WO2022199590A1 - 一种靶向bcma的纳米抗体及其应用 - Google Patents
一种靶向bcma的纳米抗体及其应用 Download PDFInfo
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- WO2022199590A1 WO2022199590A1 PCT/CN2022/082351 CN2022082351W WO2022199590A1 WO 2022199590 A1 WO2022199590 A1 WO 2022199590A1 CN 2022082351 W CN2022082351 W CN 2022082351W WO 2022199590 A1 WO2022199590 A1 WO 2022199590A1
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Definitions
- the invention belongs to the field of biopharmaceuticals, and relates to a nanobody targeting BCMA and an application thereof.
- Single-domain antibodies differ from traditional 4-chain antibodies by having a single monomeric antibody variable domain.
- camelids and sharks produce antibodies that naturally lack light chains, which are called heavy chain-only antibodies (HcAbs, or simply heavy chain antibodies).
- Antigen-binding fragments in each arm of camelid heavy chain-only antibodies have a single heavy chain variable domain (VHH) that can have high affinity for antigen without the help of a light chain.
- VHHs or Nanobodies are known as the smallest functional antigen-binding fragments with a molecular weight of approximately 15kD.
- Nanobodies have the natural advantages of high stability, strong penetration, and broad binding epitopes (Muyldermans S. Annu Rev Biochem. 2013; 82:775-97). Since its discovery, nanobodies have gradually attracted people's attention, and the basic research on them has been relatively mature. In terms of application, autoimmune diseases, blood diseases, viral infections and orthopedic diseases have entered the clinic. And neurodegenerative diseases have also shown great advantages (Morrison C. Nat Rev Drug Discov. 2019Jul; 18(7):485-487).
- MM Multiple myeloma
- the main pathology of MM is the indefinite expansion and enrichment of plasma cells in the bone marrow (BM), leading to osteonecrosis.
- Patients affected by MM may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychological burden of a cancer diagnosis.
- the main treatment options are chemotherapy and stem cell transplantation.
- the chemotherapy drugs are mainly steroids, thalidomide, lenalidomide, bortezomib or a combination of various cytotoxic agents. For younger patients, high-dose chemotherapy can be used.
- MM disease relapse remains a major obstacle due to the continuous emergence and evolution of drug-resistant clones, and significant adverse effects are unavoidable. Therefore, more effective and less toxic treatments are urgently needed to overcome drug resistance, improve quality of life and prolong survival in patients with relapsed and refractory MM (Cho, S.-F., Frontiers in Immunology, 9).
- BCMA B-cell maturation antigen
- B-cell maturation antigen is a B cell maturation antigen, a type III transmembrane protein composed of 184 amino acid residues, belonging to the TNF receptor superfamily, and its ligands belong to the TNF superfamily, such as proliferation-inducing ligands (APRIL). ), B lymphocyte stimulating factor (BAFF), BCMA can activate NF- ⁇ B and MAPK8/JNK signaling to mediate the proliferation and survival of B cells when BCMA binds to its ligand, which plays an important role in the survival of long-lived plasma cells.
- APRIL proliferation-inducing ligands
- BAFF B lymphocyte stimulating factor
- BCMA can activate NF- ⁇ B and MAPK8/JNK signaling to mediate the proliferation and survival of B cells when BCMA binds to its ligand, which plays an important role in the survival of long-lived plasma cells.
- BCMA is specifically highly expressed on the surface of advanced B cells, short-lived proliferating plasmablasts and long-lived plasma cells, and to a certain extent on memory B cells, while on naive B cells, CD34-positive hematopoietic stem cells and other normal tissue cells. No reports were found. Studies have shown that the expression of BCMA is associated with hematological malignancies, most notably multiple myeloma (MM). Target (Tai, Immunotherapy, 7(11), 1187-1199). High affinity antibodies block the binding between BCMA and its natural ligands BAFF and APRIL. Anti-BCMA sdAbs can be used in combination with cellular immunotherapy using CAR-T cells, for example to enhance cytotoxicity against tumor cells. There is currently a lack of anti-BCMA nanobodies with good efficacy and low toxicity.
- the present invention provides a BCMA-targeting nanobody and applications thereof.
- the first aspect of the technical solution of the present invention is: a nanobody, which comprises a heavy chain variable region containing CDR1, CDR2 and CDR3, and has the function of recognizing and binding BCMA, and:
- the CDR1 is selected from the following group:
- the CDR2 is the amino acid sequence shown in SEQ ID NO: 23, or the substitution, deletion or increase of 1 to 3 amino acid residues occurs in the amino acid sequence shown in SEQ ID NO: 23;
- the CDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 27-52.
- the substitution is selected from G1E, T3I, S5R and S6P/I; Described CDR1 is when 1 ⁇ 3 kinds of amino acid residues are replaced on the amino acid sequence shown in SEQ ID NO:6, the replacement is selected from F4S/D, S5R and I6F; and/or, when described CDR2 is When 1-3 amino acid residues are replaced in the amino acid sequence shown in SEQ ID NO: 23, the replacement is selected from I1V, Y2T, S3P/G/T, D4G/E/A, G5S/N, S6R /N/G/T and T7A/P/S.
- the CDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 2-7; the CDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 8-26; and/or, The CDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 27-52.
- the heavy chain variable region comprises CDR1, CDR2 and CDR3 shown in any of the following groups 1 to 26:
- the amino acid sequence of the heavy chain variable region is shown in SEQ ID NOs: 53-78, or has at least 80% of the amino acid sequence shown in SEQ ID NO: 53-78 , 90%, 95%, 96%, 97%, 98% or 99% identity.
- the present invention also provides Nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that bind to the same epitope of BCMA as any of the Nanobodies of the invention, ie capable of cross-competing with BCMA with any of the Nanobodies of the invention The bound Nanobody, heavy chain antibody, antibody or antigen-binding fragment thereof.
