[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2022037528A1 - 结合bcma的单可变结构域及抗原结合分子 - Google Patents

结合bcma的单可变结构域及抗原结合分子 Download PDF

Info

Publication number
WO2022037528A1
WO2022037528A1 PCT/CN2021/112758 CN2021112758W WO2022037528A1 WO 2022037528 A1 WO2022037528 A1 WO 2022037528A1 CN 2021112758 W CN2021112758 W CN 2021112758W WO 2022037528 A1 WO2022037528 A1 WO 2022037528A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
single variable
variable domain
Prior art date
Application number
PCT/CN2021/112758
Other languages
English (en)
French (fr)
Inventor
甄子朋
谢美娟
杜秀贞
马毅敏
张兵
徐同杰
张喜全
Original Assignee
正大天晴药业集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 正大天晴药业集团股份有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to JP2023510435A priority Critical patent/JP2023539453A/ja
Priority to US18/020,584 priority patent/US20230265181A1/en
Priority to CN202180041402.4A priority patent/CN115698077A/zh
Priority to EP21857623.9A priority patent/EP4201960A4/en
Priority to KR1020237008715A priority patent/KR20230045095A/ko
Publication of WO2022037528A1 publication Critical patent/WO2022037528A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to antigen-binding molecules, in particular to single variable domains and antigen-binding molecules that bind BCMA.
  • BCMA B cell maturation antigen
  • TFRS17 tumor necrosis factor receptor superfamily member 17
  • BAFF and APRIL are ligands of BCMA, among which BAFF (also known as BLyS, TALL-1, THANK, zTNF4, TNFSF20, D8Ertd387e) is a high-affinity ligand of BCMA, APRIL (proliferation-inducing ligand, also known as TNFSF13, TALL) -2. TRDL-1) is a low affinity ligand for BCMA.
  • BAFF and APRIL are also members of the tumor necrosis factor receptor (TNFR) superfamily B-cell activation factor receptor (BAFF-R), transmembrane activator and calcineurin ligand-interacting molecule (transmembrane activator and calcium modulator and cyclophilin ligand interactor; TACI).
  • TNFR tumor necrosis factor receptor
  • BAFF-R tumor necrosis factor receptor superfamily B-cell activation factor receptor
  • TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • Multiple myeloma is a malignant plasma cell disease in which the expression level of BCMA is significantly increased in multiple myeloma cells.
  • BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma.
  • Immunotherapeutic agents such as antibodies that bind to BCMA can block the binding between BCMA and its natural ligands BAFF or/and APRIL. As potential therapeutic targets, some antibodies targeting BCMA have been developed, but they are still limited and more available options are needed.
  • IgG antibodies in camelid animals in addition to the traditional 4-chain structure antibody IgG1 including light chain and heavy chain, also naturally exist heavy chain-only antibodies (HcAb) IgG2 and IgG3 that do not contain light chain.
  • HcAb heavy chain-only antibodies
  • Only the single variable domain ( VHH or single variable domain) in the heavy chain antibody has the characteristics of specific binding to the antigen and has a high affinity for the antigen, which is called single domain antibody (sdAb).
  • VHH domains are highly soluble and have no tendency to aggregate, small molecules show high tissue penetration, single domain antibodies do not require pairing with light chains, There is no problem of light and heavy chain mismatches when composing bispecific or multispecific antibodies, etc.
  • the present invention provides single variable domains and antigen-binding molecules that bind BCMA.
  • the present invention also provides related nucleotides, vectors, cells, compositions, construction methods and uses capable of encoding the provided single variable domains and antigen-binding molecules.
  • the invention provides an isolated single variable domain that binds BCMA, wherein the single variable domain comprises CDR1, CDR2 and CDR3 selected from the group consisting of:
  • the single variable domain comprises a CDR1, a CDR2, and a CDR3 selected from any one of the following:
  • CDR1 comprising the amino acid sequence of SEQ ID NO:7; CDR2 comprising the amino acid sequence of SEQ ID NO:8; and CDR3 comprising the amino acid sequence of SEQ ID NO:9;
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 10
  • CDR2 comprising the amino acid sequence of SEQ ID NO: 11
  • CDR3 comprising the amino acid sequence of SEQ ID NO: 12;
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 13; CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and CDR3 comprising the amino acid sequence of SEQ ID NO: 15; or
  • CDR1 comprising the amino acid sequence of SEQ ID NO: 16; CDR2 comprising the amino acid sequence of SEQ ID NO: 17; and CDR3 comprising the amino acid sequence of SEQ ID NO: 18.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91% of the sequence of SEQ ID NO: 19, 21, 23 or 25 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of amino acid sequences.
  • the invention provides an isolated single variable domain, wherein the single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence of SEQ ID NO: 19, 21, 23 or 25.
  • the invention provides an isolated single variable domain, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO: 19, 21, 23 or 25.
  • the invention provides an isolated single variable domain that binds the extracellular region of BCMA at one of the Gln7-His19, Pro23-Ser30 and Asn31-Ser44 positions of the BCMA extracellular region or Multiple amino acids, the amino acid sequence of the extracellular region of BCMA is shown in SEQ ID NO:35 or SEQ ID NO:36. The positions described here are numbered sequentially starting from the first amino acid (position 1) in the extracellular domain of human BCMA.
  • the invention provides an isolated single variable domain, wherein the single variable domain binds the same epitope as the single variable domain described in any of the embodiments herein.
  • the invention provides an isolated single variable domain, wherein the single variable domain competes with the single variable domain described in any of the embodiments herein for binding to BCMA.
  • the single variable domains described above herein are camelid or humanized. In some embodiments, the single variable domains described herein above are VHHs , preferably camelid or humanized VHHs .
  • the invention provides uses of the single variable domains described herein, including for the construction of antigen binding molecules, preferably antibodies, monospecific antibodies, multispecific antibodies or immunoconjugates.
  • the invention provides isolated antigen binding molecules that bind BCMA and comprise at least one single variable domain as described herein.
  • the present invention provides a composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a single variable domain as described herein or an antigen binding molecule as described herein.
  • the antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 27, 29, 31 or 33.
  • the invention provides an isolated nucleic acid encoding a single variable domain described herein or an antigen binding molecule described herein.
  • the invention provides a vector comprising the isolated nucleic acid described herein.
  • the invention provides host cells comprising the vectors described herein.
  • the invention provides a method of detecting or measuring BCMA in a sample comprising contacting the sample with a single variable domain described herein or an antigen binding molecule described herein and detecting or measuring the binding complex .
  • the present invention provides a method for preparing a single variable domain described herein, comprising: culturing the host cell, isolating the expressed single variable domain, wherein the vector is an expression vector, the The host cell contains nucleic acid encoding the single variable domain.
  • the present invention provides a method for preparing the antigen-binding molecule, comprising: culturing the host cell, and isolating the expressed antigen-binding molecule, wherein the vector is an expression vector, and the host cell contains the encoding nucleic acid of the antigen-binding molecule.
  • the invention provides a method of treating a subject with a BCMA-expressing tumor comprising administering to the subject a therapeutically effective amount of the single variable domain, the antigen binding molecule or said composition.
  • the invention provides a method of inhibiting, reducing or blocking BCMA signaling in a cell comprising administering to the cell an effective amount of the single variable domain, the antigen binding molecule or the described composition.
  • the present invention provides a method for killing BCMA-expressing tumor cells or inhibiting the growth of BCMA-expressing tumor cells, comprising combining the tumor cells with the single variable domain, the antigen binding molecule or the The composition is contacted.
  • the present invention provides a method of treating a subject with an autoimmune disease comprising administering to the subject a therapeutically effective amount of a single variable domain described herein, An antigen binding molecule or a composition described herein.
  • Fig. 1 is the curve that ELISA detects anti-human BCMA VH H -Fc chimeric antibody binding to antigen
  • Figure 2A is a flow cytometry detection of the binding curve of anti-human BCMA VH H -Fc chimeric antibody to CHO-hBCMA cells;
  • Figure 2B is a flow cytometry detection of the binding curve of anti-human BCMA VH H -Fc chimeric antibody to U266 cells;
  • Figure 2C is a flow cytometry detection of the binding curve of anti-human BCMA VH H -Fc chimeric antibody to RPMI8226 cells;
  • Figure 2D is a flow cytometry detection of the binding curve of anti-human BCMA VH H -Fc chimeric antibody to HUVEC cells;
  • Figure 3A is a flow cytometry detection of the binding curves of 1A10-Fc, 1A11-Fc chimeric antibodies and HEK293T-CynoBCMA;
  • Figure 3B shows the binding curves of 1A10-Fc, 1A11-Fc chimeric antibodies and HEK293T detected by flow cytometry;
  • Figure 4 shows the results of competition ELISA detection of human BCMA VH H -Fc chimeric antibody and ligand APRIL;
  • Figure 5A is a comparison diagram of the amino acid sequence of the extracellular region of human and cynomolgus monkey BCMA;
  • Figure 5B is the crystal structure of the extracellular region of human BCMA
  • Figure 5C shows the epitope relationship of 1A1, 1A10, 1A11 and 1B10.
  • antigen-binding molecule in its broadest sense refers to a molecule that specifically binds an antigenic determinant.
  • antigen binding molecules are antibodies, fusion proteins, antibody conjugates.
  • immunoglobulin refers to a protein having the structure of a naturally occurring antibody.
  • immunoglobulins of the human IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a heavy chain variable region (VH), followed by a hinge region (HR) and three constant domains (CH1, CH2 and CH3), also referred to as heavy chain constant regions.
  • VH heavy chain variable region
  • HR hinge region
  • CH1, CH2 and CH3 constant domains
  • the heavy chain also has a CH4 domain.
  • an immunoglobulin heavy chain is a polypeptide consisting of the following domains in the N-terminal to C-terminal direction: VH-CH1-HR-CH2-CH3-(CH4).
  • each light chain has a light chain variable region (VL) followed by a constant light chain domain, also referred to as a light chain constant region (CL).
  • VL light chain variable region
  • CL constant light chain domain
  • an immunoglobulin light chain is a polypeptide consisting in the N-terminal to C-terminal direction of the following domains: VL-CL.
  • Human immunoglobulins consist essentially of two Fab and Fc domains linked by an immunoglobulin hinge region.
  • antibody is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies), and antibody fragments, as long as They show the desired antigen binding activity.
  • variable domain refers to the domain of an antibody that is involved in the binding of the antibody to an antigen.
  • Each variable domain of a native antibody consists essentially of four "framework regions” and three complementarity determining regions.
  • the four "framework regions” are referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region”, respectively 4" or "FR4";
  • the framework regions are referred to in the art and hereinafter as “complementarity determining region 1" or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR2", respectively.
  • variable domain The three “complementarity determining regions” or “CDRs” of CDR3" are spaced apart.
  • CDRs complementarity determining regions
  • the general structure or sequence of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Variable domains confer antigen specificity to an antibody by having an antigen-binding site.
  • single variable domain refers to a variable domain capable of specifically binding an epitope without pairing with other variable domains.
  • the antigen binding site of a single variable domain is usually formed by three CDRs (CDR1, CDR2 and CDR3), present on a single domain.
  • the single variable domain may be a heavy chain variable domain (eg, VH) or a suitable fragment thereof; so long as it is capable of forming a single antigen-binding unit (ie, functionally consisting essentially of a single variable domain) antigen-binding unit so that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).
  • Another example of a single variable domain is the camelid "VHH domain” (or simply "VHH" or " VHH ").
  • VHH domains also known as VHH, VHH , VHH domains, or single-domain antibodies, were originally described as “heavy chain-only antibodies” (ie, "antibodies lacking light chains") with the ability to bind an antigen. variable domain.
  • VHH domain is used to relate these variable domains to the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "VH domains” or “VH”) and to those present in conventional 4-chain antibodies
  • VH domains heavy chain variable domains
  • VL domains the light chain variable domains in 4-chain antibodies are distinguished.
  • VHH domain binds specifically to the epitope without the need for additional antigen binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain).
  • VHH domains are small stable and efficient antigen recognition units formed from a single domain.
  • CDR complementarity determining region
  • HVR hypervariable region
  • Native tetrabodies typically contain six CDRs, three in the heavy chain variable region (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) and three in the light chain variable region (light chain CDR1, light chain CDR2 and light chain CDR3).
  • Heavy chain-only antibodies or single variable domains typically have three CDRs (CDR1 (or HVR1), CDR2 (or HVR2), and CDR3 (or HVR3)).
  • CDR3 shows the most diversity of the three CDRs and is believed to play a unique role in conferring fine specificity to antibodies.
  • CDRs There are currently many ways to divide and define CDRs. Among them, the Kabat definition divides CDRs based on sequence variability, and is the most commonly used (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. ( 1991)); while the Chothia definition is based on the position of the structural loops (Cyrus Chothia, et al, Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917 (1987)).
  • the AbM definition is a compromise between the Kabat definition and the Chothia definition, and is used by Oxford Molecular's AbM antibody modeling software. "Contact" defines CDRs based on the analysis of available complex crystal structures. The residues of each of these CDRs are reported in Table S1 below.
  • CDRs complementarity determining regions
  • HVRs are used to refer to the CDRs as defined by Kabat (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th ed. , Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Based on the amino acid sequence of the variable region of the antibody, those skilled in the art can routinely determine which amino acid residues a CDR of any definition contains, and CDRs of other arbitrary definitions (eg, Chothia, AbM definitions, etc.) will also be included within the scope of the present invention. .
  • Amino acid residue numbering for single variable domains is according to Kabat et al. (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda , Md. (1991)) given the VH general numbering system, as in the article by Riechmann et al (L Riechmann, et al, Single domain antibodies: comparison of camel VH and camelized human VH domains, J.Immunol.Methods. (1999)) applied to the VHH domain of camelid.
  • FR1 of VHH comprises amino acid residues at positions 1-30
  • CDR1 of VHH comprises amino acid residues at positions 31-35
  • FR2 of VHH comprises amino acid residues at positions 36-49
  • CDR2 of VHH contains amino acid residues at positions 50-65
  • FR3 of VHH contains amino acid residues at positions 66-94
  • CDR3 of VHH contains amino acid residues at positions 95-102
  • VH FR4 of H contains amino acid residues at positions 103-113.
  • the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e. , one or more positions in the actual sequence according to Kabat numbering may not be occupied, or the actual sequence may contain a greater number of amino acid residues than the Kabat numbering allows).
  • “Variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat” and variations thereof refer to Kabat et al. (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th ed. , Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) numbering system for encoding heavy or light chain variable domains of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of the FRs or CDRs of the variable domains.
  • a heavy chain variable domain may comprise a single amino acid insertion following amino acid residue 52 of CDR2 (amino acid residue 52a according to Kabat) and an inserted amino acid residue following amino acid residue 82 of a heavy chain FR (eg according to Kabat's amino acid residue 82) amino acid residues 82a, 82b and 82c, etc.).
  • the Kabat numbering of amino acid residues of a given antibody can be determined by aligning the antibody sequence with the sequence of homologous regions in "standard" Kabat numbering.
  • frame region or "FR” residues are those amino acid residues of a variable domain other than the CDR residues as defined herein.
  • human consensus framework region or "acceptor human framework” is a framework representing the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework region sequences.
  • human immunoglobulin VL or VH sequences are selected from a subset of variable domain sequences.
  • a subset of sequences is that in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include: for VL, the subgroups may be subgroups Kl, KII, KIII, or KIV as described by Kabat et al., supra.
  • the subgroup can be subgroup I, subgroup II or subgroup III as described by Kabat et al.
  • the human consensus framework can be derived from the residues specified above, such as when based on the identity of the human framework residues to the donor framework by aligning the donor framework sequences with a collection of various human framework sequences.
  • the acceptor human framework "derived from" a human consensus framework region when selecting human framework region residues by origin may comprise its identical amino acid sequence, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less.
  • Fc domain or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least part of the constant region.
  • the term includes native sequence Fc and variant Fc.
  • the C-terminal lysine of the Fc (Lys447 according to the EU numbering system) may or may not be present.
  • EC50 refers to an effective concentration, the 50% maximal response of an antigen binding molecule.
