term “naked antibody” herein refers to an antibody that is not linked, fused or conjugated to another agent or molecule (for example, label or drug), peptide or polypeptide. In specific embodiments, the naked antibody expressed by a mammalian host cell can be glycosylated by the host cell's glycosylation machinery (for example, glycosylase). In certain embodiments, the naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (for example, glycosylase). In certain embodiments, the naked antibody is an intact antibody, while in other embodiments, the naked antibody is the antigen-binding fragment of an inact antibody, such as Fab antibody.
The term “conjugated antibody” herein refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent and can be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.
The term “diabody” herein refers to bivalent bispecific antibody that can bind to different epitopes on the same or different antigens.
As used herein, the term “percent (%) sequence identity” refers to the percentage of amino acid (or nucleotide) residues of the candidate sequence that are identical to those of the reference sequence after aligning the sequences and introducing gaps (if necessary) in order to achieve maximum percentage sequence identity (for example, for optimal alignment, gaps can be introduced in one or both of the candidate sequence and the reference sequence, and non-homologous sequences can be ignored for comparison purpose). For purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence shows sequence identity from 50% to 100% in the full length of the candidate sequence or in the selected part of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purpose is at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.
The term “conservative amino acid” herein generally refers to amino acids that belong to the same class or have similar characteristics (for example, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
The term “Kabat numbering system” herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; which is incorporated herein by reference).
The term “Chothia numbering system” herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; which is incorporated herein by reference).
The term “IMGT numbering system” herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; which is incorporated herein by reference).
As used herein, the term “specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, the proteins and other biomolecules are for example specifically recognized by an antibody or an antigen-binding fragment thereof. An antibody or an antigen-binding fragment thereof that specifically binds to an antigen would bind to the antigen with a KD of less than 100 nM. For example, an antibody or an antigen-binding fragment thereof that specifically binds to an antigen would bind to the antigen with a KD of up to 100 nM (for example, between 1 pM and 100 nM). An antibody or an antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or an epitope thereof would exhibit a KD for the particular antigen or the epitope thereof of greater than 100 nM (for example, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM). Various immunoassays are available to select for an antibody that reacts specifically with a specific protein or carbohydrate. For example, solid-phase ELISA immunoassay is routinely used to select for an antibody that reacts specifically with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describes immunoassay methods and conditions that can be used to determine specific immunoreactivity.
As used herein, the term “antibody conjugate” refers to a coupled entity/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker. Examples include antibody-drug conjugate (ADC), in which the drug molecule is said another molecule.
The term “chimeric antigen receptor (CAR)” herein refers to a recombinant protein comprising at least (1) an extracellular antigen-binding domain, such as a variable heavy or light chain of an antibody, and (2) a transmembrane domain used to make the anchored CAR enter immune effector cells, and (3) an intracellular signaling domain. In certain embodiments, the extracellular antigen binding domain of the CAR comprises a scFv. The scFv can be derived from the variable heavy and light regions of a fusion antibody. Alternatively or additionally, the scFv may be derived from Fab's (rather than an antibody, for example obtained from a Fab library). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.
Herein the term “nucleic acid” includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, the nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually expressed as 5′ to 3′. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and a polymer containing a mixture of two or more of these molecules. The nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense strand and antisense strand, as well as single-stranded form and double-stranded form. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. The nucleic acid molecule also encompasses DNA and RNA molecules which are suitable as vectors for direct expression of the antibody of the invention in vitro and/or in vivo, for example in a host or patient. Such DNA (for example cDNA) or RNA (for example mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate an antibody in vivo (see, for example, Stadler et al., Nature Medicine 2017, Published online Jun. 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
As used herein, the term “vector” includes a nucleic acid vector, such as a DNA vector (such as a plasmid), an RNA vector, a virus or other suitable replicons (such as viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vector of the invention contains polynucleotide sequences together with additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibody and the antibody fragment of the invention include plasmids that contain regulatory sequences (such as promoter and enhancer regions) that direct transcription of the gene. Other useful vectors for expressing the antibody and the antibody fragment contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA transcripted from the genes. These sequence elements include, for example, 5′ and 3′ untranslated regions, internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct the efficient transcription of the genes carried on the expression vector. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothyrcin.