- the second aspect of the technical solution of the present invention is: a BCMA binding molecule, wherein the BCMA binding molecule comprises the nanobody as described in the first aspect, for example, one, two or more Monovalent or multivalent Nanobodies, multispecific Nanobodies, heavy chain antibodies or antigen-binding fragments thereof of the Nanobodies according to the first aspect.
- the BCMA-binding molecule is a heavy chain antibody.
- amino acid sequences of CH2 and CH3 of the heavy chain antibody are shown in SEQ ID NO: 1, or have at least 80%, 90% and 90% with the amino acid sequence shown in SEQ ID NO: 1. %, 95%, 96%, 97%, 98% or 99% identity.
- Nanobody represents the variable region of a heavy chain antibody (heavy chain variable region).
- Nanobodies, VHHs, single domain antibodies (sdAbs) represent the same molecule in the present invention.
- the term “heavy chain antibody” includes, in addition to Nanobodies, the CH2 and CH3 regions of conventional heavy chains.
- the term "BCMA binding molecule” can be a Nanobody, a heavy chain antibody, or a multivalent Nanobody/heavy chain antibody comprising one, two or more Nanobodies of the same specificity, comprising two or more Nanobodies of different specificities Nanobodies, multispecific antibodies, conventional antibodies or antigen-binding fragments thereof, etc.
- the third aspect of the technical solution of the present invention is: an isolated nucleic acid, wherein the isolated nucleic acid encodes the Nanobody as described in the first aspect of the present invention, or as described in the second aspect of the present invention. BCMA-binding molecules described above.
- the fourth aspect of the technical solution of the present invention is: a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid according to the third aspect of the present invention.
- the backbone of the recombinant expression vector is pCDNA3.4.
- the fifth aspect of the technical solution of the present invention is: a transformant, characterized in that the transformant comprises the isolated nucleic acid according to any one of the third aspects of the present invention, or the nucleic acid as described in the third aspect of the present invention.
- the host of the transformant is a prokaryotic cell or a eukaryotic cell. More preferably, the eukaryotic cells are CHO cells.
- the sixth aspect of the technical solution of the present invention is: a composition, characterized in that it comprises one, two or more Nanobodies according to the first aspect of the present invention, a second The BCMA-binding molecule of the present invention, the isolated nucleic acid of the third aspect of the present invention, the recombinant expression vector of the fourth aspect of the present invention, or the transformant of the fifth aspect of the present invention.
- the composition further includes pharmaceutically acceptable excipients, so that the composition is a pharmaceutical composition.
- the seventh aspect of the technical solution of the present invention is: a BCMA detection agent, wherein the BCMA detection agent comprises the nanobody according to any one of the first aspect of the present invention and/or the nanobody according to the first aspect of the present invention.
- the eighth aspect of the technical solution of the present invention is: the nanobody according to the first aspect of the present invention, the BCMA binding molecule according to the second aspect of the present invention, the isolated nanobody according to the third aspect of the present invention
- the disease is preferably a hematological malignancy, more preferably multiple myeloma.
- the ninth aspect of the technical solution of the present invention is: a method for treating a subject in need, comprising combining the nanobody according to the first aspect of the present invention, the nanobody described in the second aspect of the present invention
- the BCMA binding molecule of the present invention, the isolated nucleic acid described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, the transformant described in the fifth aspect of the present invention, or the composition described in the sixth aspect of the present invention is administered to a subject in need thereof; wherein the subject in need thereof has a disease associated with BCMA.
- the disease is preferably a hematological malignancy, more preferably multiple myeloma.
- the reagents and raw materials used in the present invention are all commercially available.
- Nanobodies, BCMA-binding molecules and compositions of the present invention can recognize and bind to BCMA, and have the potential to treat BCMA-related diseases;
- the BCMA detection agent of the present invention has the function of rapidly and efficiently detecting BCMA.
- Fig. 1 is the detection result of alpaca serum titer;
- a and B in Fig. 1 are the serum titers of A1 and A2 alpaca after different immunizations, respectively;
- Figure 2 shows the binding of IgG subtypes and antigens purified by serum before and after immunization of alpaca;
- a and B of Figure 2 show the changes of IgG subtypes in serum before and after immunization of A1 alpaca and A2 alpaca respectively;
- Fig. 3 is ELISA to detect the binding situation of clone supernatant and BCMA protein
- Fig. 4 is the structure after nanobody (VHH) transformation
- Figure 5 is a ligand competition binding experiment at the CHO-K1/BCMA cell level;
- a in Figure 5 is NB-67, NB-84, NB-90, NB-100, NB-122, NB-192, NB-257, MFI and IC50 of NB-265 and NB-367
- B in Figure 5 is NB-27, NB-29, NB-34, NB-36, NB-51, NB-53, NB-65, NB-71, NB -83, NB-102, NB-216, NB-217, NB-170, NB-222 and NB-352 MFI and IC50;
- Figure 6 is the affinity detection at the CHO-K1/BCMA cell level;
- BMK2-H4 is the positive control antibody, and
- Isotype1 is the isotype control antibody;
- a in Figure 6 is NB-1, NB-27, NB-29, NB-34, MFI and EC50 of NB-36, NB-51, NB-53, NB-65, NB-67, NB-71, NB-82, NB-83 and NB-84
- B in Figure 6 is NB-90, NB - MFI and EC50 for 100, NB-102, NB-122, NB-192, NB-216, NB-217, NB-257, NB-265 and NB-367;
- Figure 7 is the detection of the affinity level of the antibody of the present invention binding to target cells; wherein A, B and C in Figure 7 are the binding affinity levels of each antibody to RPMI8226 cells, Jeko-1 cells and Daudi cells respectively; BMK1 and BMK2-H4 are Positive control antibodies, Isotype1 and Isotype2 are isotype control antibodies (ie isotype antibodies), the source and sequence of the control antibodies are shown in Table 6, and the unit of EC50 is nM;
- Figure 8 is a flow chart of MPA screening, and the detection results of 4 antibodies; wherein A in Figure 8 is the MPA screening process, and B, C, D, and E in Figure 8 are NB-1, NB-29, NB-257, MPA assay results of NB-367 antibody.