  • IC50 refers to the inhibitory concentration, the 50% maximal response of an antigen-binding molecule. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • KD refers to the equilibrium dissociation constant, expressed in molarity (M).
  • M molarity
  • the KD value of an antigen-binding molecule can be determined using methods known in the art.
  • One method of determining the KD of an antigen-binding molecule is to use surface plasmon resonance, such as using a biosensor system, such as the Biacore system.
  • treating refers to the purpose of treating, curing, alleviating, alleviating, altering, remediating, ameliorating, ameliorating or affecting a disorder (eg, a disease), a symptom of a disorder, or to prevent or delay a symptom, complication, biochemical The onset of an indicator, or the use of measures that otherwise retard or inhibit the further progression of a disease, disorder, or condition.
  • terapéuticaally effective amount refers to the amount of an antigen binding molecule or composition or other administration necessary to provide a therapeutic and/or prophylactic benefit to a subject.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, anchors, horses, cattle, chickens, amphibians, reptiles, and the like.
  • the subject according to the present invention is a human.
  • the terms “patient” or “subject” are used interchangeably.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • isolated refers to a compound of interest (eg, a VHH, antigen-binding molecule, antibody, or nucleic acid) that has been isolated from its natural environment.
  • a compound of interest eg, a VHH, antigen-binding molecule, antibody, or nucleic acid
  • epitopes or the term “antigenic determinant” used interchangeably refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopes typically contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear” "Epitope” or “Conformational” Epitope.
  • sequence identity also called identity.
  • the comparison of sequences and the determination of percent identity between two sequences can be accomplished using mathematical algorithms, as shown in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl.
  • “about” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, ie, limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, “about” can mean a range of up to 5% (ie, ⁇ 5%), eg, within ⁇ 2%, ⁇ 1%, or ⁇ 0.5% of the particular numerical range given. Furthermore, particularly with respect to biological systems or methods, the term can mean up to an order of magnitude or up to 5 times a value. When a particular value is given in this application or in the claims, unless stated otherwise, the meaning of "about” is to be construed as being within an acceptable error range for that particular value.
  • One aspect of the invention provides isolated single variable domains that bind to BCMA, such as human BCMA.
  • Single variable domains provide more available options for drug development or drug construction targeting BCMA.
  • the single variable domains have many desirable therapeutic properties, such as good affinity for human BCMA and the ability to block the binding of the proliferation-inducing ligand APRIL to BCMA.
  • the single variable domain does not bind to human TACI and BAFFR proteins, showing specificity; in some embodiments, it cross-reacts with monkey BCMA, since monkeys are ideal for drug toxicology experiments Animals, cross-reaction with monkeys will facilitate the development of drug toxicology experiments.
  • a BCMA-binding single variable domain comprising one, two or all three CDRs of the single variable domain set forth in SEQ ID NO:19 is provided.
  • a specific embodiment provides a BCMA-binding single variable domain comprising CDR1, CDR2 and CDR3 of the single variable domain set forth in SEQ ID NO: 19.
  • a BCMA-binding single variable domain comprising one, two or all three CDRs of the single variable domain set forth in SEQ ID NO:21 is provided.
  • a specific embodiment provides a BCMA-binding single variable domain comprising CDR1, CDR2 and CDR3 of the single variable domain shown in SEQ ID NO: 21.
  • a BCMA-binding single variable domain comprising one, two or all three CDRs of the single variable domain set forth in SEQ ID NO:23 is provided.
  • a specific embodiment provides a BCMA-binding single variable domain comprising CDR1, CDR2 and CDR3 of the single variable domain set forth in SEQ ID NO: 23.
  • a BCMA-binding single variable domain comprising one, two or all three CDRs of the single variable domain set forth in SEQ ID NO:25 is provided.
  • a specific embodiment provides a BCMA-binding single variable domain comprising CDR1, CDR2 and CDR3 of the single variable domain set forth in SEQ ID NO: 25.
  • the single variable domain is camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • a BCMA-binding single variable domain comprising at least one, at least two or all three CDRs selected from: (a) comprising SEQ ID NOs: 7, 10, 13 and CDR1 of the amino acid sequence of 16; (b) CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 11, 14 and 17; and (c) comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 12, 15 and 18 sequence of CDR3.
  • the single variable domain is camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2, and CDR3), the CDRs comprising: (a) with a combination selected from the group consisting of SEQ ID NOs: 7, 10, 13 and The amino acid sequence of 16 has at least about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, A CDR1 having a sequence identity of any one of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%; (b) with a CDR1 selected from the group consisting of SEQ The amino acid sequences of ID NOs: 8, 11, 14 and 17 have at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93% , about 94%, about 95%, about 96%, about 97%, about 98%, about
  • the amino acid sequences of 9, 12, 15 and 18 have at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about CDR3s with any of 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2, and CDR3), the CDRs comprising: (a) having at least the amino acid sequence of SEQ ID NO:7 about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% %, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity of any one of CDR1s; (b) having the amino acid sequence of SEQ ID NO:8 at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about A CDR2 having any one of 97%, about 98%, about 99%, or about 100% sequence identity; and (c) having at least about 85%, about 86%, about 87%
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: (a) having at least the amino acid sequence of SEQ ID NO: 10 about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% %, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity of any one of CDR1; (b) having the amino acid sequence of SEQ ID NO: 11 at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about A CDR2 having a sequence identity of any one of 97%, about 98%, about 99%, or about 100%; and (c) having at least about 85%, about 86%, about
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: (a) having at least the amino acid sequence of SEQ ID NO: 13 about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% %, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity of any one of CDR1; (b) having the amino acid sequence of SEQ ID NO: 14 at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about A CDR2 having any one of 97%, about 98%, about 99%, or about 100% sequence identity; and (c) having at least about 85%, about 86%, about 100% of the
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: (a) having at least the amino acid sequence of SEQ ID NO: 16 about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% %, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity of any one of the CDR1s; (b) having the amino acid sequence of SEQ ID NO: 17 at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about A CDR2 having any one of 97%, about 98%, about 99%, or about 100% sequence identity; and (c) having at least about 85%, about 86%, about 100%
  • At least about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92% CDRs with any of %, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity contain substitutions relative to the reference sequence ( For example, conservative substitutions), insertions or deletions, but the single variable domain comprising this sequence retains the ability to bind to BCMA.
  • a single variable domain comprising three CDRs (CDR1, CDR2 and CDR3) comprising: (a) an amino acid selected from the group consisting of SEQ ID NOs: 7, 10, 13 and 16 A CDR1 having about 1 or about 2 amino acid substitutions (e.g., conservative substitutions), insertions or deletions in sequence comparison; CDR2 with any one of about 1, about 2, or about 3 amino acid substitutions (e.g., conservative substitutions), insertions or deletions compared to the amino acid sequence of CDR3 having any of about 1, about 2, or about 3 amino acid substitutions (eg, conservative substitutions), insertions, or deletions compared to the amino acid sequences of 9, 12, 15, and 18.
  • the single variable domains are affinity matured.
  • the single variable domain is camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: CDR1 comprising the amino acid sequence of SEQ ID NO:7; comprising SEQ ID NO: : CDR2 of the amino acid sequence of SEQ ID NO: 8; and CDR3 comprising the amino acid sequence of SEQ ID NO: 9.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2, and CDR3), the CDRs comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 10; comprising SEQ ID NO: : CDR2 of the amino acid sequence of SEQ ID NO: 11; and CDR3 comprising the amino acid sequence of SEQ ID NO: 12.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 13; comprising SEQ ID NO : CDR2 of the amino acid sequence of SEQ ID NO: 14; and CDR3 comprising the amino acid sequence of SEQ ID NO: 15.
  • a BCMA-binding single variable domain comprising three CDRs (CDR1, CDR2 and CDR3), the CDRs comprising: CDR1 comprising the amino acid sequence of SEQ ID NO: 16; comprising SEQ ID NO : CDR2 of the amino acid sequence of SEQ ID NO: 17; and CDR3 comprising the amino acid sequence of SEQ ID NO: 18.
  • the single variable domain is camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDRl, CDR2, and/or CDR3) comprises and is selected from the group consisting of SEQ ID NOs: 19, 21, 23 and
  • the amino acid sequence of 25 has at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, VHH of any of about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDRl, CDR2, and/or CDR3) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, VHH of any of about 97%, about 98%, about 99%, or about 100% sequence identity.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDRl, CDR2, and/or CDR3) comprises an amino acid sequence having at least the same amino acid sequence as SEQ ID NO:21 about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% VHH of any of %, about 98%, about 99%, or about 100% sequence identity.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDR1, CDR2, and/or CDR3) comprises an amino acid sequence having at least the same amino acid sequence as SEQ ID NO:23 about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% VHH domains of any of %, about 98%, about 99%, or about 100% sequence identity.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDR1, CDR2, and/or CDR3) comprises an amino acid sequence having at least one of the amino acid sequence of SEQ ID NO:25 about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% VHH of any of %, about 98%, about 99%, or about 100% sequence identity.
  • the single variable domain comprises at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, V of any of about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity H H, and V H H include: CDR1 comprising the amino acid sequence of SEQ ID NO:7, CDR2 comprising the amino acid sequence of SEQ ID NO:8, and CDR3 comprising the amino acid sequence of SEQ ID NO:9.
  • the single variable domain comprises at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, V of any of about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity H H, and V H H include: CDR1 comprising the amino acid sequence of SEQ ID NO:10, CDR2 comprising the amino acid sequence of SEQ ID NO:11, and CDR3 comprising the amino acid sequence of SEQ ID NO:12.
  • the single variable domain comprises at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, V of any of about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity H H, and V H H include: CDR1 comprising the amino acid sequence of SEQ ID NO:13, CDR2 comprising the amino acid sequence of SEQ ID NO:14, and CDR3 comprising the amino acid sequence of SEQ ID NO:15.
  • the single variable domain comprises at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, V of any of about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity H H, and V H H include: CDR1 comprising the amino acid sequence of SEQ ID NO:16, CDR2 comprising the amino acid sequence of SEQ ID NO:17, and CDR3 comprising the amino acid sequence of SEQ ID NO:18.
  • VH sequence that is any one of %, about 96%, about 97%, about 98%, or about 99% identical contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but includes the The single variable domain of the sequence retains the ability to bind to BCMA.
  • 1-18, 1-16, 1-14, 1-12, 1-10, 1-9, 1 in total in the amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, 23, and 25 -8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acids are substituted, inserted and/or deleted.
  • substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs).
  • the substitution, insertion or deletion occurs in a CDR region, eg, one, two or three of CDRl, CDR2, CDR3 occur.
  • substitutions, insertions or deletions occur in CDR regions and non-CDR regions.
  • the single variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 21, 23 and 25, including post-translational modifications of the sequence.
  • isolated single variable domains of VHH comprising the amino acid sequence of SEQ ID NO: 19, 21, 23 or 25 are provided.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a particular CDRl, CDR2, and/or CDR3) is VHH .
  • the basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, where CDR1 to CDR3 refer to complementarity determining Zones 1 to 3.
  • the single variable domain (including any of the above-described embodiments, eg, the above-described single variable domain comprising a specific CDRl, CDR2, and/or CDR3) is a humanized VHH .
  • Non-human derived single variable domains can be obtained by replacing one or more of the amino acid sequences of the original single variable domain sequence with one or more amino acid residues present at corresponding positions in the VH domain of a conventional human 4-chain antibody. amino acid residues are "humanized”. Humanization can desirably reduce immunogenicity.
  • a single variable domain may comprise, alone or in combination, one or more of the following property characteristics:
  • the single variable domain binds human BCMA. In some embodiments, the single variable domain binds human BCMA and monkey BCMA such as cynomolgus monkey BCMA.
  • the single variable domain blocks the binding of APRIL to BCMA.
  • the single variable domains do not bind to human TACI or/and BAFFR proteins, exhibiting good specificity.
  • the single variable domain combines property features (i)-(ii) above. In some embodiments, the single variable domain combines properties (i) and (iii) above; in some embodiments, the single variable domain combines properties (i), (iii) above ) and (iv); in some embodiments, the single variable domain combines the above property features (i)-(iv).
  • the invention provides single variable domains that bind the same epitope as any of the single variable domains described herein.
  • a single variable domain is provided that binds the same epitope as the single variable domain comprising the amino acid sequence of SEQ ID NO: 19, 21, 23 or 25.
  • the single variable domains that bind the same epitope are camelid, or humanized.
  • Single variable domains can be screened for competition for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain single variable domains that compete with each other or cross-compete for antigen binding. Accordingly, in some embodiments, the present invention provides a single variable domain that competes with any of the single variable domains described herein for binding to BCMA. In some specific embodiments, a single variable domain is provided that competes with a single variable domain comprising the amino acid sequence of SEQ ID NO: 19, 21, 23 or 25 for binding to BCMA. Binding to BCMA can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art. In some embodiments, the single variable domain that competes for binding to BCMA is camelid, or humanized.
  • the present invention provides some exemplary single variable domains that bind BCMA.
  • the amino acid sequences of the CDRs (CDRl, CDR2 and CDR3) of the exemplary single variable domains provided herein are provided in Table S2 below.
  • the full-length amino acid sequences of exemplary single variable domains are provided in Table S3 below.
  • the single variable domains of the present invention can be used to construct any desired antigen binding molecule, thereby conferring on the antigen binding molecule targeted binding properties to BCMA or other properties of the single variable domain. Accordingly, the present invention provides isolated antigen binding molecules comprising at least one (one or more) single variable domains of the present invention.
  • the antigen binding molecule comprises at least one single variable domain comprising CDR1, CDR2 and CDR3 selected from any of the following group:
  • the single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 19, 21, 23 or 25. In some more specific embodiments, the amino acid sequence of the single variable domain is set forth in SEQ ID NO: 19, 21, 23 or 25.
  • the antigen-binding molecule comprises two or more single variable domains
  • the same or different single variable domains can be selected.
  • the antigen binding molecules include antibodies, monospecific antibodies, multispecific antibodies or immunoconjugates.
  • the antigen binding molecule is an antibody.
  • the antibody is a monospecific antibody, and in another specific example, the antibody is a bispecific antibody.
  • the antibody or immunoconjugate comprises an immunoglobulin constant region.
  • the antibody or immunoconjugate comprises human immunoglobulin Fc.
  • the Fc is a human IgG1, IgG2, IgG3 or IgG4 Fc.
  • the antigen binding molecule is camelid, chimeric or humanized.
  • an antigen binding molecule may comprise one or more of the following property characteristics, alone or in combination:
  • the antigen binding molecule binds human BCMA.
  • the antigen binding molecule has the following binding affinity (KD) for human BCMA at about 1E-12M to about 1E-08M, about 1E-11M to about 1E-08M, about 8.12E-10M In the range of about 1.29E-10M, or about 8.12E-10M to about 3.13E-10M.
  • the antigen binding molecule has the following binding affinity (KD) for human BCMA: about 1E-08M or less, about 1E-09M or less, about 8.12E-10M or less, about 6.49 E-10M or less, about 3.13E-10M or less, or, about 1.29E-10M or less.
  • the binding affinity KD of the antigen binding molecules provided herein is measured by Biacore.
  • the antigen binding molecule binds human BCMA and monkey BCMA.
  • 1A10 single variable domain, 1A11 single variable domain, 1A10-Fc, and 1A11-Fc not only have good affinity for human BCMA, but also bind to cynomolgus monkey BCMA.
  • the cross-reaction with monkey BCMA will be beneficial to the development and conduct of drug toxicology experiments.
  • the antigen binding molecule blocks the binding of APRIL to BCMA.
