WO2022095801A1 - Anticorps ciblant lir1 et son utilisation - Google Patents

Anticorps ciblant lir1 et son utilisation Download PDF

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WO2022095801A1
WO2022095801A1 PCT/CN2021/127564 CN2021127564W WO2022095801A1 WO 2022095801 A1 WO2022095801 A1 WO 2022095801A1 CN 2021127564 W CN2021127564 W CN 2021127564W WO 2022095801 A1 WO2022095801 A1 WO 2022095801A1
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antibody
seq
cells
lir1
cdr
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PCT/CN2021/127564
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Chinese (zh)
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周亚丽
陈功
姜小燕
任江涛
贺小宏
王延宾
韩露
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南京北恒生物科技有限公司
江苏浦珠生物医药科技有限公司
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Publication of WO2022095801A1 publication Critical patent/WO2022095801A1/fr

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Definitions

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to antibodies targeting LIR1 and their use in the prevention and/or treatment and/or diagnosis of diseases.
  • LIR Leukocyte Immunoglobulin-like Receptor
  • ITIMs Monocyte-macrophage inhibitory receptor
  • the present invention aims to provide an antibody targeting LIR1 and its use in disease prevention and/or treatment and/or diagnosis.
  • the present invention provides a LIR1 antibody, comprising: (1) CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L2 as shown in SEQ ID NO: 2 : CDR-L3 shown in 3, CDR-H1 shown in SEQ ID NO:4, CDR-H2 shown in SEQ ID NO:5, CDR-H3 shown in SEQ ID NO:6; or ( 2) CDR-L1 as shown in SEQ ID NO: 10, CDR-L2 as shown in SEQ ID NO: 11, CDR-L3 as shown in SEQ ID NO: 12, as shown in SEQ ID NO: 13 CDR-H1, CDR-H2 as shown in SEQ ID NO:14, CDR-H3 as shown in SEQ ID NO:15.
  • the LIR1 antibody is at least 90% identical to a light chain variable region selected from the group consisting of amino acid sequences set forth in SEQ ID NOs: 7 and 16, and with a variable region selected from the group consisting of SEQ ID NOs: 8 and 17
  • the amino acid sequences shown have heavy chain variable regions that are at least 90% identical.
  • the LIR1 antibody comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 7 and 16 and a heavy chain variable region selected from the group consisting of SEQ ID NOs: 8 and 17. In one embodiment, the LIR1 antibody comprises a light chain variable region as set forth in SEQ ID NO:7 and a heavy chain variable region as set forth in SEQ ID NO:8; or as set forth in SEQ ID NO:16 The light chain variable region and the heavy chain variable region shown in SEQ ID NO: 17.
  • amino acid sequence of the LIR1 antibody is selected from SEQ ID NOs: 9 and 18.
  • the present invention also provides nucleic acid molecules encoding the above-mentioned LIR1 antibodies.
  • the nucleic acid molecule encoding the LIR1 antibody has at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 19-20 , 96%, 97%, 98%, 99%, 100% sequence identity, and the LIR1 antibody encoded by it can specifically bind to the LIR1 antigen.
  • the nucleic acid molecule encoding the LIR1 antibody is selected from the group consisting of SEQ ID NOs: 19-20.
  • the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising the LIR1 antibody as described above and one or more second antibodies that specifically bind to other antigens An antibody or antigen-binding portion thereof.
  • a multispecific antibody preferably a bispecific or trispecific antibody
  • the second antibody or antigen-binding portion thereof may be in the form of any antibody or antibody fragment, such as a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, Diabodies or sdAbs.
  • the present invention also provides vectors comprising nucleic acid molecules encoding the above-mentioned LIR1 antibodies or multispecific antibodies, and host cells expressing the LIR1 antibodies or multispecific antibodies.
  • the present invention also provides a chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain and a signaling domain, wherein the NK inhibitory ligand comprises as described above
  • the chimeric receptor comprises two NK inhibitory ligands, wherein the first NK inhibitory ligand is a LIR1 antibody as described above and the second NK inhibitory ligand is selected from: (1) Antibodies or fragments thereof targeting the following NK inhibitory receptors: NKG2A, NKG2B, CD94, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1; or (2) HLA-E, HLA-F, HLA-G, cadherin, collagen, OCIL, sialic acid, PD-L1, PD-L2, CD155, CD112, CD113, Gal-9, FGL1, and the NK inhibitory receptor binding domains they contain.