- BCMA-binding molecule is a protein that has the function of recognizing and binding BCMA, including, but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, receptor targets Binding domains, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.
- antibody includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (eg, bispecific antibodies), Diabodies and single chain molecules, and antibody fragments, especially antigen-binding fragments, eg, Fab, F(ab')2 and Fv.
- immunoglobulin Ig
- antibody is used interchangeably.
- the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
- IgM antibodies are composed of 5 basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; while IgA antibodies contain 2-5 basic 4-chain units, which can be combined with the J chain Polymerization forms multivalent assemblies.
- the 4-chain unit is typically about 150,000 Daltons.
- Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the isotype of the heavy chain.
- Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
- Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain, CH1, CH2 and CH3) and four (for the mu and epsilon isoforms, CH1, CH2, CH3 and CH4) constant domains (CH) and a hinge region (Hinge) between the CH1 and CH2 domains.
- VH variable domain
- CH1 alpha and gamma chain
- CH1 constant domain
- CH1 constant domain
- Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end.
- VL is aligned with VH and CL is aligned with the first constant domain (CH1) of the heavy chain.
- Particular amino acid residues are thought to form the interface between the light and heavy chain variable domains.
- the paired VH and VL together form an antigen binding site.
- Light chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda based on their constant domain amino acid sequences.
- Immunoglobulins can be assigned to different classes or isotypes based on their heavy chain constant domain (CH) amino acid sequences.
- immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called alpha, delta, epsilon, gamma, and mu, respectively.
- the gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function, eg humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
- Heavy chain antibodies as used herein are antibodies derived from camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of the variable region (VHH) and other constant regions. The variable region has a structure similar to the hinge region. linked to the constant region. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of chondroid heavy chain antibodies contains 1 variable region and 5 Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.
- Nanobodies are the variable regions of heavy chain antibodies. Typically, Nanobodies contain three CDRs and four FRs. Nanobodies are the smallest functional antigen-binding fragments. Usually, an antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a Nanobody consisting of only one heavy chain variable region.
- CH1 light chain and heavy chain constant region 1
- a binding molecule comprising two or more Nanobodies is a multivalent Nanobody; a binding molecule comprising two or more Nanobodies of different specificity is a multispecific Nanobody.
- Multivalent Nanobodies or multispecific Nanobodies are linked to multiple Nanobodies by linkers.
- the linker typically consists of 1-15 amino acids selected from G and S, eg ( G4S)3 .
- heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to the structural similarity between the two, the following structural descriptions for antibodies apply to heavy chain antibodies in addition to light chains.
- variable region refers to the amino-terminal domain of the heavy or light chain of an antibody.
- variable domains of heavy and light chains may be referred to as "VH” and “VL”, respectively. These domains are usually the most variable part of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.
- variable refers to situations where certain segments of the variable domains differ widely in antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (both in the light and heavy chain variable domains), namely HCDR1, HCDR2, HCDR3 (heavy chain variable regions), respectively.
- HVRs hypervariable regions
- Chain antibodies may be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3 of the light chain variable region.
- FRs framework regions
- variable domains of native heavy and light chains each comprise four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a beta-sheet conformation, connected by forming loops and in some cases beta-sheet structures Part of the three HVR connections.
- the HVRs in each chain are held together in close proximity by the FR regions, and together with the HVRs of the other chain contribute to the formation of the antigen-binding site of the antibody.
- the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4
- the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
- the constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity.
- Fc region region of crystallizable fragments or “Fc domain” or “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including those located in the immune system Binding of Fc receptors on various cells (eg, effector cells) of , or to the first component (C1q) of the classical complement system.
- the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody.
- an Fc region of an immunoglobulin heavy chain is generally defined as the stretch from the amino acid residue at position C226 or P230 of the heavy chain to the carboxy terminus, where this numbering is according to the EU index, as in Same as in Kabat.
- an Fc region can be a native sequence Fc or a variant Fc.
- Antibody fragments comprise a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. Antibody fragments are preferably antigen-binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; Or any fragment that should be able to increase half-life by incorporation into liposomes. Digestion of the antibody with papain yields two identical antigen-binding fragments called "Fab" fragments, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
- the Fab fragment consists of the entire light and heavy chain variable domains (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of the antibody produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen-binding activities and still capable of cross-linking the antigen.
- Fab' fragments differ from Fab fragments by the addition of a few additional residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the CH1 domain.
- F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between the Fab' fragments. Other chemical conjugations of antibody fragments are also known. Fc fragments comprise the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.
- FcRs Fc receptors
- Fv is the smallest antibody fragment that contains complete antigen recognition and binding sites.
- the fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association.
- Six hypervariable loops (3 loops each for the heavy and light chains) protrude from the folding of these two domains, contributing amino acid residues for antigen binding and conferring antigen binding specificity to the antibody.
- a single variable domain or half an Fv containing only three HVRs specific for the antigen has the ability to recognize and bind antigen, albeit with lower affinity than the intact binding site.
- a “single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment comprising the VH and VL domains of an antibody linked into a single polypeptide chain.
- the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen binding structure.
- the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerization, amidation, which may be present in small amounts) ), the individual antibodies that make up the population are identical. Monoclonal antibodies are highly specific, directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins.
- monoclonal indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
- monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and methods for extracting human immunoglobulin loci or encoding human The technology of producing human or human-like antibodies from animals of immunoglobulin sequence genes, single-cell sequencing method.
- Monoclonal antibodies are also included herein as "chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remainder are identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
- “Humanized” forms of non-human (eg, murine) antibodies refer to chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
- a “humanized antibody” generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in human antibodies.
- the entire antibody except the CDRs
- CDRs some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the beta-sheet backbone of the variable regions of human antibodies to generate antibodies, the specificity of which is determined by the grafted CDRs.