The term “host cell” herein refers to a cell into which a foreign nucleic acid is introduced, including the progenys of such a cell. The host cell includes “transformant” and “transformed cell” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical to the parental cell in nucleic acid content, and may contain a mutation. The mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells are included herein.
As used herein, the term “pharmaceutical composition” refers to a preparation that is present in a form which allows the active ingredients contained therein to be biologically effective and does not contain additional ingredients that would be unacceptably toxic to the subject to which the pharmaceutical composition is administered.
As used herein, the terms “subject”, “object” and “patient” refer to an organism receiving treatment for a particular disease or condition, such as a cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovid family (cattle, bison, buffalo, elk, yak, etc.), sheep, and horses receiving treatment for a disease or a condition (for example, a cell proliferative disorder, such as a cancer or an infectious disease).
As used herein, the term “treatment” refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject being treated, such as the progress of a cell proliferative disorder (such as a cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial response or complete response), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a condition or a disease, subjects who are prone to a condition or a disease or subjects who intend to prevent a condition or a disease. When referring to the terms slow down, alleviation, diminishment, palliation, remission, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
The term “effective amount” herein refers to an amount of a therapeutic agent effective to prevent or relieve a disease or a condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. “Effective amount” also refers to an amount of a compound sufficient to relieve symptoms, for example, treat, cure, prevent or relieve the associated medical conditions, or to increase the rate of treatment, cure, prevent or relieve such conditions. When the active ingredient is administered to an individual alone, a therapeutically effective dose refers to the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
The term “appropriate condition” herein refers to a condition suitable for culturing various host cells, including eukaryotic cells and prokaryotic cells.
The term “cancer” herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
The term “tumor” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “tumor” are not mutually exclusive when referred to herein.
The term “anti-tumor agent” herein refers to an anti-tumor drug, which is a class of drugs for the treatment of tumor diseases, including a chemotherapy drug, a biological agent and the like.
The term “EC50” herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and which can be measured by methods known in the art.
The term “EC80” herein refers to the concentration of an antibody that elicits 80% of the maximal effect.
Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.
The present invention will be further described below in conjunction with specific examples, and the advantages and characteristics of the present invention will become clearer along with the description. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by a manufacturer are followed. The reagents or instruments used therein for which manufacturers are not specified are all conventional products that are commercially available.
The examples of the present invention are merely exemplary, and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.
1.1 Immunization and Serum Titer Detection of Alpaca
The human CD22 (Asp20-Arg687)-His protein used for immunization was purchased from ACRO Biosystems (catalog number: CD2-H52H8). Two alpacas (Llama) were selected for immunization, and each alpaca was immunized four times with an interval of 3 weeks. After the third immunization and after the fourth immunization, peripheral blood was collected and serum was separated, and enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) were used to detect the antibody titer and specificity against human CD22 in serum, and the results are shown in
1.2 Library Construction
A total of 100 mL of alpaca peripheral blood was collected after three immunizations and after four immunizations; Lymphocyte separation medium was used to isolate PBMC, RNAiso Plus reagent (Takara, catalog number: #9108/9109) was used to extract total RNA, and PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, catalog number: 6210A) was used to reverse transcribe the extracted RNA into cDNA. Nested PCR was used to amplify the variable region nucleic acid fragment encoding the nanobody:
The nucleic acid fragment of the nanobody of interest was recovered and cloned into the phage display vector pcomb3XSS (from Sichuan NB Biolab Co., Ltd) using the restriction endonuclease SfiI (NEB, catalog number: R0123S). The product was then electrotransformed into Escherichia coli electroporation competent cell TG1, and a nanobody phage display library target against CD22 was constructed and tested. By serial dilution plating, the calculated capacity is 2.0×109. To test the insertion rate of the library, 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
1.3 Panning of Nanobody VHH Against CD22
The plate was coated with human CD22-llama Fc fusion protein (ACRO Biosystems, catalog number: 512-H525a) at 0.5 μg/well, and placed at 4° C. overnight; The next day, after blocking with 3% BSA-PBS at 37° C. for 1 hour, 100 μl of phage display library was added and incubated at 37° C. for 1 hour; After that, the plate was washed 6 times with PBST and 2 times with PBS to wash away unbound phages. Finally, 100 μL of Gly-HCl eluent was added to elute the phages that specifically bind to CD22 to enrich positive clones.
