WO2025146128A1 - Anticorps anti-liv-1 et son utilisation - Google Patents

Anticorps anti-liv-1 et son utilisation Download PDF

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Publication number
WO2025146128A1
WO2025146128A1 PCT/CN2025/070392 CN2025070392W WO2025146128A1 WO 2025146128 A1 WO2025146128 A1 WO 2025146128A1 CN 2025070392 W CN2025070392 W CN 2025070392W WO 2025146128 A1 WO2025146128 A1 WO 2025146128A1
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antibody
antigen
seq
sequence
binding fragment
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Chinese (zh)
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陈常艳
付雅媛
唐任宏
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Hainan Simcere Zaiming Pharmaceutical Co Ltd
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Hainan Simcere Zaiming Pharmaceutical Co Ltd
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    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to the fields of bioengineering and biomedicine, and in particular, to an anti-human LVI-1 antibody or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their use in treating diseases, such as treating tumors.
  • Zinc transporter LIV-1 also known as SLC39A6 or ZIP6, has a gene encoding zinc transporter LIV-1 located on chromosome 18q12.2. It is a multi-transmembrane protein with zinc transporter and metalloproteinase activity. LIV-1 is a member of the SLC39A family (ZIP family). Its main physiological function is to promote the transport of zinc ions from outside the cell to the cytoplasm, so that other family members can jointly maintain the homeostasis of zinc ions in the cell.
  • LIV-1 has been shown to interact with transcription factors STAT3 and Snail, thereby downregulating the expression of E-cadherin to promote EMT (epithelial-mesenchymal transition), thereby playing a role in the transport and migration of tumor cells (Trends Endocrinol Metab 2004; 15: 461-3; Curr Opin Cell Biol 2005; 17: 548-58.).
  • LIV-1 is not expressed in most normal tissues, but is positively expressed only in the breast, testis, and prostate. Studies have shown that LIV-1 is significantly highly expressed in a variety of tumors, especially in breast cancer, with a positive expression rate of up to 93%. In addition to breast cancer, LIV-1 is also highly expressed in a variety of solid tumors, such as melanoma (82%), prostate cancer (72%), ovarian cancer (48%), uterine cancer, lung cancer, etc. (Molecular Cancer Therapeutics, 2014, 13(12).).
  • the ADC drug ladiratuzumab vdotin (SGN-LIV1A) targeting LIV-1 is in Phase 2 clinical trials and has been proven to bring clinical benefits to patients with breast cancer, especially triple-negative breast cancer, further verifying that LIV-1 is a potential target for advanced solid tumors.
  • the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to LIV-1, nucleic acids encoding these antibodies and antigen-binding fragments thereof, pharmaceutical compositions and kits comprising the antibodies and antigen-binding fragments thereof, and the preparation of drugs that can be used to treat tumors, etc.
  • the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to LIV-1, wherein the antibody or the antigen-binding fragment thereof comprises: (a) HCDR1, HCDR2 and HCDR3 of VH described in any one of SEQ ID NO: 11, 12, 13, 38, 45, 46, 47, 53, 54 or 55; and/or, (b) LCDR1, LCDR2 and LCDR3 of VL described in any one of SEQ ID NO: 14, 15, 16, 35, 36, 37, 44 or 52; preferably, the HCDR1-3 and/or the LCDR1-3 are encoded according to the common analysis method of Kabat or IMGT.
  • the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:
  • the LCDR1, LCDR2 and LCDR3 have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:
  • the substitution is a conservative amino acid substitution.
  • it comprises a combination of heavy chain CDRs and light chain CDRs selected from the following: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL2, VH5+VL2, VH6+VL4, VH7+VL4 or VH8+VL4, and a CDRs combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the heavy chain and light chain CDRs combination; preferably, the substitution is a conservative amino acid substitution.
  • the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are derived from a human germline heavy chain template and a human germline light chain template, wherein:
  • the framework region sequence is derived from the combined sequence of human germline heavy chains IGHV3-7*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV3-7*01 as shown in SEQ ID NO: 42 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 43;
  • the framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-3*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-3*01 as shown in SEQ ID NO: 57 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 43;
  • the framework region sequence is derived from the combined sequence of human germline light chain IGKV3-11*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV3-11*01 as shown in SEQ ID NO: 39 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 41;
  • the framework region sequence is derived from the combined sequence of human germline light chains IGKV4-1*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV4-1*01 shown in SEQ ID NO: 56 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 41.
  • framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof further comprise one or more mutations selected from the following group, according to the Kabat or IMGT numbering system:
  • the framework region of the heavy chain variable region includes: Q1E, V24T, M34I, W50R, N55Q, G56A, I70L, R72A, A76S, S77N, R98K, T98R or G101A; preferably includes R98K; or preferably includes T98R; or preferably includes V24T, G56A, I70L, T98R; or preferably includes V24T, G56A, I70L, T98R, G101A; or preferably includes Q1E, M34I, W50R, R72A; or preferably includes Q1E, M34I, W50R, G56A, R72A, S77N; or preferably includes Q1E, M34I, W50R, N55Q, R72A, A76S, S77N;
  • the framework region of the light chain variable region includes: A40T, A43S, L46P, L47I, L48V, Y49K, V58I or T85M; preferably includes L46P, Y49K; or preferably includes L46P, L47I, V58I; or preferably includes L46P, L47I, V58I, T85M; or preferably includes A43S, L48V; or preferably includes A40T.
  • the antibody or antigen-binding fragment comprises:
  • the heavy chain variable region has the sequence shown in SEQ ID NO: 11, 12, 13, 38, 45, 46, 47, 53, 54 or 55;
  • the light chain variable region has the sequence shown in SEQ ID NO: 14, 15, 16, 35, 36, 37, 44 or 52;
  • amino acid sequence that is at least 90% identical to any of the sequences described in (1) to (2) above, preferably an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of the sequences described in (1) to (2) above.
  • the heavy chain variable region and the light chain variable region are selected from the following groups:
  • VH and VL combination having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequence combinations of (1) to (12) above.
  • the antibody or antigen-binding fragment thereof binds to human LIV-1 with a dissociation constant (KD) of no greater than 2 ⁇ 10 -9 M.
  • the antibody or antigen-binding fragment thereof is:
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab') 2 , Fd, Fv, scFv, a diabody, or a single domain antibody.
  • the antibody comprises the sequence of any one of the constant regions of human or mouse antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises the sequence of the constant region of human or mouse antibodies IgG1, IgG2, IgG3 or IgG4.
  • the antigen-binding fragment is selected from one or more of F(ab) 2 , Fab', Fab, Fv, scFv, bispecific antibody, nanobody and the minimum recognition unit of an antibody.
  • the antibody or antigen-binding fragment thereof is further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.
  • the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator
  • the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.
  • the present disclosure provides a multispecific antigen-binding molecule, comprising a first antigen-binding module and a second antigen-binding module, wherein the first antigen-binding module comprises the antibody or antigen-binding fragment thereof described in the first aspect above, and the second antigen-binding module specifically binds to other antigens other than LIV-1 or binds to a LIV-1 antigen epitope different from the LIV-1 antigen epitope bound by the first antigen-binding module; preferably, the other antigens are selected from CD3, CD28, CD137, CD134, CD27, ICOS, CD16, CD56, CD335, CD336, CD337, NKG2A, NKG2D, KIR, DNAM-1 or CD161; preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.
  • the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof described in the first aspect above.
  • CAR chimeric antigen receptor
  • the present disclosure provides an immune effector cell, which comprises the chimeric antigen receptor described in the third aspect or comprises a nucleic acid fragment encoding the chimeric antigen receptor described in the third aspect; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells; preferably, the immune effector cells are allogeneic immune effector cells or autologous immune cells.
  • the present disclosure provides an isolated nucleic acid molecule encoding the antibody, antigen-binding fragment, or any combination thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, or the chimeric antigen receptor described in the third aspect.
  • the present disclosure provides an expression vector comprising the nucleic acid molecule described in the fifth aspect.
  • the present disclosure provides an isolated host cell of the nucleic acid molecule described in the fifth aspect above, or the expression vector described in the sixth aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and/or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.
  • the present disclosure provides a method for preparing the immune effector cell described in the fourth aspect, the method comprising introducing a nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cell, and optionally, the method also comprises starting the immune effector cell to express the CAR described in the third aspect.
  • the present disclosure provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect in the preparation of a medicament for preventing and/or treating a tumor disease; preferably, the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive breast ductal carcinoma, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.