- Methods of producing such antibodies are well known in the art, eg, using mice with genetically engineered immune systems.
- antibodies, Nanobodies, heavy chain antibodies, etc. all include humanized variants of each of the antibodies.
- Human antibody refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
- the invention also includes derivatives and analogs of the various antibodies (eg, Nanobodies, heavy chain antibodies or antigen-binding fragments thereof, multivalent Nanobodies, multispecific Nanobodies, antibodies or antigen-binding fragments thereof).
- “Derivatives” and “analogs” refer to polypeptides that retain substantially the same biological function or activity of the various antibodies of the invention.
- Derivatives or analogs of the invention may be (i) a polypeptide having a substituent group in one or more amino acid residues, or (ii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene Diol) the polypeptide formed by fusion, or (iii) the polypeptide formed by fusion of additional amino acid sequence to this polypeptide sequence (such as leader sequence or secretory sequence or sequence used to purify this polypeptide or proprotein sequence, or with 6His tag formed fusion protein).
- additional amino acid sequence such as leader sequence or secretory sequence or sequence used to purify this polypeptide or proprotein sequence, or with 6His tag formed fusion protein.
- One of skill in the art can alter the sequences of the invention by one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) without substantially affecting the activity of the antibody. multiple) amino acids to obtain a variant of the antibody or functional fragment sequence thereof. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. In the art, conservative substitution of amino acids with similar or similar properties usually does not change the function of the protein.
- amino acids with similar properties are substituted in the FR and/or CDR regions of the variable region.
- Amino acid residues that can be conservatively substituted are well known in the art.
- Such substituted amino acid residues may or may not be encoded by the genetic code.
- the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein. All of them are considered to be included in the scope of protection of the present invention.
- Variant forms of the various antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, those capable of interacting with the various antibodies of the invention under conditions of high or low stringency Proteins encoded by DNAs that encode DNA hybridization, and polypeptides or proteins obtained using antisera against various antibodies of the present invention.
- sequence of the variant of the invention may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it was derived.
- Sequence identity according to the present invention can be measured using sequence analysis software. For example the computer programs BLAST using default parameters, especially BLASTP or TBLASTN.
- the present invention also includes those molecules having CDR-bearing antibody heavy chain variable regions, so long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein .
- Antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma technology well known in the art.
- the Nanobodies and heavy chain antibodies of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art.
- the various antibodies of the invention can be expressed in other cell lines.
- Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the invention. Transformation can be performed by any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used will depend on the host to be transformed.
- Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , encapsulation of polynucleotides in liposomes and direct microinjection of DNA into the nucleus.
- Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO).
- HeLa cells HeLa cells
- BHK baby hamster kidney
- COS monkey kidney cells
- human hepatocellular carcinoma cells eg, HepG2
- Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial BCMA binding properties.
- the present invention also provides polynucleotides encoding the above-mentioned various antibodies or fragments thereof.
- the polynucleotides of the present invention may be in the form of DNA or RNA.
- DNA forms include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be the coding or non-coding strand.
- nucleic acids that hybridize to the above polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
- the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
- stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
- the full-length nucleotide sequences of the various antibodies of the present invention or fragments thereof can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
- a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
- the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
- Biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.
- the DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
- the present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
- sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing sequence and acceptor splice sites complete intronic sequences, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, polylinkers for insertion of nucleic acids encoding antibodies to be expressed zone and optional marker elements.
- Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
- prokaryotic cells such as bacterial cells
- lower eukaryotic cells such as yeast cells
- higher eukaryotic cells such as mammalian cells.
- Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
- host cells may be various functional cells known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced cells Killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), ⁇ T cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (anti-CD3 Monoclonal antibody killer cells) and CAR-T/TCR-T cells.
- the killer cells are T cells or NK cells.
- Exemplary NK cells include, but are not limited to, primary NK cells, NK cell lines (eg, NK92), and NKT cells.
- the NK cells are primary NK cells.
- T cells include, but are not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokine-induced killer cells (CIK), tumor infiltrating cells.
- T cells of mixed cell populations such as lymphocytes (TILs).
- TILs lymphocytes
- the T cells are peripheral blood T lymphocytes and TIL-derived T cells.
- Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
- the host is a prokaryotic organism such as E. coli
- competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
- transformation can also be performed by electroporation.
- the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
- the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
- the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
- polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
- recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- the various antibodies described herein are useful in the manufacture of medicaments for the prevention or treatment of the various conditions and diseases described herein, particularly those associated with BCMA-expressing cells.
- the conditions and diseases are cancer, preferably hematological malignancies, more preferably multiple myeloma.
- compositions herein contain the binding molecules described herein, together with pharmaceutically acceptable adjuvants, including, but not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants.
- pharmaceutically acceptable adjuvants including, but not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants.
- excipients are preferably nontoxic to recipients at the doses and concentrations employed. Such excipients include, but are not limited to: saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
- compositions may contain ingredients for improving, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or The release rate, absorption or penetration of the substance. These substances are known from the prior art.
- the optimal pharmaceutical composition may be determined by the intended route of administration, mode of delivery, and desired dosage.
- compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using this method before or after lyophilization and reconstitution.
- the pharmaceutical compositions of the present invention may be selected for parenteral delivery.
- Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually presented in containers with sterile access ports, such as intravenous solution strips or vials with a hypodermic needle pierceable stopper.
- compositions may be selected for inhalation or delivery through the digestive tract, such as orally.
- preparation of such pharmaceutically acceptable compositions is within the skill of the art.
- Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art.
- kits for producing single-dose administration units may each contain a first container with dried protein and a second container with an aqueous formulation.
- kits are provided containing single-lumen and multi-lumen pre-filled syringes (eg, liquid syringes and lyophilized syringes).
- the present invention also provides methods of treating a patient, particularly a patient with a BCMA-related disorder, by administering a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
- a patient particularly a patient with a BCMA-related disorder
- a binding molecule according to any embodiment of the present invention or a pharmaceutical composition thereof.