  • the antigen-binding molecule does not bind to human TACI or/and BAFFR protein and exhibits good specificity.
  • the antigen binding molecule combines the above property features (i)-(ii). In some embodiments, the antigen binding molecule combines property features (i) and (iii) above; in some embodiments, the antigen binding molecule combines property features (i), (iii) and (iv) above ); in some embodiments, the antigen-binding molecule combines the above-mentioned property features (i)-(iv).
  • the present invention provides exemplary antigen binding molecules, such as monospecific antibodies (including 1A1-Fc, 1A10-Fc, 1A11-Fc and 1B10-Fc antibodies), by fusing a single variable domain to the Fc of human IgG1 by Fc forms homodimers.
  • monospecific antibodies including 1A1-Fc, 1A10-Fc, 1A11-Fc and 1B10-Fc antibodies
  • the amino acid sequences of exemplary monospecific antibodies are provided in Table S4 below.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a single variable domain as described herein or an antigen binding molecule as described herein.
  • any one or more of the 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc antibodies, and a pharmaceutically acceptable carrier are formulated into a pharmaceutical composition.
  • Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.
  • the present invention provides isolated nucleic acids encoding the single variable domains described herein or the antigen binding molecules described herein.
  • the nucleic acid encodes a single variable domain, such as a single variable domain of 1A1-Fc, 1A10-Fc, 1A11-Fc, or 1B10-Fc.
  • the nucleic acid encodes an antigen binding molecule, such as 1A1-Fc, 1A10-Fc, 1A11-Fc, or 1B10-Fc.
  • the sequence listing exemplifies the nucleic acid sequences of some single variable domains, antigen binding molecules.
  • the present invention provides vectors comprising the isolated nucleic acids.
  • the vector is a cloning vector; in other embodiments, the vector is an expression vector.
  • the expression vector can be any expression vector capable of expressing the single variable domain or antigen-binding molecule described herein, a specific example, the expression vector is pcDNA3.1.
  • host cells comprising the vectors described herein, the host cells being suitable host cells for cloning or expressing single variable domains or antigen-binding molecules.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antigen-binding molecules.
  • Mammalian cells are, for example, Chinese hamster ovary (CHO) cells and CHO-S cells.
  • provided herein is a method of making a single variable domain, the method comprising: culturing a host cell comprising a nucleic acid encoding a single variable domain described herein, from the host cell or host cell culture medium The single variable domain was recovered in.
  • a method of making an antigen binding molecule the method comprising: culturing a host cell comprising a nucleic acid encoding an antigen binding molecule described herein, recovering the host cell or host cell culture medium Antigen binding molecules.
  • the nucleic acid encoding the single variable domain or antigen binding molecule is inserted into a vector for further cloning and/or expression in host cells.
  • the nucleic acid can be obtained by various methods well known in the art, such as gene splicing and chemical synthesis.
  • the present invention provides the use of single variable domains or antigen binding molecules.
  • the present invention provides methods of treating a subject with a BCMA-expressing tumor comprising administering to the subject a therapeutically effective amount of a single variable domain described herein, an antigen binding molecule described herein, or an antigen binding molecule described herein or herein. the described composition.
  • Subjects in need of treatment include those already suffering from the disease or condition, as well as subjects likely to suffer from the disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition.
  • the invention also provides use of a single variable domain described herein, an antigen binding molecule described herein, or a composition described herein in the manufacture of a medicament for the treatment of a subject having a BCMA-expressing tumor.
  • the present invention provides methods of inhibiting, reducing or blocking BCMA signaling in a cell comprising administering to the cell an effective amount of a single variable domain described herein, an antigen binding molecule described herein, or an antigen binding molecule described herein Compositions.
  • the present invention also provides the use of a single variable domain or antigen binding molecule in the manufacture of a medicament for inhibiting, reducing or blocking BCMA signaling in a cell.
  • the cells are tumor cells.
  • the present invention provides methods of killing BCMA-expressing tumor cells or inhibiting the growth of BCMA-expressing tumor cells, comprising binding the tumor cells to a single variable domain described herein, an antigen binding molecule described herein, or an antigen binding molecule described herein. contact with the composition.
  • the present invention also provides the use of the single variable domain, the antigen binding molecule or the composition described herein in preparing a medicament for killing BCMA-expressing tumor cells or inhibiting the growth of BCMA-expressing tumor cells.
  • the above-mentioned tumor may be a B-cell malignancy, specific examples such as lymphoma, myeloma, multiple myeloma or leukemia.
  • the present invention provides methods of treating a subject with an autoimmune disease comprising administering to the subject a therapeutically effective amount of a single variable domain described herein, an antigen binding molecule described herein, or an antigen binding molecule described herein or herein. the described composition.
  • the present invention also provides the use of a single variable domain described herein, an antigen binding molecule described herein, or a composition described herein in the manufacture of a medicament for the treatment of a subject with an autoimmune disease.
  • the autoimmune disease may be systemic lupus erythematosus.
  • methods of detecting or measuring BCMA in a sample comprising contacting the sample with a single variable domain or antigen binding molecule described herein and detecting or measuring the binding complex.
  • the recombinant human BCMA-Fc fusion protein (ACRO, product catalog No. BC7-H5254) was mixed and emulsified with complete Freund's adjuvant according to the volume ratio of 1:1, and the Bactrian camel was immunized by subcutaneous multi-point injection for the first time; Weekly, the recombinant human BCMA-Fc fusion protein and incomplete Freund's adjuvant were mixed and emulsified at a volume ratio of 1:1 for boosting immunization. Serum was taken after the 4th or 5th immunization to measure the titer of anti-human BCMA antibodies. After multiple rounds of immunization, the peripheral blood of Bactrian camel was collected, and peripheral blood mononuclear cells (PBMC) were isolated.
  • PBMC peripheral blood mononuclear cells
  • Total RNA was extracted from PBMC (from 1.1) using TRIzol TM reagent. The quality of the extracted total RNA was assessed by 1% agarose gel electrophoresis and quantified by measuring the absorbance at 260nm and 280nm , the ratio of OD260nm/OD280nm should be between 1.8-2.0.
  • RNA was reverse transcribed into cDNA using the cDNA synthesis kit PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, Catalog No. 6210A) according to the instructions.
  • the variable region sequence of camelid antibody was amplified by nested PCR method, and the specific method was as follows. Using cDNA as a template, primers Call001 (SEQ ID NO: 1) and Call002 (SEQ ID NO: 2) were used to carry out the first round of PCR amplification, and the short fragments of the amplified DNA products were recovered with a gel recovery kit (QIAGEN, product catalogue). No. 28706) purification.
  • V-Back (SEQ ID NO: 3): GATGTGCAGCTGCAGGAGTCTGGRGGAGG
  • V-Fwd (SEQ ID NO: 4): CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
  • the PCR reaction program of the first and second rounds was as follows: pre-denaturation at 94°C for 6 min, followed by denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 30 cycles, and finally extension at 72°C for 10 min.
  • VHH coding fragment amplified by nested PCR was digested with PstI/NotI endonuclease, and inserted into the phagemid vector pMECS (NTCC plasmid vector strain cell gene collection center, product catalog No.pMECS), and constructed into a recombinant vector, and electroporated into Escherichia coli TG1 (Lucigen, product catalog No. 60502-1).
  • pMECS NTCC plasmid vector strain cell gene collection center, product catalog No.pMECS
  • the rest of the bacterial solution was coated on a selective plate containing 100 ⁇ g/ml ampicillin, and the bacterial lawn of the colony was scraped from the plate, supplemented with glycerol, and frozen at -80 °C as the library stock.
  • the VHH library stock was amplified to logarithmic growth phase, M13KO7 helper phage (New England Biolabs, catalog No. N0315S) was added for library amplification, and the library was shaken at 28°C and 200 rpm overnight.
  • the single-domain antibody phage display library was panned by solid-phase panning.
  • Recombinant BCMA-Avitage TM (ACRO, product catalogue No. BCA-H82E4) was immobilized on a high-adsorption ELISA plate, and after blocking, the phage obtained in 1.4 was added to the well plate and incubated at 37°C for 1-2h.
  • Non-specifically bound phages were removed by washing with phosphate Tween buffered saline (PBST) 10 times. After washing with PBS, bound phages were eluted with trypsin enzyme and 4-(2-aminoethyl)benzenesulfonyl fluoride.
  • the positive clones that only bind to human BCMA-His and have a relatively high signal value were selected for conservation and sequencing.
  • the positive clones 1A1, 1A10, 1A11 and 1B10 were obtained by screening.
  • the VHH nucleotide sequence of 1A1 is SEQ ID NO:20, and the amino acid sequence is SEQ ID NO:19;
  • the VHH nucleotide sequence of 1A10 is SEQ ID NO:22, and the amino acid sequence is SEQ ID NO:22.
  • VHH nucleotide sequence of 1A11 is SEQ ID NO:24, and the amino acid sequence is SEQ ID NO:23
  • VHH nucleotide sequence of 1B10 is SEQ ID NO:26, and the amino acid sequence is SEQ ID NO:26 NO: 25.
  • VHH -Fc chimeric antibodies were constructed by linking the VHH sequences of the screened positive clones with the human Fc region. Specifically, the VHH sequence obtained by sequencing in 2.2 or the anti-human BCMA VHH control antibody (BM) sequence (the amino acid sequence is the same as SEQ ID NO: 125 in CN109153731A) was inserted into a human IgG1 constant region (the amino acid sequence is SEQ ID NO: 125) These VHH -Fc chimeric antibodies ( BM -Fc is the control).
  • BM anti-human BCMA VHH control antibody
  • VH H-Fc chimeric antibody to human BCMA protein was detected by indirect ELISA method against human BCMA-His protein (see 3.2 for the method), and surface plasmon resonance technology was used to detect Binding of antibodies to human TACI and BAFFR proteins (see 3.3 for methods). It was detected that 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc could specifically bind to BCMA-His protein, but not to human TACI and BAFFR proteins.
  • the full-length amino acid sequence of 1A1-Fc is such as SEQ ID NO: 27, and the nucleotide sequence is such as SEQ ID NO: 28; the full-length amino acid sequence of 1A10-Fc is such as SEQ ID NO: 29, and the nucleotide sequence is such as SEQ ID NO:30;
  • the full-length amino acid sequence of 1A11-Fc is as SEQ ID NO:31, and the nucleotide sequence is as SEQ ID NO:32;
  • the full-length amino acid sequence of 1B10-Fc is as SEQ ID NO:33, and the nucleotide sequence is as SEQ ID NO:33.
  • the sequence is as SEQ ID NO:34.
  • Affinity detection of anti-human BCMA VH H -Fc chimeric antibodies was performed using a Biomolecular Interaction Analysis System (GE, Biacore T200). Amino-conjugated Anti-hIgG(Fc) Antibody (GE, Catalog No. BR-1008-39) to a CM5 sensor chip using running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ⁇ 12H 2 O , 1.8 mM KH 2 PO 4 , 0.05% surfactant P-20 (w/v), pH 7.4) diluted anti-human BCMA VH H -Fc chimeric antibody to 1 ⁇ g/ml, 30 ⁇ l/min flow rate was captured through the experimental channel.
  • GE Biomolecular Interaction Analysis System
  • the software BiaControl Software 2.0 collects data signals in real time, the software BiaEvaluation Software 2.0 analyzes the data, uses the Langmuir 1:1 model to fit, calculates the association rate constant Ka (1/Ms), the dissociation rate constant Kd (1/s), and the equilibrium constant KD (M) value.
  • Ka (1/Ms the association rate constant
  • Kd the dissociation rate constant
  • M the equilibrium constant KD
  • AKD001A is a stable cell line with high expression of BCMA
  • U266 cells Basic Medicine Cell Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences, product catalog No.3111C0001CCC000684
  • PRMI8226 cells China Basic Medical Cell Center, Institute of Basic Medicine, Academy of Medical Sciences, Product Catalog No. 3111C0001CCC000083
  • HUVEC cells ScienCell Research Laboratories, Product Catalog No.
  • AKD001A 8000 are people who do not express BCMA Umbilical vein endothelial cell line. Incubate 2 ⁇ 10 5 target cells with anti-human BCMA VH H -Fc chimeric antibody at serial dilution (initial concentration 126nM, 5-fold serial dilution, 6 concentrations), incubate on ice for 1 hour, wash cells, add PE-labeled Anti-human IgG Fc antibody (Jackson Immuno Research, catalog No. 109-116-170) was incubated on ice for 0.5 hours, and cells were washed and detected by flow cytometry (Thermo Fisher Scientific Inc., Attune NXT).
  • 1A1-Fc, 1A10-Fc, 1A11-Fc and 1B10-Fc can effectively target and bind to CHO-hBCMA cells with high BCMA expression levels and U266 cells with intermediate BCMA expression levels , and the RPMI8226 cells with a low level of BCMA expression also had significant binding, and 1A10-Fc and 1A11-Fc had better binding to U266 cells than BM-Fc.
  • 1A10-Fc and 1A11-Fc did not bind to HUVEC cells that did not express BCMA, but 1A1-Fc and 1B10-Fc bound to HUVEC cells that did not express BCMA.
  • Cynomolgus monkey BCMA full-length (SEQ ID NO: 6) gene was synthesized in vitro and inserted into the pCDNA3.1 eukaryotic expression vector.
  • the 1A10-Fc, 1A11-Fc and BM-Fc antibodies were serially diluted (initial concentration 126nM, 5-fold serial dilution, 5 concentrations, the sample dilution was the negative control group), and incubated with 2 ⁇ 10 5 transiently transgenic crabs respectively.
  • Monkey BCMA and untransfected HEK293T cells incubated on ice for 1 hour, washed the cells, added PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, product catalog No. 109-116-170), incubated on ice for 0.5 hours, Cells were washed and detected using a flow cytometer (Thermo Fisher Scientific Inc., Attune NXT).
  • Figure 3A uses HEK293T cells transiently transfected with cynomolgus monkey BCMA (HEK293T-CynoBCMA)
  • Figure 3B uses HEK293T cells that were not transfected with cynomolgus monkey BCMA
  • Table 4 shows the binding of Fc chimeric antibodies to HEK293T cells transiently transfected with cynomolgus monkey BCMA EC50 value and maximum binding MFI value (Bmax). The results showed that 1A10-Fc and 1A11-Fc bound to cynomolgus monkey BCMA in cells, but BM-Fc antibody did not bind to cynomolgus monkey BCMA.
  • the anti-human BCMA VH H -Fc chimeric antibodies 1A10-Fc and 1A11-Fc of serial dilution were respectively mixed with 100ng/ml biotin-conjugated recombinant BCMA-His
  • the protein ACRO, catalogue No.BCA-H522y
  • ACRO catalogue No.APL-H5244
  • FIG. 5A the positions where the amino acid sequences of the extracellular domain of human BCMA differ from those of the extracellular domain of cynomolgus monkey BCMA are Gly6, Ala20, Ile22, Asn31, Val45 and Thr52.
  • Clones 1A10 and 1A11 cross-react with cynomolgus monkey BCMA protein, so it is speculated that the possible epitopes of these two clones are located at Gln7 ⁇ His19, and/or Pro23 ⁇ Ser30, and/or Asn31 ⁇ Ser44, and/or Thr46 ⁇ Gly51 .
  • Figure 5B shows the structure of the extracellular domain of human BCMA in the PDB database (PDB number: 2kn1).
  • the extracellular region of human BCMA contains three pairs of disulfide bonds, Cys8-Cys21, Cys24-Cys37 and Cys28-Cys41; human BCMA and APRIL are mainly bound to the ⁇ -hairpin structure (Bossen, C. et al. Semin. Immunol. 2006, 18(5): 263-275), it is speculated that the main binding sites of clones 1A10-Fc and 1A11-Fc are between Gln7 ⁇ His19, and/or Pro23 ⁇ Ser30 between, and/or between Asn31 ⁇ Ser44.
  • the human BCMA VH H -Fc chimeric antibody was diluted to 2 ⁇ g/ml, coated in a high-adsorption microtiter plate and blocked after washing; 20 ⁇ g/ml of the human BCMA VH H -Fc chimeric antibody was mixed with biotin
  • the conjugated BCMA-His protein (ACRO, product catalog No. BCA-H522y) was incubated at room temperature for 0.5 hours to obtain the antigen-antibody mixture.
  • the incubated antibody-antigen mixture or only biotin-conjugated BCMA- His protein (control group) was added to the well plate at 100 ⁇ l/well and incubated at 37°C for 1 hour; the unbound biotin-conjugated BCMA-His protein was washed away, and HRP-conjugated streptavidin (eBioscience, product catalog No. .18-4100-51), wash the plate 5 times for color development. Detect the light absorption signal value at 450nm wavelength and 650nm reference wavelength.
  • blocking rate (signal value of control group ⁇ signal value of sample group)/signal value of control group ⁇ 100%
  • the blocking rate of one antibody to the binding signal of another antibody to BCMA-Bio was calculated.
  • the results are shown in Table 6.
  • the diagonal position of " ⁇ " in the chessboard (marked in gray) is a positive control group with antibody self-competition, and the blocking rate is above 99%.
  • the blocking rate of 1A10-Fc to the binding of 1A11-Fc to human BCMA (32.6%) was less than 50%, and the blocking rate of 1A11-Fc to the binding of 1A10-Fc to human BCMA (21.6%) was also less than 50%, indicating that 1A10 -There is no obvious competitive relationship between Fc and 1A11-Fc, indicating that 1A10-Fc and 1A11-Fc can simultaneously bind to different epitopes of BCMA protein.
  • Epitope competition analysis was performed using the Biomolecular Interaction Analysis System (GE, Biacore T200). Amino-conjugated Anti-His Antibody (GE, Catalog No. 28995056) was applied to the CM5 sensor chip, and BCMA-His protein (ACRO, Catalog No. BCA-H522y) was diluted with running buffer to about 1 ⁇ g/ml, 30 ⁇ l/ The min flow rate was captured through the experimental channel, and the capture signal was controlled at 180RU-190RU by adjusting the binding time.
  • GE Biomolecular Interaction Analysis System
  • the signal value of 1A10-Fc antibody reaching saturation after binding to BCMA is 472.5RU; at this time, 1A11-Fc is injected again, and the saturation signal value is 579.0RU, which is the same as the saturation signal value of single injection of 1A11-Fc.
  • the signal value is 653.1RU equivalent.
  • the target cells were U266 cells.
  • incubate 2 ⁇ 10 5 target cells incubate on ice for 1 hour, wash the cells, add PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, product catalog No. 109-116-170), and incubate on ice for 0.5 hours , cells were washed and detected using a flow cytometer (Thermo Fisher Scientific Inc., Attune NXT).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供了一种结合BCMA的免疫球蛋白单可变结构域及抗原结合分子，具体地包括免疫球蛋白单可变结构域及抗原结合分子、编码它们的核酸、包含该核酸的载体、包含该载体的细胞、包含它们的药物组合物，还提供了其治疗用途。

