WO2023098813A1 - Antibodies binding cd38 and uses thereof - Google Patents

Antibodies binding cd38 and uses thereof Download PDF

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WO2023098813A1
WO2023098813A1 PCT/CN2022/135904 CN2022135904W WO2023098813A1 WO 2023098813 A1 WO2023098813 A1 WO 2023098813A1 CN 2022135904 W CN2022135904 W CN 2022135904W WO 2023098813 A1 WO2023098813 A1 WO 2023098813A1
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seq
amino acid
acid sequence
identity
antibody
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PCT/CN2022/135904
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French (fr)
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Huan Lin
Xiao Huang
Jianming SUN
Yurong QIN
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Nanjing Leads Biolabs Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to humanized anti CD38 antibodies (including biparatopic antibodies) , methods for their production, pharmaceutical compositions containing these antibodies, and uses thereof.
  • CD38 was first identified in 1980 as a membrane-bound protein of 45 kDa [1] . It was described as a single-chain transmembrane type II glycoprotein encoded by a gene mapped on chromosome 4 (4p15) [2] . CD38 includes a long C-terminal extracellular domain (258 amino acids, aa) , a transmembrane segment (21 aa) and a short N-terminal cytoplasmic tail (21 aa) .
  • CD38 expression in the immune system varies during lymphocyte development, activation and differentiation. Indeed, human CD38 is highly expressed on medullary thymocytes, downregulated in the majority of circulating mature T cells and up-regulated upon activation of T cells. CD38 is expressed on immunosuppressive cells, such as T regulatory cells, B regulatory cells, and myeloid-derived suppressor cells. In solid tissues, the protein is expressed by epithelial cells in the prostate, beta-cells in pancreas, Purkinje cells and neurofibrillary tangles in brain, muscle cells, renal tubules, retinal gangliar cells and corneal cells in the eye [3] .
  • CD38 performs as a marker of ontogenesis in B lymphocytes, and more importantly, it is highly expressed on normal plasma cells as well as on plasma cells derived from patients affected by Multiple myeloma (MM) . Indeed, >90%of the malignant plasma cells from patients with MM show surface expression of CD38 [4] .
  • CD38 may discourage the development of immunotherapies directed against this molecule; however, the analysis of CD38 expression levels across different cellular populations clearly shows that normal and malignant plasma cells express the highest levels of CD38 followed by NK cells and other B and T cell subpopulations.
  • CD38 is functionally pleiotropic, working as an ectoenzyme and as a receptor simultaneously.
  • the identification of CD31 (also known as PECAM-1) as a non-substrate specific ligand was key in recognizing CD38 as a receptor.
  • the CD38/CD31 crosstalk is an important step in the regulation of cytoplasmic calcium fluxes and secretion of cytokines such as IL-6 and IL-10. This interaction probably regulates the migration of leukocytes and CD38 positive cancer cells through the endothelial cell wall [5] .
  • CD38 was described as part of the leukocyte ectonucleotidases family, characterized by two main substrates: NAD+ and NADP+. These two substrates were converted by CD38 and following enzymes, such as CD39 and CD73, to adenosine, that was widely recognized as an immunosuppressive effector [6] .
  • CD38 protein can be used as a unique therapeutic target for malignant plasma cell tumors.
  • the mechanism of action of anti-CD38 agents does not rely only on CD38 expression on tumor cells, but also rely on mAbs that have Fc effect, such as antibody-dependent cellular cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , and CDC.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC mAbs that have Fc effect
  • Fc functions of therapeutic antibody depend on NK cells, macrophages, and the complement system, respectively. Therefore, the combination of immunomodulators and anti-CD38 antibodies can significantly improve the therapeutic effect.
  • Daratumumab is a human IgG1, ⁇ mAb that specifically binds a unique epitope on the CD38 molecule. It can induce CDC of CD38-overexpressing cell lines, as well as patient-derived MM cells in vitro. While in xenograft models, Daratumumab interrupted tumor growth at very low doses. Binding of daratumumab to CD38 on the surface of tumor cells may induce complement activation, CDC, ADCC, ADCP , programmed cell death (PCD) , and modulation of CD38 enzyme activity. [8]
  • Isatuximab also known as SAR650984, is another anti-CD38 antibody with potent proapoptotic activity, which is probably related to its binding to a specific epitope on the CD38 molecule.
  • Isatuximab has Fc-dependent effector functions including CDC, ADCP and ADCC [9] .
  • isatuximab induced apoptosis without the use of crosslinking agents.
  • isatuximab was shown to trigger lysosomal cell death, another mechanism of cell killing, in MM cell lines. Pomalidomide will enhance this lysosomal killing effect in MM cell lines.
  • MOR202 is another antibody that targets the CD38 molecule and induce MM cell killing via CDC, ADCC and ADCP.
  • MOR202 was well tolerated at doses up to 16 mg/kg with the maximum tolerated dose not reached. It combined with low-dose dexamethasone resulted in a OR or better in 29%of patients [11] .
  • Nanobodies are soluble single domain antibody fragment derived from the variable domain of heavy chain antibodies (VHH) naturally occurring in camelids.
  • VHH variable domain of heavy chain antibodies
  • Bispecific/biparatopic antibodies was obtained by fusion two anti-CD38 VHH domains binding different epitope on CD38 into one human IgG-like structure, these bispecific/biparatopic antibodies showed enhancement of CDC effects to CD38 expressing cells [12] .
  • the CDC effects is critical for the efficacy of anti-CD38 antibodies.
  • Antibody-dependent cell-mediated cytotoxicity a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc ⁇ receptors with both activating and inhibitory activities [13] .
  • the ADCC effect is one of the most important MOA of daratumumab and isatuximab for MM. Therefore, improving the ADCC effect through engineering is an important step in the development of therapeutic antibodies against MM.
  • biparatopic nanobody-based bispecific antibody We sought to exploit the nanobodies to construct highly soluble biparatopic nanobody-based bispecific antibody and enhanced ADCC effect by the amino-acid mutation. Remarkably, these biparatopic antibody show higher CDC and ADCC potency than daratumumab and therefore hold promise as novel therapeutics for the treatment of multiple myeloma.
  • humanized anti-CD38 antibodies including bispecific antibodies which are useful as a therapeutic agent for treatment of multiple myeloma and other hematological tumors.
  • the present invention relates to antibodies binding to CD38, which derived from anti CD38 VHH in camelids.
  • the anti CD38 antibodies are humanized.
  • the anti CD38 antibodies are biparatopic antibodies or antigen-binding fragment thereof, or are humanized biparatopic antibodies or antigen-binding fragment thereof.
  • the CD38 is derived from human or cynomolgus.
  • the CD38 comprises SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention relates to an anti-CD38 VHH, which comprises three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3.
  • the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3 which is included in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein:
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 39
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 41
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 43; or
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 45
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 47
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 49
  • the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein:
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 40
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 42
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 44; or
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 46
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 48
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 50
  • the anti-CD38 VHH comprises an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10, or the VHH consists of amino acid sequences as shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the present invention relates to a humanized anti-CD38 VHH, which comprises three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3.
  • the humanized anti-CD38 VHH nanobody comprises HCDR1, HCDR2 and HCDR3 which is included in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the humanized anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise or consist of 1) amino acid sequences as shown in SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively; or 2) amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, respectively; or 3) amino acid sequences as shown in SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; or 4) amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.
  • the humanized anti-CD38 VHH comprises back-mutations in human framework region.
  • the humanized anti-CD38 VHH nanobody comprises an amino acid sequence as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22, or the VHH consists of amino acid sequences as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22.
  • the present invention relates to an anti-CD38 heavy chain antibody (HcAb) or an antigen-binding fragment thereof, which comprises the humanized anti-CD38 VHH or anti-CD38 VHH described in the above aspects.
  • HcAb heavy chain antibody
  • the anti-CD38 heavy chain antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3,
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 39
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 41
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 43; or
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 45
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 47
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 49
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 40
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 42
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 44; or
  • HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 46
  • HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 48
  • HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 50.
  • the anti-CD38 heavy chain antibody and the antigen-binding fragment thereof comprises VHH as described in the above aspects.
  • the anti-CD38 heavy chain antibody and the antigen-binding fragment thereof comprises VHH comprising an amino acid sequence as shown in any one of SEQ ID NO: 9-22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 9-22, or consisting of amino acid sequences as shown in any one of SEQ ID NO: 9-22.
  • the present invention relates to an anti-CD38 heavy chain antibody or the antigen-binding fragment thereof, which comprises:
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 9 and having the same CDRs as SEQ ID NO: 9, or consisting of SEQ ID NO: 9;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 10 and having the same CDRs as SEQ ID NO: 10, or consisting of SEQ ID NO: 10;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 11 and having the same CDRs as SEQ ID NO: 11, or consisting of SEQ ID NO: 11;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 12 and having the same CDRs as SEQ ID NO: 12, or consisting of SEQ ID NO: 12;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 13 and having the same CDRs as SEQ ID NO: 13, or consisting of SEQ ID NO: 13;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 14 and having the same CDRs as SEQ ID NO: 14, or consisting of SEQ ID NO: 14;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 15 and having the same CDRs as SEQ ID NO: 15, or consisting of SEQ ID NO: 15;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 16 and having the same CDRs as SEQ ID NO: 16, or consisting of SEQ ID NO: 16;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 17 and having the same CDRs as SEQ ID NO: 17, or consisting of SEQ ID NO: 17;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 18 and having the same CDRs as SEQ ID NO: 18, or consisting of SEQ ID NO: 18;
  • a VHH comprising an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 19 and having the same CDRs as SEQ ID NO: 19, or consisting of SEQ ID NO: 19;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 20 and having the same CDRs as SEQ ID NO: 20, or consisting of SEQ ID NO: 20;
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 21 and having the same CDRs as SEQ ID NO: 21, or consisting of SEQ ID NO: 21; or
  • VHH comprising an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 22 and having the same CDRs as SEQ ID NO: 22, or consisting of SEQ ID NO: 22.
  • the anti-CD38 heavy chain antibody or the antigen-binding fragment thereof further comprises Fc region.
  • the Fc region is or is derived from human IgG constant region, e.g., IgG1, IgG2, IgG3 or IgG4, preferable IgG1 constant region.
  • the anti-CD38 antibody and the antigen-binding fragment thereof comprises IgG Fc.
  • the IgG Fc is IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc.
  • the IgG Fc comprises amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 23, or the IgG Fc consists of SEQ ID NO: 23.
  • the IgG Fc comprises mutation, for example the mutation is amino acids substitution, deletion, and addition.
  • the mutation can improve ADCC, ADCP effect.
  • the mutation is one or more amino acids substitution, for example S239D and/or I332E (EU numbering) substitution.
  • the IgG Fc comprises knob mutation (s) or hole mutation (s) , for example, IgG Fc comprises amino acid sequence as shown in SEQ ID NO: 34 or 35; or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 34 or 35; or IgG Fc consists of SEQ ID NO: 34 or 35.
  • the anti-CD38 antibody of the present invention is a bispecific antibody, preferablely a biparatopic antibody.
  • the biparatopic anti-CD38 antibody comprises the humanized anti-CD38 VHH or anti-CD38 VHH in previous aspects.
  • the bispecific anti-CD38 antibody is selected from:
  • each chain comprises two anti-CD38 VHHs (VHH1 and VHH2) , the hinge, CH2 and CH3 domain;
  • chain 1 comprises anti-CD38 VHH1, the hinge, CH2 and CH3 domain
  • chain 2 comprises anti-CD38 VHH2, hinge, CH2 and CH3 domain;
  • IgG-Like symmetrical bispecific antibody having two arms, each arm consists of two chains, from N terminal to C terminal, chain 1 comprises anti-CD38 VHH1 and heavy chain constant region (i.e., CH1, hinge, CH2 and CH3 domain) , chain 2 comprises anti-CD38 VHH2 and light chain constant region,
  • chain 1 comprises anti-CD38 VHH1 and heavy chain constant region (i.e., CH1, hinge, CH2 and CH3 domain)
  • chain 2 comprises anti-CD38 VHH2 and light chain constant region
  • VHH1 is different from VHH2.
  • VHH1 and VHH2 in the symmetrical bispecific antibody are linked by a linker, wherein the linker can be any well-known linker in the art, it is generally a flexible short peptide sequence, for example, (G 4 S) n, where n is an integer number, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9; GGGSG; GGSGG; GSGGG; SGGGG; GGGTS; GTSPGG; GNGGGS; G4S-GGSGG-G4S-SGGGG; GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; KESGSVSGSSE QLAQFRSLD; GGRRGGSLRQQ.
  • the linker is GGSGGS (SEQ ID NO: 53) .
  • the VHH of the bispecific antibody is derived from Camelid’s anti-CD38 VHHs, or is derived from humanized anti-CD38 VHHs.
  • the VHH is any one of the VHH described in any one of previous aspects.
  • heavy constant region of the bispecific antibody is derived from IgG1, IgG2, IgG3 or IgG4, preferably derived from IgG1 isotype.
  • light chain constant region is derived from ⁇ light chain or ⁇ light chain.
  • Fc region of the bispecific antibody have mutations, for example substitution, deletion and addition of one or more amino acid residues.
  • the Fc region comprises mutations improving ADCC effect and/or ADCP effect of the bispecific antibody.
  • the Fc region comprises mutations S239D/I332E (according to EU numbering) .
  • Fc region of the bispecific antibody has knob-in-hole mutation.
  • the Fc region comprise substitutions N384D, Q418E, N421D, Y407V, T366S, L368A and Y349C (according to EU numbering) .
  • the Fc region comprise substitutions T366W and S354C (according to EU numbering) .
  • the bispecific antibody comprises two set of HCDR1, HCDR2 and HCDR3, the first set of HCDR1, HCDR2 and HCDR3 comprise 3 HCDRs contained in SEQ ID NO: 9, and the second set of HCDR1, HCDR2 and HCDR3 comprise 3 HCDRs contained in SEQ ID NO: 10.
  • the first set of HCDR1, HCDR2 and HCDR3 comprise or consist of 1) amino acid sequences as shown in SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively; or 2) amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, respectively, and the second set of HCDR1, HCDR2 and HCDR3 comprise or consist of 3) amino acid sequences as shown in SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; or 4) amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.
  • the bispecific antibody comprises VHH1 and VHH2, when VHH1 comprises the first set of HCDR1, HCDR2 and HCDR3, then VHH2 comprises the second set of HCDR1, HCDR2 and HCDR3, vice versa.
  • the bispecific antibody comprises VHH1 and VHH2 selected from SEQ ID NO: 9 or SEQ ID NO: 10, wherein VHH1 and VHH2 is selected different sequence.
  • the bispecific antibody comprises humanized VHH1 and VHH2 of the present invention, wherein the humanized VHH1 and VHH2 is independently from each other selected from humanized VHH180 (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) and humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) , provided that humanized VHH1 differs from VHH2.
  • humanized VHH1 and VHH2 is independently from each other selected from humanized VHH180 (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) and humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) , provided that humanized VHH1 differs from VHH2.
  • the bispecific antibody comprises humanized VHH1 and VHH2 of the present application, wherein when the humanized VHH1 is selected from humanized VHH180 (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) , then VHH2 is selected from humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) , vice versa.
  • the bispecific antibody is selected from:
  • a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprise or consist of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;
  • an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 36 and another chain comprises or consists of SEQ ID NO: 37; or
  • an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 38 and the chain 2 comprises or consists of SEQ ID NO: 30.