1.4 Screening of Specific Single Positive Clones by Phage Enzyme-Linked Immunoassay
After panning, blank Escherichia coli were infected with the obtained CD22-binding positive phages and plated. Then 96 single colonies were selected for proliferation and culture. The plates were coated with human CD22-llama Fc and human CD22-His proteins respectively at 4° C. overnight, the phage culture supernatant was added, and incubated at 37° C. for 1 hour. After washing, TMB chromogenic solution was added to develop color, and the optical density was measured at a wavelength of 450 nm. Human CD22-llama Fc and human CD22-His double-positive clones were selected for sequencing. The sequencing results were analyzed using MOE software, and the evolutionary tree was constructed according to the amino acid sequence coding the VHH protein. According to the sequence similarity, 18 clones were obtained by eliminating the sequences that were close to each other on the evolutionary tree, and the CDR sequences of the 18 clones were analyzed by KABAT, Chothia or IMGT software respectively, and the corresponding sequence information was shown in the following table 2-4. Wherein Table 2 showed the antibody sequences represented by amino acids of 18 nanobody molecules, and Table 3 showed the antibody sequences represented by nucleotides of 18 nanobody molecules, and Table 4 showed the results of IMGT, Kabat and Chothia analysis of the CDRs of 18 nanobody molecules. Subsequently, the production and identification of VHH nanobody Fc fusion protein were carried out.
The VHH variable region sequence was recombined into the expression vector BI3.4-huIgG1 containing a signal peptide and human IgG1 Fc (the human IgG1 Fc sequence was shown in SEQ ID NO: 14, the hinge region sequence was shown in SEQ ID NO: 15) by Taizhou Biointron Biotechnology Co., Ltd, and plasmids were prepared according to the established standard molecular biology methods. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, catalog number: 24765-1), and the transfected cells were continuously cultured at 37° C. for 5 days using FreeStyle™ 293 (Thermo Fisher Scientific, catalog number: 12338018), and the cell components were removed by centrifugation to obtain the culture supernatant containing the VHH antibody. The culture supernatant was loaded onto the protein A chromatography column (the protein A filler AT Protein A Diamond and the chromatography column BXK 16/26 were both purchased from Bestchrom (Shanghai) Biosciences Ltd., and the catalog numbers were: AA0273 and B-1620, respectively), the column was washed with PBS phosphate buffer (pH 7.4), then washed with 20 mM PB, 1M NaCl (pH 7.2), and finally eluted with citric acid buffer (pH 3.4). The antibody with Fc label eluted from the protein A chromatography column was collected, neutralized with 1/10 volume of 1M Tris (pH 8.0), and dialyzed with PBS at 4° C. overnight, and the dialyzed protein was aseptically filtered by 0.22 micron filter membrane and then subpackaged for storage at −80° C.
2.2 Preparation of Control Antibody
The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane. HA22, m971 and hL22 are antibodies that recognize human CD22, wherein the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain 5-7. The heavy chain variable region and light chain variable region sequences of HA22 were obtained according to U.S. Pat. No. 9,580,461 B (which is incorporated herein by reference), the heavy chain variable region and light chain variable region sequences of m971 were obtained according to U.S. Pat. No. 8,591,889 B (which is incorporated herein by reference), and the heavy chain variable region and light chain variable region amino acid sequences of hL22 were obtained according to U.S. Pat. No. 5,789,554 B (which is incorporated herein by reference). The VH and VL of the antibody HA22, m971 and hL22 that recognize human CD22 and the human IgG1 Fc were linked in order from the N-terminal to the C-terminal, wherein the VH and VL were linked by three GGGGS linkers to form scFv-hFc, and the corresponding amino acid sequence information was shown in Table 5 below. The corresponding nucleotide sequences were respectively cloned into the pTT5 vector by GENERAL Biosystems (Anhui) Corporation Limited and expressed in HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) and purified according to the method of Example 2.1.