Description

结合BCMA的单可变结构域及抗原结合分子 技术领域
本发明涉及抗原结合分子，尤其涉及结合BCMA的单可变结构域及抗原结合分子。
背景技术
B细胞成熟抗原(BCMA)，也称为肿瘤坏死因子受体超家族成员17(TNFRS17)，是一种在人体中由TNFRSF17基因编码的蛋白质。
BAFF、APRIL是BCMA的配体，其中BAFF(也称为BLyS、TALL-1、THANK、zTNF4、TNFSF20、D8Ertd387e)是BCMA的高亲和力配体，APRIL(增殖诱导配体，也称为TNFSF13、TALL-2、TRDL-1)是BCMA的低亲和力配体。此外，BAFF、APRIL还是肿瘤坏死因子受体(TNFR)超家族成员B细胞活化因子受体(B-cell activationfactor receptor；BAFF-R)、跨膜激活剂及钙调亲环素配体相互作用分子(transmembrane activator and calcium modulator and cyclophilin ligand interactor；TACI)的配体。BCMA与BAFF-R、TACI一起调控B细胞的增殖、存活、成熟与分化。
多发性骨髓瘤是一种恶性浆细胞病，在多发性骨髓瘤细胞中，BCMA的表达水平显著增加。BCMA可以是针对多发性骨髓瘤的免疫治疗剂的合适肿瘤抗原靶标。免疫治疗剂如与BCMA结合的抗体可阻断BCMA和其天然配体BAFF或/和APRIL之间的结合。作为潜在的治疗靶点，已有一些靶向BCMA的抗体被开发，但仍然是有限的，需要更多可用的选择。
骆驼科动物体内的IgG抗体，除传统包含轻链和重链的4链结构抗体IgG1外，还天然存在不含有轻链的仅重链抗体(HcAb)IgG2和IgG3。仅重链抗体中的单个可变区结构域(V HH或单可变结构域)具备特异性结合抗原的特点，且对抗原具有较高的亲和力，被称为单域抗体(sdAb)。基于其独特性，使用V HH结构域-单独或作为较大抗原结合分子的一部分，具有一些比常规scFv、抗体片段如Fab等更加显著的优势，例如仅需要单一结构域以高亲和力特异结合抗原、可容易地改造成多价及多特异性形式、V HH结构域高度可溶且无聚集趋势、分子小从而显示较高的组织渗透性、单域抗体不需要与轻链配对，在组成双特异性抗体或多特异性抗体时不存在轻重链错配问题，等等。
发明内容
本发明提供了结合BCMA的单可变结构域及抗原结合分子。本发明还提供了能够编码所提供的单可变结构域及抗原结合分子的相关核苷酸、载体、细胞、组合物、构建方法及用途。
在一方面，本发明提供了分离的单可变结构域，其结合BCMA，其中所述单可变结构域包含选自以下的CDR1、CDR2和CDR3：
(a)与选自SEQ ID NO:7、10、13和16的氨基酸序列具有至少70％的序列同一性的CDR1；
(b)与选自SEQ ID NO:8、11、14和17的氨基酸序列具有至少85％的序列同一性的CDR2；和
(c)与选自SEQ ID NO:9、12、15和18的氨基酸序列具有至少85％的序列同一性的CDR3。
在一些实施方案中，所述单可变结构域包含选自以下任一一组的CDR1、CDR2和CDR3：
(i)包含SEQ ID NO:7的氨基酸序列的CDR1；包含SEQ ID NO:8的氨基酸序列的CDR2；和包含SEQ ID NO:9的氨基酸序列的CDR3；
(ii)包含SEQ ID NO:10的氨基酸序列的CDR1；包含SEQ ID NO:11的氨基酸序列的CDR2；和包含SEQ ID NO:12的氨基酸序列的CDR3；
(iii)包含SEQ ID NO:13的氨基酸序列的CDR1；包含SEQ ID NO:14的氨基酸序列的CDR2；和包含SEQ ID NO:15的氨基酸序列的CDR3；或
(iv)包含SEQ ID NO:16的氨基酸序列的CDR1；包含SEQ ID NO:17的氨基酸序列的CDR2；和包含SEQ ID NO:18的氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO：19、21、23或25的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性的氨基酸序列。
在一方面，本发明提供了分离的单可变结构域，其中所述单可变结构域包含SEQ ID NO:19、21、23或25的氨基酸序列的CDR1、CDR2和CDR3。
在一方面，本发明提供了分离的单可变结构域，其中所述单可变结构域包含SEQ ID NO:19、21、23或25的氨基酸序列。
在一方面，本发明提供了分离的单可变结构域，其结合BCMA胞外区，结合位点处于所述BCMA胞外区的Gln7-His19、Pro23-Ser30以及Asn31-Ser44位置中的一个或多个氨基酸，BCMA胞外区的氨基酸序列如SEQ ID NO:35或SEQ ID NO:36所示。该处所述位置从人BCMA胞外区的第一个氨基酸(位置1)开始顺次编号。
在一方面，本发明提供了分离的单可变结构域，其中所述单可变结构域与本文任一实施方案中所述的单可变结构域结合相同表位。
在一方面，本发明提供了分离的单可变结构域，其中所述单可变结构域与本文任一实施方案中所述的单可变结构域竞争结合BCMA。
在一些实施方案中，本文上述单可变结构域是骆驼科动物的或人源化的。在一些实施方案中，本文上述单可变结构域是V HH，优选是骆驼科动物的或人源化的V HH。
在一方面，本发明提供了本文所述的单可变结构域的用途，包括用于构建抗原结合分子优选抗体、单特异性抗体、多特异性抗体或免疫偶联物。
在一方面，本发明提供了分离的抗原结合分子，其结合BCMA且包含至少一个本文所述的单可变结构域。
在一方面，本发明提供了组合物，其包含活性成分以及药学上可接受的载体，其中活性成分为本文所述的单可变结构域或根据本文所述的抗原结合分子。在一些实施方案中，所述抗原结合分子包含如SEQ ID NO：27、29、31或33所示氨基酸序列。
在一方面，本发明提供了分离的核酸，其编码本文所述的单可变结构域或本文所述的抗原结合分子。
在一方面，本发明提供了载体，其包含本文所述的分离的核酸。
在一方面，本发明提供了宿主细胞，其包含本文所述的载体。
在一方面，本发明提供了检测或测量样品中的BCMA的方法，其包括使所述样品与本文所述的单可变结构域或本文所述的抗原结合分子接触并且检测或测量结合复合物。
在一方面，本发明提供了制备本文所述的单可变结构域的方法，包括：培养所述的宿主细胞，分离所表达的所述单可变结构域，其中载体为表达载体，所述宿主细胞包含编码所述单可变结构域的核酸。
在一方面，本发明提供了制备所述的抗原结合分子的方法，包括：培养所述的宿主细胞，分离所表达的所述抗原结合分子，其中载体为表达载体，所述宿主细胞包含编码所述抗原结合分子的核酸。
在另一方面，本发明提供了治疗患有表达BCMA的肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的所述的单可变结构域、所述的抗原结合分子或所述的组合物。
在另一方面，本发明提供了抑制、减少或阻断细胞中的BCMA信号传导的方法，其包括向所述细胞施用有效量的所述的单可变结构域、所述的抗原结合分子或所述的组合物。
在另一方面，本发明提供了杀伤表达BCMA的肿瘤细胞或抑制表达BCMA的肿瘤细胞生长的方法，其包括使所述肿瘤细胞与所述的单可变结构域、所述的抗原结合分子或所述的组合物接触。
在另一方面，本发明提供了治疗患有自身免疫性疾病的受试者的方法，其包括向所述受试者施用治疗有效量的本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物。
附图说明
图1为ELISA检测抗人BCMA V HH-Fc嵌合抗体结合抗原的曲线；
图2A为流式细胞术检测抗人BCMA V HH-Fc嵌合抗体与CHO-hBCMA细胞的结合曲线；
图2B为流式细胞术检测抗人BCMA V HH-Fc嵌合抗体与U266细胞的结合曲线；
图2C为流式细胞术检测抗人BCMA V HH-Fc嵌合抗体与RPMI8226细胞的结合曲线；
图2D为流式细胞术检测抗人BCMA V HH-Fc嵌合抗体与HUVEC细胞的结合曲线；
图3A为流式细胞术检测1A10-Fc、1A11-Fc嵌合抗体与HEK293T-CynoBCMA的结合曲线；
图3B为流式细胞术检测1A10-Fc、1A11-Fc嵌合抗体与HEK293T的结合曲线；
图4显示了人BCMA V HH-Fc嵌合抗体与配体APRIL的竞争ELISA检测结果；
图5A为人、食蟹猴BCMA的胞外区氨基酸序列对比图；
图5B为人BCMA的胞外区晶体结构；
图5C为1A1、1A10、1A11和1B10的表位关系。
具体实施方式
术语
术语“抗原结合分子”在其最广泛的含义上指特异性结合抗原决定簇的分子。抗原结合分子的一些实例是抗体、融合蛋白、抗体偶联物。
术语“免疫球蛋白”指具有天然存在的抗体的结构的蛋白质。例如，人IgG类的免疫球蛋白是约150,000道尔顿的异四聚体糖蛋白，由二硫键连接的两条轻链和两条重链组成。从N端至C端，每条重链具有重链可变区(VH)，随后是铰链区(HR)和三个恒定结构域(CH1、CH2和CH3)，也称为重链恒定区。在IgE类免疫球蛋白的情况下，重链还具有CH4结构域。因此，免疫球蛋白重链是在N端至C端方向上由以下结构域组成的多肽：VH-CH1-HR-CH2-CH3-(CH4)。类似地，从N端至C端，每条轻链具有轻链可变区(VL)，随后是恒定轻链结构域，也称为轻链恒定区(CL)。因此，免疫球蛋白轻链是在N端至C端方向上由以下结构域组成的多肽：VL-CL。人的免疫球蛋白基本上由通过免疫球蛋白铰链区连接的两个Fab和Fc结构域组成。
术语“抗体”以最广泛的含义使用，涵盖包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体、三特异性抗体)以及抗体片段的多种抗体结构，只要它们显示希望的抗原结合活性。
术语“可变结构域”或“可变区”是指抗体的涉及该抗体与抗原结合的结构域。天然抗体的每个可变结构域基本上由四个“框架区”和三个互补决定区组成。四个“框架区”在本领域中和在下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”；所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
术语“单可变结构域”是指能够在不与其他可变结构域配对的情况下特异性结合抗原表位的可变结构域。单可变结构域的抗原结合位点通常由三个CDR(CDR1、CDR2和CDR3)形成，存在于单个结构域上。在一些情况下，单可变结构域可以是重链可变结构域(例如VH)或其合适的片段；只要它能够形成单个抗原结合单元(即基本上由单可变结构域组成的功能性抗原结合单元，这样单抗原结合结构域就不需要与另一个可变结构域相互作用即可形成功能性抗原结合单元)。单可变结构域的另一个实例为骆驼科的“VHH结构域”(或简称为“VHH”或“V HH”)。
“VHH结构域”，亦称为VHH、V HH、V HH结构域或单域抗体，最初被描述为“仅重链抗体”(即“缺乏轻链的抗体”)的结合抗原的可变结构域。使用术语“VHH结构域”以将这些可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”或“VH”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”或“VL”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域不同，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一结构域形成的小型稳定及高效的抗原识别单元。
“CDR”(互补决定区)，也称为“高变区(HVR)”通常是指在序列上高度可变和/或形成结构限定的环的抗体可变区的每个区域。天然四链抗体通常包含六个CDR，三个在重链可变区中(重链CDR1、重链CDR2和重链CDR3)，三个在轻链可变区中(轻链CDR1、轻链 CDR2和轻链CDR3)。仅重链抗体或单可变结构域通常具有三个CDR(CDR1(或HVR1)、CDR2(或HVR2)和CDR3(或HVR3))。CDR3显示出在三种CDR中最具多样性，并且据信在赋予抗体精细特异性方面发挥了独特的作用。
当前有许多方法来划分定义CDR。其中，Kabat定义基于序列可变性划分CDR，并且是最常用的(Elvin A.Kabat,et al，Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991))；而Chothia定义则基于结构环的位置(Cyrus Chothia,et al,Canonical Structures for the Hypervariable Regions of Immunoglobulins,J.Mol.Biol.196:901-917(1987))。AbM定义是Kabat定义和Chothia定义之间的折衷方案，并且被Oxford Molecular的AbM抗体建模软件使用。“接触(contact)”定义CDR的基础是对可用的复合物晶体结构的分析。这些CDR中的每个的残基记录于下表S1。
表S1 CDR划分
Figure PCTCN2021112758-appb-000001
在本文的某些特定语境下，“互补决定区”、“CDR”、或“HVR”用于指Kabat定义的CDR(Elvin A.Kabat,et al，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。基于抗体的可变区氨基酸序列，本领域技术人员可以常规地确定任意定义的CDR包含哪些氨基酸残基，对于其他任意定义(例如Chothia、AbM定义等)的CDR也将涵盖在本发明的范围内。
单可变结构域(诸如V HH)的氨基酸残基编号根据Kabat等人(Elvin A.Kabat,et al，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,Md.(1991))给出的VH通用编号系统进行，如在Riechmann等人的文章(L Riechmann,et al，Single domain antibodies:comparison of camel VH and camelised human VH domains,J.Immunol.Methods.(1999))中应用于骆驼科动物的V HH结构域。根据该编号，V HH的FR1包含位置1-30处的氨基酸残基，V HH的CDR1包含位置31-35处的氨基酸残基，V HH的FR2包含位置36-49处的氨基酸，V HH的CDR2包含位置50-65处的氨基酸残基，V HH的FR3包含位置66-94处的氨基酸残基，V HH的CDR3包含位置95-102处的氨基酸残基，V HH的FR4包含位置103-113处的氨基酸残基。在这方面，应该注意的是，正如本领域熟知的VH和V HH，每个CDR中氨基酸残基的总数是可以变化的，并且可能不对应于Kabat编号所指示的氨基酸残基总数(即，在实际序列中根据Kabat编号的一个或多个位置可以不被占用，或实际序列可能含有比Kabat编号允许的数量更多的氨基酸残基)。
“如Kabat中的可变结构域残基编号”或“如Kabat中的氨基酸位置编号”及其变化是指Kabat等人(Elvin A.Kabat,et al，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,Md.(1991))用于编码抗体的重链可变结构域或轻链可变结构域的编号系统。使用该编号系统，实际线性氨基酸序列可以含有更少或另外的氨基酸，这些氨基酸对应于可变结构域的FR或CDR的缩短或插入。例如，重链可变结构域可包括CDR2的氨基酸残基52之后的单个氨基酸插入物(根据Kabat的氨基酸残基52a)和重链FR氨基酸残基82之后的插入氨基酸残基(例如根据Kabat的氨基酸残基82a、82b和82c等)。对于给定抗体而言，可以通过抗体序列与“标准”Kabat编号的同源区序列比对来确定给定抗体的氨基酸残基的Kabat编号。
术语“框架区”或“FR”残基是除了本文定义的CDR残基之外的那些可变结构域的氨基酸残基。
术语“人共有框架区”或“受体人框架”是代表人免疫球蛋白VL或VH框架区序列的 选择中最常出现的氨基酸残基的框架。一般而言，人免疫球蛋白VL或VH序列的选择来自可变结构域序列的亚组。一般而言，序列的亚组是Kabat等人,Sequences of Proteins of ImmunologicalInterest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991)中的亚组。实例包括：对于VL，亚组可以是Kabat等人(出处同上)所述的亚组κI、κII、κIII或κIV。另外，对于VH，亚组可以是Kabat等人所述的亚组I、亚组II或亚组III。或者，人共有区框架可衍生自上文特定的残基，比如当通过将供体框架区序列与各种人框架区序列的集合进行比对根据人框架区残基与供体框架区的同源性来选择人框架区残基时“衍生自”人共有框架区的受体人框架可包含其相同的氨基酸序列，或它可含有预先存在的氨基酸序列变化。在一些实施方案中，预先存在的氨基酸变化的数目为10或更小、9或更小、8或更小、7或更小、6或更小、5或更小、4或更小、3或更小或2或更小。