- the terms "patient”, “subject”, “individual”, and “subject” are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog , cats, rabbits, etc.), and most preferably humans.
- Treatment refers to the administration of a therapeutic regimen described herein to a subject to achieve at least one positive therapeutic effect (eg, reduction in cancer cell number, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth). rate decreases).
- Treatment regimens that effectively treat a patient can vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anticancer response in the subject.
- the therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the invention to be employed will depend, for example, on the extent and purpose of treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will depend in part on the molecule being delivered, the indication, the route of administration and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient status) changes. In certain embodiments, the clinician can titrate the dose and alter the route of administration to obtain optimal therapeutic effect. For example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
- compositions may thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter.
- the route of administration of the pharmaceutical composition is according to known methods, such as injection by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional routes; Via a sustained release system or via an implanted device.
- the binding molecules of the present invention are useful in assays, eg, binding assays, to detect and/or quantify BCMA expressed in tissues or cells due to their high affinity for BCMA. Binding molecules such as single domain antibodies can be used in further studies investigating the role of BCMA in disease.
- the method for detecting BCMA is roughly as follows: obtaining a sample of cells and/or tissue; detecting the level of BCMA in the sample.
- the BCMA binding molecules of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with BCMA.
- the present invention provides detection of the presence of BCMA in a sample using classical immunohistological methods known to those skilled in the art. Detection of BCMA can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of BCMA include ELISA, FACS, RIA, and the like.
- binding molecules such as single domain antibodies are typically labeled with detectable labeling groups.
- Suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (eg, 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (eg, FITC, rhodamine) luminescence, lanthanide phosphors), enzymatic groups (eg, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (Computed Tomography) contrast agent.
- MRI magnetic resonance imaging
- CT Computed Tomography
- Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to BCMA.
- An example of one such assay would involve detecting the amount of free antibody in a solution containing an amount of BCMA in the presence or absence of the test molecule.
- An increased amount of free antibody ie, antibody that does not bind BCMA
- the antibody is labeled with a labeling group.
- the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
- the present invention also provides a detection kit for detecting the level of BCMA, the kit includes an antibody that recognizes BCMA protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers, detection Labeling, detection substrates, etc.
- the detection kit may be an in vitro diagnostic device.
- the alpacas were immunized with recombinant human BCMA protein with a C-terminal Fc tag (Acrobiosystems, Cat. No.: BC7-H5254) purchased from Acro Company, and two alpacas (A1 and A2) were immunized in total.
- Acrobiosystems Cat. No.: BC7-H5254
- two alpacas (A1 and A2) were immunized in total.
- 400 ⁇ g of immunogen and CFA (complete Freund's adjuvant) were mixed, and 10 points of immunization were selected subcutaneously along the scapula and back, with 200 ⁇ L per point.
- IFA Incomplete Freund's adjuvant
- the second immunization was carried out at an interval of 3 weeks after the first immunization, and then once every two weeks; the immunogen of the second to fifth immunization was 200 ⁇ g, and the immunogen of the sixth immunization was 100 ⁇ g.
- the immunogen of the second to fifth immunization was 200 ⁇ g
- the immunogen of the sixth immunization was 100 ⁇ g.
- IgG subclass fractionation was performed according to standard operating procedures. IgG subclasses were fractionated from alpaca serum using protein G and protein A resins. 5ml serum samples were loaded onto a 5ml protein G HP column and the column was washed with phosphate buffer (20mM, pH 7.4). The IgG3 (MW 90,000 Da) fraction was eluted with 0.15 M NaCl, 0.58% acetic acid (pH 3.5), and the eluate was neutralized to pH 7.4 with 1 M Tris-HCl (pH 8.2).
- the IgG1 (MW 150,000 Da) fraction was eluted with 0.1 M Glycine-HCl (pH 2.7), and the eluate was neutralized to pH 7.4 with 1 M Tris-HCl (pH 8.2).
- the effluent from the Protein G HP column was then loaded onto a 5 ml Protein A HP column, and the column was washed with 20 ml of phosphate buffer (20 mM, pH 7.0).
- the IgG2 (MW 80,000 Da) fraction was eluted with 0.15 M NaCl, 0.58% acetic acid (pH 4.5), and the eluate was neutralized to pH 7.4 with 1 M Tris-HCl (pH 8.2).
- the concentration of purified IgG1, IgG2 and IgG3 antibodies was determined by OD280 and the respective purity was assessed by reducing and non-reducing SDS-PAGE analysis.
- the immune response of alpacas was assessed by ELISA in which the binding of serum samples and purified IgG to the immobilized immunogen was determined. Serum collected prior to immunization and during bleeds one week after each immunization was assessed. ELISA plates were coated with 2 ⁇ g/mL human recombinant BCMA his tag diluted antigen (Acrobiosystem, BCA-H522y) overnight at 4°C; plates were then washed 3 times with wash buffer, followed by blocking at room temperature for 2 hours. The plate was then washed three times with wash buffer, and the serially diluted serum was added to the wells of the ELISA plate and incubated at 37°C for 1 hour.
- BCMA his tag diluted antigen Acrobiosystem, BCA-H522y
- FIG. 1 The experimental results (Fig. 1-Fig. 2) showed that after 6 times of immunization, the titers of the two alpacas were maintained at a high level (titer>5 ⁇ 10 5 ), which was expected to meet the subsequent phage library construction need.
- a and B in Figure 1 are the serum titers of A1 and A2 alpacas after different immunizations, respectively.
- a and B of Figure 2 show the changes of IgG subtypes in serum before and after immunization of A1 alpaca and A2 alpaca, respectively.
- RNA as template and oligo dT as primer, the first strand of cDNA was synthesized according to the instructions of TAKARA reverse transcriptase.