  • the bispecific antibody comprises mutations improving ADCC effect and/or ADCP effect and is selected from:
  • a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprise or consist of SEQ ID NO: 31 or SEQ ID NO: 32;
  • an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 28 and another chain comprises or consists of SEQ ID NO: 29; or
  • an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 33 and the chain 2 comprises or consists of SEQ ID NO: 30.
  • the bispecific antibodies are humanized bispecific antibodies, wherein the camelids VHHs had been replaced by corresponding humanized VHHs disclosed above.
  • the humanized bispecific antibody has two same chains comprising amino acid sequence as shown in SEQ ID NO: 51.
  • the humanized bispecific antibody comprises mutations improving ADCC effect and/or ADCP effect.
  • the humanized bispecific antibody comprises a Fc region comprising mutations S239D/I332E (numbering according to EU index) .
  • the humanized bispecific antibody has two same chains comprising an amino acid sequence as shown in SEQ ID NO: 52.
  • the antigen-binding fragment of the present invention is an antibody fragment comprising Fab, Fab’, Fab’-SH, Fv, single chain antibody (e.g., scFv) , (Fab’) 2 , single domain antibody (e.g., VHH) , domain antibody (dAb) or linear antibody.
  • the present invention relates to an isolated nucleic acid encoding anyone or more chains of anti-CD38 antibody of the above aspects.
  • the present invention relates to a vector comprising the nucleic acid of the Fifth aspect.
  • the vector is an expression vector.
  • the present invention relates to a host cell comprising the nucleic acid of the Fifth aspect or the vector of the Sixth aspect.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., CHO cells or CHO-S cells or 293 cells or 293T cells) , or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof.
  • the present invention relates to a method of preparing an anti-CD38 antibody or the antigen-binding fragment thereof comprising culturing the host cell of the Seventh aspect, under conditions suitable for expression of a nucleic acid encoding an anti-CD38 antibody or the antigen-binding fragment thereof of the present invention, optionally said method further comprises recovering the anti-CD38 antibody or the antigen-binding fragment thereof from the host cell.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-CD38 antibody or the antigen-binding fragment thereof according to the present invention, and optionally pharmaceutically acceptable adjuvant (s) .
  • the present invention relates to a combination product, comprising the anti-CD38 antibody or the antigen-binding fragment thereof of the present invention and one or more other therapeutic agents, e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
  • a chemotherapeutic agent e.g., other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
  • the present invention relates to a method of treating cancer in a subject comprising administering to said subject the anti-CD38 antibody or the antigen-binding fragment thereof, or the pharmaceutical composition, or the combination product of the present invention.
  • the cancer is that with abnormal level of CD38 (e.g., the high protein or nucleic acid level of CD38) .
  • said cancer is hematological tumors, malignant plasma cell tumors, multiple myeloma, relapsed/refractory multiple myeloma.
  • the method of the present invention further comprises administering to said subject in combination with one or more therapies, e.g., a therapeutic modality and/or other therapeutic agent, preferably the therapeutic modality includes surgery and/or radiation therapy, and/or the other therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
  • a therapeutic modality includes surgery and/or radiation therapy
  • the other therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
  • the present invention relates to a use of the anti-CD38 antibody or the antigen-binding fragment thereof, or the pharmaceutical composition, or the combination product of the present invention in the manufacture of a medicament for treating cancer in a subject.
  • the cancer is that with abnormal level of CD38 (e.g., the high protein or nucleic acid level of CD38) .
  • said cancer is hematological tumors, malignant plasma cell tumors, multiple myeloma, relapsed/refractory multiple myeloma.
  • Figure 1 shows humanized anti-human CD38 heavy chain antibodies bound to human CD38 protein, in which EC50 and representative binding curves of each anti-human CD38 antibodies comprising corresponding VHH in table 1 were show.
  • VHH180 represents Camelids heavy chain antibody VHH180-Ab
  • VHH194 represents Camelids heavy chain antibody VHH194- Ab
  • VHH180_n represents humanized heavy chain antibody VHH180_n-Ab
  • VHH180_1 represents humanized heavy chain antibody VHH180_1-Ab
  • VHH194_n represents humanized heavy chain antibody VHH194_n-Ab
  • VHH194_1 represents humanized heavy chain antibody VHH194_1-Ab.
  • FIG. 2 shows humanized heavy chain antibodies of VHH180 and VHH194 bound to human CD38 on cell surface
  • VHH180 represents Camelids heavy chain antibody VHH180-Ab
  • VHH194 represents Camelids heavy chain antibody VHH194-Ab
  • VHH180_n represents humanized heavy chain antibody VHH180_n-Ab
  • VHH194_n represents humanized heavy chain antibody VHH194_n-Ab .
  • Figure 3 shows epitope binning of two anti-human CD38 heavy chain antibodies.
  • VHH180_3 and VHH194_2 represent humanized anti-human CD38 antibodies VHH180_3-Ab and VHH194_2-Ab, respectively;
  • B-180-3 and B-194-2 represent biotinylated humanized anti-human CD38 antibodies VHH180_3-Ab and VHH194_2-Ab, respectively.
  • FIG 4 shows ADCC activity of anti-CD38 antibodies, in which VHH180_2, VHH180_3, VHH194_2 and VHH194_4 represent VHH180_2-Ab, VHH180_3-Ab, VHH194_2-Ab and VHH194_4-Ab, respectively.
  • Figure 5 shows schematic diagram of three biparatopic antibody structures.
  • Figure 6 shows CDC activity of different structure of biparatopic antibodies on Daudi cells.
  • Figure 7 shows ADCC activity of different structure of biparatopic antibodies on Daudi cells.
  • Figure 8 shows ADCC activity of ADCC-enhancement mutants of biparatopic antibodies.
  • Figure 9 shows CDC activity of ADCC-enhancement mutant of biparatopic antibodies.
  • Figure 10 shows ADCC activity of ADCC-enhancement mutant of humanized biparatopic antibodies.
  • FIG. 11 shows ADCP activity of ADCC-enhancement mutant of humanized biparatopic antibodies
  • antibody broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , heavy chain antibodies, nanobodies and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • Ig immunoglobulin
  • anti-CD38 antibody refers to an antibody which is capable of binding to (e.g., human or Cynomolgus) CD38 protein or a fragment thereof with sufficient affinity such that the antibody can be used as diagnostic and/or therapeutic agent in targeting (e.g., human or Cynomolgus) CD38.
  • the extent of binding of an anti-CD38 antibody to an unrelated, non-CD38 protein is less than about 10 %of the binding of the antibody to CD38 as measured, for example, by Surface Plasmon Resonance.
  • an antibody that specifically binds to human CD38 has a dissociation constant (K D ) of 10 -8 M or less.
  • HcAb heavy chain antibody
  • VHH and term “nanobody” can be used interchangeably, and refer to heavy chain variable region of HcAb, from N terminal to C terminal, comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • a “humanized” antibody refers to an antibody comprising human framework region (FR) and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95%or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs and few residues in the heavy chain constant region if modulation of the effector functions is needed, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a “humanized form” of an antibody e.g., a non-human antibody, refers to an antibody that has undergone humanization.
  • a “humanized” VHH refers to a VHH comprising amino acid residues derived from non-human CDRs and amino acid residues from human FRs.
  • humanized antibodies may retain non-human residues within the human framework regions to enhance proper binding characteristics and/or some amino acid mutations may be introduced within the CDRs in order to improve the binding affinity and/or to reduce the immunogenicity and/or to increase the degree of humanness.
  • some residues in human framework should be back-mutations to the original non-human sequence.
  • an “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include but are not limited to Fv, Fab, Fab', Fab'-SH, F (ab') 2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv) ; single domain antibody; and multispecific antibodies formed from antibody fragments.
  • epitope determinant refers to a portion of an antigen (e.g., CD38) that specifically interacts with an antibody molecule.
  • This portion referred to herein as an epitope determinant
  • an epitope determinant typically comprises an element such as an amino acid side chain or a sugar side chain or a component thereof.
  • Epitope determinants can be defined according to methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen-deuterium exchange) . Some epitopes are linear epitopes, while others are conformational epitopes.
  • a “multispecific antibody” refers an antibody that has binding specificities for at least two different epitopes on the same antigen or different antigens.
  • bispecific antibody anti-CD38 bispecific antibody
  • biparatopic antibody anti-CD38 antibody
  • anti-CD38 biparatopic antibody anti-CD38 biparatopic antibody
  • similar expressions can be used interchangeably and refer to an anti-CD38 antibody having binding specificities for two different epitopes of CD38, in which each antigen binding domain recognizes unique, non-overlapping epitopes of CD38.
  • An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the reference antibody to its antigen in a competition assay.
  • An antibody that inhibits (e.g., competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits binding of 50%, 60%, 70%, 80%, 90%, or 95%or more of the reference antibody to its antigen.
  • CDR region is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ( “hypervariable loop” ) and/or comprises antigen-contacting residues ( “antigen contact point” ) .
  • CDRs are primarily responsible for binding to epitopes.
  • the CDRs of the heavy or light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chains are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chains are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • CDRs can also be determined based on having the same Kabat numbering positions as a reference CDR sequence (e.g., any of the exemplary CDRs of the invention) .
  • CDR or “CDR sequence” encompasses CDR sequences determined by any of the manners described above.
  • the boundaries of the CDRs of the antibodies of the invention are determined by Kabat scheme or IMGT scheme.
  • Antibodies with different specificities have different CDRs (under the same assignment system) .
  • CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The smallest overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thereby providing a “minimal binding unit” for antigen binding.
  • the minimal binding unit may be a sub-portion of the CDR.
  • residues of the rest CDR sequences can be determined by antibody structure and protein folding. Therefore, any variants of the CDRs given herein will also be considered in the invention.
  • the amino acid residues in the minimal binding unit may remain unchanged, while the other CDR residues defined by Kabat or Chothia may be substituted by conservative amino acid residues.
  • IgA immunoglobulin 1
  • IgG 2 immunoglobulin 2
  • IgG 3 immunoglobulin 3
  • IgG 4 immunoglobulin 1
  • IgA 2 immunoglobulin 2
  • ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ respectively.
  • a person skilled in the art can select and obtain the antibody in an appropriate class of the present invention according to the practical desire.
  • Binding affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) .
  • binding affinity refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) .
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D ) . Affinity can be measured by common methods known in the art, including those described herein.
  • ADCC antibody-dependent cellular cytotoxicity
  • complement-dependent cytotoxicity refers to lysis of cells induced by the antibody as reported herein in the presence of complement.
  • ADCP antibody-dependent cellular phagocytosis
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) , NIH Publication 91-3242.
  • isolated nucleic acid refers to a nucleic acid molecule which has been separated from components of its natural environment.
  • the isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells” , which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared.
  • composition refers to a formulation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • pharmaceutically acceptable adjuvants refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete) ) , excipient or vehicle co-administered with the therapeutic agent.
  • adjuvant e.g., Freund's adjuvant (complete and incomplete)
  • excipient or vehicle co-administered with the therapeutic agent.
  • treatment refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • therapeutic agent encompasses any substance effective in preventing or treating tumors (such as cancer) and infections, including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against to the immune checkpoint molecule) , active anti-infective agents, immunomodulators, small entities.
  • combination product refers to a fixed or non-fixed combination of dosage unit forms or a kit of parts for combined administration in which two or more therapeutic agents can be administered independently at the same time or administered separately within a time interval, especially when these time intervals allow the combined partner to demonstrate collaboration, for example, synergistic effects.
  • fixed combination means that the antibody of the invention and the combination partner (e.g., other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are administered to a patient simultaneously in the form of a single entity or dose.
  • non-fixed combination means that the antibodies and combination partners of the present invention (e.g., other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are administered to patients simultaneously, concurrently, or sequentially as separate entities, and there is no specific time limitation, where such administration provides therapeutically effective levels of the two compounds in the patient.
  • other therapeutic agents such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents
  • cocktail therapy such as the administration of three or more therapeutic agents.
  • the drug combination is a non-fixed combination.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • tumor tumor necrosis
  • the term “effective amount” refers to an amount or dosage of the antibody or fragment or conjugate or composition of the invention which generates expected effects in a patient in need of treatment or prevention after administered to the patient in a single or multiple doses.
  • the effective amount can be easily determined by an attending physician as a person skilled in the art by considering a variety of factors as follows: species such as mammals; its size, age, and general health; the specific disease involved; the extent or severity of the disease; response in an individual patient; specific antibody administered; route of administration; bioavailability characteristics of the administered formulation; selected dose regimen; and use of any concomitant therapy.
  • “Therapeutically effective amount” refers to an amount effective to achieve a desired therapeutic outcome at a required dosage for a desired period of time.
  • the therapeutically effective amount of an antibody or an antibody fragment, or conjugate or composition thereof can vary depending on a variety of factors such as morbid state, age, sex, and weight of an individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • the therapeutically effective amount is also such an amount in which any toxic or undesired effect of the antibody or antibody fragment, or conjugate or composition thereof is inferior to the therapeutically beneficial effect.
  • “Therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80%or 90%, relative to untreated subjects.
  • a measurable parameter e.g., tumor growth rate
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, goat, cat, dog, and horse) , primates (e.g., human and non-human primates such as monkey) , rabbit, and rodents (e.g., mouse and rat) . In some embodiments, the individual or subject is human.
  • administering refers to the physical introduction of a composition comprising a therapeutic agent (e.g., anti-CD38 antibody) to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • the amino acid change described herein includes amino acid substitution, insertion, or deletion.
  • the amino acid change described herein is amino acid substitution, for example conservative substitution.
  • the present application provides a Camelids VHH, a humanized VHH, or an antibody comprising the Camelids VHH or the humanized VHH, wherein the above said molecules comprise the following HCDRs:
  • the amino acid change described herein occurs in region (s) outside the CDRs (e.g., in FRs) .
  • the amino acid change occurs in FR region (s) of the VHH or VH, e.g., in FR1, FR2, FR3 and/or FR4 of the VHH or VH.
  • the substitution is a conservative substitution.
  • a conservative substitution refers to a replacement of an amino acid by another amino acid of the same class, e.g., an acidic amino acid replacement by another acidic amino acid, a basic amino acid replacement by another basic amino acid, or a neutral amino acid replacement by another neutral amino acid. Exemplary substitutions are shown in Table below:
  • one or more amino acid modifications can be introduced into an Fc region of an antibody provided herein, thereby producing an Fc region variant such that, for example, the efficacy of the antibody in treating cancer or a cell proliferative disease is enhanced.
  • modification can be incorporated to increase the ADCC and/or ADCP function of the antibody.
  • the invention provides a nucleic acid encoding any of the above anti-CD38 antibodies or fragments thereof.
  • the nucleic acid can encode an amino acid sequence comprising the light chain variable region and/or the heavy chain variable region of the antibody, or an amino acid sequence comprising the light chain and/or the heavy chain of the antibody.
  • the vector is an expression vector, such as an eukaryotic expression vector.
  • the vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC) . Numerous vector systems can be used.
  • the expression vector of the present invention is a pcDNA, e.g., pcDNA3.1 expression vector.
  • the present invention provides a host cell containing a nucleic acid encoding the region of antibody described herein or the vector described herein.
  • Suitable host cells for cloning or expressing the nucleic acid encoding the antibody or the vector include prokaryotic or eukaryotic cells as described herein.
  • the antibody can be produced, for example, in bacteria. After expression, the antibody can be isolated from bacterial paste in soluble fraction and can be further purified.
  • the host cell is E. coli.
  • the host cell is eukaryotic.