术语“Fc结构域”或“Fc”用来定义免疫球蛋白重链的C端区域，其包含至少部分恒定区。该术语包括天然序列Fc和变体Fc。Fc的C末端赖氨酸(根据EU编号系统的Lys447)可存在或不存在。
术语“EC50”是指有效浓度，抗原结合分子的50％最大应答。术语“IC50”是指抑制浓度，抗原结合分子的50％最大应答。EC50和IC50两者均可以通过ELISA或FACS分析或本领域已知的任何其他方法进行测量。
术语“KD”在用于本文时指平衡解离常数，以摩尔浓度(M)表示。抗原结合分子的KD值可以使用本领域公知的方法测定。一种测定抗原结合分子KD的方法是使用表面等离振子共振(surface plasmon resonance)，如使用生物传感器系统，例如Biacore系统。
术语“治疗”是指为了以统计学显著的方式治疗、治愈、减轻、缓解、改变、补救、改善、改进或影响疾患(例如疾病)、疾患的症状，或预防或延缓症状、并发症、生化指标的发作，或以其他方式阻滞或抑制疾病、疾患或病症的进一步发展而使用措施。
术语“治疗有效量”是指向受试者提供治疗性和/或预防性益处所必需的抗原结合分子或组合物或其他施用物的量。
术语“受试者”包括任何人类或非人动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、锚、马、牛、鸡、两栖动物、爬行动物等。优选地，根据本发明的受试者是人。除非标明，术语“患者”或“受试者”可以互换使用。
术语“特异性的结合”或“特异性结合”意为结合对于抗原而言是选择性的并且可以与不需要或非特异性的相互作用区别开。抗原结合分子或单可变结构域与特定抗原决定簇结合的能力可通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其它技术测量，例如表面等离子体共振(SPR)技术(在Biacore仪器上分析)。
术语“分离的”是指已经从其天然环境中分离的目标化合物(例如，VHH、抗原结合分子、抗体或核酸)。
术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
术语序列“同一性(identity)”，也称一致性。两序列间的百分比同一性为序列共有的相同位置数的函数(即％同一性＝相同位置数/位置总数×100)，其中需考虑产生两序列的最优比对需要引入的缺口数和每个缺口的长度。如下述非限制性实施例所示，可以使用数学算法完成序列的比较和两序列间百分比同一性的测定。可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))测定两氨基酸序列间的百分比同一性，该算法已收入到ALIGN程序(版本2.0)中，其使用PAM120残基权重表，缺口长度罚分为12，缺口罚分为4。此外，可以使用Needleman和Wunsch的算法(J.Mol.Biol.484-453(1970))测定两氨基酸序列的百分比同一性，该算法已掺入到GCG软件包(可在www.gcg.com获得)中的GAP程序中， 其使用Blossum 62矩阵或PAM250矩阵，缺口权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，“约”可以表示多至5％(即±5％)的范围，例如在所给定的具体数值范围±2％范围内、±1％范围内或±0.5％范围内波动。此外，特别对于生物学系统或方法，该术语可以表示多至一个数量级或多至某值的5倍。当本申请或权利要求中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。
在本文中，除非另有说明，术语“包括”或其等同物(例如含有、包含等)为开放式术语，表示包括但不限于所指明的要素、步骤或者组成，而是对于未指明的要素、步骤或者组成是开放的。
在本文中，除非另有说明，否则单数术语涵盖复数的指代物，反之亦然。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本文中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。本发明的各个方面将在下述分部中进一步详细描述。
单可变结构域
本发明的一个方面提供结合至BCMA(诸如人BCMA)的经分离的单可变结构域。单可变结构域为靶向BCMA药物的开发或药物构建提供了更多可用的选择。所述单可变结构域具有诸多期望的治疗特性，例如对人BCMA具有良好的亲和力，能够阻断增殖诱导配体APRIL与BCMA的结合。特别的，在一些方案中，单可变结构域与人TACI和BAFFR蛋白不结合，表现了特异性；在一些实施方案中与猴BCMA有交叉反应，由于猴是药物毒理实验中的理想实验动物，与猴的交叉反应将利于药物毒理实验的开展。
在一些实施方案中，提供了包含SEQ ID NO:19所示单可变结构域的一个、两个或全部三个CDR的结合BCMA的单可变结构域。一个具体的实施方案，提供了包含SEQ ID NO:19所示单可变结构域的CDR1、CDR2和CDR3的结合BCMA的单可变结构域。在一些实施方案中，提供了包含SEQ ID NO:21所示单可变结构域的一个、两个或全部三个CDR的结合BCMA的单可变结构域。一个具体的实施方案，提供了包含SEQ ID NO:21所示单可变结构域的CDR1、CDR2和CDR3的结合BCMA的单可变结构域。在一些实施方案中，提供了包含SEQ ID NO:23所示单可变结构域的一个、两个或全部三个CDR的结合BCMA的单可变结构域。一个具体的实施方案，提供了包含SEQ ID NO:23所示单可变结构域的CDR1、CDR2和CDR3的结合BCMA的单可变结构域。在一些实施方案中，提供了包含SEQ ID NO:25所示单可变结构域的一个、两个或全部三个CDR的结合BCMA的单可变结构域。一个具体的实施方案，提供了包含SEQ ID NO:25所示单可变结构域的CDR1、CDR2和CDR3的结合BCMA的单可变结构域。在一些实施方案中，单可变结构域是骆驼科动物的。在一些实施方案中，单可变结构域是人源化的。在一些实施方案中，单可变结构域包含受体人框架。
在一些实施方案中，提供了包含选自以下的至少一个、至少两个或全部三个CDR的结合BCMA的单可变结构域：(a)包含选自SEQ ID NO:7、10、13和16的氨基酸序列的CDR1；(b)包含选自SEQ ID NO:8、11、14和17的氨基酸序列的CDR2；和(c)包含选自SEQ ID NO:9、12、15和18的氨基酸序列的CDR3。在一些实施方案中，单可变结构域是骆驼科动物的。在一些实施方案中，单可变结构域是人源化的。在一些实施方案中，单可变结构域包含受体人框架。
在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：(a)与选自SEQ ID NO:7、10、13和16的氨基酸序列具有 至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR1；(b)与选自SEQ ID NO:8、11、14和17的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR2；和(c)与选自SEQ ID NO:9、12、15和18的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR3。在一些具体的实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：(a)与SEQ ID NO:7的氨基酸序列具有至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR1；(b)与SEQ ID NO:8的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR2；和(c)与SEQ ID NO:9的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR3。在一些具体的实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：(a)与SEQ ID NO:10的氨基酸序列具有至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR1；(b)与SEQ ID NO:11的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR2；和(c)与SEQ ID NO:12的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR3。在一些具体的实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：(a)与SEQ ID NO:13的氨基酸序列具有至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR1；(b)与SEQ ID NO:14的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR2；和(c)与SEQ ID NO:15的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR3。在一些具体的实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：(a)与SEQ ID NO:16的氨基酸序列具有至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR1；(b)与SEQ ID NO:17的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约 99％或约100％中的任一者的序列同一性的CDR2；和(c)与SEQ ID NO:18的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的CDR3。在一些实施方案中，具有至少约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的同一性的CDR相对于参考序列含有取代(例如，保守取代)、插入或缺失，但包含该序列的单可变结构域保留了结合至BCMA的能力。在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的单可变结构域，所述CDR包括：(a)与选自SEQ ID NO:7、10、13和16的氨基酸序列相比，具有约1个或约2个中的任一者的氨基酸取代(例如，保守取代)、插入或缺失的CDR1；(b)与选自SEQ ID NO:8、11、14和17的氨基酸序列相比，具有约1个、约2个或约3个中的任一者的氨基酸取代(例如，保守取代)、插入或缺失的CDR2；和(c)与选自SEQ ID NO:9、12、15和18的氨基酸序列相比，具有约1个、约2个或约3个中的任一者的氨基酸取代(例如，保守取代)、插入或缺失的CDR3。在一些实施方案中，单可变结构域是亲和力成熟的。在一些实施方案中，单可变结构域是骆驼科动物的。在一些实施方案中，单可变结构域是人源化的。在一些实施方案中，单可变结构域包含受体人框架。
在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：包含SEQ ID NO:7的氨基酸序列的CDR1；包含SEQ ID NO:8的氨基酸序列的CDR2；和包含SEQ ID NO:9的氨基酸序列的CDR3。在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：包含SEQ ID NO:10的氨基酸序列的CDR1；包含SEQ ID NO:11的氨基酸序列的CDR2；和包含SEQ ID NO:12的氨基酸序列的CDR3。在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：包含SEQ ID NO:13的氨基酸序列的CDR1；包含SEQ ID NO:14的氨基酸序列的CDR2；和包含SEQ ID NO:15的氨基酸序列的CDR3。在一些实施方案中，提供了包含三种CDR(CDR1、CDR2和CDR3)的结合BCMA的单可变结构域，所述CDR包括：包含SEQ ID NO:16的氨基酸序列的CDR1；包含SEQ ID NO:17的氨基酸序列的CDR2；和包含SEQ ID NO:18的氨基酸序列的CDR3。在一些实施方案中，单可变结构域是骆驼科动物的。在一些实施方案中，单可变结构域是人源化的。在一些实施方案中，单可变结构域包含受体人框架。
在一些实施方案中，单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)包含与选自SEQ ID NO:19、21、23和25的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH。在一些具体的实施方案中，单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)包含与选自SEQ ID NO:19的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH。在一些具体的实施方案中，单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)包含与SEQ ID NO:21的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH。在一些具体的实施方案中，单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构 域)包含与SEQ ID NO:23的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH域。在一些具体的实施方案中，单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)包含与SEQ ID NO:25的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH。在一些更具体的实施方案中，单可变结构域包含与SEQ ID NO:19的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH，并且V HH包括：包含SEQ ID NO：7的氨基酸序列的CDR1，包含SEQ ID NO：8的氨基酸序列的CDR2，和包含SEQ ID NO：9的氨基酸序列的CDR3。在一些更具体的实施方案中，单可变结构域包含与SEQ ID NO:21的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH，并且V HH包括：包含SEQ ID NO：10的氨基酸序列的CDR1，包含SEQ ID NO：11的氨基酸序列的CDR2，和包含SEQ ID NO：12的氨基酸序列的CDR3。在一些更具体的实施方案中，单可变结构域包含与SEQ ID NO:23的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH，并且V HH包括：包含SEQ ID NO：13的氨基酸序列的CDR1，包含SEQ ID NO：14的氨基酸序列的CDR2，和包含SEQ ID NO：15的氨基酸序列的CDR3。在一些更具体的实施方案中，单可变结构域包含与SEQ ID NO:25的氨基酸序列具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或约100％中的任一者的序列同一性的V HH，并且V HH包括：包含SEQ ID NO：16的氨基酸序列的CDR1，包含SEQ ID NO：17的氨基酸序列的CDR2，和包含SEQ ID NO：18的氨基酸序列的CDR3。在一些实施方案中，具有至少约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％中的任一者的同一性的V HH序列相对于参考序列含有取代(例如，保守取代)、插入或缺失，但包含该序列的单可变结构域保留了结合至BCMA的能力。在一些实施方案中，在选自SEQ ID NO:19、21、23和25的氨基酸序列中总共1-18、1-16、1-14、1-12、1-10、1-9、1-8、1-7、1-6、1-5、1-4、1-3或1-2个氨基酸被取代、插入和/或缺失。在一些实施方案中，取代、插入或缺失发生在CDR之外的区域中(即，FR中)。在一些实施方案中，取代、插入或缺失发生在CDR区域，例如发生CDR1、CDR2、CDR3中的一个、两个或三个。在一些实施方案中，取代、插入或缺失发生在CDR区域和非CDR区域。任选地，单可变结构域包含选自SEQ ID NO:19、21、23和25的氨基酸序列，包括该序列的翻译后修饰。