- VHH coding gene of VHH was obtained by nested PCR. Amplify the variable region fragment of VHH by nested PCR:
- Upstream primer 1 GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 84)
- Downstream Primer 1 GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO:85)
- Upstream primer 2 GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO:86)
- Downstream primer 2 GGACTAGTGCGGCCGCTGGAGACGGTGACCTGG GT (SEQ ID NO: 87)
- the phagemid pME207 and PCR amplification products were digested with Sfi I and Not I respectively (NEB), recovered and quantified, and the two fragments were ligated with T4 DNA ligase (TaKaRa) at a molar ratio of 1:3 , at 16 °C, overnight ligation.
- T4 DNA ligase T4 DNA ligase
- the ligation product was dissolved in 100 ⁇ L of sterile water, and electroporation was performed ten times to transform Escherichia coli TG1. Take 100 ⁇ L of the electroporated and cultured bacterial liquid to double-dilute, spread it on an ampicillin LB culture plate, and calculate the storage capacity. After scraping and washing the bacterial fur on the culture plate with 10 mL of 2 ⁇ YT medium, add 25% glycerol with a final concentration, pack in aliquots, and store at -80°C for later use. The size of the storage capacity is 4.3 ⁇ 10 9 . In order to detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached more than 90%.
- the culture was centrifuged, and the pellet was resuspended with 200 mL of 2 ⁇ YT (containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL kanamycin).
- PEG/NaCl solution placed on ice for 60 min, centrifuged at 8000 rpm for 30 min, resuspended and precipitated in 5 mL of PBS to obtain an anti-BCMA VHH immune library, 10 ⁇ L was taken to measure the titer, and the rest were aliquoted and stored at -80 °C for later use.
- Target antigen-specific clones were identified by ELISA and FACS techniques (see Figure 3).
- the ELISA plate was coated with BCMA his, and the supernatant was taken for ELISA detection.
- Figure 3 shows the results of BCMA full-length protein ELISA screening, and some positive clones were selected for sequencing. After sequence analysis, clones with different CDR3 regions were selected for reinduction, and the binding to CHO-K1/BCMA cell line and RPMI8226 cell line overexpressing human BCMA protein was detected by FACS. Finally, 27 clones as shown in Table 1 were screened with Human BCMA target antigen-specific clone.
- ELISA method TG1 strains from a single output phage clone were induced to grow overnight in a 96-well deep-well plate. In order to identify clones bound to antigenic proteins, recombinant human BCMA his tag protein and PD-1his control protein were used in coating buffer, respectively. 96-well ELISA microtiter plates were coated overnight at 4°C and then blocked with blocking buffer. After blocking, approximately 100 ⁇ L/well of phage supernatant from overnight cell cultures was added to the plate and incubated for 1 hour at room temperature. Plates were washed four times and HRP-conjugated anti-c-myc antibody was added to the plate and incubated for 30 minutes at room temperature. Plates were washed five more times and substrate solution was added to wells for color development. Measure the absorbance of each well at 450 nm.
- FACS method Take the cloned supernatant and add it to a 96-well plate containing 5 ⁇ 10 5 CHO-K1/BCMA cells or RPMI8226 cells, incubate at 4°C for 40 min; centrifuge at 220g for 5 min, discard the supernatant, and add 200 ⁇ L of pre-cooling to each well resuspended in DPBS and centrifuged to discard the supernatant; add 150 ⁇ L of 2.5 ⁇ g/mL biotin-anti-his antibody (GenScript, A00186-100) to each well to resuspend the cells, incubate at 4°C for 40 min; wash once with pre-cooled DPBS After that, 150 ⁇ L of PE Streptavidin (Biolegend) diluted with DPBS according to 1:500 was added to each well, the cells were resuspended, and incubated at 4° C.
- PE Streptavidin Biolegend
- the screened nanobody VHH sequence was inserted into the pCDNA3.4-IgG4 plasmid vector, constructed into a VHH-hIgG4 format ( Figure 4), and expressed through the ExpiCHO TM (Thermo Fisher) expression system. After one week of expression, the supernatant was collected and purified by Protein A (GE), and then the protein quality was detected by Nanodrop, and the protein purity was detected by HPLC. As shown in Table 1, the obtained antibody purity (buffer is PBS with pH 7.4) and yield meet the needs of subsequent experiments.
- the purified antibody and the control antibody were diluted to 1000 nM with DPBS, and then serially diluted to 8 concentration points according to a 3-fold ratio.
- Biotin-APRIL was diluted to 4 ⁇ g/mL with DPBS.
- One-step diluted APRIL was mixed and incubated at 4°C for 40min.
- Cross-reactivity was determined by ELISA. Plates (Corning) were coated with 2 ⁇ g/mL cynomolgus BCMA (Acrobiosystems, BCA-C52H7-100ug) and mouse BCMA (Acrobiosystems, BCA-M52H3) overnight at 4°C. After blocking and washing, 10 ⁇ g/mL, 2 ⁇ g/mL, and 0.4 ⁇ g/mL of antibodies were added to BCMA antigen-coated well plates, respectively, and incubated at 37°C for 1 hour.
- Cross-reactivity was determined by ELISA method. Plate (Corning), respectively, 27 test antibodies and BMK control antibodies, anti-TACI antibodies (Abcam, ab89744), anti-BAFFR antibodies (Abcam, ab16232) were diluted with coating solution to 5 ⁇ g/mL, 100 ⁇ L/well Add to 96-well plates and incubate overnight at 4°C. After blocking and washing, 100 ⁇ L of 4 ⁇ g/mL biotin-TACI (Acrobiosystems, TAI-H82F6-25ug), biotin-BAFFR (Acrobiosystems, BAR-H5257-100ug) or control protein were added to the plates coated with different antibodies, respectively. and incubated at 37°C for 1 hour.
- VHH-hIgG4 antibody The binding affinity of VHH-hIgG4 antibody to human BCMA his tag protein was detected by surface plasmon resonance (SPR).
- SPR surface plasmon resonance
- the process is as follows: Biacore T200 (GE) was used in this experiment, the detection temperature was 25°C, and the buffer was 1 ⁇ HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% v/v Surfactant P20, GE).