  • the tumor disease is breast cancer, prostate cancer, cervical cancer, melanom
  • the present disclosure provides the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect for preventing and/or treating a tumor disease; preferably, the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometrial cancer, invasive breast ductal carcinoma, non-small cell lung cancer, pancreatic cancer, lung cancer, or squamous cell carcinoma.
  • the tumor disease is breast cancer, prostate cancer, cervical cancer, melanoma, ovarian cancer, endometri
  • the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, the chimeric antigen receptor described in the third aspect, the immune effector cell described in the fourth aspect, the isolated nucleic acid molecule described in the fifth aspect, the expression vector described in the sixth aspect, the host cell described in the seventh aspect, the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect; optionally, also comprising instructions for use.
  • the present disclosure aims to obtain antibody sequences targeting LIV-1 to produce more drug forms targeting LIV-1.
  • the LIV-1 antibody disclosed in the present disclosure is obtained by screening cells that highly express LIV-1 protein, has good endocytosis effect, PK, PD activity and anti-tumor effect, low toxicity, and can be used to prepare ADC molecules, and the prepared ADC molecules have better advantages in binding, endocytosis, plasma stability, anti-tumor and other aspects compared with positive references.
  • antibody refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterojunction antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies, antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab') 2 , Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab') 2 fragments which lack the Fc fragment of an intact antibody (cleared more quickly from animal circulation) and therefore lack Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein in their entirety).
  • antibodies herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).
  • LIV-1 herein is also referred to as Zip6 and refers to a zinc transporter belonging to a subfamily of ZIP zinc transporters known as LZT. In GeneCard, LIV-1 is also referred to as SLC39A6 (solute carrier protein family 39 (zinc transporter), member 6). Although for the purpose of brevity, the present disclosure is mainly exemplified by LIV-1, it should be understood that the definitions and embodiments involving LIV-1 can also be used for other zinc transporters disclosed herein.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • Fully length antibody and “intact antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody.
  • the term "antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.
  • An antibody fragment can be a Fab, F(ab') 2 , scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody.
  • binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) combinations of two or more isolated CDRs
  • the two domains of the Fv fragment, VL and VH are encoded by independent genes, the two domains can be joined by a linker using recombinant methods, which enables it to be made into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., Science 242: 423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art. (The above content is incorporated herein in its entirety).
  • single domain antibody (sdAb), "VHH", and “nanobody” have the same meaning and are used interchangeably, referring to cloning the variable region of the heavy chain of an antibody to construct a single domain antibody consisting of only one heavy chain variable region (VH), which is the smallest antigen-binding fragment with complete functions.
  • the heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, and the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • minimum recognition unit of an antibody refers to an antibody that contains only one complementary determining region (CDR) polypeptide, also known as a hypervariable region polypeptide.
  • CDR complementary determining region
  • the molecular mass of this unit is only about 1% of the complete antibody, but it can still bind to the corresponding antigen.
  • the term "bispecific antibody” refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificities for at least two different antigens.
  • one of the binding specificities can be detected for an antigenic epitope of LIV1, and the other can be detected for another antigenic epitope of LIV1 or any other antigen other than LIV1, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial-derived protein, or a bacterial surface protein, etc.
  • chimeric antibody refers to an antibody having variable sequences of an immunoglobulin derived from one source organism (e.g., rat or mouse) and constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.
  • CDR complementarity determining region
  • FR framework region
  • variable domains of native heavy and light chains each include four framework regions that mainly adopt a lamella configuration, which are connected by three CDRs (CDR1, CDR2 and CDR3), which form a loop connecting the lamella structure, and form a part of the lamella structure in some cases.
  • CDR1, CDR2 and CDR3 CDR1, CDR2 and CDR3
  • the CDR in each chain is closely held together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contributes to the formation of the antigen binding site of the antibody with the CDR from other antibody chains.
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.
  • VH refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).
  • heavy chain constant region herein refers to the carboxyl terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interactions with Fc receptors, which have a more conservative amino acid sequence relative to the variable domains of the antibody.
  • the "heavy chain constant region” comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • the “heavy chain constant region” includes a “full-length heavy chain constant region” and a “heavy chain constant region fragment”, the former having a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a portion of a full-length heavy chain constant region”.