在一些实施方案中，提供了包含SEQ ID NO:19、21、23或25的氨基酸序列的V HH的经分离的单可变结构域。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是V HH。基本V HH从N-末端至C-末端具有以下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中FR1至FR4分别指框架区1至4，其中CDR1至CDR3是指互补决定区1至3。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是人源化的V HH。源自非人的单可变结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始单可变结构域序列的氨基酸序列中的一个或多个氨基酸残基而经“人源化”。人源化可以期望的降低免疫原性。
在一些实施方案中，根据上述实施方案中的任一种的单可变结构域可单独地或组合地包括下述性质特征中的一种或几种：
(i)结合人BCMA；
(ii)结合猴BCMA；
(iii)阻断APRIL与BCMA的结合；或
(iv)与人TACI或/和BAFFR蛋白不结合。
在一些实施方案中，所述的单可变结构域结合人BCMA。在一些实施方案中，所述的单可变结构域结合人BCMA和猴BCMA如食蟹猴BCMA。
在一些实施方案中，所述的单可变结构域阻断APRIL与BCMA的结合。
在一些实施方案中，所述的单可变结构域与人TACI或/和BAFFR蛋白不结合，表现了良好的特异性。
在一些实施方案中，所述的单可变结构域组合上述性质特征(i)-(ii)。在一些实施方案中，所述的单可变结构域组合上述性质特征(i)和(iii)；在一些实施方案中，所述的单可变结构域组合上述性质特征(i)、(iii)和(iv)；在一些实施方案中，所述的单可变结构域组合上述性质特征(i)-(iv)。
在一些实施方案中，本发明提供了与本文所述的单可变结构域中的任一者结合相同表位的单可变结构域。在一些具体的实施方案中，提供与包含SEQ ID NO:19、21、23或25的氨基酸序列的单可变结构域结合相同的表位的单可变结构域。在一些实施方案中，所述结合相同表位的单可变结构域是骆驼科动物的、或人源化的。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选单可变结构域。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的单可变结构域。因此，在一些实施方案中，本发明提供了与本文所述的单可变结构域中的任一者竞争结合至BCMA的单可变结构域。在一些具体的实施方案中，提供与包含SEQ ID NO:19、21、23或25的氨基酸序列的单可变结构域竞争结合至BCMA的单可变结构域。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其他方法测量与BCMA的结合。在一些实施方案中，所述竞争结合至BCMA的单可变结构域是骆驼科动物的、或人源化的。
本发明提供了一些示例性的结合BCMA的单可变结构域。本文提供的示例性的单可变结构域的CDR(CDR1、CDR2和CDR3)的氨基酸序列提供于下表S2中。示例性的单可变结构域的全长氨基酸序列提供于下表S3中。
表S2：单可变结构域的CDR序列
Figure PCTCN2021112758-appb-000002
表S3：单可变结构域全长序列
Figure PCTCN2021112758-appb-000003
抗原结合分子
本发明的单可变结构域可以用于构建任何需要的抗原结合分子，从而赋予抗原结合分子靶向结合BCMA的特性或其他所述单可变结构域的特性。因此，本发明提供了包含至少一个(一个或多个)本发明单可变结构域的分离的抗原结合分子。
在一些实施方案中，所述抗原结合分子包含至少一个单可变结构域，所述单可变结构域包含选自以下任意一组的CDR1、CDR2和CDR3：
(i)SEQ ID NO：7所示的CDR1，SEQ ID NO：8所示的CDR2，和SEQ ID NO：9所示的CDR3；
(ii)SEQ ID NO：10所示的CDR1，SEQ ID NO：11所示的CDR2，和SEQ ID NO：12所示的CDR3；
(iii)SEQ ID NO：13所示的CDR1，SEQ ID NO：14所示的CDR2，和SEQ ID NO：15所示的CDR3；或
(iv)SEQ ID NO：16所示的CDR1，SEQ ID NO：17所示的CDR2，和SEQ ID NO：18所示的CDR3。
在一些实施方案中，所述单可变结构域包含SEQ ID NO：19、21、23或25所示的氨基酸序列。一些更具体的实施方案，所述单可变结构域的氨基酸序列如SEQ ID NO：19、21、23或25所示。
当抗原结合分子包含的单可变结构域是两个或两个以上时，可以选择相同或不同的单可变结构域。
所述的抗原结合分子包括抗体、单特异性抗体、多特异性抗体或免疫偶联物。在一个实施方案中，所述的抗原结合分子是抗体。一个具体的例子，抗体是单特异性抗体，另一个具体的例子，抗体是双特异性抗体。在一个实施方案中，所述抗体或免疫偶联物包含免疫球蛋白恒定区。一个更具体的实施方案，所述的抗体或免疫偶联物包含人免疫球蛋白Fc。优选的，所述Fc为人IgG1、IgG2、IgG3或IgG4的Fc。
在一些实施方案中，所述抗原结合分子是骆驼科动物的、嵌合的或人源化的。
在一些实施方案中，根据上述实施方案中的任一种的抗原结合分子可单独地或组合地包括下述性质特征中的一种或几种：
(i)结合人BCMA；
(ii)结合猴BCMA；
(iii)阻断APRIL与BCMA的结合；或
(iv)与人TACI或/和BAFFR蛋白不结合。
在一些实施方案中，所述的抗原结合分子结合人BCMA。在一些实施方案中，所述的抗原结合分子对人BCMA具有下述结合亲和力(KD)：在约1E-12M至约1E-08M、约1E-11M至约1E-08M、约8.12E-10M至约1.29E-10M、或约8.12E-10M至约3.13E-10M的范 围内。在一些实施方案中，所述抗原结合分子对人BCMA具有下述结合亲和力(KD)：约1E-08M或更小、约1E-09M或更小、约8.12E-10M或更小、约6.49E-10M或更小、约3.13E-10M或更小，或，约1.29E-10M或更小。在一些实施方案中，通过Biacore来测量本文提供的抗原结合分子的结合亲和力KD。
在一些实施方案中，所述的抗原结合分子结合人BCMA和猴BCMA。例如1A10单可变结构域、1A11单可变结构域、1A10-Fc、1A11-Fc不仅对人BCMA具有良好的亲和力，还与食蟹猴BCMA有结合。与猴BCMA的交叉反应将有利于药物毒理实验的开展与进行。
在一些实施方案中，所述的抗原结合分子阻断APRIL与BCMA的结合。
在一些实施方案中，所述的抗原结合分子与人TACI或/和BAFFR蛋白不结合，表现了良好的特异性。
在一些实施方案中，所述的抗原结合分子组合上述性质特征(i)-(ii)。在一些实施方案中，所述的抗原结合分子组合上述性质特征(i)和(iii)；在一些实施方案中，所述的抗原结合分子组合上述性质特征(i)、(iii)和(iv)；在一些实施方案中，所述的抗原结合分子组合上述性质特征(i)-(iv)。
本发明提供了示例性的抗原结合分子，如单特异性抗体(包括1A1-Fc、1A10-Fc、1A11-Fc和1B10-Fc抗体)，将单可变结构域与人IgG1的Fc融合，通过Fc形成同二聚体。示例性的单特异性抗体的氨基酸序列提供于下表S4中。
表S4：示例性抗原结合分子的全长序列
Figure PCTCN2021112758-appb-000004
Figure PCTCN2021112758-appb-000005
组合物
本发明提供了药物组合物，其包含活性成分以及药学上可接受的载体，其中活性成分为本文所述的单可变结构域或本文所述的抗原结合分子。在一些实施方案中，将1A1-Fc、1A10-Fc、1A11-Fc和1B10-Fc抗体的任意一个或几个，以及药学上可接受的载体制成药物组合物。药学上可接受的载体包括，例如，赋形剂、稀释剂、包封材料、填充剂、缓冲剂或其他试剂。
分离的核酸
本发明提供了分离的核酸，其编码本文所述的单可变结构域或本文所述的抗原结合分子。在一些实施方案中，所述核酸编码单可变结构域，如1A1-Fc、1A10-Fc、1A11-Fc或1B10-Fc的单可变结构域。在一些实施方案中，所述核酸编码抗原结合分子，如1A1-Fc、1A10-Fc、1A11-Fc或1B10-Fc。序列表中示例性的列举了一些单可变结构域、抗原结合分子的核酸序列。
载体
本发明提供了包含所述的分离的核酸的载体。在一些实施方案中，所述的载体为克隆载体；在另一些实施方案中，所述的载体为表达载体。所述表达载体可选的能够表达本文所述单可变结构域或抗原结合分子的任意表达载体，一个具体的例子，表达载体为pcDNA3.1。
宿主细胞
在一些实施方案中，本文提供包含本文所述载体的宿主细胞，宿主细胞为用于克隆或表达单可变结构域或抗原结合分子的适当宿主细胞。在一些实施方案中，宿主细胞为原核细胞。在另一些实施方案中，宿主细胞为真核细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳细胞或适用于制备抗原结合分子的其他细胞。哺乳细胞例如为中国仓鼠卵巢(CHO)细胞、CHO-S细胞。
制备单可变结构域或抗原结合分子的方法
在一些实施方案中，本文提供了制备单可变结构域的方法，所述方法包括：培养包含编码本文所述单可变结构域的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述单可变结构域。在一些实施方案中，本文提供了制备抗原结合分子的方法，所述方法包括：培养包含编码本文所述抗原结合分子的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述抗原结合分子。
为了产生所述的单可变结构域或抗原结合分子，将编码所述单可变结构域或抗原结合分子的核酸插入载体，用于在宿主细胞中进一步克隆或/和表达。所述核酸可以采用基因拼接、化学合成等多种本领域所熟知的方法获取。
用途
本发明提供了单可变结构域或抗原结合分子的用途。
本发明提供了治疗患有表达BCMA的肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物。需要治疗的受试者包括那些已经患有疾病或病状的受试者，以及可能患疾病或病状并且其目的是预防、延迟或减弱疾病或病状的受试者。本发明还提供了本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物在制备治疗患有表达BCMA的肿瘤的受试者的药物中的用途。
本发明提供了抑制、减少或阻断细胞中的BCMA信号传导的方法，其包括向所述细胞施用有效量的本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物。本发明还提供了单可变结构域或抗原结合分子在制备抑制、减少或阻断细胞中的BCMA信号传导的药物中的用途。一些实施方案中，细胞为肿瘤细胞。
本发明提供了杀伤表达BCMA的肿瘤细胞或抑制表达BCMA的肿瘤细胞生长的方法，其包括使所述肿瘤细胞与本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的 组合物接触。本发明还提供了所述的单可变结构域、抗原结合分子或本文所述的组合物在制备杀伤表达BCMA的肿瘤细胞或抑制表达BCMA的肿瘤细胞生长的药物中的用途。
上述肿瘤可以为B细胞恶性肿瘤，具体的实例例如淋巴瘤、骨髓瘤、多发性骨髓瘤或白血病。
本发明提供了治疗患有自身免疫性疾病的受试者的方法，其包括向所述受试者施用治疗有效量的本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物。本发明还提供了本文所述的单可变结构域、本文所述的抗原结合分子或本文所述的组合物在制备治疗患有自身免疫性疾病的受试者的药物中的用途。所述自身免疫性疾病可以为系统性红斑狼疮。
在一些实施方案中，提供了检测或测量样品中的BCMA的方法，其包括使所述样品与本文所述的单可变结构域或抗原结合分子接触并且检测或测量结合复合物。
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本发明的教义，本领域的普通技术人员将显而易见的是，可另外对本发明进行某些改变和修改而不背离所附权利要求的精神和范围。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。
具体实施方式
实施例1抗BCMA单域抗体噬菌体展示文库构建
1.1动物免疫
将重组人BCMA-Fc融合蛋白(ACRO，产品目录No.BC7-H5254)与完全弗氏佐剂按照体积比1:1混合乳化，对双峰驼进行首次皮下多点注射免疫；之后每间隔2周，将重组人BCMA-Fc融合蛋白与不完全弗氏佐剂按照体积比1:1混合乳化后进行加强免疫。在第4或第5次免疫之后取血清检测抗人BCMA抗体的滴度。在多轮免疫后取双峰驼外周血，分离外周血单个核细胞(PBMC)。
1.2 RNA提取
使用TRIzol TM试剂从PBMC(来自1.1)中提取总RNA。提取的总RNA用1％琼脂糖凝胶电泳来评估质量，并通过测量260nm和280nm下的吸光度来定量，OD 260nm/OD 280nm的比值应在1.8-2.0之间。
1.3 V HH扩增
用cDNA合成试剂盒PrimeScript TM II 1st Strand cDNA Synthesis Kit(TAKARA，产品目录No.6210A)按照说明，将总RNA逆转录成cDNA。使用巢式PCR法扩增骆驼抗体的可变区序列，具体方法如下。以cDNA为模板，使用引物Call001(SEQ ID NO:1)和Call002(SEQ ID NO:2)进行第一轮PCR扩增，扩增得到的DNA产物短片段用胶回收试剂盒(QIAGEN，产品目录No.28706)纯化。以第一轮PCR产物为模板，使用引物V-Back(SEQ ID NO:3)和V-Fwd(SEQ ID NO:4)进行第二轮PCR扩增，扩增得到的DNA产物即为V HH编码片段，用胶回收试剂盒(QIAGEN，产品目录No.28706)进行纯化。
Call001(SEQ ID NO:1)：GTCCTGGCTGCTCTTCTACAAGG
Call002(SEQ ID NO:2)：GGTACGTGCTGTTGAACTGTTCC
V-Back(SEQ ID NO:3)：GATGTGCAGCTGCAGGAGTCTGGRGGAGG
V-Fwd(SEQ ID NO:4)：CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
第一轮及第二轮的PCR反应程序为：94℃预变性6min，随后进行94℃变性30s、55℃退火30s、72℃延伸30s，共反应30个循环，最后进行72℃延伸10min。
1.4 V HH噬菌体文库构建
将巢式PCR扩增得到的V HH编码片段经PstI/NotI内切酶酶切后，插入噬菌粒载体pMECS(NTCC质粒载体菌种细胞基因保藏中心，产品目录No.pMECS)中，构建成重组载体，并电转入大肠杆菌TG1(Lucigen，产品目录No.60502-1)中。取一小部分转化后的菌液稀释后 涂布含有100μg/ml氨苄青霉素的选择性平板，通过菌落计数法来计算文库容量，并随机挑取100个克隆测序来评估文库质量。其余菌液涂布含有100μg/ml氨苄青霉素的选择性平板，从平板上刮下菌落的菌苔，补充甘油后冻存于-80℃作为文库原种。将V HH文库原种扩增到对数生长期，加入M13KO7辅助噬菌体(New England Biolabs，产品目录No.N0315S)进行文库扩增，28℃、200转/分钟振摇过夜。将菌液离心取上清，加入上清1/4体积的PEG6000/NaCl溶液(20％PEG6000(w/v)，2.5M NaCl)，冰上孵育1-2小时沉淀噬菌体，离心收取噬菌体沉淀，用PBS重悬后加20％甘油保存于-80℃作为单域抗体噬菌体展示文库。
实施例2抗人BCMA单域抗体筛选
2.1淘选
采用固相淘选的方式对单域抗体噬菌体展示文库进行淘选。将重组BCMA-Avitage TM(ACRO，产品目录No.BCA-H82E4)固定在高吸附酶标板上，封闭后将1.4中获得的噬菌体加入孔板中37℃孵育1-2h。采用磷酸盐吐温缓冲液(PBST)清洗10次以去除非特异性结合的噬菌体，PBS清洗后，用trypsin酶将结合的噬菌体洗脱下来，用4-(2-氨乙基)苯磺酰氟盐酸盐(AEBSF)将酶中和后，感染大肠杆菌TG1扩增后进行下一轮文库淘选。通过2-3轮淘选富集后，将收集的噬菌体感染对数生长期大肠杆菌TG1，涂布含20％(w/v)葡萄糖和100μg/ml氨苄青霉素的选择性平板。挑取单克隆进行培养，异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达制备上清。
2.2阳性克隆鉴定
通过针对人BCMA-His(ACRO，产品目录No.BCA-H522y)的间接ELISA方法对挑选的克隆进行阳性鉴定。将人BCMA-His或对照蛋白人Fc蛋白(ACRO，产品目录No.FCC-H5214)包被在高吸附酶标板上，然后用封闭液封闭，加2.1中制备的上清37℃孵育1小时，清洗后加HRP缀合的抗HA标签二抗(GenScript，产品目录No.A01296)，37℃孵育0.5小时，清洗5次，加显色底物进行显色，检测450nm波长及650nm参比波长处光吸收信号值。挑选仅结合人BCMA-His且信号值比较高的阳性克隆进行保种并测序。筛选获得阳性克隆1A1、1A10、1A11和1B10。经序列分析，1A1的V HH核苷酸序列为SEQ ID NO:20，氨基酸序列为SEQ ID NO:19；1A10的V HH核苷酸序列为SEQ ID NO:22，氨基酸序列为SEQ ID NO:21；1A11的V HH核苷酸序列为SEQ ID NO:24，氨基酸序列为SEQ ID NO:23；1B10的V HH核苷酸序列为SEQ ID NO:26，氨基酸序列为SEQ ID NO:25。