  • the first NK inhibitory ligand is a LIR1 antibody as described above and the second NK inhibitory ligand is selected from: (1) Antibodies or fragments thereof targeting the following NK inhibitory receptors: NKG2A, NKG2B, CD94,
  • the signaling domain in a chimeric receptor of the invention consists of one or more costimulatory domains. That is, do not comprise primary signaling domains, eg, primary signaling domains from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the signaling domain of the chimeric receptors of the invention may further comprise a primary signaling domain, eg, the CD3 ⁇ intracellular region.
  • the present invention also provides nucleic acid molecules encoding a chimeric receptor targeting LIR1 as defined above, and vectors comprising said nucleic acid molecules.
  • the present invention also provides an engineered immune cell expressing the chimeric receptor of the present invention comprising an LIR1 antibody, and wherein the expression of at least one MHC-related gene is inhibited or silenced.
  • the MHC-related genes are selected from the group consisting of: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof are preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and combinations thereof.
  • the engineered immune cells expressing the chimeric receptor comprising the LIR1 antibody of the present invention further comprise that the expression of at least one TCR/CD3 gene is inhibited or silenced, examples of which include For example TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ .
  • the engineered immune cells provided by the present invention further express chimeric antigen receptors targeting tumor antigens.
  • the immune cells are selected from T cells, NK cells, NKT cells, macrophages, dendritic cells.
  • the present invention also provides an antibody conjugate comprising the LIR1 antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, half-life prolonging Structural moieties, detectable labels and drugs.
  • the half-life extending moiety is selected from: the half-life extending moiety is selected from the binding structure of albumin, the binding structure of transferrin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, Human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind human serum albumin.
  • the detectable label is selected from the group consisting of fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents.
  • the drug is selected from cytotoxins and immunomodulators.
  • the present invention also provides a detection kit comprising the antibody, multispecific antibody, antibody conjugate or chimeric receptor of the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody, chimeric receptor, multispecific antibody, engineered cell or antibody conjugate of the present invention, and one or more pharmaceutical agents acceptable excipients.
  • the present invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with LIR1 expression, comprising administering to a subject an antibody, chimeric receptor, multispecific antibody as described above , antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • antibody has the broadest meaning as understood by those skilled in the art, and includes monoclonal antibodies (including whole antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies) ), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting the desired biological activity.
  • the antibodies of the invention can be of any class (eg, IgG, IgE, IgM, IgD, IgA, etc.) or subclass (eg, IgGl, IgG2, IgG2a, IgG3, IgG4, IgAl, IgA2, etc.).
  • Antibodies of the present invention also include recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and antigen-binding portions thereof.
  • an intact antibody comprises two heavy chains and two light chains linked together by disulfide bonds, each light chain being disulfide bonded to its respective heavy chain in a "Y"-shaped structure.
  • Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain variable region comprises three complementarity determining regions (CDRs): CDR-H1, CDR-H2 and CDR-H3, the heavy chain constant
  • the region contains three constant domains: CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (VL) and a light chain constant region, wherein the light chain variable region includes three CDRs: CDR-L1, CDR-L2 and CDR-L3, and the light chain constant region includes a constant structure Domain CL.
  • the CDRs are separated by more conserved framework regions (FRs).
  • FRs conserved framework regions
  • the variable region of the heavy/light chain is responsible for antigen recognition and binding, while the constant region mediates the binding of antibodies to host tissues or factors, including various cells of the immune system (such as effector cells) and the first of the classical complement system. one component.
  • the boundaries of a given CDR or FR may vary depending on the protocol used for identification.
  • the Kabat scheme is based on structural alignment
  • the Chothia scheme is based on structural information.
  • Both Kabat and Chothia's scheme numbering is based on the most common antibody region sequence lengths, where insertions are provided by intervening letters (eg "30a") and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels”) in different positions, resulting in different numbers.
  • the Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM protocol is a compromise between the Kabat and Chothia definitions and is based on the protocol used by Oxford Molecular's AbM antibody modeling software.
  • CDRs of a given antibody or regions thereof (eg, variable regions thereof) encompass the CDRs defined by any of the above schemes or other known schemes.
  • CDR a particular CDR (eg, CDR3) is designated to contain a given amino acid sequence
  • FRs of a given antibody or region thereof encompass the FRs defined by any of the above schemes or other known schemes.
  • antibody fragment or "antigen-binding portion” encompasses only a portion of an intact antibody, typically comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen.
  • antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), heavy Chain variable region (VH) or light chain variable region (VL), linear antibodies, "diabodies” with two antigen binding sites, single domain antibodies, nanobodies, natural ligands for said antigen or functional fragments, etc. Accordingly, "antibodies” of the present invention encompass antibody fragments as defined above.
  • the LIR1 antibody of the invention is an scFv.
  • Single-chain antibody and “scFv” are used interchangeably herein and refer to an antibody comprising an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids).
  • linker size and composition see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A.
  • the scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.
  • the LIR1 antibody of the invention comprises (1) CDR-L1 as set forth in SEQ ID NO: 1, CDR-L2 as set forth in SEQ ID NO: 2, CDR-L2 as set forth in SEQ ID NO: 3 CDR-L3, CDR-H1 as set forth in SEQ ID NO:4, CDR-H2 as set forth in SEQ ID NO:5, CDR-H3 as set forth in SEQ ID NO:6; or (2) as SEQ ID NO:6 NO: CDR-L1 shown in SEQ ID NO: 10, CDR-L2 shown in SEQ ID NO: 11, CDR-L3 shown in SEQ ID NO: 12, CDR-H1 shown in SEQ ID NO: 13, CDR-H2 shown in SEQ ID NO: 14, CDR-H3 shown in SEQ ID NO: 15.
  • the LIR1 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A light chain variable region of 98%, 99%, 100% sequence identity, and having at least 90%, 91%, 92%, 93%, 94%, 90%, 91%, 92%, 93%, 94%, Heavy chain variable regions of 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the LIR1 antibody comprises a light chain variable region selected from the group consisting of SEQ ID NOs: 7 and 16 and a heavy chain variable region selected from the group consisting of SEQ ID NOs: 8 and 17.
  • the LIR1 antibody comprises a light chain variable region as shown in SEQ ID NO:7 and a heavy chain variable region as shown in SEQ ID NO:8; or a light chain as shown in SEQ ID NO:16 chain variable region and heavy chain variable region as set forth in SEQ ID NO:17.
  • amino acid sequence of the LIR1 antibody is set forth in SEQ ID NO: 9 or 18.
  • sequence identity refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical.
  • sequence identity can be determined using several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.
  • the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising the LIR1 antibody as described above, which further comprises one or more secondary antibodies that specifically bind to other antigens .
  • a multispecific antibody preferably a bispecific or trispecific antibody
  • the LIR1 antibody as described above, which further comprises one or more secondary antibodies that specifically bind to other antigens .
  • multispecific refers to an antigen binding protein that has polyepitopic specificity (ie, is capable of specifically binding to two, three or more different epitopes on a biomolecule or capable of specific binds epitopes on two, three or more different biomolecules).
  • bispecific means that an antigen binding protein has two different antigen binding specificities.
  • the secondary antibody may be in any antibody or antibody fragment format, eg, a full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, diabody, or sdAb.
  • the invention relates to nucleic acid molecules encoding the LIR1 antibodies or multispecific antibodies of the invention.
  • the nucleic acid of the present invention may be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acid molecule encoding the LIR1 antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 19-20 %, 97%, 98%, 99%, 100% sequence identity, and the LIR1 antibody encoded by it can specifically bind to LIR1 antigen.
  • the nucleic acid molecules encoding the LIR1 antibodies are shown in SEQ ID NOs: 19-20.
  • the nucleic acid of the present invention may also be in the form of, may be present in and/or be part of a vector, such as a plasmid, cosmid or YAC.
  • the vector may in particular be an expression vector, ie a vector that provides for expression of the LIR1 antibody in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). Selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the LIR1 antibodies of the invention include, but are not limited to, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.
  • the present invention also provides host cells expressing the LIR1 antibodies, multispecific antibodies of the present invention and/or containing the nucleic acids or vectors of the present invention.
  • Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains).
  • gram-negative bacterial strains eg, Escherichia coli, Proteus, and Pseudomonas strains
  • gram-positive bacterial strains eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains.
  • Suitable fungal cells include cells of species of Trichoderma, Neurospora and Aspergillus; or Saccharomyces (eg Saccharomyces cerevisiae), Saccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of the genus Hansenula.
  • Saccharomyces eg Saccharomyces cerevisiae
  • Saccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing heterologous proteins may also be used in the present invention.
  • the present invention also provides a chimeric receptor comprising the LIR1 antibody as described above. Since LIR1 is an NK inhibitory receptor, chimeric receptors comprising LIR1 antibodies can be used to inhibit the killing of NK cells.