  • the host cell can be selected from the group consisting of yeasts, mammalian cells (e.g., a human cell) , insect cells, plant cells, or other cells suitable for preparation of an antibody or an antigen-binding fragment thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the vector encoding the antibody, including fungus and yeast strains.
  • useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40, human embryonic kidney lines (293HEK or 293 or 293T cells) and the like.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, and myeloma cell lines such as Y0, NS0, and Sp2/0.
  • the present invention provides a method for preparing an anti-CD38 antibody, wherein the method comprises steps of incubating host cells containing an nucleic acid encoding the antibody or antigen binding fragment of the present invention (anyone or more regions or chains of the antibody) , under conditions suitable for expressing antibodies or the antigen binding fragments, and optionally, recovering the antibody or the fragments from the host cells (or the host cell cultures) .
  • nucleic acids encoding the antibody are isolated and inserted into one or more vectors for further cloning and/or expression in the host cells.
  • the nucleic acid is readily isolated and sequenced by using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding heavy and light chains of antibodies) .
  • Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., protein A) , size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art.
  • the purity of the antibody molecule of the present invention can be determined by any of a variety of well-known analysis methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the invention further provides a pharmaceutical composition, or a pharmaceutical formulation, or combination product comprising an anti-CD38 antibody or antigen-binding thereof, or the nucleic acid encoding the anti-CD38 antibody or the fragment thereof.
  • composition, formulation or combination product may further contain suitable pharmaceutical adjuvants such as a pharmaceutical carrier, an excipient, and the like known in the art, including buffers.
  • suitable pharmaceutical adjuvants such as a pharmaceutical carrier, an excipient, and the like known in the art, including buffers.
  • the pharmaceutical carrier suitable for use in the invention can be sterile liquid, such as water and oil, including petroleum, or oil of animal, vegetable, or synthetic source, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly used for injectable solutions.
  • compositions may take the form of a solution, a suspension, an emulsion, a tablet, a pill, a capsule, a powder, a sustained release preparation, and the like.
  • composition or formulation of the invention may also contain more than one active ingredient required by a particular indication to be treated, preferably active ingredients having complementary activities without adversely affecting one another.
  • the present invention further provides a combination product, comprising the anti-CD38 antibody or the antigen binding fragment thereof of the present invention, and other therapeutic agents, e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immunomodulator.
  • other therapeutic agents e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immunomodulator.
  • the invention in another aspect, relates to a method for treating a cancer in a subject, the method comprises administering therapeutically effective amount of the antibody or antigen binding fragments, the pharmaceutical composition or formulation, the combination product or the nucleic acid of the present invention.
  • the method above further comprises administering to said subject in combination with one or more therapies, e.g., a therapeutic modality and/or other therapeutic agent.
  • therapies e.g., a therapeutic modality and/or other therapeutic agent.
  • the invention provides uses of the anti-CD38 antibody or the fragment thereof in manufacture or preparation of a medicine for treatment of the above-mentioned related diseases or conditions.
  • VHH180 and VHH194 were obtained from Y-CLONE Medical Sciences Co., Ltd, then VHH180 (SEQ ID NO: 09) and VHH194 (SEQ ID NO: 10) were humanized through CDR Grafting.
  • IMGT –GENE database http: //www. imgt. org was used to search germline sequences, two human germline sequences IGHV3-66*01 and IGHV3-23*02 were selected as CDR accepter for VHH180 and VHH194, respectively.
  • VHH180 According to grafting CDR region annotated by IMGT, three CDRs of the VHH180 were grafted from the original variable chain into human IGHV3-66*01 germline framework, and three CDRs of the VHH194 were grafted from the original variable chain into human IGHV3-23*02 germline framework.
  • Table 1 shows some obtained humanized anti-CD38 VHHs named as VHH180_n and VHH194_n, respectively, n is selected from 1-6.
  • Table 1 lists obtained humanized anti-CD38 VHH with specific mutated amino acids residues of each of VHH.
  • Nucleotide sequences encoding the above humanized VHH were directly fused to nucleotide sequence encoding the human IgG1-Fc sequence with C220S mutation (EU numbering) (SEQ ID NO: 23) , then were synthesized by General Biosystems (Anhui) Co., Ltd.
  • the synthesized genes express heavy chains named as VHH180_n-Fc or VHH194_n-Fc, respectively.
  • heavy chain VHH180_1-Fc consists of VHH180_1 and human IgG1-Fc.
  • nucleotide sequences encoding Camelids-derived VHH180 and VHH194 were also fused to nucleotide sequence encoding the human IgG1-Fc sequence with C220S mutation (EU numbering) , then were synthesized by General Biosystems (Anhui) Co., Ltd. Encoded heavy chains are named as VHH180-Fc (SEQ ID NO: 54) and VHH194-Fc (SEQ ID NO: 55) .
  • Nucleotide sequences encoding the heavy chains were inserted into the expression vector pcDNA3.1, respectively.
  • Each of successfully constructed vectors encoding heavy chains was transfected into CHO-S cells using ExpiCHO TM Expression System (ThermoFisher) , according to manufacturer's instructions.
  • the transfected cells were cultured in ExpiCHO TM Expression Medium for 12 days to express each humanized anti-CD38 heavy chains.
  • Two identical humanized anti-CD38 heavy chain interacted with each other to form humanized anti-CD38 heavy chain antibody named as VHH180_n-Ab, or VHH194_n-Ab.
  • VHH180_1-Fcs interacted with each other to form heavy chain antibody of VHH180_1, and named as VHH180_1-Ab. Then culture supernatants were harvested and purified by using Protein A affinity chromatography (GE healthcare) , according to manufacturer's instructions.
  • GE healthcare Protein A affinity chromatography
  • Example 1 To test the function of the humanized anti-CD38 heavy chain antibodies, all humanized anti-CD38 heavy chain antibodies obtained in Example 1 were analyzed for binding potency, affinity, ADCC effect in this Example.
  • Humanized anti-CD38 heavy chain antibodies generated as described above were detected for binding potency to human CD38 in an ELISA format.
  • ELISA assay was performed generally as described in Baker et al. (Trends Biotechnol. 20: 149-156, 2002) . Briefly, 96-well flat bottom plates (Hangzhou Xinyou, Cat#100096H) were coated with 50 ⁇ l Human CD38-his tag protein (Sino biological, Cat#10808-H08H) at a concentration of 0.5 mg/mL in PBS (Hyclone, Cat#SH30256.01) overnight at 4°C. After removing the coating solution, the plates were then blocked by incubating with 1%BSA in PBS for one hour at 37°C.
  • PBST PBS containing 0.05 %Tween20
  • 6 points, 3-folds serial dilution solutions were prepared in binding buffer (PBS containing 0.05 %Tween 20 and 0.5%BSA) with initiative concentration of 8 nM.
  • the diluted antibody solutions were added into individual wells of the plates, and the plates were incubated for one hour at 37°C, Daratumumab and Isatuximab were used as positive control, since Daratumumab and Isatuximab were expressed by the inventors of the present application in house according to well-known amino acid sequences of Daratumumab and Isatuximab, they are also named as Daratumumab analog and Isatuximab analog.
  • RPMI-8226 cells (NCACC, Cat#TCHU234) were used. RPMI-8226 cells were cultured in completely RPMI 1640 Medium (Gibco, Cat#22400089) with 10%fetal bovine serum (FBS) , 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. Eight-point 4-fold series dilutions of humanized anti-CD38 antibodies were made in PBS with initiative concentrations of 3 ⁇ g/mL, and then added to cells.
  • FBS fetal bovine serum
  • the obtained cell mixtures were incubated at 4°C for 30 minutes, followed by washing cells with PBS twice to remove unbound molecules.
  • 100 ⁇ L 1: 10000 diluted R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098) was incubated with RPMI-8226 cells at 4°C for 30 minutes. Then, cells were washed twice with PBS, re-suspended in PBS. Analysis of CD38 binding was carried out with the BD Accuri C5 flow cytometer (BD Bioscience) , according to manufacturer's instructions.
  • Figure 2 shows results of binding potency of humanized anti-human CD38 heavy chain antibodies to human CD38 protein expressed on the cell surface, in which all generated humanized anti-human CD38 antibodies of the present invention can bind to human CD38 protein.
  • the results are substantially consistent with the results of ELISA binding assay in section 1 above.
  • cross reactivity and binding kinetics of generated humanized anti-human CD38 heavy chain antibodies was determined by detecting affinity (monovalent KD) of anti-human CD38 heavy chain antibodies to human CD38 or cynomolgus monkey CD38 through the ForteBio Octet RED 96 (bio-layer interferometry) .
  • AHC Anti-Human IgG Fc Capture
  • PBST PBS, 0.1%Tween-20
  • a kinetic assay was performed in 96-well plates and ran using following times for each step: a) balance baseline for 100 seconds in PBST, b) loading with humanized anti-human CD38 antibodies to at least 1 nanometer, c) balance baseline for 300 seconds, d) association with His-tagged human CD38 (SEQ ID NO: 01) or His-tagged cynomolgus monkey CD38 (SEQ ID NO: 02) at concentration of 100 nM for 100 seconds, and e) dissociation in PBST for 600 seconds.
  • the obtained data sets were fitted with a 1: 1 Local fitting Model using Octet software.
  • VHH180-Ab and VHH194-Ab represent Camelids anti CD38 heavy chain antibodies
  • VHH180_3-Ab and VHH194_2-Ab were biotinylated by EZ-Link TM Sulfo-NHS-LC-LC-biotin kit (Thermo, Cat#21338) .
  • the biotinylated antibodies were named B-180-3 and B-194-2, respectively.
  • the ADCC reporter assay system was developed by using a Daudi (NCACC, Cat#TCHu140) cell line as target cells and a Jurkat (NCACC, Cat#TCHU123) cell line as effector cells.
  • This Jurkat effector cell line was engineered to stable express human Fc ⁇ IIIa receptor (V158 variant) and a NFAT-RE driven luciferase reporter (Promega, pGL4.30 [luc2P/NFAT-RE/Hygro] , Cat#E8481) .
  • cultured Jurkat cells were con-transfected with NFAT-luc2P reporter plasmids (Promega, Cat#E8481) and plasmids comprising CD16a F genes (SEQ ID NO: 2 discribed in WO2012121911 A2) by using Gene Pul Ser X Cell TM kit (BIO-RAD) , according to manufacturer's instructions.
  • single cell clone stable expressing NFAT-luc2P and CD16a F was screened with 400 ⁇ g/mL Hygromycin B and 0.2 ⁇ g/mL puromycin.
  • ADCC reporter assay 20 ⁇ L Daudi target cells in assay buffer containing 90%RPMI 1640 (Gibco, Cat#22400-089) and 10%FBS (Gibco, Cat#10099-141) were added to a 384-well assay plate (Corning, Cat#3570) , with final concentration of 10,000 cells/well. 10 ⁇ l/well of series dilutions of anti-CD38 antibodies were added to individual wells (30nM initiative solution was subjected to 3-fold gradient dilution, and total 9 series dilution solutions were generated) . Then 20 ⁇ l effector cells in assay buffer were added to each well, with final concentration of 50,000 cells/well.
  • the 384-well plate was incubated for 4 ⁇ 6 hours at 37°C, 5%CO 2 . After incubation, the plate was cooled to room temperature for 15 minutes, and then 30 ⁇ l/well of One-Glo TM Reagent (Promega, Cat#E6130) was added to each well. The plate was incubated for 5 minutes at room temperature, then read using Tecan F200 (Tecan, F200 Pro) luminescence mode. Curves and half maximal effective concentration (EC50) were analyzed using the Graphpad Prism software with the “Dose-response Stimulation ⁇ Log (agonist) vs. response-Variable slope (four parameters) ” model.
  • VHH180_2-Ab, VHH180_3-Ab, VHH194_2-Ab and VHH194_4-Ab were selected for ADCC function detecting, Daratumumab and Isatuximab used as positive control.
  • the results were shown in Figure 4 and suggested that all tested humanized anti-CD38 antibodies have comparable ADCC activity with Daratumumab and Isatuximab, in which ADCC function of VHH194_2-Ab and VHH194_4-Ab are relatively stronger than controls.
  • VHH180 and VHH194 to construct three different structures of bispecific antibodies: symmetrical, asymmetric, IgG-like symmetrical bispecific antibody, schematic diagram of these bispecific antibodies are shown in Figure 5.
  • each chain comprises two VHHs of anti-CD38 VHH (VHH1 and VHH2) fused via GGSGGS linker, the hinge, CH2 and CH3 domain of human IgG1.
  • chain 1 comprises VHH1 of anti-CD38 VHH, the hinge, CH2 and CH3 domain of human IgG1 (SEQ ID NO: 35 IgG1-Fc-Hole) with substitutions N384D, Q418E, N421D, Y407V, T366S, L368A and Y349C (numbering according to EU index)
  • chain 2 comprises VHH2 of anti-CD38 VHH, hinge, CH2 and CH3 domain of human IgG1 (SEQ ID NO: 34 IgG1-Fc-Knob) with substitutions T366W and S354C (numbering according to EU index) .
  • IgG-Like symmetrical bispecific antibody has two arms, each arm is consisted of two chains, chain 1, from N terminal to C terminal, comprises VHH1 of anti-CD38 VHH and human IgG1 constant region (i.e., CH1, hinge, CH2 and CH3 domain) , chain 2, from N terminal to C terminal, comprises VHH2 of anti-CD38 VHH, human kappa light chain.
  • Table 3A lists the composited sequence of three types of bispecific antibodies.
  • CDC Complement-Dependent Cytotoxicity Assay on Daudi cells (NCACC, Cat#TCHU140) were performed to evaluate the potency of anti-CD38 antibodies of the present application. Daudi cells were seeded at a density of 5,000 cells per well and were pre-incubated with each antibody at 300 nM, 30 nM and 3 nM for 30 min. Serum from a healthy donor was added into cells with 20%final concentration. The effect of Rituxan (SEQ ID NO: 3 &4) , Daratumumab (SEQ ID NO: 5 &6) and Isatuximab (SEQ ID NO: 7 &8) on Daudi CDC was used as an internal control to assure the assay quality.
  • Rituxan SEQ ID NO: 3 &4
  • Daratumumab SEQ ID NO: 5 &6
  • Isatuximab SEQ ID NO: 7 &8
  • %Cell lysis 100 ⁇ (1- (RLUsample) / (RLUcell+NHS) ) , in which NHS (Schbio biotech, Cat#PBSR005C) is used for normal human serum.
  • Results are recorded in Figure 7.
  • the results show that the symmetrical bispecific antibody VHH180_194 show the strongest ADCC effect than other biparatopic antibodies, its EC50 value is 0.031 nM.
  • ADCC-enhanced bispecific antibodies by mutating amino acid residues in Fc region of one or two chains of bispecific antibodies in table 3, which amino acid residues play a role in binding of Fc of antibodies to Fc receptors and are disclosed in the prior art.
  • this example carries out two amino acids substitution (S239D/I332E) in the Fc region of one or two chains of the antibody (numbering according to EU index) , such two substitutions can increase the affinity of Fc for Fc ⁇ RIIa and Fc ⁇ RIIIa.
  • Table 4 lists substituted bispecific antibodies in table 3.
  • ADCC activity of mutated bispecific antibodies in table 4 were detected following procedure disclosed in Example 2 section 5, except RPMI8226 (NCACC, Cat#TCHU234) as the target cell.
  • Results in Figure 8 shows that different structures also reflect different ADCC enhancement effects.