实施例3制备抗人BCMA V HH-Fc嵌合抗体
3.1 V HH-Fc嵌合抗体的制备
将筛选的阳性克隆的V HH序列与人Fc区的连接，构建V HH-Fc嵌合抗体。具体的，将2.2中测序获得的V HH序列或抗人BCMA V HH对照抗体(BM)序列(氨基酸序列与CN109153731A中SEQ ID NO:125相同)插入含有人IgG1恒定区(氨基酸序列为SEQ ID NO:5)的pCDNA3.1真核表达载体中，使用Expifectamine TM CHO Transfection Kit瞬转表达系统(Thermo Fisher Scientific Inc.，产品目录No.A29129)表达这些V HH-Fc嵌合抗体(BM-Fc为对照)。使用蛋白A亲和柱纯化后，采用针对人BCMA-His蛋白的间接ELISA法(方法参见3.2)检测V HH-Fc嵌合抗体与人BCMA蛋白的结合活性，并采用表面等离子技术共振技术检测抗体与人TACI、BAFFR蛋白的结合情况(方法参见3.3)。经检测1A1-Fc、1A10-Fc、1A11-Fc、和1B10-Fc均可特异性结合BCMA-His蛋白，而不结合人TACI、BAFFR蛋白。经序列分析，1A1-Fc的全长氨基酸序列如SEQ ID NO:27，核苷酸序列如SEQ ID NO:28；1A10-Fc的全长氨基酸序列如SEQ ID NO:29，核苷酸序列如SEQ ID NO:30；1A11-Fc的全长氨基酸序列如SEQ ID NO:31，核苷酸序列如SEQ ID NO:32；1B10-Fc的全长氨基酸序列如SEQ ID NO:33，核苷酸序列如SEQ  ID NO:34。
3.2间接ELISA法检测抗体与人BCMA-His蛋白的结合
将2μg/ml人BCMA-His蛋白(ACRO，产品目录No.BCA-H522y)包被在高吸附酶标板上，然后用3％(w/v)BSA封闭液封闭，加入100μl/孔梯度稀释(初始浓度126nM，5倍梯度稀释，7个浓度)的抗人BCMA V HH-Fc嵌合抗体，37℃孵育1小时，清洗后加HRP缀合的抗人IgG Fc标签二抗(PerkinElmer，产品目录No.NEF802001EA)，37℃孵育0.5小时，清洗5次，加100μl/孔TMB底物溶液(TIANGEN，产品目录No.PA107)进行显色，检测450nm波长及650nm参比波长处光吸收信号值，拟合曲线(图1)并计算EC50值(表1)。
表1 V HH-Fc嵌合抗体与人BCMA-His蛋白亲和力
Figure PCTCN2021112758-appb-000006
3.3表面等离子技术共振技术检测抗体与人TACI、BAFFR蛋白的结合
使用生物分子相互作用分析系统(GE，Biacore T200)检测抗人BCMA VHH-Fc嵌合抗体的特异性。氨基偶联Anti-hIgG(Fc)Antibody(GE，产品目录No.BR-1008-39)至CM5传感芯片，使用运行缓冲液(137mM Nacl，2.7mM KCl，10mM Na 2HPO 4·12H 2O，1.8mM KH 2PO 4，0.05％surfactant P-20(w/v)，pH 7.4)稀释抗人BCMA V HH-Fc嵌合抗体至2μg/ml，30μl/min流速通过实验通道进行捕获90s。使用运行缓冲液稀释人TACI或BAFFR蛋白至100nM，50μl/min流速结合，观察结合信号曲线。1A1-Fc、1A10-Fc、1A11-Fc和1B10-Fc均未观察到结合曲线，与TACI、BAFFR蛋白均不结合。
实施例4抗人BCMA V HH-Fc嵌合抗体与人、食蟹猴BCMA的亲和力
4.1表面等离子技术共振技术测定抗体与人、食蟹猴BCMA的亲和力
使用生物分子相互作用分析系统(GE，Biacore T200)进行抗人BCMA V HH-Fc嵌合抗体的亲和力检测。氨基偶联Anti-hIgG(Fc)Antibody(GE，产品目录No.BR-1008-39)至CM5传感芯片，使用运行缓冲液(137mM Nacl，2.7mM KCl，10mM Na 2HPO 4·12H 2O，1.8mM KH 2PO 4，0.05％surfactant P-20(w/v)，pH 7.4)稀释抗人BCMA V HH-Fc嵌合抗体至1μg/ml，30μl/min流速通过实验通道进行捕获。使用运行缓冲液稀释人BCMA-His(ACRO，产品目录No.BCA-H522y)或食蟹猴BCMA-His(ACRO，产品目录No.BCA-C52H7)至100nM、50nM、25nM、12.5nmol/L、6.25nM、3.125nM，50μl/min流速结合，结合时间设置为200s，之后停止进样进行解离，解离时间设置为800s。软件BiaControl Software 2.0实时采集数据信号，软件BiaEvaluation Software 2.0数据分析，用Langmuir 1：1模型拟合，计算结合速率常数Ka(1/Ms)、解离速率常数Kd(1/s)、平衡常数KD(M)值。检测结果如表2所示，1A1-Fc、1A10-Fc、1A11-Fc和1B10-Fc与人BCMA蛋白均有较高的亲和力，其中1A10-Fc和1A11-Fc与食蟹猴BCMA蛋白有交叉反应。
表2 V HH-Fc嵌合抗体与人、食蟹猴BCMA的亲和力
Figure PCTCN2021112758-appb-000007
Figure PCTCN2021112758-appb-000008
4.2流式细胞术测定抗体与细胞的结合
采用流式细胞术检测抗人BCMA V HH-Fc嵌合抗体与BCMA不同表达水平的靶细胞的结合，其中CHO-hBCMA细胞(爱康得生物医学技术(苏州)有限公司，产品目录No.AKD001A)为BCMA高表达的稳转细胞系，U266细胞(中国医学科学院基础医学研究所基础医学细胞中心，产品目录No.3111C0001CCC000684)为BCMA中等表达水平的天然人骨髓瘤细胞系，PRMI8226细胞(中国医学科学院基础医学研究所基础医学细胞中心，产品目录No.3111C0001CCC000083)为BCMA低表达水平的天然人骨髓瘤细胞系，HUVEC细胞(ScienCell Research Laboratories，产品目录No.AKD001A 8000)为不表达BCMA的人脐静脉内皮细胞系。用梯度稀释(初始浓度126nM，5倍梯度稀释，6个浓度)的抗人BCMA V HH-Fc嵌合抗体孵育2×10 5靶细胞，冰上孵育1小时后清洗细胞，加PE标记的抗人IgG Fc抗体(Jackson Immuno Research，产品目录No.109-116-170)，冰上孵育0.5小时，清洗细胞后使用流式细胞仪(Thermo Fisher Scientific Inc.，Attune NXT)检测。
如图2A～2C，表3所示，1A1-Fc、1A10-Fc、1A11-Fc和1B10-Fc均可有效靶向结合BCMA表达水平较高的CHO-hBCMA细胞以及BCMA中等表达水平的U266细胞，与BCMA表达水平偏低的RPMI8226细胞也有明显的结合，1A10-Fc和1A11-Fc对U266细胞的结合优于BM-Fc。如图2D所示，1A10-Fc和1A11-Fc与不表达BCMA的HUVEC细胞无结合，但1A1-Fc和1B10-Fc与不表达BCMA的HUVEC细胞有结合。
表3抗人BCMA V HH-Fc嵌合抗体与靶细胞的亲和力
Figure PCTCN2021112758-appb-000009
4.3流式细胞术测定抗体与瞬转食蟹猴BCMA的HEK293T细胞的结合
体外合成食蟹猴BCMA全长(SEQ ID NO:6)基因并插入到pCDNA3.1真核表达载体中。根据制造商方案，使用Lipofectamine TM 3000(Thermo Fisher Scientific Inc.，产品目录No.L3000015)将表达载体转染到HEK293T细胞中。简而言之，将HEK293T细胞按照5×10 5/孔铺入6孔板。约24小时后吸去含胎牛血清培养基并用PBS清洗细胞，之后按照每孔DNA：Lipo3000＝5μg:7.5μl的比例加入转染试剂。约6小时后更换为含10％胎牛血清(v/v)的培养基。转染约24小时后取细胞进行流式细胞术检测。
将1A10-Fc、1A11-Fc及BM-Fc抗体进行梯度稀释(初始浓度126nM，5倍梯度稀释，5个浓度，样品稀释液为阴性对照组)，分别孵育2×10 5个瞬转食蟹猴BCMA和未转染的HEK293T细胞，冰上孵育1小时后清洗细胞，加PE标记的抗人IgG Fc抗体(Jackson Immuno Research，产品目录No.109-116-170)，冰上孵育0.5小时，清洗细胞后使用流式细胞仪(Thermo Fisher Scientific Inc.，Attune NXT)检测。图3A采用瞬转食蟹猴BCMA的HEK293T细胞(HEK293T-CynoBCMA)，图3B采用未转染食蟹猴BCMA的HEK293T细胞，表4为Fc嵌合抗体与瞬转食蟹猴BCMA的HEK293T细胞结合的EC50值和最大结合MFI值(Bmax)。结果表明，1A10-Fc、 1A11-Fc结合细胞的食蟹猴BCMA，BM-Fc抗体不结合食蟹猴BCMA。
表4抗人BCMA V HH-Fc嵌合抗体与瞬转食蟹猴BCMA的HEK293T细胞的结合
Figure PCTCN2021112758-appb-000010
实施例5抗人BCMA V HH-Fc嵌合抗体阻断APRIL与BCMA的结合
将梯度稀释(初始浓度252nM，5倍梯度稀释，7个浓度)的抗人BCMA V HH-Fc嵌合抗体1A10-Fc、1A11-Fc分别与100ng/ml生物素缀合的重组BCMA-His蛋白(ACRO，产品目录No.BCA-H522y)按照1:1的体积比混合并室温孵育1小时后，加入到固定有重组APRIL蛋白(ACRO，产品目录No.APL-H5244)的酶标板中，同时设置对照组仅加入50ng/ml生物素缀合的重组BCMA-His蛋白；37℃孵育1小时，清洗掉未结合的生物素缀合的BCMA-His蛋白，加入HRP缀合的链霉亲和素(eBioscience，产品目录No.18-4100-51)，洗板5次后进行显色。检测450nm波长及650nm参比波长处光吸收信号值。按照公式结合量＝样品信号值/对照组信号值×100％，计算各抗体的各浓度样品对BCMA与APRIL结合量，并进行曲线拟合。结果如图4和表5所示，1A1-Fc、1B10-Fc、1A10-Fc和1A11-Fc均可有效阻断APRIL与BCMA的结合。
如图5A所示，人BCMA胞外区与食蟹猴BCMA胞外区氨基酸序列存在差异的位置为Gly6、Ala20、Ile22、Asn31、Val45和Thr52。克隆1A10和1A11与食蟹猴BCMA蛋白有交叉反应，据此推测这两个克隆可能的表位位于Gln7～His19，和/或Pro23～Ser30，和/或Asn31～Ser44，和/或Thr46～Gly51。图5B为PDB数据库中人BCMA的胞外区结构(PDB编号：2kn1)。如图5A、5B所示，人BCMA的胞外区含有3对二硫键，分别为Cys8-Cys21、Cys24-Cys37和Cys28-Cys41；人BCMA与APRIL主要结合在β-发卡结构上(Bossen,C.et al.Semin.Immunol.2006,18(5):263-275)，据此推测克隆1A10-Fc和1A11-Fc的主要结合位点在Gln7～His19之间，和/或Pro23～Ser30之间，和/或Asn31～Ser44之间。
表5抗人BCMA V HH-Fc阻断APRIL与BCMA的结合
Figure PCTCN2021112758-appb-000011
实施例6抗人BCMA V HH不同克隆间的表位差异分析
6.1 ELISA(棋盘法)检测抗人BCMA V HH不同克隆间的竞争关系
将人BCMA V HH-Fc嵌合抗体稀释至2μg/ml，包被在高吸附酶标板中并洗板后封闭；将20μg/ml的人BCMA V HH-Fc嵌合抗体与生物素缀合的BCMA-His蛋白(ACRO，产品目录No.BCA-H522y)室温孵育0.5小时得抗原抗体混合物，按照棋盘排布方法，按顺序将孵育的抗体抗原混合物或仅生物素缀合的BCMA-His蛋白(对照组)按100μl/孔加入孔板37℃孵育1小时；清洗掉未结合的生物素缀合的BCMA-His蛋白，加入HRP缀合的链霉亲和素(eBioscience，产品目录No.18-4100-51)，洗板5次后进行显色。检测450nm波长及650nm参比波长处光吸收信号值。按照公式阻断率＝(对照组信号值-样品组信号值)/对照组信号值×100％，计算一个抗体对另一抗体与BCMA-Bio结合信号的阻断率。结果如表6所示，棋盘中“\”对角线位置(灰 色标记)为抗体自我竞争的阳性对照组，阻断率均达到99％以上。1A10-Fc对1A11-Fc与人BCMA结合的阻断率(32.6％)小于50％，且1A11-Fc对1A10-Fc与人BCMA结合的阻断率(21.6％)亦小于50％，说明1A10-Fc与1A11-Fc之间不存在明显竞争关系，表明1A10-Fc与1A11-Fc可同时结合BCMA蛋白的不同表位。同理，1A10-Fc与1A1-Fc，1A10-Fc与1B10-Fc之间也均不存在明显竞争关系，表明1A10-Fc与1A1-Fc可同时结合BCMA蛋白的不同表位，1A10-Fc与1B10可同时结合BCMA蛋白的不同表位。1A1-Fc、1A11-Fc和1B10-Fc相互间的阻断率均超过99％。
表6 ELISA(棋盘法)表位竞争
棋盘法 1A1-Fc 1A10-Fc 1A11-Fc 1B10-Fc
1A1-Fc 100.1％ 7.6％ 99.4％ 101.2％
1A10-Fc 36.9％ 100.4％ 32.6％ 46.4％
1A11-Fc 100.6％ 21.6％ 99.7％ 99.4％
1B10-Fc 100.3％ 26.7％ 99.5％ 100.6％
6.2表面等离子技术共振技术表位差异分析
使用生物分子相互作用分析系统(GE，Biacore T200)进行表位竞争关系分析。氨基偶联Anti-His Antibody(GE，产品目录No.28995056)至CM5传感芯片，使用运行缓冲液稀释BCMA-His蛋白(ACRO，产品目录No.BCA-H522y)至1μg/ml左右，30μl/min流速通过实验通道进行捕获，通过调整结合时间将捕获信号控制在180RU-190RU。使用运行缓冲液稀释抗人BCMA V HH-Fc嵌合抗体V HH-Fc1至10μg/ml(饱和浓度，浓度增加后结合信号值不变)进样至信号到达平台，进样结束后立即进另一抗人BCMA V HH-Fc嵌合抗体V HH-Fc2。观察抗体结合曲线并分别记录两个抗体的结合信号值。信号值变化如表7所示，1A10-Fc抗体与BCMA结合达到饱和的信号值为472.5RU；此时再进样1A11-Fc，饱和信号值为579.0RU，与单独进样1A11-Fc的饱和信号值653.1RU相当。反之亦然，且正反两种进样顺序累积信号值相当，说明1A10-Fc和1A11-Fc可以同时结合BCMA蛋白上的不同表位。
表7 V HH-Fc抗体SPR信号值变化及分析
Figure PCTCN2021112758-appb-000012
6.3流式细胞术表位差异分析
由于细胞膜上表达的BCMA蛋白可能与游离蛋白在表位的有效暴露方面存在差异，因此采用流式细胞术进一步确认1A10、1A11两个克隆是否可以同时结合细胞膜上的BCMA蛋白。靶细胞为U266细胞。将20μg/ml或10μg/ml的1A10-Fc、1A11-Fc抗体单独孵育2×10 5靶细胞，或将20μg/ml的1A10-Fc抗体与20μg/ml的1A11-Fc抗体按照1:1体积比混合后孵育2×10 5靶细胞，冰上孵育1小时后清洗细胞，加PE标记的抗人IgG Fc抗体(Jackson Immuno Research，产品目录No.109-116-170)，冰上孵育0.5小时，清洗细胞后使用流式细胞仪(Thermo Fisher Scientific Inc.，Attune NXT)检测。结果如表8所示，当增加10μg/ml相同抗体，1A10-Fc样品的荧光均值增加百分比为902.0/823.5-1＝9.5％，1A11-Fc样品的荧光均值增加百分比为702.0/697.5-1＝0.6％，说明两个抗体10μg/ml均接近饱和浓度。在此基础上增加10μg/ml不同抗体，即10μg/ml的1A10-Fc增加10μg/ml的1A11-Fc，荧光均值增加百分比为1443.5/823.5-1＝75.3％；10μg/ml的1A11-Fc增加10μg/ml的1A10-Fc，荧光均值增加百分比为1443.5/697.5-1＝107.0％，说明两个抗体1A10-Fc、1A11-Fc可以同时结合细胞 膜上BCMA蛋白的不同表位。
推测1A1、1A10、1A11和1B10的表位关系如图5C所示：1A10结合人BCMA的表位与1A1、1A11、1B10的表位无重叠或有小部分重叠，而1A1、1A11、1B10结合人BCMA的表位有大部分重叠或完全重叠。
表8抗人BCMA VHH嵌合抗体与U266细胞的结合
Figure PCTCN2021112758-appb-000013