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region fragment” can be selected from a CH1, Fc or CH3 domain.
  • Fc refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of antibodies.
  • the Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of immunoglobulin heavy chains can vary slightly, the Fc region of human IgG heavy chains is generally defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal.
  • the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be, for example, removed during the production or purification of antibodies, or by recombinant engineering of nucleic acids encoding antibody heavy chains, and therefore, the Fc region may include or may not include Lys447.
  • humanized antibody refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and/or constant region) comes from a human immunoglobulin (recipient antibody).
  • donor antibody non-human antibody
  • non-CDR region e.g., variable region FR and/or constant region
  • human immunoglobulin residual antibody
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies” herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.
  • another mammalian species e.g., mouse
  • naked antibody herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), a peptide or a polypeptide.
  • a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell.
  • the naked antibody when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylase), the naked antibody is not glycosylated.
  • the naked antibody is a complete antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of a complete antibody, such as a Fab antibody.
  • conjugated antibody refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • diabody herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.
  • percent (%) sequence identity refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored).
  • alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence.
  • the length of the candidate sequence compared for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence.
  • conservative amino acid generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).
  • amino acids within each of the following groups are conservative amino acid residues
  • the replacement of amino acid residues within the group is a replacement of conservative amino acids:
  • Acidic amino acids Asp (D) and Glu (E);
  • Aromatic amino acids Phe (F), Tyr (Y) and Trp (W).
  • Kabat numbering system in this article generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat.
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or its antigen-binding fragment.
  • An antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM).
  • An antibody or its antigen-binding fragment that does not show specific binding to a specific antigen or its epitope will show a KD of greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) to the specific antigen or its epitope.
  • a variety of immunoassays can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate.
  • solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • antibody conjugate refers to a coupling/conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), in which the drug molecule is the other molecule.
  • ADC antibody-drug conjugate
  • chimeric antigen receptor herein refers to a recombinant protein that comprises at least (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain.
  • the extracellular antigen binding domain of CAR comprises scFv.
  • ScFv can be derived from the variable heavy and light regions of a fusion antibody.
  • scFv can be derived from Fab (rather than an antibody, e.g., obtained from a Fab library).
  • scFv is fused to a transmembrane domain and then fused to an intracellular signaling domain.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules.
  • Nucleic acid molecules can be linear or cyclic.
  • nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms.
  • nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for direct expression of the antibodies disclosed herein in vitro and/or in vivo, for example in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • mice with high antibody titers in serum were selected for spleen cell fusion. Three days before spleen cell fusion, the immunization was boosted by subcutaneous, plantar and intraperitoneal injection of 50 ⁇ g/mouse antigen solution prepared with physiological saline.
  • mice After the mice were killed, spleen cells and lymphocytes were collected. After centrifugation at 1500 rpm, the supernatant was discarded, and ACK lysis solution (Gibco, A1049201) was added to the cells to lyse the red blood cells mixed in the cells to obtain a cell suspension.
  • ACK lysis solution Gibco, A1049201
  • the cells were washed 3 times with DMEM basal medium (Gibco, 10569044) at 1500 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC, Cat.CRL-1581) at a ratio of 2:1 in the number of live cells, and cell fusion was performed using BTX ECM2001+ high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY, VOL.220).
  • the fused cells were diluted into DMEM medium containing 20% (w/w) fetal bovine serum (ExCellBio, FND500), 1 ⁇ HAT (sigma, H0262-10VL), bovine insulin (Yeason, 40107ES25), and NEAA (Gibco, 11140050), and then added into a 96-well cell culture plate at 5 ⁇ 10 4 cells/200 ⁇ L per well, and cultured in a 5% (v/v) CO 2 , 37°C incubator.
  • DMEM medium 20% (w/w) fetal bovine serum (ExCellBio, FND500), 1 ⁇ HAT (sigma, H0262-10VL), bovine insulin (Yeason, 40107ES25), and NEAA (Gibco, 11140050)
  • the supernatant of the fusion plate was screened by ELISA to determine the binding activity to human LIV-1 protein; the supernatant of the positive clone with higher binding activity was selected, and the binding activity to monkey LIV-1 protein and cells overexpressing human LIV-1 was further determined by ELISA.