  • the biparatopic antibody 194-180SE showed the strongest ADCC effect, and 180-194SE, another ADCC-enhanced antibody showed similar ADCC effect of 194-180SE.
  • the EC50 values of these two antibodies are 0.008292 nM and 0.01065 nM, respectively.
  • the ADCC effects of the two antibodies are stronger than that of daratumumab and isatuximab.
  • the CDC effect of the mutants was also investigated according to procedure disclosed in Example 4 except 10%final concentration of human serum from the healthy donor. Results are shown in Figure 9. The results showed that the above ADCC enhancement modification would not have a substantial impact on the CDC effect of the biparatopic antibody. Compared with the ADCC enhancement modification, the CDC effect was basically unchanged; The EC50 values of 194-180SE and 180-194SE are 0.6197nM and 0.6737nM respectively, both are significantly stronger than the reference daratumumab and Isatuximab.
  • the inventors firstly humanized the constructed biparatopic antibodies of Example 3, that is to say, replacing Camelids VHH by corresponding humanized VHH, for example, replacing VHH180 by one of VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 and VHH180_6; replacing VHH194 by one of VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 and VHH194_6.
  • the obtained humanized biparatope antibody named by corresponding humanized VHH, for example symmetrical bispecific antibody 180-3_194-2 (SEQ ID NO: 51) .
  • mutant 180-3_194-2 which carries two amino acids substitution (S239D/I332E) in the Fc region of the antibody (SEQ ID NO:52) .
  • Daudi cells with endogenous high expression of CD38 were used as target cells, PBMC cells ( Cat#PBMNC025C) were used as effector cells.
  • 50 ⁇ l Daudi cells in 99%MEM- ⁇ (Gibco, Cat#41061-029) containing 1%FBS (Gibco, Cat#10099-141) assay buffer were added to a 96-well assay plate (Corning, Cat#3599) , with final concentration of 10,000 cells/well.
  • 50 ⁇ l/well of humanized biparatopic antibody (eight-point series dilutions started from 1 nM) or Daratumumab analog (5-fold series dilution, the final assay concentrations started from 1 nM) were added to cells.
  • PBMC effector cells in 99%MEM- ⁇ (Gibco, Cat#41061-029) containing 1%FBS (Gibco, Cat#10099-141) and 200 IU/ml human recombinant IL2 assay buffer were added to each well, with final concentration of 100,000 cells/well.
  • the 96-well plate was incubated for 24 hours at 37°C, 5%CO 2 . After incubation, the 96-well plate was removed and centrifuge at low speed (2000 rpm) for 3 min to allow the precipitation of cell pellet. 50 ⁇ L/well of supernatant was carefully aspirated and transferred to a new 96-well plate.
  • An equal volume of LDH cytotoxicity detection solution (Roche, Cat#11644793001) was added into the plate and incubated at room temperature for 30 min. The absorbance was read by Infinite F50 plate-reader at wavelength of 490nm.
  • ADCP reporter assay 20 ⁇ l Daudi target cells in 90%RPMI 1640 (Gibco, Cat#22400-089) containing 10%FBS (Gibco, Cat#10099-141) assay buffer were added to a 384-well assay plate (Corning, Cat#3570) , with final concentration of 10,000 cells/well. 10 ⁇ l/well of the nine-point series dilutions of humanized biparatopic antibody, Daratumumab analog or Isatuximab analog (5-fold series dilution, the final assay concentrations started from 50 nM) were added to cells.
  • Fc ⁇ IIa effector cell Jurkat cell line in 90%RPMI 1640 (Gibco, Cat#22400-089) containing 10%FBS (Gibco, Cat#10099-141) assay buffer were added to each well, with final concentration of 50,000 cells/well.
  • the 384-well plate was incubated for 4 ⁇ 6 hours at 37°C, 5%CO 2 . After incubation, the plate was cooled to room temperature for 15 minutes, and 30 ⁇ l/well of One-Glo TM Reagent (Promega, Cat#E6130) was added. The plate was incubated for 5 minutes at room temperature, then read using Tecan F200 (Tecan, F200 Pro) luminescence mode.

Abstract

Provided are humanized anti CD38 antibodies (including biparatopic antibodies), methods for their production, pharmaceutical compositions containing these antibodies, and uses thereof.

Description

ANTIBODIES BINDING CD38 AND USES THEREOF
This application claims priority to PCT International Patent Application Serial No. PCT/CN2021/135423, filed on December 3, 2021, which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
The present invention relates to humanized anti CD38 antibodies (including biparatopic antibodies) , methods for their production, pharmaceutical compositions containing these antibodies, and uses thereof.
BACKGROUND OF THE INVENTION
CD38 was first identified in 1980 as a membrane-bound protein of 45 kDa [1] . It was described as a single-chain transmembrane type II glycoprotein encoded by a gene mapped on chromosome 4 (4p15) [2] . CD38 includes a long C-terminal extracellular domain (258 amino acids, aa) , a transmembrane segment (21 aa) and a short N-terminal cytoplasmic tail (21 aa) .
CD38 expression in the immune system varies during lymphocyte development, activation and differentiation. Indeed, human CD38 is highly expressed on medullary thymocytes, downregulated in the majority of circulating mature T cells and up-regulated upon activation of T cells. CD38 is expressed on immunosuppressive cells, such as T regulatory cells, B regulatory cells, and myeloid-derived suppressor cells. In solid tissues, the protein is expressed by epithelial cells in the prostate, beta-cells in pancreas, Purkinje cells and neurofibrillary tangles in brain, muscle cells, renal tubules, retinal gangliar cells and corneal cells in the eye [3] .
CD38 performs as a marker of ontogenesis in B lymphocytes, and more importantly, it is highly expressed on normal plasma cells as well as on plasma cells derived from patients affected by Multiple myeloma (MM) . Indeed, >90%of the malignant plasma cells from patients with MM show surface expression of CD38 [4] .
The broad expression of CD38 may discourage the development of immunotherapies directed against this molecule; however, the analysis of CD38 expression levels across different cellular populations clearly shows that normal and malignant plasma cells express the highest levels of CD38 followed by NK cells and other B and T cell subpopulations. [4]
CD38 is functionally pleiotropic, working as an ectoenzyme and as a receptor simultaneously. The identification of CD31 (also known as PECAM-1) as a non-substrate specific ligand was key in recognizing CD38 as a receptor. The CD38/CD31 crosstalk is an important step in the regulation of cytoplasmic calcium fluxes and secretion of cytokines such as IL-6 and IL-10. This interaction probably regulates the migration of leukocytes and CD38 positive cancer cells through the endothelial cell wall [5] .
Recently reports show that CD38 was described as part of the leukocyte ectonucleotidases family, characterized by two main substrates: NAD+ and NADP+. These two substrates were  converted by CD38 and following enzymes, such as CD39 and CD73, to adenosine, that was widely recognized as an immunosuppressive effector [6] .
Due to the above characteristics, CD38 protein can be used as a unique therapeutic target for malignant plasma cell tumors. However, the mechanism of action of anti-CD38 agents does not rely only on CD38 expression on tumor cells, but also rely on mAbs that have Fc effect, such as antibody-dependent cellular cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , and CDC. These Fc functions of therapeutic antibody depend on NK cells, macrophages, and the complement system, respectively. Therefore, the combination of immunomodulators and anti-CD38 antibodies can significantly improve the therapeutic effect. [7]
Daratumumab is a human IgG1, κ mAb that specifically binds a unique epitope on the CD38 molecule. It can induce CDC of CD38-overexpressing cell lines, as well as patient-derived MM cells in vitro. While in xenograft models, Daratumumab interrupted tumor growth at very low doses. Binding of daratumumab to CD38 on the surface of tumor cells may induce complement activation, CDC, ADCC, ADCP , programmed cell death (PCD) , and modulation of CD38 enzyme activity. [8]
The FDA approved daratumumab as a fourth-line therapy for patients with relapsed/refractory multiple myeloma in 2015. It was the world’s first monoclonal antibody drug approved for the treatment of multiple myeloma. In 2019, it was approved by the NMPA to become the first approved monoclonal antibody drug for the treatment of multiple myeloma in China.
Isatuximab, also known as SAR650984, is another anti-CD38 antibody with potent proapoptotic activity, which is probably related to its binding to a specific epitope on the CD38 molecule. Isatuximab has Fc-dependent effector functions including CDC, ADCP and ADCC [9] . Furthermore, isatuximab induced apoptosis without the use of crosslinking agents. In addition, isatuximab was shown to trigger lysosomal cell death, another mechanism of cell killing, in MM cell lines. Pomalidomide will enhance this lysosomal killing effect in MM cell lines. [10]
MOR202 is another antibody that targets the CD38 molecule and induce MM cell killing via CDC, ADCC and ADCP. In the clinical trail phase I/II with relapsed/refractory MM patients MOR202 was well tolerated at doses up to 16 mg/kg with the maximum tolerated dose not reached. It combined with low-dose dexamethasone resulted in a OR or better in 29%of patients [11] .
Nanobodies are soluble single domain antibody fragment derived from the variable domain of heavy chain antibodies (VHH) naturally occurring in camelids. Bispecific/biparatopic antibodies was obtained by fusion two anti-CD38 VHH domains binding different epitope on CD38 into one human IgG-like structure, these bispecific/biparatopic antibodies showed enhancement of CDC effects to CD38 expressing cells [12] . As described above, the CDC effects is critical for the efficacy of anti-CD38 antibodies.
Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcγ receptors with both activating and inhibitory activities [13] . Especially in the treatment of hematological tumors, the ADCC effect is one of the most important MOA of daratumumab and isatuximab for MM. Therefore, improving the ADCC effect through engineering is an important step in the development of therapeutic antibodies against MM.
We sought to exploit the nanobodies to construct highly soluble biparatopic nanobody-based bispecific antibody and enhanced ADCC effect by the amino-acid mutation. Remarkably, these biparatopic antibody show higher CDC and ADCC potency than daratumumab and therefore hold promise as novel therapeutics for the treatment of multiple myeloma.
SUMMARY OF THE INVENTION
Herein are provided humanized anti-CD38 antibodies (including bispecific antibodies) which are useful as a therapeutic agent for treatment of multiple myeloma and other hematological tumors.
The present invention relates to antibodies binding to CD38, which derived from anti CD38 VHH in camelids. In one embodiment, the anti CD38 antibodies are humanized. In another embodiment, the anti CD38 antibodies are biparatopic antibodies or antigen-binding fragment thereof, or are humanized biparatopic antibodies or antigen-binding fragment thereof. In one embodiment, the CD38 is derived from human or cynomolgus. In another embodiment, the CD38 comprises SEQ ID NO: 1 or SEQ ID NO: 2.
In the first aspect, the present invention relates to an anti-CD38 VHH, which comprises three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3.
In an embodiment, the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3 which is included in SEQ ID NO: 9 or SEQ ID NO: 10.
In an embodiment, the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein:
1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 39, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 41, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 43; or
2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 45, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 47, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 49,
wherein the CDRs sequences are defined according to Kabat scheme.
In another embodiment, the anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein:
1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 40, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 42, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 44; or
2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 46, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 48, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 50,
wherein the CDRs sequences are defined according to IMGT scheme.
In a further embodiment, the anti-CD38 VHH comprises an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10, or the VHH consists of amino acid sequences as shown in SEQ ID NO: 9 or SEQ ID NO: 10.
In the Second aspect, the present invention relates to a humanized anti-CD38 VHH, which comprises three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3.
In an embodiment, the humanized anti-CD38 VHH nanobody comprises HCDR1, HCDR2 and HCDR3 which is included in SEQ ID NO: 9 or SEQ ID NO: 10.
In an embodiment, the humanized anti-CD38 VHH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise or consist of 1) amino acid sequences as shown in SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively; or 2) amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, respectively; or 3) amino acid sequences as shown in SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; or 4) amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.
In another embodiment, the humanized anti-CD38 VHH comprises back-mutations in human framework region.
In a further embodiment, the humanized anti-CD38 VHH nanobody comprises an amino acid sequence as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22, or the VHH consists of amino acid sequences as shown in any one of SEQ ID NO: 11-16 or any one of SEQ ID NO: 17-22.
In the Third aspect, the present invention relates to an anti-CD38 heavy chain antibody (HcAb) or an antigen-binding fragment thereof, which comprises the humanized anti-CD38 VHH or anti-CD38 VHH described in the above aspects.
In an embodiment, the anti-CD38 heavy chain antibody or the antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3,
I. according to Kabat scheme, the CDRs sequences are defined, wherein
1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 39, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 41, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 43; or
2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 45, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 47, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 49,
II. according to IMGT scheme, the CDRs sequences are defined, wherein
1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 40, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 42, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 44; or
2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 46, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 48, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 50.
In another embodiment, the anti-CD38 heavy chain antibody and the antigen-binding fragment thereof comprises VHH as described in the above aspects. In yet another embodiment, the anti-CD38 heavy chain antibody and the antigen-binding fragment thereof comprises VHH comprising an  amino acid sequence as shown in any one of SEQ ID NO: 9-22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 9-22, or consisting of amino acid sequences as shown in any one of SEQ ID NO: 9-22.
In another embodiment, the present invention relates to an anti-CD38 heavy chain antibody or the antigen-binding fragment thereof, which comprises:
(1) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 9 and having the same CDRs as SEQ ID NO: 9, or consisting of SEQ ID NO: 9;
(2) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 10 and having the same CDRs as SEQ ID NO: 10, or consisting of SEQ ID NO: 10;
(3) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 11 and having the same CDRs as SEQ ID NO: 11, or consisting of SEQ ID NO: 11;
(4) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 12 and having the same CDRs as SEQ ID NO: 12, or consisting of SEQ ID NO: 12;
(5) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 13 and having the same CDRs as SEQ ID NO: 13, or consisting of SEQ ID NO: 13;
(6) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 14 and having the same CDRs as SEQ ID NO: 14, or consisting of SEQ ID NO: 14;
(7) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 15 and having the same CDRs as SEQ ID NO: 15, or consisting of SEQ ID NO: 15;
(8) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 16 and having the same CDRs as SEQ ID NO: 16, or consisting of SEQ ID NO: 16;
(9) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 17 and having the same CDRs as SEQ ID NO: 17, or consisting of SEQ ID NO: 17;
(10) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 18 and having the same CDRs as SEQ ID NO: 18, or consisting of SEQ ID NO: 18;
(11) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 19 and having the same CDRs as SEQ ID NO: 19, or consisting of SEQ ID NO: 19;
(12) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 20 and having the same CDRs as SEQ ID NO: 20, or consisting of SEQ ID NO: 20;
(13) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 21 and having the same CDRs as SEQ ID NO: 21, or consisting of SEQ ID NO: 21; or
(14) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 22 and having the same CDRs as SEQ ID NO: 22, or consisting of SEQ ID NO: 22.
In a further embodiment, the anti-CD38 heavy chain antibody or the antigen-binding fragment thereof further comprises Fc region.
In one embodiment, the Fc region is or is derived from human IgG constant region, e.g., IgG1, IgG2, IgG3 or IgG4, preferable IgG1 constant region.