Claims (15)

  1. 一种分离的单可变结构域，其结合BCMA，其中所述单可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (a)与选自SEQ ID NO:10、13、7和16的氨基酸序列具有至少70％的序列同一性的CDR1；
    (b)与选自SEQ ID NO:11、14、8和17的氨基酸序列具有至少85％的序列同一性的CDR2；和
    (c)与选自SEQ ID NO:12、15、9和18的氨基酸序列具有至少85％的序列同一性的CDR3。
  2. 根据权利要求1所述的分离的单可变结构域，其中所述单可变结构域包含选自以下任一一组的CDR1、CDR2和CDR3：
    (ii)与SEQ ID NO:10的氨基酸序列具有至少70％的序列同一性的CDR1，与SEQ ID NO:11的氨基酸序列具有至少85％的序列同一性的CDR2，以及与SEQ ID NO:12的氨基酸序列具有至少85％的序列同一性的CDR3；
    (iii)与SEQ ID NO:13的氨基酸序列具有至少70％的序列同一性的CDR1，与SEQ ID NO:14的氨基酸序列具有至少85％的序列同一性的CDR2，以及与SEQ ID NO:15的氨基酸序列具有至少85％的序列同一性的CDR3；
    (i)与SEQ ID NO:7的氨基酸序列具有至少70％的序列同一性的CDR1，与SEQ ID NO:8的氨基酸序列具有至少85％的序列同一性的CDR2，以及与SEQ ID NO:9的氨基酸序列具有至少85％的序列同一性的CDR3；或，
    (iv)与SEQ ID NO:16的氨基酸序列具有至少70％的序列同一性的CDR1，与SEQ ID NO:17的氨基酸序列具有至少85％的序列同一性的CDR2，以及与SEQ ID NO:18的氨基酸序列具有至少85％的序列同一性的CDR3。
  3. 根据权利要求2所述的分离的单可变结构域，其中所述单可变结构域包含选自以下任一一组的CDR1、CDR2和CDR3：
    (ii)包含SEQ ID NO:10的氨基酸序列的CDR1；包含SEQ ID NO:11的氨基酸序列的CDR2；和包含SEQ ID NO:12的氨基酸序列的CDR3；
    (iii)包含SEQ ID NO:13的氨基酸序列的CDR1；包含SEQ ID NO:14的氨基酸序列的CDR2；和包含SEQ ID NO:15的氨基酸序列的CDR3；
    (i)包含SEQ ID NO:7的氨基酸序列的CDR1；包含SEQ ID NO:8的氨基酸序列的CDR2；和包含SEQ ID NO:9的氨基酸序列的CDR3；或
    (iv)包含SEQ ID NO:16的氨基酸序列的CDR1；包含SEQ ID NO:17的氨基酸序列的CDR2；和包含SEQ ID NO:18的氨基酸序列的CDR3。
  4. 一种分离的单可变结构域，其中所述单可变结构域包含SEQ ID NO：19、21、23或25的氨基酸序列的CDR1、CDR2和CDR3。
  5. 根据权利要求1-4中任一项所述的分离的单可变结构域，其中所述单可变结构域包含与SEQ ID NO：19、21、23或25的序列具有至少85％的序列同一性的氨基酸序列；或所述单可变结构域包含SEQ ID NO：19、21、23或25的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的单可变结构域，其中所述的单可变结构域是骆驼科动物的或人源化的；可选的，所述的单可变结构域是V HH。
  7. 根据权利要求1-6中任一项所述的分离的单可变结构域，其中所述单可变结构域展现出下述性质中的一种或几种的组合：
    (i)结合人BCMA；
    (ii)结合猴BCMA；
    (iii)阻断APRIL与BCMA的结合；或
    (iv)与人TACI或/和BAFFR蛋白不结合。
  8. 一种分离的抗原结合分子，其结合BCMA且包含至少一个权利要求1-7中任一项所述的单可变结构域。
  9. 根据权利要求8所述的分离的抗原结合分子，其中所述抗原结合分子是抗体、单特异性抗体、多特异性抗体或免疫偶联物，可选地，所述抗体或免疫偶联物包含免疫球蛋白恒定区；可选的，所述免疫球蛋白恒定区包含人免疫球蛋白Fc，优选为IgG1、IgG2、IgG3或IgG4的Fc；可选的，所述抗原结合分子包含如SEQ ID NO：27、29、31或33所示氨基酸序列。
  10. 根据权利要求8或9所述的分离的抗原结合分子，其展现出下述性质中的一种或几种的组合：
    (i)结合人BCMA；
    (ii)结合猴BCMA；
    (iii)阻断APRIL与BCMA的结合；和，
    (iv)与人TACI或/和BAFFR蛋白不结合。
  11. 一种组合物，其包含活性成分以及药学上可接受的载体，其中活性成分为根据权利要求1-7中任一项所述的单可变结构域或根据权利要求8-10中任一项所述的抗原结合分子。
  12. 一种分离的核酸，其编码根据权利要求1-7中任一项所述的单可变结构域或根据权利要求8-10中任一项所述的抗原结合分子。
  13. 一种治疗患有表达BCMA的肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的权利要求1-7中任一项所述的单可变结构域、权利要求8-10中任一项所述的抗原结合分子或权利要求11所述的组合物；可选的，所述的肿瘤为B细胞恶性肿瘤；可选的，所述B细胞恶性肿瘤为淋巴瘤、骨髓瘤、多发性骨髓瘤或白血病。
  14. 一种抑制、减少或阻断细胞中的BCMA信号传导的方法，其包括向所述细胞施用有效量的权利要求1-7中任一项所述的单可变结构域、根据权利要求8-10中任一项所述的抗原结合分子或权利要求11所述的组合物；可选的，细胞为B细胞恶性肿瘤细胞；可选的，所述B细胞恶性肿瘤为淋巴瘤、骨髓瘤、多发性骨髓瘤或白血病。
  15. 一种治疗患有自身免疫性疾病的受试者的方法，其包括向所述受试者施用治疗有效量的权利要求1-7中任一项所述的单可变结构域、权利要求8-10中任一项所述的抗原结合分子或权利要求11所述的组合物；可选的，所述自身免疫性疾病为系统性红斑狼疮。
PCT/CN2021/112758 2020-08-20 2021-08-16 结合bcma的单可变结构域及抗原结合分子 WO2022037528A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2023510435A JP2023539453A (ja) 2020-08-20 2021-08-16 Ｂｃｍａと結合する単一可変ドメイン及び抗原結合分子
US18/020,584 US20230265181A1 (en) 2020-08-20 2021-08-16 Single variable domain and antigen binding molecule binding bcma
CN202180041402.4A CN115698077A (zh) 2020-08-20 2021-08-16 结合bcma的单可变结构域及抗原结合分子
EP21857623.9A EP4201960A4 (en) 2020-08-20 2021-08-16 BCMA-BINDING SINGLE MOLECULE WITH VARIABLE DOMAIN AND ANTIGEN-BINDING MOLECULE
KR1020237008715A KR20230045095A (ko) 2020-08-20 2021-08-16 Bcma와 결합하는 단일 가변 도메인 및 항원 결합 분자

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010842501.X 2020-08-20
CN202010842501 2020-08-20

Publications (1)

Publication Number Publication Date
WO2022037528A1 true WO2022037528A1 (zh) 2022-02-24

Family

ID=80323414

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/112758 WO2022037528A1 (zh) 2020-08-20 2021-08-16 结合bcma的单可变结构域及抗原结合分子

Country Status (6)

Country Link
US (1) US20230265181A1 (zh)
EP (1) EP4201960A4 (zh)
JP (1) JP2023539453A (zh)
KR (1) KR20230045095A (zh)
CN (1) CN115698077A (zh)
WO (1) WO2022037528A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218380A1 (zh) * 2021-04-15 2022-10-20 正大天晴药业集团股份有限公司 靶向bcma的多特异性抗体

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104877026A (zh) * 2009-03-10 2015-09-02 比奥根Ma公司 抗-bcma抗体
CN109153731A (zh) 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
WO2019149269A1 (zh) * 2018-02-01 2019-08-08 信达生物制药(苏州)有限公司 全人源的抗b细胞成熟抗原(bcma)单链抗体及其应用
CN110891971A (zh) * 2017-06-20 2020-03-17 特尼奥生物股份有限公司 仅有重链的抗bcma抗体
US20200197529A1 (en) * 2011-05-27 2020-06-25 Glaxo Group Limited Antigen binding proteins

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112023020341A2 (pt) * 2021-04-15 2024-02-06 Chia Tai Tianqing Pharmaceutical Group Co Ltd Anticorpo multiespecífico, composição farmacêutica, método para tratar uma doença relacionada à expressão de bcma e anticorpo de ligação a bcma

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104877026A (zh) * 2009-03-10 2015-09-02 比奥根Ma公司 抗-bcma抗体
US20200197529A1 (en) * 2011-05-27 2020-06-25 Glaxo Group Limited Antigen binding proteins
CN109153731A (zh) 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
CN110891971A (zh) * 2017-06-20 2020-03-17 特尼奥生物股份有限公司 仅有重链的抗bcma抗体
WO2019149269A1 (zh) * 2018-02-01 2019-08-08 信达生物制药(苏州)有限公司 全人源的抗b细胞成熟抗原(bcma)单链抗体及其应用

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BOSSEN, C ET AL., SEMIN. IMMUNOL., vol. 18, no. 5, 2006, pages 263 - 275
CYRUS CHOTHIA ET AL.: "Canonical Structures for the Hypervariable Regions of Immunoglobulins", J. MOL. BIOL., vol. 196, 1987, pages 901 - 917, XP002619358, DOI: 10.1016/0022-2836(87)90412-8
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17
ELVIN A. KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
GAO, YOULIN ET AL.: "Correlation Between Serum BCMA and MIF Levels and Disease Activity of Systemic Lupus Erythematosus", JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 12, no. 1, 31 January 2013 (2013-01-31), pages 59 - 60, XP055902074 *
GAVRIATOPOULOU MARIA, NTANASIS-STATHOPOULOS IOANNIS, DIMOPOULOS MELETIOS ATHANASIOS, TERPOS EVANGELOS: "Anti-BCMA antibodies in the future management of multiple myeloma", EXPERT REVIEW OF ANTICANCER THERAPY, vol. 19, no. 4, 3 April 2019 (2019-04-03), GB , pages 319 - 326, XP055783034, ISSN: 1473-7140, DOI: 10.1080/14737140.2019.1586539 *
L RIECHMANN ET AL.: "Single domain antibodies: comparison of camel VH and camelised human VH domains", J. IMMUNOL. METHODS, 1999
LUAN, CHUNYAN ET AL.: "Advance of Research on the Immunotherapy Targeting B Cell Maration Antigen for Multiple Myeloma-Review", JOURNAL OF EXPERIMENTAL HEMATOLOGY, vol. 27, no. 5, 20 October 2019 (2019-10-20), pages 1701 - 1705, XP009528967 *
NEEDLEMANWUNSCH, J. MOL. BIOL., 1970, pages 484 - 453
See also references of EP4201960A4
SHIH-FENG CHO, KENNETH C. ANDERSON, YU-TZU TAI: "Targeting B Cell Maturation Antigen (BCMA) in Multiple Myeloma: Potential Uses of BCMA-Based Immunotherapy", FRONTIERS IN IMMUNOLOGY, vol. 9, XP055518135, DOI: 10.3389/fimmu.2018.01821 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218380A1 (zh) * 2021-04-15 2022-10-20 正大天晴药业集团股份有限公司 靶向bcma的多特异性抗体

Also Published As

Publication number Publication date
CN115698077A (zh) 2023-02-03
JP2023539453A (ja) 2023-09-14
EP4201960A4 (en) 2024-09-04
EP4201960A1 (en) 2023-06-28
KR20230045095A (ko) 2023-04-04
US20230265181A1 (en) 2023-08-24

Similar Documents

Publication Publication Date Title
TWI840399B (zh) 結合人il-4r的抗體、其抗原結合片段及其醫藥用途
WO2019062832A1 (zh) Tigit抗体、其抗原结合片段及医药用途
US20200369774A1 (en) Il-6r antibody and antigen binding fragment thereof and medical use
CN115109156A (zh) 一种靶向bcma的纳米抗体及其应用
WO2022127889A1 (zh) Her2抗体及其应用
WO2022127844A1 (zh) Cd5抗体及其应用
WO2022037528A1 (zh) 结合bcma的单可变结构域及抗原结合分子
WO2021197401A1 (zh) 结合cd47的抗原结合多肽及用途
CN115298216A (zh) 抗体或其抗原结合片段、其制备方法及医药用途
EP4428156A1 (en) Anti-bcma nanobody and use thereof
WO2022037527A1 (zh) 结合bcma的单可变结构域及抗原结合分子
KR20230035079A (ko) 응고인자 xi(fxi) 결합 단백질
TW202214703A (zh) 一種抗ＩｇＥ的工程化抗體及其應用
KR20220140787A (ko) 인간 cd47-표적화 단일-도메인 항체 및 그 사용
CN115521377A (zh) 人表皮生长因子受体结合分子及其应用
CN118240082A (zh) Pd-1结合分子及其应用
WO2024012434A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
WO2023078393A1 (zh) 抗c-Met抗体及其应用
CN110407942B (zh) 针对kn044的单域抗体
WO2024088386A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
WO2023078391A1 (zh) 结合c-Met的抗体及其应用
WO2024017326A1 (zh) 抗gprc5d纳米抗体及其应用
WO2024012513A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
JP2024541882A (ja) ｃ－Ｍｅｔに結合する抗体及びその使用
TW202434634A (zh) 抗IL-18Rβ抗體、其抗原結合片段及其醫藥應用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21857623

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023510435

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20237008715

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021857623

Country of ref document: EP

Effective date: 20230320