2.3 Preparation of Human CD22-His Tag Protein
The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane, the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain 5-7. The nucleotide sequences encoding the amino acid sequence of human CD22 protein (NCBI: NP_001762.2, SEQ ID NO: 1), the extracellular region (ECD, extra-cellular domain) amino acid sequence Asp 20-Arg 687 (SEQ ID NO: 2), the domain 1-4 Asp 20-Val 425 amino acid sequence (SEQ ID NO: 3) and the domain 5-7 Asp 414-Arg 687 amino acid sequence (SEQ ID NO: 4) were cloned into the pTT5 vector by GENERAL Biosystems (Anhui) Corporation Limited, respectively, and plasmids were prepared according to the established standard molecular biology methods. The corresponding amino acid sequence information was shown in Table 6 below. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) were transiently transfected (PEI, Polysciences, catalog number: 24765-1) and FreeStyle™293 (Thermofisher scientific, catalog number: 12338018) was used for scale-up culture at 37° C. After 6 days, the cell culture fluid was collected, centrifuged to remove cell components, and the culture supernatant containing the extracellular region of human CD22 protein was obtained. The culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap™ Excel (GE Healthcare, catalog number: GE17-3712-06), and the change in the ultraviolet absorbance (A280 nm) was monitored with an ultraviolet (UV) detector. After sample loading, the nickel ion affinity chromatography column was washed with 20 mM PB, 0.5M NaCl (pH 7.4) until the ultraviolet absorbance returned to the baseline, and then gradient elutions (2%, 4%, 8%, 16%, 50%, 100%) were performed with Buffer A: mM PB, 0.5M NaCl (pH 7.4) and Buffer B: 20 mM PB, 0.5M NaCl, and 500 mM imidazole. His-tagged human CD22 protein eluted from the nickel ion affinity chromatography column was collected and dialyzed against PBS phosphate buffer (pH 7.4) overnight in a refrigerator at 4° C. The dialyzed protein was aseptically filtered by 0.22 micron filter membrane and then subpackaged for storage at −80° C. to obtain purified human CD22 protein. The bands of interest of samples detected by SDS-PAGE reducing gel and non-reducing gel were shown in
3.1 Identification of Cell Line Expressing CD22 Endogenously
Raji cells (purchased from China Center for Type Culture Collection, Wuhan University) were scale-up cultured in T-25 cell culture flasks to the logarithmic growth phase, the supernatant of the medium was discarded by centrifugation, and the cell pellet was washed 2 times with PBS. The HA22 and m971 antibodies were used as primary antibodies, APC-labeled secondary antibody (purchased from Biolegend, catalog number: 409306) was used and FACS (FACS Canto™, purchased from BD company) was used for detection and result analysis. The analysis results were shown in Table 7 and
3.2 Preparation of CHO-K1 Monoclonal Cell Line Stably Transfected with Human CD22
The nucleotide sequence encoding the full-length amino acid sequence of human CD22 (NCBI: NP_001762.2, SEQ ID NO: 1) was cloned into the pcDNA3.1 vector and a plasmid was prepared by GENERAL Biosystems (Anhui) Corporation Limited. Plasmid transfection (Lipofectamine® 3000Transfection Kit, purchased from Invitrogen, catalog number: L3000-015) was performed on CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and then the transfected cells were selectively incubated for 2 weeks in DMEM/F12 medium containing 10 μg/ml of puromycin and 10% (w/w) of fetal bovine serum. The FITC-labeled anti-CD22 antibody (Thermofisher scientific, catalog number: 11-0229-42) was used to sort the positive monoclonal cells into a 96-well plate on flow cytometer FACS Ariall (BD Biosciences) and the plate was placed in a cell incubator at 37° C. and 5% (v/v) CO2 for cell culture. Some wells containing monoclonal cells were selected for amplification after approximately 2 weeks. The amplified clones were screened by flow cytometry. The monoclonal cell line with better growth and higher fluorescence intensity was selected for further scale-up culture and then freezed in liquid nitrogen.