  • the present invention provides a chimeric receptor comprising one or more NK inhibitory ligands, a transmembrane domain, and a signaling domain, wherein the NK inhibitory ligand comprises as described above
  • the chimeric receptor comprises multiple NK inhibitory ligands, eg, two NK inhibitory ligands, wherein the first NK inhibitory ligand is a LIR1 antibody as described above and the second NK inhibitory ligand Sexual ligands are antibodies or fragments thereof that target other NK inhibitory receptors, and/or natural ligands for other NK inhibitory receptors or NK inhibitory receptor binding regions they comprise.
  • the second NK inhibitory ligand is an antibody or fragment thereof that targets the following NK inhibitory receptors: NKG2/CD94 components (eg, NKG2A, NKG2B, CD94); Killer cell Ig-like Receptor (KIR) family members (eg, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); leukocyte Ig-like receptor (LIR) family members (eg, LIR2, LIR3, LIR5, and LIR8); NK cells are affected by Body protein 1 (NKR-P1) family members (eg, NKR-P1B and NKR-P1D); immune checkpoint receptors (eg, PD-1, TIGIT and CD96, TIM3, LAG3); carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1); sialic acid-binding immunoglobulin-like lectin (SIGLEC)
  • the second NK inhibitory ligand is selected from antibodies or fragments thereof targeting the following NK inhibitory receptors: NKG2A, NKG2B, CD94, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1. Still more preferably, the second NK inhibitory ligand is selected from the group consisting of antibodies or fragments thereof targeting the following NK inhibitory receptors: NKG2A, CD94, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1.
  • the second NK inhibitory ligand is a natural ligand of another NK inhibitory receptor or the NK inhibitory receptor binding region it comprises, eg, HLA-E, HLA-F, HLA-G , cadherin, collagen, OCIL, sialic acid, immune checkpoint ligands (eg PD-L1/PD-L2, CD155, CD112, CD113, Gal-9, FGL1, etc.), and the NK inhibitory receptors they contain body binding region.
  • the second NK inhibitory ligand is selected from HLA-E, HLA-F, HLA-G, cadherin, PD-L1, PD-L2, or the NK inhibitory receptor binding regions they comprise.
  • the second NK inhibitory ligand is selected from HLA-E extracellular domain, HLA-G extracellular domain, E-cadherin extracellular domain, PD-L1 extracellular domain and PD-L2 extracellular domain Area. More preferably, the second NK inhibitory ligand is the E-cadherin extracellular domain comprising EC1 and EC2, more preferably EC1, EC2, EC3, EC4 and EC5.
  • transmembrane domain refers to a polypeptide structure that enables expression of a chimeric receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells .
  • the transmembrane domain can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling when the chimeric receptor binds to the target antigen.
  • Transmembrane domains particularly useful in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain or CD28, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% of the amino acid sequence shown in SEQ ID NO: 24 or 26 % or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% with the nucleic acid molecule shown in SEQ ID NO: 25 or 27 or 99% or 100% sequence identity.
  • costimulatory domain refers to an intracellular functional signaling domain from a costimulatory molecule that comprises the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • costimulatory molecule refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell.
  • Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the invention include, but are not limited to, intracellular regions derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 ( B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28 or 4-1BB+CD28.
  • the 4-1BB costimulatory domain is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:27 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleic acid molecule shown in SEQ ID NO: 28 identity.
  • the CD28 costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:25 Sequence identity, or its coding sequence, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleic acid molecule set forth in SEQ ID NO:26 .
  • the signaling domain consists of one or more costimulatory domains (ie, does not comprise a primary signaling domain, eg, from FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a , the primary signaling domains of CD79b and CD66d).
  • the signaling domain of a chimeric receptor of the invention may also comprise a primary signaling domain, such as the CD3 ⁇ intracellular region.
  • the CD3 ⁇ intracellular region is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence shown in SEQ ID NO: 29 or 31 or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the nucleic acid molecule shown in SEQ ID NO: 30 or 32 or 100% sequence identity.
  • the chimeric receptors of the invention may further comprise a hinge region between the antibody and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain and an antibody. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain.
  • the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region comprises a CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1 hinge region portion, more preferably a CD8 ⁇ , CD28 or IgG4 hinge, which is set forth in SEQ ID NO: 37, 39 or 41
  • the amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence is identical to that of SEQ ID NO: 38, 40 or 42
  • the nucleotide sequences shown have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the CR of the invention may also comprise a signal peptide such that when expressed in a cell, eg, a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain a long segment of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptidases At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases.
  • the signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, for example signal peptides derived from B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , and the like.