In an embodiment, the anti-CD38 antibody and the antigen-binding fragment thereof comprises IgG Fc. In another embodiment, the IgG Fc is IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc. In another embodiment, the IgG Fc comprises amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 23, or the IgG Fc consists of SEQ ID NO: 23. In another embodiment, the IgG Fc comprises mutation, for example the mutation is amino acids substitution, deletion, and addition. In a further embodiment, the mutation can improve ADCC, ADCP effect. In a specific embodiment, the mutation is one or more amino acids substitution, for example S239D and/or I332E (EU numbering) substitution. In yet another embodiment, the IgG Fc comprises knob mutation (s) or hole mutation (s) , for example, IgG Fc comprises amino acid sequence as shown in SEQ ID NO: 34 or 35; or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 34 or 35; or IgG Fc consists of SEQ ID NO: 34 or 35.
In the Fourth aspect, the anti-CD38 antibody of the present invention is a bispecific antibody, preferablely a biparatopic antibody. In an embodiment, the biparatopic anti-CD38 antibody comprises the humanized anti-CD38 VHH or anti-CD38 VHH in previous aspects.
In an embodiment, the bispecific anti-CD38 antibody is selected from:
1) symmetrical bispecific antibody consisting of two same chains: from N terminal to C terminal, each chain comprises two anti-CD38 VHHs (VHH1 and VHH2) , the hinge, CH2 and CH3 domain;
2) asymmetric bispecific antibody consisting of two different chains: from N terminal to C terminal, chain 1 comprises anti-CD38 VHH1, the hinge, CH2 and CH3 domain, chain 2 comprises anti-CD38 VHH2, hinge, CH2 and CH3 domain; or
3) IgG-Like symmetrical bispecific antibody having two arms, each arm consists of two chains, from N terminal to C terminal, chain 1 comprises anti-CD38 VHH1 and heavy chain constant region (i.e., CH1, hinge, CH2 and CH3 domain) , chain 2 comprises anti-CD38 VHH2 and light chain constant region,
wherein VHH1 is different from VHH2.
In another embodiment, VHH1 and VHH2 in the symmetrical bispecific antibody are linked by a linker, wherein the linker can be any well-known linker in the art, it is generally a flexible short peptide sequence, for example, (G 4S) n, where n is an integer number, for example, n is 1, 2, 3, 4, 5, 6,  7, 8, 9; GGGSG; GGSGG; GSGGG; SGGGG; GGGTS; GTSPGG; GNGGGS; G4S-GGSGG-G4S-SGGGG; GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; KESGSVSGSSE QLAQFRSLD; GGRRGGSLRQQ. In an embodiment, the linker is GGSGGS (SEQ ID NO: 53) .
In another embodiment, the VHH of the bispecific antibody is derived from Camelid’s anti-CD38 VHHs, or is derived from humanized anti-CD38 VHHs. In a further embodiment, the VHH is any one of the VHH described in any one of previous aspects.
In an embodiment, heavy constant region of the bispecific antibody is derived from IgG1, IgG2, IgG3 or IgG4, preferably derived from IgG1 isotype. In another embodiment, light chain constant region is derived from κ light chain or λ light chain.
In another embodiment, Fc region of the bispecific antibody have mutations, for example substitution, deletion and addition of one or more amino acid residues. In an embodiment, the Fc region comprises mutations improving ADCC effect and/or ADCP effect of the bispecific antibody. In a further embodiment, the Fc region comprises mutations S239D/I332E (according to EU numbering) .
In a further embodiment, Fc region of the bispecific antibody has knob-in-hole mutation. In still another embodiment, the Fc region comprise substitutions N384D, Q418E, N421D, Y407V, T366S, L368A and Y349C (according to EU numbering) . In still another embodiment, the Fc region comprise substitutions T366W and S354C (according to EU numbering) .
In an embodiment, the bispecific antibody comprises two set of HCDR1, HCDR2 and HCDR3, the first set of HCDR1, HCDR2 and HCDR3 comprise 3 HCDRs contained in SEQ ID NO: 9, and the second set of HCDR1, HCDR2 and HCDR3 comprise 3 HCDRs contained in SEQ ID NO: 10. For example, the first set of HCDR1, HCDR2 and HCDR3 comprise or consist of 1) amino acid sequences as shown in SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively; or 2) amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, respectively, and the second set of HCDR1, HCDR2 and HCDR3 comprise or consist of 3) amino acid sequences as shown in SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; or 4) amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.
In another embodiment, the bispecific antibody comprises VHH1 and VHH2, when VHH1 comprises the first set of HCDR1, HCDR2 and HCDR3, then VHH2 comprises the second set of HCDR1, HCDR2 and HCDR3, vice versa.
In a further embodiment, the bispecific antibody comprises VHH1 and VHH2 selected from SEQ ID NO: 9 or SEQ ID NO: 10, wherein VHH1 and VHH2 is selected different sequence.
In another embodiment, the bispecific antibody comprises humanized VHH1 and VHH2 of the present invention, wherein the humanized VHH1 and VHH2 is independently from each other selected from humanized VHH180 (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) and humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) , provided that humanized VHH1 differs from VHH2.
In a further embodiment, the bispecific antibody comprises humanized VHH1 and VHH2 of the present application, wherein when the humanized VHH1 is selected from humanized VHH180  (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) , then VHH2 is selected from humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) , vice versa.
In another embodiment, the bispecific antibody is selected from:
1) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprise or consist of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;
2) an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 36 and another chain comprises or consists of SEQ ID NO: 37; or
3) an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 38 and the chain 2 comprises or consists of SEQ ID NO: 30.
In another embodiment, the bispecific antibody comprises mutations improving ADCC effect and/or ADCP effect and is selected from:
1) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprise or consist of SEQ ID NO: 31 or SEQ ID NO: 32;
2) an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 28 and another chain comprises or consists of SEQ ID NO: 29; or
3) an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 33 and the chain 2 comprises or consists of SEQ ID NO: 30.
In another embodiment, the bispecific antibodies are humanized bispecific antibodies, wherein the camelids VHHs had been replaced by corresponding humanized VHHs disclosed above. In a specific embodiment, the humanized bispecific antibody has two same chains comprising amino acid sequence as shown in SEQ ID NO: 51.
In another embodiment, the humanized bispecific antibody comprises mutations improving ADCC effect and/or ADCP effect. In a further embodiment, the humanized bispecific antibody comprises a Fc region comprising mutations S239D/I332E (numbering according to EU index) . In a specific embodiment, the humanized bispecific antibody has two same chains comprising an amino acid sequence as shown in SEQ ID NO: 52.
In one embodiment of any one of the above aspects, the antigen-binding fragment of the present invention is an antibody fragment comprising Fab, Fab’, Fab’-SH, Fv, single chain antibody (e.g., scFv) , (Fab’)  2, single domain antibody (e.g., VHH) , domain antibody (dAb) or linear antibody.
In the Fifth aspect, the present invention relates to an isolated nucleic acid encoding anyone or more chains of anti-CD38 antibody of the above aspects.
In the Sixth aspect, the present invention relates to a vector comprising the nucleic acid of the Fifth aspect. In one embodiment, the vector is an expression vector.
In the Seventh aspect, the present invention relates to a host cell comprising the nucleic acid of the Fifth aspect or the vector of the Sixth aspect. Preferably the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., CHO cells or CHO-S cells or 293  cells or 293T cells) , or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof.
In the Eighth aspect, the present invention relates to a method of preparing an anti-CD38 antibody or the antigen-binding fragment thereof comprising culturing the host cell of the Seventh aspect, under conditions suitable for expression of a nucleic acid encoding an anti-CD38 antibody or the antigen-binding fragment thereof of the present invention, optionally said method further comprises recovering the anti-CD38 antibody or the antigen-binding fragment thereof from the host cell.
In the Ninth aspect, the present invention relates to a pharmaceutical composition comprising the anti-CD38 antibody or the antigen-binding fragment thereof according to the present invention, and optionally pharmaceutically acceptable adjuvant (s) .
In the Tenth aspect, the present invention relates to a combination product, comprising the anti-CD38 antibody or the antigen-binding fragment thereof of the present invention and one or more other therapeutic agents, e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
In the Eleventh aspect, the present invention relates to a method of treating cancer in a subject comprising administering to said subject the anti-CD38 antibody or the antigen-binding fragment thereof, or the pharmaceutical composition, or the combination product of the present invention.
In one embodiment, the cancer is that with abnormal level of CD38 (e.g., the high protein or nucleic acid level of CD38) . In another embodiment, said cancer is hematological tumors, malignant plasma cell tumors, multiple myeloma, relapsed/refractory multiple myeloma.
In one embodiment, the method of the present invention further comprises administering to said subject in combination with one or more therapies, e.g., a therapeutic modality and/or other therapeutic agent, preferably the therapeutic modality includes surgery and/or radiation therapy, and/or the other therapeutic agent is selected from the group consisting of a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immune-regulating agent.
In the Twelfth aspect, the present invention relates to a use of the anti-CD38 antibody or the antigen-binding fragment thereof, or the pharmaceutical composition, or the combination product of the present invention in the manufacture of a medicament for treating cancer in a subject.
In one embodiment, the cancer is that with abnormal level of CD38 (e.g., the high protein or nucleic acid level of CD38) . In another embodiment, said cancer is hematological tumors, malignant plasma cell tumors, multiple myeloma, relapsed/refractory multiple myeloma.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows humanized anti-human CD38 heavy chain antibodies bound to human CD38 protein, in which EC50 and representative binding curves of each anti-human CD38 antibodies comprising corresponding VHH in table 1 were show. In figure 1, VHH180 represents Camelids heavy chain antibody VHH180-Ab, VHH194 represents Camelids heavy chain antibody VHH194- Ab, VHH180_n represents humanized heavy chain antibody VHH180_n-Ab, for example, VHH180_1 represents humanized heavy chain antibody VHH180_1-Ab, VHH194_n represents humanized heavy chain antibody VHH194_n-Ab , for example, VHH194_1 represents humanized heavy chain antibody VHH194_1-Ab.
Figure 2 shows humanized heavy chain antibodies of VHH180 and VHH194 bound to human CD38 on cell surface, VHH180 represents Camelids heavy chain antibody VHH180-Ab, VHH194 represents Camelids heavy chain antibody VHH194-Ab, VHH180_n represents humanized heavy chain antibody VHH180_n-Ab, VHH194_n represents humanized heavy chain antibody VHH194_n-Ab .
Figure 3 shows epitope binning of two anti-human CD38 heavy chain antibodies. VHH180_3 and VHH194_2 represent humanized anti-human CD38 antibodies VHH180_3-Ab and VHH194_2-Ab, respectively; B-180-3 and B-194-2 represent biotinylated humanized anti-human CD38 antibodies VHH180_3-Ab and VHH194_2-Ab, respectively.
Figure 4 shows ADCC activity of anti-CD38 antibodies, in which VHH180_2, VHH180_3, VHH194_2 and VHH194_4 represent VHH180_2-Ab, VHH180_3-Ab, VHH194_2-Ab and VHH194_4-Ab, respectively.
Figure 5 shows schematic diagram of three biparatopic antibody structures.
Figure 6 shows CDC activity of different structure of biparatopic antibodies on Daudi cells.
Figure 7 shows ADCC activity of different structure of biparatopic antibodies on Daudi cells.
Figure 8 shows ADCC activity of ADCC-enhancement mutants of biparatopic antibodies.
Figure 9 shows CDC activity of ADCC-enhancement mutant of biparatopic antibodies.
Figure 10 shows ADCC activity of ADCC-enhancement mutant of humanized biparatopic antibodies.
Figure 11 shows ADCP activity of ADCC-enhancement mutant of humanized biparatopic antibodies
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
For the purpose of interpreting the specification, the following definitions will be used, and the terms used in the singular may also include the plural, and vice versa, if appropriate. It is understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be restrictive.
The term “and/or” where used herein is to be taken as specific disclosure of each of the two or more specified features or components with or without the other. Thus, the term “and/or” as used in a phrase, such as “A and/or B” herein, is intended to include “A and B” , “A or B” , “A” (alone) , and “B” (alone) .
It is understood that wherever aspects are described herein with the language “comprising, ” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.
The term “about” and “approximately” when used in connection with a numerical value is meant to encompass numerical values within the range between the lower limit of 10%or 5%less than the specified numerical value and the upper limit of 10%or 5%greater than the specified numerical value.
The term “antibody” broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , heavy chain antibodies, nanobodies and antibody fragments so long as they exhibit the desired antigen-binding activity.
The term “anti-CD38 antibody” , “anti-CD38” , “CD38 antibody” or “antibody binding to CD38” as used herein refers to an antibody which is capable of binding to (e.g., human or Cynomolgus) CD38 protein or a fragment thereof with sufficient affinity such that the antibody can be used as diagnostic and/or therapeutic agent in targeting (e.g., human or Cynomolgus) CD38. In one embodiment, the extent of binding of an anti-CD38 antibody to an unrelated, non-CD38 protein is less than about 10 %of the binding of the antibody to CD38 as measured, for example, by Surface Plasmon Resonance. In certain embodiments, an antibody that specifically binds to human CD38 has a dissociation constant (K D) of 10 -8 M or less.
Term “heavy chain antibody (HcAb) ” means a kind of antibody that naturally lacks light chain and consists of two heavy chains, each heavy chain generally comprises heavy chain variable region (VHH) , hinge region, CH2 and CH3 of heavy chain constant region.
Term “VHH” and term “nanobody” can be used interchangeably, and refer to heavy chain variable region of HcAb, from N terminal to C terminal, comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
A “humanized” antibody refers to an antibody comprising human framework region (FR) and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95%or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs and few residues in the heavy chain constant region if modulation of the effector functions is needed, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A “humanized” VHH refers to a VHH comprising amino acid residues derived from non-human CDRs and amino acid residues from human FRs. In some instances, humanized antibodies may retain non-human residues within the human framework regions to enhance proper binding characteristics and/or some amino acid mutations may be introduced within the CDRs in order to improve the binding affinity and/or to reduce the immunogenicity and/or to increase the degree of humanness.  For example, to restore the binding activity of antibody and/or VHH, some residues in human framework should be back-mutations to the original non-human sequence.
An “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody, include but are not limited to Fv, Fab, Fab', Fab'-SH, F (ab') 2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv) ; single domain antibody; and multispecific antibodies formed from antibody fragments.
As used herein, the term “epitope” refers to a portion of an antigen (e.g., CD38) that specifically interacts with an antibody molecule. This portion (referred to herein as an epitope determinant) typically comprises an element such as an amino acid side chain or a sugar side chain or a component thereof. Epitope determinants can be defined according to methods known in the art or disclosed herein (e.g., by crystallography or by hydrogen-deuterium exchange) . Some epitopes are linear epitopes, while others are conformational epitopes.
A “multispecific antibody” refers an antibody that has binding specificities for at least two different epitopes on the same antigen or different antigens.
In the context of the present application, term “bispecific antibody” , “anti-CD38 bispecific antibody” , “biparatopic antibody” , “biparatope antibody” , “anti-CD38 biparatopic antibody” and similar expressions can be used interchangeably and refer to an anti-CD38 antibody having binding specificities for two different epitopes of CD38, in which each antigen binding domain recognizes unique, non-overlapping epitopes of CD38.
An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%or 95%or more of the binding of the reference antibody to its antigen in a competition assay.
An antibody that inhibits (e.g., competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits binding of 50%, 60%, 70%, 80%, 90%, or 95%or more of the reference antibody to its antigen.
"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ( "hypervariable loop" ) and/or comprises antigen-contacting residues ( "antigen contact point" ) . CDRs are primarily responsible for binding to epitopes. The CDRs of the heavy or light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chains are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chains are referred to as LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of a light chain variable region or a heavy chain variable region, the exact amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins" , Journal of Molecular Biology, 273: 927-948 (1997) ) , Kabat  based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987) ) , AbM (University of Bath) , Contact (University College London) , International ImMunoGeneTics database (IMGT) (imgt. cines. fr/on the World Wide Web) , and North CDR definition based on the affinity propagation clustering using a large number of crystal structures.