The specific selection results were shown in Table 8 and
4.1 Binding of VHH-Fc Antibodies to Human CD22 Protein Detected by Enzyme-Linked Immunosorbent Assay (ELISA)
In order to detect the binding activity of VHH-Fc to human CD22 protein, the purified human CD22-ECD-His protein obtained in Example 2 was diluted with PBS to a final concentration of 2 μg/mL, and then added to 96-well ELISA plate at 100 μl/well. The plate was sealed with plastic film and incubated overnight at 4° C., the plate was washed 2 times with PBS the next day, and then a blocking solution [PBS+2% (w/w) BSA] was added for blocking at room temperature for 2 hours. The blocking solution was poured off, and 100 nM of serially diluted VHH-Fc antibody or negative control antibody was added at 50 μl/well. After incubation at 37° C. for 2 hours, the plate was washed 3 times with PBS. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma, catalog number: A0170) was added, and incubated at 37° C. for 2 hours, and the plate was washed 5 times with PBS. TMB substrate was added at 50 μl/well, and incubated at room temperature for 30 minutes, then a stop solution (1.0 N HCl) was added at 50 μl/well. An ELISA plate reader (Multimode Plate Reader, EnSight, purchased from PerkinElmer) was used to read the OD450nm value, and the ELISA results of VHH-Fc and human CD22-ECD are shown in
4.2 The Binding of Antibody to Different CD22 Expressing Cells Detected by Flow Cytometry (FACS)
The required cells were scale-up cultured in a T-75 cell culture flask to the logarithmic growth phase. For the adherent cell CHO-K1, the medium was aspirated, the cells were washed 2 times with PBS buffer, and then digested with trypsin. After the digestion was terminated, the cells were washed 2 times with PBS buffer. For suspension cell Raji, the medium supernatant was directly centrifuged and discarded, and the cell pellet was washed 2 times with PBS. After counting the cells in the previous step, the cell pellet was resuspended with [PBS+2% (w/w) BSA] blocking solution to 2×10 6 cells/ml, and added to a 96-well FACS reaction plate at 50 μl/well, and then the VHH-Fc antibody test sample was added at 50 μl/well, and incubated on ice for 2 hours. The mixture was centrifuged and washed 3 times with PBS buffer, Alexa Flour 488-labeled secondary antibody (purchased from Invitrogen, catalog number: A-11013) was added at 50 μl/well, and incubated on ice for 1 hour. The obtained mixture was centrifuged and washed 5 times with PBS, and FACS (FACS Canto™, purchased from BD Company) was used for detection and result analysis. Data analysis was performed by software (CellQuest) to obtain the mean fluorescence density (MFI) of the cells. And then software (GraphPad Prism8) was used for analysis, data fitting, and EC50 value calculation. The analysis results are shown in Table 10 and
5.1 Binding of VHH-Fc Antibodies to Murine CD22 Protein Detected by ELISA
In order to detect the species cross-binding activity of the VHH-Fc antibodies, an ELISA plate was coated with commercial mouse CD22 (ACROBiosystems, catalog number: SI2-M52Ha), and the ELISA detection was performed according to the method in Example 4.1. The ELISA results of VHH-Fc and murine CD22-ECD are shown in
5.2 Binding of VHH-Fc Antibodies to Peripheral Blood B Cells of Cynomolgus Monkey (Latin Name: Macaca fascicularis) Detected by FACS
The monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Inc.)