  • the signal peptide useful in the present invention is from B2M or CD8 ⁇ , which is at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO: 33 or 35 , 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the CR comprises a LIR1 antibody as provided herein or a multispecific antibody comprising the LIR1 antibody, a CD8 ⁇ transmembrane region, and a signaling domain comprising the group consisting of CD28 and The costimulatory domain of 4-1BB.
  • the signaling domain consists of a costimulatory domain selected from CD28 and 4-1BB.
  • the signaling domain further comprises the CD3 ⁇ intracellular domain.
  • the CAR may further comprise a signal peptide from B2M, CD8 ⁇ , IgG1 or GM-CSFR ⁇ .
  • the present invention also provides nucleic acid molecules encoding a chimeric receptor targeting LIR1 as defined above, and vectors comprising said nucleic acid molecules.
  • vector is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell, in which the nucleic acid molecule can eg be replicated and/or expressed.
  • Vectors generally include targeting vectors and expression vectors.
  • a "targeting vector” is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a specific targeting sequence at the site.
  • An “expression vector” is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric receptor polypeptides of the invention, in suitable host cells, and the translation of their mRNAs.
  • the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs).
  • the vector itself is usually a nucleic acid molecule, usually a DNA sequence containing an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • the VL vector typically also contains an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS).
  • the vector may additionally contain a promoter, multiple elements such as polyadenylated tails (polyA), 3'UTRs, enhancers, terminators, insulators, operons, selectable markers, reporter genes, targeting sequences and/or protein purification tags.
  • polyA polyadenylated tails
  • the vector is an in vitro transcribed vector.
  • LIR1 can bind to HLA-I molecules, it inhibits the activation of immune cells such as NK cells. Therefore, the introduction of exogenous LIR1 antibodies can inhibit the killing effect of NK cells by binding to LIR1, which is especially useful in certain situations (such as the deletion of HLA-I molecules or the preparation of universal CAR-T cells). .
  • the present invention also provides an engineered immune cell expressing a chimeric receptor of the present invention comprising a LIR1 antibody, and wherein the expression of at least one MHC-related gene is inhibited or silenced.
  • MHC-related genes include MHC genes themselves (eg, MHC-class I molecules and MHC-class II molecules), as well as genes that interact with or regulate expression of MHC genes.
  • MHC-class I molecules include, but are not limited to, HLA-A, HLA-B, HLA-C, B2M.
  • MHC-class II molecules include, but are not limited to, HLA-DPAl, HLA-DQA1, and HLA-DRA.
  • genes that interact with or regulate expression of MHC genes include, but are not limited to, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, and CIITA.
  • suppressing or silencing the expression of MHC-related genes refers to suppressing or silencing the expression of one or more genes selected from the group consisting of: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5 , RFXAP, RFXANK, CIITA, and combinations thereof.
  • the engineered immune cells expressing the chimeric receptor comprising the LIR1 antibody of the present invention further comprise that the expression of at least one TCR/CD3 gene is inhibited or silenced, examples of which include For example TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ .
  • the engineered immune cells expressing the chimeric receptors of the present invention comprise suppressed or silenced expression of at least one TCR/CD3 gene and at least one MHC-related gene, wherein the at least one A kind of TCR/CD3 gene is selected from TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and their combination; Said at least one MHC-related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA , HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA and combinations thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA and their combination.
  • the at least one TCR/CD3 gene is selected from the group consisting of TRAC, TRBC, and combinations thereof
  • the at least one MHC-related gene is selected from the group consisting of B2M, RFX5, RFXAP, RFXANK, CIITA, and combinations thereof .
  • the expression of TRAC or TRBC, and B2M of the engineered immune cells is inhibited or silenced.
  • the expression of TRAC or TRBC, and CIITA of the engineered immune cells is inhibited or silenced.
  • the expression of TRAC or TRBC, B2M and CIITA of the engineered immune cells is inhibited or silenced.
  • the expression of TRAC or TRBC, B2M and RFX5 of the engineered immune cells is inhibited or silenced.
  • Methods of inhibiting gene expression or silencing genes include, but are not limited to, DNA fragmentation mediated by, for example, meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, or by Antisense oligonucleotides, RNAi, shRNA and other technologies inactivate genes.
  • the engineered immune cells provided by the present invention further express chimeric antigen receptors targeting tumor antigens.
  • the chimeric antigen receptor targets a tumor antigen selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr -abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3,
  • the target is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2(Her2/neu), MUCl, EGFR, CAIX, WT1, NY- ESO-1, CD79a, CD79b, GPC3, Claudin18.2, NKG2D and any combination thereof.