For example, according to different CDR determination schemes, the residues of each CDR are as follows.
Figure PCTCN2022135904-appb-000001
CDRs can also be determined based on having the same Kabat numbering positions as a reference CDR sequence (e.g., any of the exemplary CDRs of the invention) .
Unless otherwise stated, in the invention, the term “CDR” or “CDR sequence” encompasses CDR sequences determined by any of the manners described above.
Unless otherwise stated, in the invention, when referring to the position of residues in an antibody variable region (including heavy chain variable region residues and light chain variable region residues) , it refers to the numbering positions according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ) .
In one embodiment, the boundaries of the CDRs of the antibodies of the invention are determined by Kabat scheme or IMGT scheme.
It should be noted that boundaries of CDRs of variable regions of an antibody obtained by different assignment systems may differ. That is, CDR sequences of variable regions of an antibody defined by different assignment systems differ. Therefore, when it comes to defining an antibody with specific CDR sequences defined in the invention, the scope of the antibody also encompasses such antibody whose variable region sequences comprise the specific CDR sequences, but having claimed CDR boundaries different from the specific CDR boundaries defined by the invention as a different protocol (e.g., different assignment system rules or their combinations) is applied.
Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system) . However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The smallest overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thereby providing a “minimal binding unit” for antigen binding. The minimal binding unit may be a sub-portion of the CDR. As will be clear to those skilled in the art, residues of the rest CDR sequences can be determined by antibody structure and protein folding. Therefore, any variants of the CDRs given herein will also be considered in the invention. For example, in one CDR variant, the amino acid residues in the minimal binding unit may remain unchanged, while the other CDR residues defined by Kabat or Chothia may be substituted by conservative amino acid residues.
There are five major classes of antibodies known in the art: IgA, IgD, IgE, IgG and IgM, and several of these antibodies can be further divided into subclasses (isotypes) , for example, IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. A person skilled in the art can select and obtain the antibody in an appropriate class of the present invention according to the practical desire.
“Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) . The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K D) . Affinity can be measured by common methods known in the art, including those described herein.
The term “antibody-dependent cellular cytotoxicity (ADCC) ” is a function mediated by Fc receptor binding and refers to lysis of target cells by an antibody as reported herein in the presence of effector cells.
The term “complement-dependent cytotoxicity (CDC) ” refers to lysis of cells induced by the antibody as reported herein in the presence of complement.
The term “antibody-dependent cellular phagocytosis (ADCP) ” refers to a cellular response in which an antibody that binds to a target cell binds to a corresponding receptor on the surface of a macrophage to induce activation of the macrophage, thereby the target cell is internalized and degraded.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) , NIH Publication 91-3242.
An “isolated” nucleic acid refers to a nucleic acid molecule which has been separated from components of its natural environment. The isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
The term “vector” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .
The terms “host cell” refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells” , which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared.
When percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. The calculation relative to the full length of the longer sequence applies to both the nucleic acid sequence and the polypeptide sequence.
The term “pharmaceutical composition” refers to a formulation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
The term “pharmaceutically acceptable adjuvants” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete) ) , excipient or vehicle co-administered with the therapeutic agent.
As used herein, “treatment” (or “treat” or “treating” ) refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
The term “therapeutic agent” as described herein encompasses any substance effective in preventing or treating tumors (such as cancer) and infections, including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies (e.g., antibodies against to the immune checkpoint molecule) , active anti-infective agents, immunomodulators, small entities.
The term “combination product” refers to a fixed or non-fixed combination of dosage unit forms or a kit of parts for combined administration in which two or more therapeutic agents can be administered independently at the same time or administered separately within a time interval, especially when these time intervals allow the combined partner to demonstrate collaboration, for example, synergistic effects. The term “fixed combination” means that the antibody of the invention and the combination partner (e.g., other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are administered to a patient simultaneously in the form of a single entity or dose. The term “non-fixed combination” means that the antibodies and combination partners of the present invention (e.g., other therapeutic agents, such as immunomodulators, such as immunosuppressive agents or anti-inflammatory agents) are administered to patients simultaneously, concurrently, or sequentially as separate entities, and there is no specific time limitation, where such administration provides therapeutically effective levels of the two compounds in the patient. The latter also applies to cocktail therapy, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.
The terms “cancer” , “cancerous” and “tumor” can be used interchangeably and refer to or describe a physiological disease in mammals that is typically characterized by unregulated cell growth.
The term “effective amount” refers to an amount or dosage of the antibody or fragment or conjugate or composition of the invention which generates expected effects in a patient in need of treatment or prevention after administered to the patient in a single or multiple doses. The effective amount can be easily determined by an attending physician as a person skilled in the art by considering a variety of factors as follows: species such as mammals; its size, age, and general health; the specific disease involved; the extent or severity of the disease; response in an individual patient; specific antibody administered; route of administration; bioavailability characteristics of the administered formulation; selected dose regimen; and use of any concomitant therapy.
“Therapeutically effective amount” refers to an amount effective to achieve a desired therapeutic outcome at a required dosage for a desired period of time. The therapeutically effective amount of an antibody or an antibody fragment, or conjugate or composition thereof can vary depending on a variety of factors such as morbid state, age, sex, and weight of an individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. The therapeutically effective amount is also such an amount in which any toxic or undesired effect of the antibody or antibody fragment, or conjugate or composition thereof is inferior to the therapeutically beneficial effect. “Therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80%or 90%, relative to untreated subjects.
“Individual” or “subject” includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, goat, cat, dog, and horse) , primates (e.g., human and non-human primates such as  monkey) , rabbit, and rodents (e.g., mouse and rat) . In some embodiments, the individual or subject is human.
As used herein, the term “administering” , “administration” or “administered” refers to the physical introduction of a composition comprising a therapeutic agent (e.g., anti-CD38 antibody) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
Antibody of the Invention
In one embodiment of the invention, the amino acid change described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid change described herein is amino acid substitution, for example conservative substitution.
In one embodiment, the present application provides a Camelids VHH, a humanized VHH, or an antibody comprising the Camelids VHH or the humanized VHH, wherein the above said molecules comprise the following HCDRs:
Figure PCTCN2022135904-appb-000002
In a preferred embodiment, the amino acid change described herein occurs in region (s) outside the CDRs (e.g., in FRs) . In one embodiment, the amino acid change occurs in FR region (s) of the VHH or VH, e.g., in FR1, FR2, FR3 and/or FR4 of the VHH or VH.
In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to a replacement of an amino acid by another amino acid of the same class, e.g., an acidic amino acid replacement by another acidic amino acid, a basic amino acid replacement by another basic amino acid, or a neutral amino acid replacement by another neutral amino acid. Exemplary substitutions are shown in Table below:
Figure PCTCN2022135904-appb-000003
Figure PCTCN2022135904-appb-000004
In certain embodiments, one or more amino acid modifications can be introduced into an Fc region of an antibody provided herein, thereby producing an Fc region variant such that, for example, the efficacy of the antibody in treating cancer or a cell proliferative disease is enhanced. For example, modification can be incorporated to increase the ADCC and/or ADCP function of the antibody.
Nucleic acid of the invention and host cell comprising same
In one aspect, the invention provides a nucleic acid encoding any of the above anti-CD38 antibodies or fragments thereof. The nucleic acid can encode an amino acid sequence comprising the light chain variable region and/or the heavy chain variable region of the antibody, or an amino acid sequence comprising the light chain and/or the heavy chain of the antibody.
In one embodiment, one or more vectors containing anyone of the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as an eukaryotic expression vector. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC) . Numerous vector systems can be used. In a preferred embodiment, the expression vector of the present invention is a pcDNA, e.g., pcDNA3.1 expression vector.
In one embodiment, the present invention provides a host cell containing a nucleic acid encoding the region of antibody described herein or the vector described herein. Suitable host cells for cloning or expressing the nucleic acid encoding the antibody or the vector include prokaryotic or eukaryotic cells as described herein. The antibody can be produced, for example, in bacteria. After expression, the  antibody can be isolated from bacterial paste in soluble fraction and can be further purified. In one embodiment, the host cell is E. coli.
In another embodiment, the host cell is eukaryotic. The host cell can be selected from the group consisting of yeasts, mammalian cells (e.g., a human cell) , insect cells, plant cells, or other cells suitable for preparation of an antibody or an antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the vector encoding the antibody, including fungus and yeast strains. Examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40, human embryonic kidney lines (293HEK or 293 or 293T cells) and the like. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, and myeloma cell lines such as Y0, NS0, and Sp2/0.
Method for preparing and purifying antibody or antigen-binding fragment thereof of the present invention
In one embodiment, the present invention provides a method for preparing an anti-CD38 antibody, wherein the method comprises steps of incubating host cells containing an nucleic acid encoding the antibody or antigen binding fragment of the present invention (anyone or more regions or chains of the antibody) , under conditions suitable for expressing antibodies or the antigen binding fragments, and optionally, recovering the antibody or the fragments from the host cells (or the host cell cultures) .
For recombinant production of the antibody, nucleic acids encoding the antibody (e.g., the nucleic acids encoding VH or VL or the heavy chain or the light chain or chains of the bispecific antibodies) are isolated and inserted into one or more vectors for further cloning and/or expression in the host cells. The nucleic acid is readily isolated and sequenced by using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding heavy and light chains of antibodies) .
Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., protein A) , size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibody molecule of the present invention can be determined by any of a variety of well-known analysis methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
Pharmaceutical composition, formulation or combination product
The invention further provides a pharmaceutical composition, or a pharmaceutical formulation, or combination product comprising an anti-CD38 antibody or antigen-binding thereof, or the nucleic acid encoding the anti-CD38 antibody or the fragment thereof.
Such composition, formulation or combination product may further contain suitable pharmaceutical adjuvants such as a pharmaceutical carrier, an excipient, and the like known in the art, including buffers.
The pharmaceutical carrier suitable for use in the invention can be sterile liquid, such as water and oil, including petroleum, or oil of animal, vegetable, or synthetic source, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly used for injectable solutions.
The compositions may take the form of a solution, a suspension, an emulsion, a tablet, a pill, a capsule, a powder, a sustained release preparation, and the like.
The pharmaceutical composition or formulation of the invention may also contain more than one active ingredient required by a particular indication to be treated, preferably active ingredients having complementary activities without adversely affecting one another.
The present invention further provides a combination product, comprising the anti-CD38 antibody or the antigen binding fragment thereof of the present invention, and other therapeutic agents, e.g., a chemotherapeutic agent, other antibody, cytotoxic agent, vaccine, anti-infection agents, small molecular entity, or immunomodulator.
Use and method
In another aspect, the invention relates to a method for treating a cancer in a subject, the method comprises administering therapeutically effective amount of the antibody or antigen binding fragments, the pharmaceutical composition or formulation, the combination product or the nucleic acid of the present invention.
In some embodiments, the method above further comprises administering to said subject in combination with one or more therapies, e.g., a therapeutic modality and/or other therapeutic agent.
In another aspect, the invention provides uses of the anti-CD38 antibody or the fragment thereof in manufacture or preparation of a medicine for treatment of the above-mentioned related diseases or conditions.
EXAMPLES
Example 1. Preparation and Expression of humanized Anti-CD38 antibodies
To achieve humanized Anti-CD38 antibodies, firstly, two anti-human CD38 Camelids-derived Nanobodies VHH180 and VHH194 were obtained from Y-CLONE Medical Sciences Co., Ltd, then VHH180 (SEQ ID NO: 09) and VHH194 (SEQ ID NO: 10) were humanized through CDR Grafting. Briefly, IMGT –GENE database (http: //www. imgt. org) was used to search germline sequences, two human germline sequences IGHV3-66*01 and IGHV3-23*02 were selected as CDR accepter for VHH180 and VHH194, respectively. According to grafting CDR region annotated by IMGT, three CDRs of the VHH180 were grafted from the original variable chain into human IGHV3-66*01 germline framework, and three CDRs of the VHH194 were grafted from the original variable chain into human IGHV3-23*02 germline framework. To restore the binding activity of Nanobodies VHH180 and VHH194, some residues in human germline framework should be back-mutations to the original alpaca sequence. Table 1 shows some obtained humanized anti-CD38 VHHs named as VHH180_n and VHH194_n, respectively, n is selected from 1-6.
Table 1 lists obtained humanized anti-CD38 VHH with specific mutated amino acids residues of each of VHH.
Table 1A: humanized VHH180 clones with mutations
  H35 H37 H44 H45 H47 H49 H78 H108
VHH180_1 (SEQ ID NO: 11) S35G         S49A L78V L108Q
VHH180_2 (SEQ ID NO: 12) S35G V37Y G44Q L45R W47F S49A L78V L108Q
VHH180_3 (SEQ ID NO: 13) S35G V37Y       S49A L78V L108Q
VHH180_4 (SEQ ID NO: 14) S35G V37Y G44Q L45R W47F S49A L78V  
VHH180_5 (SEQ ID NO: 15) S35G V37Y     W47F S49A L78V  
VHH180_6 (SEQ ID NO: 16) S35G V37F G44E L45R W47G S49A L78V  
Table 1B: humanized VHH194 clones with mutations
  H35 H37 H44 H45 H47 H49 H50 H58 H78 H108
VHH194_1 (SEQ ID NO: 17) S35A         S49A A50T Y58D L78V  
VHH194_2 (SEQ ID NO: 18) S35A V37F G44E L45R W47F S49A A50T Y58D L78V  
VHH194_3 (SEQ ID NO: 19) S35A V37F G44E L45R W47F S49A A50T Y58D L78V M108Q
VHH194_4 (SEQ ID NO: 20) S35A V37F     W47F S49A A50T Y58D L78V M108Q
VHH194_5 (SEQ ID NO: 21) S35A V37F       S49A A50T Y58D L78V  
VHH194_6 (SEQ ID NO: 22) S35A V37F G44E L45R W47G S49A A50T Y58D L78V  
Note: Amino acid residue numbering in table 1 is according to Kabat (Kabat et al. 1991) .
Nucleotide sequences encoding the above humanized VHH were directly fused to nucleotide sequence encoding the human IgG1-Fc sequence with C220S mutation (EU numbering) (SEQ ID NO: 23) , then were synthesized by General Biosystems (Anhui) Co., Ltd. The synthesized genes express heavy chains named as VHH180_n-Fc or VHH194_n-Fc, respectively. For example, heavy chain VHH180_1-Fc consists of VHH180_1 and human IgG1-Fc.
As a control, nucleotide sequences encoding Camelids-derived VHH180 and VHH194 were also fused to nucleotide sequence encoding the human IgG1-Fc sequence with C220S mutation (EU numbering) , then were synthesized by General Biosystems (Anhui) Co., Ltd. Encoded heavy chains are named as VHH180-Fc (SEQ ID NO: 54) and VHH194-Fc (SEQ ID NO: 55) .
Nucleotide sequences encoding the heavy chains were inserted into the expression vector pcDNA3.1, respectively. Each of successfully constructed vectors encoding heavy chains was transfected into CHO-S cells using ExpiCHO TM Expression System (ThermoFisher) , according to manufacturer's instructions. The transfected cells were cultured in ExpiCHO TM Expression Medium for 12 days to express each humanized anti-CD38 heavy chains. Two identical humanized anti-CD38 heavy chain interacted with each other to form humanized anti-CD38 heavy chain antibody named as VHH180_n-Ab, or VHH194_n-Ab. For example, two VHH180_1-Fcs interacted with each other to form heavy chain antibody of VHH180_1, and named as VHH180_1-Ab. Then culture supernatants  were harvested and purified by using Protein A affinity chromatography (GE healthcare) , according to manufacturer's instructions.