  • the positive clones that meet the conditions were selected and subcloned using semi-solid culture medium (purchased from stemcell, Cat.03810).
  • the grown clones were picked one by one into 96-well culture plates and expanded in DMEM culture medium containing 10% (w/w) fetal bovine serum and 1 ⁇ HT (Sigma, H0137-10VL).
  • ELISA was used for preliminary screening, and the monoclonal clones with positive binding activity to human LIV-1 protein were selected and expanded to 24-well plates for further culture. After 3 days, the culture supernatant was further tested to evaluate its binding activity to monkey LIV-1 protein and mouse LIV-1 protein.
  • the best clone was selected and expanded in DMEM medium containing 10% FBS at 37°C and 5% CO 2. The supernatant was collected after 7 days and purified with protein A to obtain purified hybridoma monoclonal antibodies.
  • DNA sequences encoding heavy and light chain immunoglobulins were obtained from the hybridoma mouse antibodies, and chimeric antibodies were constructed after sequencing.
  • the light chain variable region was cloned into the vector PTT5-huIgGLC (Kappa) containing the human light chain constant region (SEQ ID NO: 10) and regulatory elements to express the complete IgG light chain in mammalian cells. After correct sequencing, it was transfected into Expi-293 mammalian cells, and the protein was secreted into the culture medium after expression. The supernatant was collected and filtered for purification. Protein A chromatography was used to purify the protein. The culture supernatant was loaded onto a Protein A column of appropriate size and eluted with a high salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3 to 5 column volumes.
  • a high salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4
  • Enzyme-linked immunosorbent assay was used to detect the binding of chimeric antibodies to human/monkey LIV-1 protein.
  • Human LIV1-mFc produced in-house as in Section 1.1 of the Examples
  • monkey LIV1-mFc produced in-house as in Section 1.1 of the Examples
  • the chimeric antibody CHI-LIV1- 1, CHI-LIV1-2, CHI-LIV1-3 were diluted to 100nM, 10-fold gradient dilution was performed at 8 points, 50 ⁇ l/well was added to the blocked 96-well plate, incubated at room temperature for 1 hour, washed 5 times with PBST, HRP (horseradish peroxidase) labeled secondary antibody (purchased from Jackson, 109-035-088) was added, incubated at room temperature for 1 hour, washed 5 times with PBST, TMB substrate 50 ⁇ l/well was added, incubated at room temperature for 5-10 minutes, and stop solution (1.0N HCl) 50 ⁇ l/well was added.
  • HRP horseradish peroxidase labeled secondary antibody
  • the OD 450nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer).
  • the detection results of the chimeric antibody and protein are shown in Table 4, indicating that the chimeric antibody can bind to human LIV1 protein and monkey LIV1 protein.
  • the isotype control antibody is anti-FITC antibody (produced in-house), and the data in the table are OD 450nm values.
  • the reaction signal was detected in real time using a Biacore 8K instrument (GE) to obtain the binding and dissociation curves.
  • GE Biacore 8K instrument
  • the antigen human LIV-1 was diluted 1:1 with running buffer from the starting concentration (the actual concentration tested is shown in the detailed results), and a series of concentration gradients were set.
  • the binding process was detected by injecting different concentrations of antigen for 240s, with a flow rate of 30 ⁇ L/min and a dissociation time of 600s. After each cycle of dissociation was completed, the antigen-antibody complex was washed with pH 1.5 glycine-HCl regeneration solution (Cat.#BR-1003-54, Cytiva) at a flow rate of 30 ⁇ L/min for 30s to complete the regeneration of the chip surface.
  • the heavy chain and light chain variable region germline genes with high homology to the mouse antibody were selected as templates, and the CDRs of the mouse antibody were transplanted into the corresponding human templates to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the buried residues, the residues that directly interact with the CDR region, and the residues in the framework region that have an important influence on the conformation of VL and VH are back-mutated to obtain humanized monoclonal antibodies.