  • the CAR of the present invention can be designed to include a ligand binding domain specific for that antigen.
  • an antibody to CD19 can be used as the ligand binding domain of the invention.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and/or NKT cells.
  • the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, eg, primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.
  • Nucleic acid sequences encoding chimeric receptors can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.).
  • Transfection is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell.
  • An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • RNA transfection the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell.
  • the term is mainly used for non-viral methods in eukaryotic cells.
  • transduction is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material.
  • Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells).
  • transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means.
  • the cells or bacteria must be in a competent state.
  • techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation.
  • the present invention provides a plurality of engineered immune cells, wherein one immune cell expresses a chimeric receptor described herein and another immune cell expresses a second chimeric receptor targeting other NK inhibitory receptors combined receptors.
  • the second chimeric receptor comprises a second NK inhibitory ligand, a transmembrane domain, and a signaling domain, wherein the second NK inhibitory ligand, the transmembrane domain, and a signaling domain Conductive domains are defined as described in the "Chimeric Receptors" section.
  • one immune cell is engineered to express a chimeric receptor comprising a LIR1 antibody
  • another immune cell is engineered to express a chimeric antigen receptor targeting a tumor antigen.
  • the plurality of engineered immune cells can be administered together or separately.
  • the plurality of immune cells may be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.
  • the present invention provides an antibody conjugate comprising a LIR1 antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, a radioisotope, a half-life extending moiety , detectable markers and drugs.
  • the present invention provides an antibody conjugate comprising a LIR1 antibody as defined in the present invention and an Fc.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc.
  • Native Fc refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, produced by digestion of an intact antibody.
  • the source of immunoglobulins for the production of native Fc is preferably derived from humans.
  • Native Fc fragments are composed of monomeric polypeptides that can be linked in dimeric or multimeric form by covalent linkages (eg, disulfide bonds) and non-covalent linkages.
  • native Fc the disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10:4071-9).
  • native Fc the disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10:4071-9).
  • native Fc as used herein generally refers to monomeric, dimeric and multimeric forms.
  • “Variant Fc” refers to an amino acid sequence that differs from that of a “native” or “wild-type” Fc due to at least one "amino acid modification” as defined herein, also referred to as an "Fc variant".
  • “Fc” also includes single-chain Fc (scFc), ie, a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, which is capable of naturally folding into a functional dimeric Fc region.
  • the Fc is preferably a human immunoglobulin Fc, more preferably a human IgGl Fc.
  • the present invention provides an antibody conjugate comprising a LIR1 antibody as defined in the present invention and a radioisotope.
  • radioisotopes useful in the present invention include, but are not limited to, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.
  • the present invention provides an antibody conjugate comprising the LIR1 antibody as defined in the present invention and a half-life extending moiety selected from the group consisting of the binding structure of albumin, the Binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and white polypeptides (including antibodies) that bind human serum albumin.
  • the present invention provides an antibody conjugate comprising a LIR1 antibody as defined in the present invention and a detectable label.
  • detectable label herein means a compound that produces a detectable signal.
  • the detectable label can be an MRI contrast agent, a scintigraphic contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent.
  • detectable labels examples include fluorophores (such as fluorescein, Alexa, or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase), antibiotics (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes and contrast agents (such as nanoparticles or gadolinium).
  • fluorophores such as fluorescein, Alexa, or cyanine
  • chemiluminescent compounds such as luminol
  • bioluminescent compounds such as luciferase or alkaline phosphatase
  • enzymes such as Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase
  • antibiotics such as kanamycin, ampicillin, chloramphenicol, t
  • the present invention provides an antibody conjugate comprising a LIR1 antibody as defined in the present invention and a drug, such as a cytotoxin or an immunomodulatory agent (ie, an antibody drug conjugate), conjugated to the LIR1 antibody thing).
  • a drug such as a cytotoxin or an immunomodulatory agent (ie, an antibody drug conjugate), conjugated to the LIR1 antibody thing).
  • the drug is covalently attached to the antibody and usually relies on a linker.
  • the drug is a cytotoxin.
  • the drug is an immunomodulatory agent.
  • cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, benzidine Nitrogen mustard, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, chain Zocin, mitomycin, cis-dichlorodiamineplatinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, caminomycin, Darubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, glaremycin, atramycin (AMC), vincristine, vinblastine, Paclitaxel,
  • immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vortexporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine , mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony stimulating factors such as G-CSF and (GM-CSF), interferons such as interferon-alpha, interferon interferon-beta and interferon-gamma), stem cell growth factors designated "S1 factor", erythropoietin and thrombopoietin, or a combination thereof.
  • TNF tumor nec
  • the present invention also provides a detection kit comprising the antibody, multispecific antibody, chimeric receptor or antibody conjugate of the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody, multispecific antibody, chimeric receptor, engineered immune cell or antibody conjugate of the present invention, and one or more Pharmaceutically acceptable excipients.
  • the term "pharmaceutically acceptable excipient” means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration.
  • Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.
  • compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, act
  • the present invention also provides a method of treating and/or preventing and/or diagnosing a disease associated with LIR1 expression, comprising administering to a subject an antibody, chimeric receptor, multispecific antibody as described above , antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • diseases associated with LIR1 expression include, but are not limited to: acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, erythroleukemia, acute megakaryocytic leukemia; chronic leukemia, such as Chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia; and other special types of leukemia such as hairy cell leukemia, prolymphocytic leukemia, plasma cell leukemia, adult T-cell leukemia, eosinophilic leukemia, basophilic leukemia myeloid leukemia, etc.), blastic plasmacytoid dendritic cell tumor, malignant lymphoproliferative disorders, Waldenstrom macroglobulinemia, lymphomas (including Hodgkin lymphoma and non-Hodgkin lymphoma such as B Cell lymphomas (including low-grade/follicular non-Hodgkin lymphom
  • small cell lung cancer non-small cell lung cancer, glandular lung and squamous lung cancer
  • melanoma myeloma, neuroblastoma
  • oral cancer e.g., lips, tongue, mouth, and pharynx
  • ovarian cancer pancreatic cancer, prostate cancer, mesothelioma, retinoblastoma, rhabdoid Sarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell cancer, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumor of the urinary system, vulvar cancer.
  • the diseases treated by the engineered immune cells or the pharmaceutical composition of the present invention are selected from: monocyte/macrophage dysfunction diseases, leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.
  • the anti-LIR1 antibody-containing chimeric receptor of the invention and the tumor antigen-targeting chimeric antigen receptor are co-expressed in immune cells or are administered in combination with immune cells expressing both, respectively.
  • the diseases that can be treated depend on the tumor antigen targeted by the chimeric antigen receptor.
  • a chimeric receptor of the invention comprising an anti-LIR1 antibody
  • the diseases that can be treated are diseases associated with CD19 expression, such as B-cell malignancies, including Acute lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), B-cell Hodgkin's lymphoma (B-HL) and non-Hodgkin's lymphoma (B-NHL), etc.
  • a chimeric receptor comprising an anti-LIR1 antibody is used to inhibit killing of reinfused engineered immune cells by NK cells, while a chimeric antigen receptor is used to target cell killing through binding to tumor antigens .
  • Figure 1 shows the level of scFv expression of UNKi-T cells containing LIR1 antibody.
  • Figure 2 Shows the inhibitory effect of LIR1 antibody-containing UNKi-T cells on NK cell killing. Two-way ANOVA was used for analysis and statistical analysis was performed with T test. ** Indicates that the P value is less than 0.01, reaching a significant level.
  • the sequence containing the extracellular region of the LIR1 protein was cloned into a pCP vector with a His tag, and the correctly sequenced plasmid was transiently infected with CHO cells. After culturing for 8-10 days, the cell culture medium was harvested, and the protein was purified by affinity chromatography to obtain The extracellular domain of human LIR1 protein as an immunogen.
  • mice 6-8 week old female Balb/c mice were used for primary and booster immunization, and ELISA and FACS were used to detect the antibody titer and specificity of protein immunogen in serum. After comprehensive evaluation, mice were given the last booster immunization. Mice were sacrificed after 3-4 days, spleen cells were collected, mixed with mouse myeloma cells (SP2/0) in a certain proportion, and the proliferated hybridoma cells were identified by FACS and ELISA, and positive cell lines were screened. . After several rounds of subcloning identification, two hybridoma cells that can stably secrete LIR1 antibody were finally obtained, and the LIR1-1 and KIR1-2 antibodies were obtained after the antibodies in the supernatants of the two cell lines were purified.