Example 2. Screening and characterization of humanized Anti-CD38 heavy chain antibodies
To test the function of the humanized anti-CD38 heavy chain antibodies, all humanized anti-CD38 heavy chain antibodies obtained in Example 1 were analyzed for binding potency, affinity, ADCC effect in this Example.
1. ELISA assay of binding of anti-CD38 heavy chain antibodies to human CD38
Humanized anti-CD38 heavy chain antibodies generated as described above were detected for binding potency to human CD38 in an ELISA format. ELISA assay was performed generally as described in Baker et al. (Trends Biotechnol. 20: 149-156, 2002) . Briefly, 96-well flat bottom plates (Hangzhou Xinyou, Cat#100096H) were coated with 50 μl Human CD38-his tag protein (Sino biological, Cat#10808-H08H) at a concentration of 0.5 mg/mL in PBS (Hyclone, Cat#SH30256.01) overnight at 4℃. After removing the coating solution, the plates were then blocked by incubating with 1%BSA in PBS for one hour at 37℃. After blocking, the plates were washed three times with PBST (PBS containing 0.05 %Tween20) . For each of humanized anti-CD38 antibodies, 6 points, 3-folds serial dilution solutions were prepared in binding buffer (PBS containing 0.05 %Tween 20 and 0.5%BSA) with initiative concentration of 8 nM. The diluted antibody solutions were added into individual wells of the plates, and the plates were incubated for one hour at 37℃, Daratumumab and Isatuximab were used as positive control, since Daratumumab and Isatuximab were expressed by the inventors of the present application in house according to well-known amino acid sequences of Daratumumab and Isatuximab, they are also named as Daratumumab analog and Isatuximab analog. After the plates were washed three times with PBST, 50 μl 1: 10,000 dilution of Peroxidase AffiniPure Donkey Anti-Human IgG (H+L) (Jackson ImmunoResearch, Cat#709-035-149) in binding buffer were added into each well. The plates were incubated at 37℃ for one hour, and then washed with PBST again. Wells of the plates were developed by adding 50 μL/well tetramethylbenzidine (TMB) (Thermo, Cat#34028) and incubating at room temperature for 15 minutes. Enzymatic color development reaction was stopped by adding 50 μl 1M H 2SO 4 to each well. The absorbance at 450 nm-620 nm was determined. EC50 value of each humanized anti-human CD38 heavy chain antibodies was calculated by using four-parameter fitting model and was used to evaluate relative potency of each antibody.
The EC50 and representative binding curves of each anti-human CD38 heavy chain antibodies comprising corresponding VHH in table 1 are shown in Figure 1. Results show that all constructed humanized heavy chain antibodies can bind to human CD38 protein, and EC50 values of most antibodies are comparable with that of parent heavy chain antibodies (VHH180-Ab or VHH194-Ab) .
2. Humanized anti-CD38 antibodies bound to human CD38 expressed on the cell surface
A cell based binding assay was established to determine whether humanized anti-human CD38 heavy chain antibodies of the present invention can bind to human CD38 protein expressed on cell surface. In this experiment, RPMI-8226 cells (NCACC, Cat#TCHU234) were used. RPMI-8226 cells were cultured in completely RPMI 1640 Medium (Gibco, Cat#22400089) with 10%fetal  bovine serum (FBS) , 100 U/ml penicillin, and 100 μg/ml streptomycin. Eight-point 4-fold series dilutions of humanized anti-CD38 antibodies were made in PBS with initiative concentrations of 3 μg/mL, and then added to cells. The obtained cell mixtures were incubated at 4℃ for 30 minutes, followed by washing cells with PBS twice to remove unbound molecules. 100 μL 1: 10000 diluted R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098) was incubated with RPMI-8226 cells at 4℃ for 30 minutes. Then, cells were washed twice with PBS, re-suspended in PBS. Analysis of CD38 binding was carried out with the BD Accuri C5 flow cytometer (BD Bioscience) , according to manufacturer's instructions.
Figure 2. shows results of binding potency of humanized anti-human CD38 heavy chain antibodies to human CD38 protein expressed on the cell surface, in which all generated humanized anti-human CD38 antibodies of the present invention can bind to human CD38 protein. The results are substantially consistent with the results of ELISA binding assay in section 1 above.
3. Binding kinetics and cross-reactivity of generated humanized anti-human CD38 heavy chain antibodies
In this experiment, cross reactivity and binding kinetics of generated humanized anti-human CD38 heavy chain antibodies was determined by detecting affinity (monovalent KD) of anti-human CD38 heavy chain antibodies to human CD38 or cynomolgus monkey CD38 through the ForteBio Octet RED 96 (bio-layer interferometry) .
Briefly, Anti-Human IgG Fc Capture (AHC) biosensor (ForteBio, Cat#18-5060) were hydrated for 10 minutes at room temperature in PBST (PBS, 0.1%Tween-20) . A kinetic assay was performed in 96-well plates and ran using following times for each step: a) balance baseline for 100 seconds in PBST, b) loading with humanized anti-human CD38 antibodies to at least 1 nanometer, c) balance baseline for 300 seconds, d) association with His-tagged human CD38 (SEQ ID NO: 01) or His-tagged cynomolgus monkey CD38 (SEQ ID NO: 02) at concentration of 100 nM for 100 seconds, and e) dissociation in PBST for 600 seconds. The obtained data sets were fitted with a 1: 1 Local fitting Model using Octet software.
Affinity of non-humanized (negative control) and humanized antibodies were determined in this experiment and results are listed in Table 2 as follows.
Table 2. Affinities of humanized antibodies to recombinant human and cynomolgus monkey CD38
Figure PCTCN2022135904-appb-000005
Figure PCTCN2022135904-appb-000006
Note: VHH180-Ab and VHH194-Ab represent Camelids anti CD38 heavy chain antibodies
having VHH180 and VHH194, respectively.
The results suggested that all humanized heavy chain antibodies maintain high affinity to antigen. The results also show that all humanized antibodies can specifically bind to human CD38 and cynomolgus monkey CD38, suggested they have similar cross-activity to parent antibody VHH180-Ab or VHH194-Ab.
4. Epitope binning of two humanized anti-human CD38 heavy chain antibodies by ELISA
In this experiment, we tested whether humanized anti-human CD38 heavy chain antibodies competitively bind to the same epitope on human CD38.
Two humanized anti-human CD38 heavy chain antibodies VHH180_3-Ab and VHH194_2-Ab were biotinylated by EZ-Link TM Sulfo-NHS-LC-LC-biotin kit (Thermo, Cat#21338) . The biotinylated antibodies were named B-180-3 and B-194-2, respectively.
Two 96-well flat bottom plates (Hangzhou Xinyou, Cat#100096H) were coated with 50 μl Human CD38-his tag protein (Sino biological, Cat#10808-H08H) at a concentration of 0.5 mg/mL in PBS (Hyclone, Cat#SH30256.01) overnight at 4℃. After removing the coating solution, the plates  were then blocked by incubation with 1%BSA in PBS for one hour at 37℃. After blocking, the plates were washed three times with PBST (PBS containing 0.05 %Tween20) . Eight points, 4-fold serial dilution solutions of VHH180_3-Ab and VHH194_2-Ab were prepared in binding buffer, with initiative concentration of 250nM, respectively. Each of 50 μl serial dilution solutions of VHH180_3-Ab and VHH194_2-Ab was mixed with the same volume of 0.016μg/mL B-180-3, added to the wells of plate 1, and then incubated at 37℃ for 1h. 50 μl 0.008μg/mL B-180-3 is used as a control. On plate 2, the same assay was carried out, except that B-180-3 was replaced by B-194-2 with the same concentration. Other conditions are the same as those used in plate 1. Then, the two plates were washed three times with PBST, incubated for one hour at 37℃ with Peroxidase Streptavidin (Jackson ImmunoResearch, Cat#016-030-084) diluted 1/20,000 in the binding buffer, and then washed with PBST again. Wells of the plates were developed by adding 50 μL/well TMB (Thermo, Cat#34028) and incubating at room temperature for 10 minutes. Enzymatic color development reaction was stopped by adding 50 μl 1M H 2SO 4 to each well. The absorbance at 450 nm-620 nm was determined.
Results are shown in Figure 3, and suggest that VHH180_3-Ab and VHH194_2-Ab antibodies do not compete binding to human CD38.
5. Characterization of ADCC activity of humanized anti-CD38 antibodies
To detect the biological activity of humanized anti-CD38 antibodies, the ADCC reporter assay system was developed by using a Daudi (NCACC, Cat#TCHu140) cell line as target cells and a Jurkat (NCACC, Cat#TCHU123) cell line as effector cells.
This Jurkat effector cell line was engineered to stable express human FcγIIIa receptor (V158 variant) and a NFAT-RE driven luciferase reporter (Promega, pGL4.30 [luc2P/NFAT-RE/Hygro] , Cat#E8481) . Briefly, cultured Jurkat cells were con-transfected with NFAT-luc2P reporter plasmids (Promega, Cat#E8481) and plasmids comprising CD16a F genes (SEQ ID NO: 2 discribed in WO2012121911 A2) by using Gene Pul Ser X Cell TM kit (BIO-RAD) , according to manufacturer's instructions. Then, single cell clone stable expressing NFAT-luc2P and CD16a F was screened with 400μg/mL Hygromycin B and 0.2 μg/mL puromycin.
In the ADCC reporter assay, 20μL Daudi target cells in assay buffer containing 90%RPMI 1640 (Gibco, Cat#22400-089) and 10%FBS (Gibco, Cat#10099-141) were added to a 384-well assay plate (Corning, Cat#3570) , with final concentration of 10,000 cells/well. 10μl/well of series dilutions of anti-CD38 antibodies were added to individual wells (30nM initiative solution was subjected to 3-fold gradient dilution, and total 9 series dilution solutions were generated) . Then 20μl effector cells in assay buffer were added to each well, with final concentration of 50,000 cells/well. The 384-well plate was incubated for 4～6 hours at 37℃, 5%CO 2. After incubation, the plate was cooled to room temperature for 15 minutes, and then 30 μl/well of One-Glo TM Reagent (Promega, Cat#E6130) was added to each well. The plate was incubated for 5 minutes at room temperature, then read using Tecan F200 (Tecan, F200 Pro) luminescence mode. Curves and half maximal effective concentration (EC50) were analyzed using the Graphpad Prism software with the “Dose-response Stimulation →Log (agonist) vs. response-Variable slope (four parameters) ” model.
Four candidate heavy chain antibodies VHH180_2-Ab, VHH180_3-Ab, VHH194_2-Ab and VHH194_4-Ab were selected for ADCC function detecting, Daratumumab and Isatuximab used as positive control. The results were shown in Figure 4 and suggested that all tested humanized anti-CD38 antibodies have comparable ADCC activity with Daratumumab and Isatuximab, in which ADCC function of VHH194_2-Ab and VHH194_4-Ab are relatively stronger than controls.
Example 3 Construction of biparatopic antibodies
We have used VHH180 and VHH194 to construct three different structures of bispecific antibodies: symmetrical, asymmetric, IgG-like symmetrical bispecific antibody, schematic diagram of these bispecific antibodies are shown in Figure 5.
The symmetrical bispecific antibody was consisted of two same chains. From N terminal to C terminal, each chain comprises two VHHs of anti-CD38 VHH (VHH1 and VHH2) fused via GGSGGS linker, the hinge, CH2 and CH3 domain of human IgG1.
The asymmetric bispecific antibody was consisted of two different chains. From N terminal to C terminal, chain 1 comprises VHH1 of anti-CD38 VHH, the hinge, CH2 and CH3 domain of human IgG1 (SEQ ID NO: 35 IgG1-Fc-Hole) with substitutions N384D, Q418E, N421D, Y407V, T366S, L368A and Y349C (numbering according to EU index) , chain 2 comprises VHH2 of anti-CD38 VHH, hinge, CH2 and CH3 domain of human IgG1 (SEQ ID NO: 34 IgG1-Fc-Knob) with substitutions T366W and S354C (numbering according to EU index) .
IgG-Like symmetrical bispecific antibody has two arms, each arm is consisted of two chains, chain 1, from N terminal to C terminal, comprises VHH1 of anti-CD38 VHH and human IgG1 constant region (i.e., CH1, hinge, CH2 and CH3 domain) , chain 2, from N terminal to C terminal, comprises VHH2 of anti-CD38 VHH, human kappa light chain. Table 3A lists the composited sequence of three types of bispecific antibodies.
Table 3
Figure PCTCN2022135904-appb-000007
Example 4 Complement-Dependent Cytotoxicity (CDC) of biparatopic antibody
The CDC effect of biparatopic antibodies were tested in this example.
Complement-Dependent Cytotoxicity (CDC) Assay on Daudi cells (NCACC, Cat#TCHU140) were performed to evaluate the potency of anti-CD38 antibodies of the present application. Daudi  cells were seeded at a density of 5,000 cells per well and were pre-incubated with each antibody at 300 nM, 30 nM and 3 nM for 30 min. Serum from a healthy donor was added into cells with 20%final concentration. The effect of Rituxan (SEQ ID NO: 3 &4) , Daratumumab (SEQ ID NO: 5 &6) and Isatuximab (SEQ ID NO: 7 &8) on Daudi CDC was used as an internal control to assure the assay quality. After incubation in a 37℃, 5%CO 2 incubator for 4 hours, cells were lysized with Cell-Titer Glo reagent (Promega, Cat#G7572) and read the RLU data on F200 (Tecan, F200 Pro) . The percentages of cell lysis were calculated according the formula below,
%Cell lysis =100 × (1- (RLUsample) / (RLUcell+NHS) ) , in which NHS (Schbio biotech, Cat#PBSR005C) is used for normal human serum.
Results are shown in Figure 6, in which the biparatopic antibody can significantly improve the effect of CDC, and biparatopic antibodies with different structures also showed different degrees of enhancement of CDC effect. Among biparatopic symmetrical bispecific antibodies, VHH194_180, shows the strongest effect of CDC, and its EC50 value is 0.1892 nM.
The ADCC effect of biparatopic antibodies and humanized antibodies were tested according to procedure in Example 2 section 5.
Results are recorded in Figure 7. The results show that the symmetrical bispecific antibody VHH180_194 show the strongest ADCC effect than other biparatopic antibodies, its EC50 value is 0.031 nM.
Example 5 Characterization of ADCC enhanced mutants of biparatopic antibody
To enhance the ADCC activity of the bispecific antibody of the present application, the inventors conceive of constructing ADCC-enhanced bispecific antibodies by mutating amino acid residues in Fc region of one or two chains of bispecific antibodies in table 3, which amino acid residues play a role in binding of Fc of antibodies to Fc receptors and are disclosed in the prior art. Specifically, this example carries out two amino acids substitution (S239D/I332E) in the Fc region of one or two chains of the antibody (numbering according to EU index) , such two substitutions can increase the affinity of Fc for FcγRIIa and FcγRIIIa. Table 4 lists substituted bispecific antibodies in table 3.
Table 4
Figure PCTCN2022135904-appb-000008
The ADCC activity of mutated bispecific antibodies in table 4 were detected following procedure disclosed in Example 2 section 5, except RPMI8226 (NCACC, Cat#TCHU234) as the target cell.