according to the instructions of Ficoll-Paque Plus (purchased from GE Healthcare, catalog number: 171440-02). After the cell suspension was centrifuged, the cells were resuspended in PBS containing 1% BSA, and then the cells were counted. At the same time, the murine antibody Brilliant Violet 605 anti-human CD20 (catalog number: 302334, purchased from Biolegend) with monkey CD20 cross-binding activity and the VHH-Fc antibodies to be tested (1 nM, 10 nM and 100 nM) were added. The mixture was incubated for 1 hour at room temperature. After washing the cells three times, APC-labeled secondary antibody anti-human IgG Fc (catalog number: 409306, purchased from Biolegend) was added. After incubation at room temperature in the dark for 30 minutes, the cells were washed 5 times, gently resuspended with PBS containing 1% BSA, and detected and analyzed by FACS (FACS Canto™, purchased from BD Company). Wherein CD20 was used as a marker of B cells, and the CD20-positive B cell population was gated, the proportion of VHH-Fc positive cells was analyzed, and the proportion of VHH-Fc positive cell population to B cell population was calculated after treatments with VHH-Fc antibodies at the concentrations of 100 nM, 10 nM and 1 nM, respectively. The results are shown in Table 12. The scatter plot of double-stained cells by Brilliant Violet 605-labeled CD20 and APC secondary antibody indirectly labeled VHH-Fc is shown in
6.1 Assay of Affinity of VHH-Fc to Human CD22-ECD-his Protein
Anti-human CD22 VHH-hFc antibody was captured using a Protein A chip (GE Healthcare; 29-127-558). Sample buffer and running buffer were HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69). The flow-through cell was set to 25° C. The sample block was set to 16° C. Both were pretreated with the running buffer. In each cycle, the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD22 antigen protein was injected. The binding and dissociation process of the antibody and the antigen protein was recorded, and finally Glycine pH 1.5 (GE Healthcare; BR-1003-54) was used to complete chip regeneration. Binding was measured by injecting different concentrations of recombinant human CD22-ECD His in solution for 240 s with a flow rate of 30 μL/min. The concentration started from 200 nM (see the detailed results for the actual concentration in the test) and was diluted at 1:1, making a total of 5 concentrations. The dissociation phase was monitored for up to 600 s and was triggered by switching from sample solution to running buffer. The surface was regenerated by washing with a 10 mM of glycine solution (pH 1.5) for 30 s at a flow rate of 30 μL/min. Bulk refractive index difference was corrected by subtracting the response obtained from the goat anti-human Fc surface. Blank injection was also subtracted (=double reference). For calculation of apparent KD and other kinetic parameters, Langmuir 1:1 model was used. The binding rate (Ka), dissociation rate (Kd) and binding affinity (KD) of the VHH-Fc antibodies to human CD22-His protein are shown in Table 13, and the antibody HA22 was used as a control. As shown in
6.2 Assay of Affinity of VHH-Fc to Cynomolgus Monkey CD22-ECD-his Protein
According to the method of Example 6.1, the affinity of VHH-Fc antibodies to cynomolgus monkey CD22-ECD-His (purchased from R&D, catalog number: 9864-SL-050) protein was determined, and the results are shown in
7.1 Identification of Antigen-Binding Region of Antibody
The CD22 protein has 7 IgG-like domains outside the cell, in which domain 1 is located at the farthest end from the membrane and domain 7 is located at the nearest end from the membrane, the antigen-binding epitopes of HA22 and hL22 are located in domain 2-3, and the antigen-binding epitope of m971 is located in domain5-7. In order to identify the antigen-binding epitope distribution of the VHH antibodies, according to the ELISA method in Example 4.1, human CD22-domain1-4-His (distal end of membrane) and human CD22 domain5-7-His (proximal end of membrane) were used for coating, respectively. The VHH antibodies were classified into the type of distal end of membrane and the type of proximal end of membrane, as shown in
7.2 Antibody Antigen-Binding Epitope Competition Experiment (Epitope Binning)
Competitive ELISA was used to classify the epitopes of VHH antibodies and control antibody with known epitopes. According to the method in Example 4.2, the ELISA plate was coated with 2 μg/mL of the antibody, and the human CD22-ECD-his protein was serially diluted starting from 30 μg/mL, and the EC80 value was calculated (Table 16). The ELISA plate was coated with 2 μg/mL of the antibody, 25 μg/mL of the antibody to be detected was added, and then human CD22-ECD-his protein corresponding to each antibody to be detected was added at the EC80 concentration, incubated for 2 hours, and washed 5 times with PBS, and then HRP-labeled anti-His antibody was added for detection. If there is no competitive relationship between the antibody coated on the plate and the antibody to be detected in the solution, the antibody coated on the plate can bind to the complex of the antibody to be detected and the human CD22-ECD-his antigen in solution, and the absorption at OD450nm was detected, and the inhibition rate between each pair of antibodies was calculated according to the absorbance at OD450nm (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011395861.6 | Dec 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/135058 | 12/2/2021 | WO |