- the chip surface was regenerated by injecting 10mM Glycine-HCl pH1.5(GE) for 30s. Data processing was performed using BIA evaluation Software 2.0 (GE), double-reference subtraction of sensorgrams, and Langmuir 1:1 model fitting to calculate kinetic constants.
- the binding affinity of the anti-BCMA VHH-hIgG4 antibody to target cells was determined by a cell-based assay. Briefly, the VHH-hlgG4 recombinant antibody was preliminarily dosed with 100 nM as the initial concentration, and the ratio was 4 times for 8 consecutive concentration points.
- CHO-K1/BCMA cells expressing human BCMA full-length protein were plated in 96-well plates, 5 ⁇ 10 5 cells per well, and the serially diluted heavy chain antibody (VHH-hlgg4 recombinant antibody) was mixed with CHO-K1/BCMA The cells were incubated at 4°C for 40 minutes.
- the secondary antibody anti-human IgG PE Jackson Immuno Research, Code: 109-117-008 was added and incubated for 30 minutes. After washing, the CytoFLEX flow cytometer was used for detection. . Curve fitting was performed by Graphprism software and the EC50 of the antibody was calculated. The results are shown in Figure 6, and the EC50 ranged from 0.6 to 2.8 nM.
- the heavy chain antibody was incubated with U-87MG cell line that does not express BCMA antigen, RPMI8226 cell that overexpresses BCMA antigen, Jeko-1 cell, and Daudi cell with serially diluted heavy chain antibody.
- the detection antibody is anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot: 145501).
- the EC50 of the antibody was calculated by fitting the curve. The results are shown in A, B, and C of Figure 7.
- the binding affinity of each antibody to RPMI8226 cells ranges from 0.8 nM to 7 nM, and the binding affinity of each antibody to Jeko-1 cells ranges from 8 nM to 100 nM.
- the binding affinity of each antibody to Daudi cells ranged from 1.5 to 300 nM. Each antibody showed no binding to U-87MG, which does not express BCMA.
- Membrane Proteome Array screening is a membrane protein array screening platform developed by Integral molecular in the United States. They displayed 5,300 different human membrane proteins on the cell surface by transfecting HEK293 cells, and detected antibodies in these cells by FACS. The binding signal on the protein is used to evaluate the specificity of the antibody to be detected (as shown in Figure 8A). Based on the detection results of affinity and binding to various BCMA overexpressing cell lines, we selected the most representative 4 antibodies for MPA screening. The results showed that the NB-1 antibody not only binds to the target protein BCMA protein, but also binds to DGCR2.
- the protein has non-specific binding (as shown in B of Figure 8); the other three antibodies including NB-29, NB-257, and NB-367 have good specificity (as shown in C, D, and E of Figure 8, respectively) , only specifically binds to the target protein BCMA.
- Tissue cross-reaction This test was mainly completed by the clinical trial testing center of Shanghai Cell Therapy Group. The purpose of the study was to evaluate whether several preferred antibodies cross-react with 34 normal human frozen tissues (each derived from 3 different individuals) using immunohistochemical staining for streptavidin and biotin (Table 1). 5).
- This test is divided into 3 test groups (NB-29 antibody-Biotin, NB-257 antibody-Biotin, NB-367 antibody-Biotin), recombinant Anti-BCMA antibody EPRBOB-R1-R1-F1-24 (abcam, cat# ab245940), 3 biomarker BMK antibodies (obtained by expression and purification in CHO cells, see Table 6 for sequence and source); and 1 NSG602_OLIGO_31 negative isotype control antibody group (see Table 6 for sequence information).
- test article group (NB-29 antibody) showed positive staining in tonsil, thyroid follicular epithelium, liver, duodenum, gastric mucosa, renal tubule and collecting duct.