  • the specific sequence of the hLIV1-1 humanized antibody variable region is as follows:
  • amino acid sequence of hLIV1-1.VL1 is shown in SEQ ID NO: 35:
  • amino acid sequence of hLIV1-1.VL4 is shown in SEQ ID NO: 36:
  • amino acid sequence of the humanized light chain template IGKV3-11*01 is shown in SEQ ID NO: 39:
  • the specific sequence of the hLIV1-2 humanized antibody variable region is as follows:
  • amino acid sequence of hLIV1-2.VH1 is shown in SEQ ID NO: 45:
  • amino acid sequence of hLIV1-2.VH3a is shown in SEQ ID NO: 46:
  • amino acid sequence of hLIV1-2.VH3b is shown in SEQ ID NO: 47:
  • amino acid sequence of the humanized light chain template IGKV1-NL1*01 is shown in SEQ ID NO: 48:
  • amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO: 41:
  • amino acid sequence of the humanized heavy chain template IGHV1-69-2*01 is shown in SEQ ID NO: 49:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 43:
  • the present invention selects different light chain and heavy chain sequences from the above-mentioned hLIV1-2 humanized antibody light chain and heavy chain variable region back mutation designs for cross-combination, and finally obtains a variety of hLIV1-2 humanized antibodies.
  • the variable region amino acid sequences of each antibody are as follows:
  • the humanized light chain templates of the mouse antibody LIV1-3 are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01.
  • the CDRs of the mouse antibody LIV1-3 are transplanted into their human templates to obtain the corresponding humanized versions.
  • the key amino acids in the FR region sequence of the humanized antibody LIV1-3 are back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the CDR amino acid residues of the antibody are determined and annotated by the IMGT numbering system.
  • LIV1-3 has sites that are prone to chemical modification, and we perform point mutations on these sites to eliminate the risk of modification. See Table 13 for specific mutation designs.

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Abstract

L'invention concerne un anticorps se liant de manière spécifique à LIV-1 et son utilisation. L'invention concerne spécifiquement un anticorps murin et humanisé qui se lie à LIV-1, son procédé de préparation et son utilisation, l'anticorps ayant une bonne affinité pour la protéine LIV-1, et pouvant par conséquent être utilisé dans la préparation de médicaments pour le traitement de tumeurs et analogues.
PCT/CN2025/070392 2024-01-05 2025-01-03 Anticorps anti-liv-1 et son utilisation Pending WO2025146128A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130259860A1 (en) * 2010-12-06 2013-10-03 Seattle Genetics, Inc. Humanized Antibodies To LIV-1 And Use Of Same To Treat Cancer
WO2023125888A1 (fr) * 2021-12-31 2023-07-06 山东先声生物制药有限公司 Anticorps gprc5d et son utilisation
WO2023241621A1 (fr) * 2022-06-16 2023-12-21 山东博安生物技术股份有限公司 Anticorps anti-liv-1 et conjugué anticorps-médicament
WO2024109944A1 (fr) * 2022-11-25 2024-05-30 江苏恒瑞医药股份有限公司 Anticorps anti-liv-1, conjugué médicamenteux associé et utilisation médicale associée
WO2025021118A1 (fr) * 2023-07-25 2025-01-30 海南先声再明医药股份有限公司 Conjugué ligand-médicament et lieur pour conjugaison

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Publication number Priority date Publication date Assignee Title
US20130259860A1 (en) * 2010-12-06 2013-10-03 Seattle Genetics, Inc. Humanized Antibodies To LIV-1 And Use Of Same To Treat Cancer
WO2023125888A1 (fr) * 2021-12-31 2023-07-06 山东先声生物制药有限公司 Anticorps gprc5d et son utilisation
WO2023241621A1 (fr) * 2022-06-16 2023-12-21 山东博安生物技术股份有限公司 Anticorps anti-liv-1 et conjugué anticorps-médicament
WO2024109944A1 (fr) * 2022-11-25 2024-05-30 江苏恒瑞医药股份有限公司 Anticorps anti-liv-1, conjugué médicamenteux associé et utilisation médicale associée
WO2025021118A1 (fr) * 2023-07-25 2025-01-30 海南先声再明医药股份有限公司 Conjugué ligand-médicament et lieur pour conjugaison

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Title
LUE HUI-WEN, YANG XIAOJIAN, WANG RUOXIANG, QIAN WEIPING, XU ROY Z. H., LYLES ROBERT, OSUNKOYA ADEBOYE O., ZHOU BINHUA P., VESSELLA: "LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling", PLOS ONE, vol. 6, no. 11, 16 November 2011 (2011-11-16), US , pages 1 - 13, XP093334387, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0027720 *

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