  • SP2/0 mouse myeloma cells
  • Antibodies were digested with trypsin, high-quality primary and secondary spectra were obtained through DNA data acquisition, and the spectra were de novo analyzed with peaks software to obtain preliminary amino acid sequence results; a variety of proteases were used to digest antibodies Solution, after MSE collects data, all peptide peaks and the completeness and fragmentation information of each valence state of each peptide can be obtained; the obtained more comprehensive mass spectrometry data is matched with the preliminary amino acid sequence, and finally two LIR1 singles are obtained.
  • the amino acid sequences of chain antibodies are shown in Table 1 below.
  • Opti-MEM 3ml Opti-MEM (Gibco, Item No. 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120ul of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 min, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells . Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, the concentrated lentivirus was added, and after culturing for 3 days, T cells expressing the chimeric receptor containing the LIR1 antibody were obtained.
  • DynaBeads CD3/CD28 CTSTM Gibco, Cat. No. 40203D
  • the CRISPR/Cas9 system was then used to knock out TCR/CD3 components (specifically TRAC genes) and MHC-related genes ( Specifically, B2M and RFX5), Mock T cells, LIR1-UNKi-1-T cells (containing the LIR1-1 plasmid) and LIR1-UNKi-2-T cells (containing the LIR1-2 plasmid) were obtained, respectively. And using FITC Mouse Anti-Human CD3 (BD Pharmingen, Cat. No. 555916) antibody, PE mouse anti-human HLA-I (R&D Cat. No. FAB7098P) and APC anti-human DR, DP, DQ (biolegend, Cat. No. 361714) antibody, by flow The expression efficiency of CD3/HLA-I/HLA-II in UNKi-T cells, Mock T cells and NT cells was detected by cytometry, and the results are shown in Table 2 below.
  • TRAC genes specifically, B2M and RFX5
  • the UNKi-T cells and Mock-T cells prepared by the present invention were labeled with Far-Red (invitrogen, Cat. No. C34564).
  • the labeled UNKi-T cells and Mock T cells were then plated into 96-well plates at a concentration of 1 ⁇ 10 4 cells/well, and NK92 cells were added at a 2:1 effector-target ratio for co-culture. After 16-18 hours, the proportion of T cells in the culture was detected by flow cytometry, and then the killing rate of NK cells to T cells was calculated. The results are shown in Figure 2.
  • the two kinds of UNKi-T cells expressing the chimeric receptor containing LIR1 antibody prepared in this example can significantly reduce the killing effect of NK cells on T cells.

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Abstract

La présente invention concerne un anticorps ciblant LIR1, et un anticorps multi-spécifique, un récepteur chimérique, un conjugué anticorps, une composition pharmaceutique et un kit les comprenant, ainsi que leur utilisation dans le diagnostic/le traitement/la prévention de maladies associées à l'expression de LIR1.
PCT/CN2021/127564 2020-11-03 2021-10-29 Anticorps ciblant lir1 et son utilisation WO2022095801A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065329A1 (fr) * 2014-10-24 2016-04-28 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés pour induire la phagocytose de cellules positives de classe i du cmh et pour contrer la résistance aux agents anti-cd47/sirpa
CN109790213A (zh) * 2016-07-29 2019-05-21 德克萨斯大学体系董事会 用于鉴定lilrb阻断抗体的方法
CN111315778A (zh) * 2017-08-23 2020-06-19 蜻蜓疗法股份有限公司 结合nkg2d、cd16和肿瘤相关抗原的蛋白质
WO2020136147A1 (fr) * 2018-12-26 2020-07-02 Innate Pharma Composés et méthodes de traitement du cancer de la tête et du cou
CN111867614A (zh) * 2018-01-18 2020-10-30 艾达奈特公司 抗lilrb抗体及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065329A1 (fr) * 2014-10-24 2016-04-28 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés pour induire la phagocytose de cellules positives de classe i du cmh et pour contrer la résistance aux agents anti-cd47/sirpa
CN109790213A (zh) * 2016-07-29 2019-05-21 德克萨斯大学体系董事会 用于鉴定lilrb阻断抗体的方法
CN111315778A (zh) * 2017-08-23 2020-06-19 蜻蜓疗法股份有限公司 结合nkg2d、cd16和肿瘤相关抗原的蛋白质
CN111867614A (zh) * 2018-01-18 2020-10-30 艾达奈特公司 抗lilrb抗体及其用途
WO2020136147A1 (fr) * 2018-12-26 2020-07-02 Innate Pharma Composés et méthodes de traitement du cancer de la tête et du cou
WO2020136145A2 (fr) * 2018-12-26 2020-07-02 Innate Pharma Anticorps neutralisant le récepteur de type immunoglobuline des leucocytes

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