- test product group (NB-367 antibody) showed positive staining in thyroid follicular epithelium, liver, duodenum, renal tubule and collecting duct.
- BMK1 and BMK3-IgG1 showed no positive binding with 34 kinds of human tissues, and BMK2-H4 showed positive binding with the germinal center of lymphoid region of tonsillar, gastric mucosa and duodenum.
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Abstract
Description
抗体 | 受体 | ka(1/Ms) | kd(1/s) | KD(M) |
NB-1 | BCMA/TNFR-His | 5.16E+06 | 1.06E-03 | 2.06E-10 |
NB-29 | BCMA/TNFR-His | 2.15E+06 | 4.13E-04 | 1.92E-10 |
NB-34 | BCMA/TNFR-His | 2.21E+06 | 1.75E-05 | 7.91E-12 |
NB-36 | BCMA/TNFR-His | 2.79E+06 | 8.14E-04 | 2.92E-10 |
NB-51 | BCMA/TNFR-His | 1.12E+07 | 4.96E-04 | 4.44E-11 |
NB-53 | BCMA/TNFR-His | 2.26E+06 | 1.79E-02 | 7.92E-09 |
NB-65 | BCMA/TNFR-His | 9.81E+06 | 1.66E-04 | 1.69E-11 |
NB-67 | BCMA/TNFR-His | 5.98E+06 | 2.96E-04 | 4.95E-11 |
NB-71 | BCMA/TNFR-His | 9.53E+06 | 2.68E-03 | 2.81E-10 |
NB-79 | BCMA/TNFR-His | 5.67E+06 | 1.64E-03 | 2.90E-10 |
NB-82 | BCMA/TNFR-His | 1.75E+06 | 5.55E-05 | 3.18E-11 |
NB-83 | BCMA/TNFR-His | 6.17E+06 | 2.99E-04 | 4.84E-11 |
NB-84 | BCMA/TNFR-His | 5.50E+06 | 6.60E-03 | 1.20E-09 |
NB-90 | BCMA/TNFR-His | 1.16E+07 | 2.75E-03 | 2.36E-10 |
NB-100 | BCMA/TNFR-His | 7.72E+06 | 3.36E-03 | 4.35E-10 |
NB-102 | BCMA/TNFR-His | 6.66E+06 | 3.42E-03 | 5.13E-10 |
NB-122 | BCMA/TNFR-His | 9.69E+06 | 9.68E-06 | 9.99E-13 |
NB-170 | BCMA/TNFR-His | 2.28E+06 | 2.15E-04 | 9.44E-11 |
NB-192 | BCMA/TNFR-His | 2.57E+06 | 2.24E-04 | 8.73E-11 |
NB-216 | BCMA/TNFR-His | 1.03E+06 | 2.70E-04 | 2.62E-10 |
NB-217 | BCMA/TNFR-His | 3.12E+06 | 2.08E-03 | 6.67E-10 |
NB-222 | BCMA/TNFR-His | 9.61E+06 | 7.02E-05 | 7.31E-12 |
NB-257 | BCMA/TNFR-His | 4.77E+06 | 1.02E-02 | 2.14E-09 |
NB-265 | BCMA/TNFR-His | 8.43E+06 | 3.92E-04 | 4.65E-11 |
NB-352 | BCMA/TNFR-His | 6.00E+06 | 1.83E-03 | 3.05E-10 |
NB-367 | BCMA/TNFR-His | 6.74E+06 | 2.06E-03 | 3.05E-10 |
Claims (12)
- 一种纳米抗体,其特征在于,其包括含CDR1、CDR2和/或CDR3的重链可变区,并具有识别并结合BCMA的功能,且其中:所述CDR1选自以下组:(1)如SEQ ID NO:4的氨基酸序列所示,或在如SEQ ID NO:4所示的氨基酸序列上发生1~3种氨基酸残基的替换、缺失或增加;(2)如SEQ ID NO:6的氨基酸序列所示,或在如SEQ ID NO:6所示的氨基酸序列上发生1~3种氨基酸残基的替换、缺失或增加;所述CDR2为如SEQ ID NO:23所示的氨基酸序列,或在如SEQ ID NO:23所示的氨基酸序列发生1~3种氨基酸残基的替换、缺失或增加;所述CDR3选自如SEQ ID NO:27~52所示的氨基酸序列。
- 如权利要求1所述的纳米抗体,其特征在于,当所述CDR1为在如SEQ ID NO:4所示的氨基酸序列上发生1~3种氨基酸残基的替换时,所述替换选自G1E、T3I、S5R和S6P/I;当所述CDR1为在如SEQ ID NO:6所示的氨基酸序列上发生1~3种氨基酸残基的替换时,所述替换选自F4S/D、S5R和I6F;和/或,当所述CDR2为在如SEQ ID NO:23所示的氨基酸序列发生1~3种氨基酸残基的替换时,所述替换选自I1V、Y2T、S3P/G/T、D4G/E/A、G5S/N、S6R/N/G/T和T7A/P/S;优选地,所述CDR1选自如SEQ ID NO:2~7所示的氨基酸序列;所述CDR2选自如SEQ ID NO:8~26所示的氨基酸序列;和/或,所述CDR3选自如SEQ ID NO:27~52所示的氨基酸序列。
- 如权利要求1~3任一项所述的纳米抗体,其特征在于,所述重链可变区的氨基酸序列如SEQ ID NO:53~78所示,或与如SEQ ID NO:53~78的氨基酸序列具有至少80%、90%、95%、96%、97%、98%或99%同一性。
- 一种BCMA结合分子,其中,所述BCMA结合分子包含如权利要求1~4任一项所述的纳米抗体;优选地,所述BCMA结合分子为包含一条、两条或多条如权利要求1~4任一项所述的纳米抗体的单价或多价纳米抗体、双特异性抗体、多特异性抗体、重链抗 体或其抗原结合片段。
- 如权利要求5所述的BCMA结合分子,其特征在于,所述BCMA结合分子为重链抗体;优选地,所述重链抗体的Fc的氨基酸序列如SEQ ID NO:1所示,或与如SEQ ID NO:1所示的氨基酸序列具有至少80%、90%、95%、96%、97%、98%或99%同一性。
- 一种分离的核酸,其特征在于,所述分离的核酸编码如权利要求1~4任一项所述的纳米抗体、或如权利要求5或6所述的BCMA结合分子。
- 一种重组表达载体,其特征在于,所述重组表达载体包含如权利要求7所述的分离的核酸;优选地,所述重组表达载体的骨架为pCDNA3.4。
- 一种转化体,其特征在于,所述转化体包含如权利要求7所述的分离的核酸,或如权利要求8所述的重组表达载体;优选地,所述转化体的宿主为原核细胞或真核细胞;更优选地,所述真核细胞为CHO细胞。
- 一种组合物,其特征在于,其包括一条、两条或多条如权利要求1~4任一项所述的纳米抗体、权利要求5或6所述的BCMA结合分子、权利要求7所述的分离的核酸、权利要求8所述的重组表达载体或权利要求9所述的转化体;优选地,所述组合物还包括药学上可接受的辅料。
- 一种BCMA检测剂,其特征在于,所述BCMA检测剂包括如权利要求1~4所述的纳米抗体,和/或,如权利要求5或6所述的BCMA结合分子;优选地,所述BCMA检测剂用于流式检测。
- 如权利要求1~4任一项所述的纳米抗体、权利要求5或6所述的BCMA结合分子、权利要求7所述的分离的核酸、权利要求8所述的重组表达载体或权利要求9所述的转化体在制备治疗与BCMA相关疾病的药物中的应用;所述疾病优选为血液恶性肿瘤,更优选为多发性骨髓瘤。
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CN117186228A (zh) * | 2022-12-06 | 2023-12-08 | 成都赛恩吉诺生物科技有限公司 | 包含长cdr3序列的抗人bcma纳米抗体的双特异性抗体及应用 |
WO2024094096A1 (en) * | 2022-11-02 | 2024-05-10 | Zhejiang Nanomab Technology Center Co. Ltd. | Selection of nanobodies using sequence features |
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