Results in Figure 8 shows that different structures also reflect different ADCC enhancement effects. The biparatopic antibody 194-180SE showed the strongest ADCC effect, and 180-194SE, another ADCC-enhanced antibody showed similar ADCC effect of 194-180SE. The EC50 values of these two antibodies are 0.008292 nM and 0.01065 nM, respectively. The ADCC effects of the two antibodies are stronger than that of daratumumab and isatuximab.
The CDC effect of the mutants was also investigated according to procedure disclosed in Example 4 except 10%final concentration of human serum from the healthy donor. Results are shown in Figure 9. The results showed that the above ADCC enhancement modification would not have a substantial impact on the CDC effect of the biparatopic antibody. Compared with the ADCC enhancement modification, the CDC effect was basically unchanged; The EC50 values of 194-180SE and 180-194SE are 0.6197nM and 0.6737nM respectively, both are significantly stronger than the reference daratumumab and Isatuximab.
Example 6 Characterization of humanized biparatopic antibody
In this Example, the inventors firstly humanized the constructed biparatopic antibodies of Example 3, that is to say, replacing Camelids VHH by corresponding humanized VHH, for example, replacing VHH180 by one of VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 and VHH180_6; replacing VHH194 by one of VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 and VHH194_6. The obtained humanized biparatope antibody named by corresponding humanized VHH, for example symmetrical bispecific antibody 180-3_194-2 (SEQ ID NO: 51) .
Furthermore, in order to improve ADCC effect, the inventors mutated the humanized biparatope antibody 180-3_194-2 according to procedure in Example 5 and obtained mutant 180-3_194-2SE which carries two amino acids substitution (S239D/I332E) in the Fc region of the antibody (SEQ ID NO:52) .
Then the inventor evaluated ADCC effect and ADCP of humanized biparatopic antibodies and mutated humanized biparatopic antibodies.
In vitro ADCC effect evaluation of the humanized biparatopic antibody and the mutant thereof
In the ADCC assay, Daudi cells with endogenous high expression of CD38 were used as target cells, PBMC cells (
Figure PCTCN2022135904-appb-000009
Cat#PBMNC025C) were used as effector cells. 50 μl Daudi cells in 99%MEM-α (Gibco, Cat#41061-029) containing 1%FBS (Gibco, Cat#10099-141) assay buffer were added to a 96-well assay plate (Corning, Cat#3599) , with final concentration of 10,000 cells/well. 50μl/well of humanized biparatopic antibody (eight-point series dilutions started from 1 nM) or Daratumumab analog (5-fold series dilution, the final assay concentrations started from 1 nM) were added to cells. Then 100 μl PBMC effector cells in 99%MEM-α (Gibco, Cat#41061-029) containing 1%FBS (Gibco, Cat#10099-141) and 200 IU/ml human recombinant IL2 assay buffer were added to each well, with final concentration of 100,000 cells/well. The 96-well plate was incubated for 24 hours at 37℃, 5%CO 2. After incubation, the 96-well plate was removed and centrifuge at low speed (2000 rpm) for 3 min to allow the precipitation of cell pellet. 50 μL/well of supernatant was carefully aspirated and transferred to a new 96-well plate. An equal volume of LDH  cytotoxicity detection solution (Roche, Cat#11644793001) was added into the plate and incubated at room temperature for 30 min. The absorbance was read by Infinite F50 plate-reader at wavelength of 490nm.
Results were showed in Figure 10.
The results show that the humanized biparatopic antibody and the mutant thereof all can induce significant ADCC effect on Daudi cells, the ADCC effect of mutant 180-3_194-2SE is better than that of Daratumumab analog.
ADCP reporter gene activity of the humanized biparatopic antibody and the mutant thereof
In order to detect the biological activity of humanized biparatopic antibody, we developed an ADCP reporter assay system using Daudi cell line as target cells and a stable Jurkat cell line as effector cells. This Jurkat effector cell line stably expresses human FcγIIa receptor, CD32aH variant and a NFAT-RE driven luciferase reporter.
In the ADCP reporter assay, 20 μl Daudi target cells in 90%RPMI 1640 (Gibco, Cat#22400-089) containing 10%FBS (Gibco, Cat#10099-141) assay buffer were added to a 384-well assay plate (Corning, Cat#3570) , with final concentration of 10,000 cells/well. 10 μl/well of the nine-point series dilutions of humanized biparatopic antibody, Daratumumab analog or Isatuximab analog (5-fold series dilution, the final assay concentrations started from 50 nM) were added to cells. Then 20 μl FcγIIa effector cell (Jurkat cell) line in 90%RPMI 1640 (Gibco, Cat#22400-089) containing 10%FBS (Gibco, Cat#10099-141) assay buffer were added to each well, with final concentration of 50,000 cells/well. The 384-well plate was incubated for 4～6 hours at 37℃, 5%CO 2. After incubation, the plate was cooled to room temperature for 15 minutes, and 30 μl/well of One-Glo TM Reagent (Promega, Cat#E6130) was added. The plate was incubated for 5 minutes at room temperature, then read using Tecan F200 (Tecan, F200 Pro) luminescence mode. Curves and half maximal effective concentration (EC 50) were analyzed using the GraphPad Prism software with the “Dose-response-Stimulation→Log (agonist) vs. response-Variable slope (four parameters) ” model. Results were showed in Figure 11.
The results show that the humanized biparatopic antibody and the mutant thereof all can induce significant ADCP effect, and ADCP effect of mutant 180-3_194-2SE is significantly higher than that of Daratumumab analog and Isatuximab analog.
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[2] Nakagawara K, Mori M, Takasawa S, et al. Assignment of CD38, the gene encoding human leukocyte antigen CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) , to chromosome 4p15. Cytogenet. Genome Res. 1995; 69: 38–39.
[3] Deaglio S, Mehta K, Malavasi F. Human CD38: a revolutionary story of enzymes and receptors. Leuk. Res. 2001; 25: 1-12
[4] Leo R, Boeker M, Peest D, et al. Multiparameter analyses of normal and malignant human plasma cells: CD38++, CD56+, CD54+, cIg+ is the common phenotype of myeloma cells. Ann. Hematol. 1992; 64: 132-139.
[5] Deaglio S, Morra M, Mallone R, et al. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J. Immunol. 1998; 160: 395-402
[6] Allard B, Beavis PA, Darcy PK, et al. Immunosuppressive activities of adenosine in cancer. Curr. Opin. Pharmacol. 2016; 29: 7-16.
[7] Francesca B, Mattia D, et al. CD38 as an immunotherapeutic target in multiple meloma. Expert Opinion on Biological Therapy. 2018; 18: 1209-1221
[8] Maarten L. Niels W. et al. Discovery, development, and mechanisms of action of the human CD38 antibody daratumumab. Successful Drug Discovery: Volume 3: 153-193
[9] Deckert J. Wetzel MC, et al. SAR650984, a novel humanized CD38-targeting antibody, demonstrates potent antitumor activity in models of multiple myeloma and other CD38+hematologic malignancies. Clin. Cancer Res, 20 (17) , 4574-4583 (2014) .
[10] Jiang H, Acharya C, An G. et al. SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide. Leukemia, 30 (2) , 399-408 (2016) .
[11] Raab M, Chatterjee M, et al. A phase I/II study of the CD38 antibody MOR202 alone and in combination with pomalidomide or lenalidomide in patients with relapsed or refractory multiple myeloma. Blood, 128 (22) , 1152 (2016)
[12] Kerstin S, Katharina P, et al. CD38-specific biparatopic heavy chan Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells. Frontiers in immunology. 2018; 9: 2553
[13] Greg A. Wei D. et al. Engineered antibody Fc variants with enhanced effector function. PNAS. 2006; 103: 4005-4010.

Claims (23)

  1. An anti-CD38 VHH nanobody comprising HCDR1, HCDR2 and HCDR3 which is included in SEQ ID NO: 9 or SEQ ID NO: 10.
  2. An anti-CD38 VHH nanobody comprising HCDR1, HCDR2 and HCDR3, wherein:
    I. the CDRs sequences are defined according to Kabat scheme, wherein
    1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 39, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 41, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 43; or
    2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 45, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 47, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 49;
    or,
    II. the CDRs sequences are defined according to IMGT scheme, wherein
    1) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 40, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 42, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 44; or
    2) HCDR1 comprises or consists of amino acid sequences as shown in SEQ ID NO: 46, HCDR2 comprises or consists of amino acid sequences as shown in SEQ ID NO: 48, and HCDR3 comprises or consists of amino acid sequences as shown in SEQ ID NO: 50.
  3. The anti-CD38 VHH nanobody according to claim 1 or 2, which comprises an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 10 and having the same CDRs as SEQ ID NO: 9 or SEQ ID NO: 10, or consists of amino acid sequences as shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  4. The anti-CD38 VHH nanobody according to any one of claims 1-3, which is a humanized anti-CD38 VHH nanobody.
  5. The anti-CD38 VHH nanobody according to claim 4, which the humanized anti-CD38 VHH nanobody comprises back-mutations in human framework region.
  6. The anti-CD38 VHH nanobody according to claim 4 or 5, which the humanized anti-CD38 VHH nanobody comprises:
    1) an amino acid sequence as shown in any one of SEQ ID NO: 11-16 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 11-16 and having the same CDRs as the corresponding one of SEQ ID NO: 11-16; or
    2) an amino acid sequence as shown in any one of SEQ ID NO: 17-22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identity with the amino acid sequence as shown in any one of SEQ ID NO: 17-22 and having the same CDRs as the corresponding one of SEQ ID NO: 17-22.
  7. An anti-CD38 heavy chain antibody or an antigen-binding fragment thereof, which comprises the anti-CD38 VHH nanobody according to any one of claims 1-6.
  8. The anti-CD38 heavy chain antibody or the antigen-binding fragment thereof according to claim 7, which comprises:
    (1) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 9 and having the same CDRs as SEQ ID NO: 9, or consisting of SEQ ID NO: 9;
    (2) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 10 and having the same CDRs as SEQ ID NO: 10, or consisting of SEQ ID NO: 10;
    (3) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 11 and having the same CDRs as SEQ ID NO: 11, or consisting of SEQ ID NO: 11;
    (4) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 12 and having the same CDRs as SEQ ID NO: 12, or consisting of SEQ ID NO: 12;
    (5) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 13 and having the same CDRs as SEQ ID NO: 13, or consisting of SEQ ID NO: 13;
    (6) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 14 and having the same CDRs as SEQ ID NO: 14, or consisting of SEQ ID NO: 14;
    (7) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 15 and having the same CDRs as SEQ ID NO: 15, or consisting of SEQ ID NO: 15;
    (8) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 16 and having the same CDRs as SEQ ID NO: 16, or consisting of SEQ ID NO: 16;
    (9) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 17 and having the same CDRs as SEQ ID NO: 17, or consisting of SEQ ID NO: 17;
    (10) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 18 and having the same CDRs as SEQ ID NO: 18, or consisting of SEQ ID NO: 18;
    (11) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 19 and having the same CDRs as SEQ ID NO: 19, or consisting of SEQ ID NO: 19;
    (12) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 20 and having the same CDRs as SEQ ID NO: 20, or consisting of SEQ ID NO: 20;
    (13) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 21 and having the same CDRs as SEQ ID NO: 21, or consisting of SEQ ID NO: 21; or
    (14) a VHH comprising an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 22 and having the same CDRs as SEQ ID NO: 22, or consisting of SEQ ID NO: 22.
  9. The anti-CD38 heavy chain antibody or the antigen-binding fragment thereof according to claim 7 or 8, which comprises a Fc region derived from IgG1, IgG2, IgG3 or IgG4, especially derived from IgG1 isotype, preferably, the Fc region
    1) comprises amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 23, or IgG Fc consists of SEQ ID NO: 23;
    2) comprises mutations, for example the mutations are amino acids substitution, deletion, and addition, preferably the mutations can improve ADCC, ADCP effect, for example, the mutation are one or more amino acids substitution, such as S239D/I332E (EU numbering) substitution; knob mutation (s) or hole mutation (s) ; or
    3) comprises amino acid sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 34 or 35, or IgG Fc consists of SEQ ID NO: 34 or 35;
    more preferably, the anti-CD38 heavy chain antibody or the antigen-binding fragment thereof comprise:
    (1) an amino acid sequence as shown in SEQ ID NO: 54, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 54 and having the same CDRs as SEQ ID NO: 54, or consisting of SEQ ID NO: 54;
    (2) an amino acid sequence as shown in SEQ ID NO: 55, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 55 and having the same CDRs as SEQ ID NO: 55, or consisting of SEQ ID NO: 55;
    (3) an amino acid sequence as shown in SEQ ID NO: 56, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 56 and having the same CDRs as SEQ ID NO: 56, or consisting of SEQ ID NO: 56;
    (4) an amino acid sequence as shown in SEQ ID NO: 57, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 57 and having the same CDRs as SEQ ID NO: 57, or consisting of SEQ ID NO: 57;
    (5) an amino acid sequence as shown in SEQ ID NO: 58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 58 and having the same CDRs as SEQ ID NO: 58, or consisting of SEQ ID NO: 58;
    (6) an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 59 and having the same CDRs as SEQ ID NO: 59, or consisting of SEQ ID NO: 59;
    (7) an amino acid sequence as shown in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 60 and having the same CDRs as SEQ ID NO: 60, or consisting of SEQ ID NO: 60;
    (8) an amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 61 and having the same CDRs as SEQ ID NO: 61, or consisting of SEQ ID NO: 61;
    (9) an amino acid sequence as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 62 and having the same CDRs as SEQ ID NO: 62, or consisting of SEQ ID NO: 62;
    (10) an amino acid sequence as shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 63 and having the same CDRs as SEQ ID NO: 63, or consisting of SEQ ID NO: 63;
    (11) an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 64 and having the same CDRs as SEQ ID NO: 64, or consisting of SEQ ID NO: 64;
    (12) an amino acid sequence as shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 65 and having the same CDRs as SEQ ID NO: 65, or consisting of SEQ ID NO: 65;
    (13) an amino acid sequence as shown in SEQ ID NO: 66, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 66 and having the same CDRs as SEQ ID NO: 66, or consisting of SEQ ID NO: 66; or
    (14) an amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 67 and having the same CDRs as SEQ ID NO: 67, or consisting of SEQ ID NO: 67.
  10. A bispecific antibody comprising the anti-CD38 VHH nanobody according to any one of claims 1-6, wherein the bispecific anti-CD38 antibody is selected from:
    1) symmetrical bispecific antibody consisting of two same chains: from N terminal to C terminal, each chain comprises anti-CD38 VHH1 and anti-CD38 VHH2, the hinge, CH2 and CH3 domain;
    2) asymmetric bispecific antibody consisting of two different chains: from N terminal to C terminal, chain 1 comprises anti-CD38 VHH1, the hinge, CH2 and CH3 domain, chain 2 comprises anti-CD38 VHH2, hinge, CH2 and CH3 domain; or
    3) IgG-Like symmetrical bispecific antibody having two chain 1 and two chain 2, wherein, from N terminal to C terminal, chain 1 comprises anti-CD38 VHH1 and heavy chain constant region, and chain 2 comprises anti-CD38 VHH2 and light chain constant region,
    wherein VHH1 is different from VHH2.
  11. The bispecific antibody of claim 10, wherein the VHH1 and VHH2 comprise independently from each other HCDR1, HCDR2 and HCDR3 selected from:
    1) amino acid sequences as shown in SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43, respectively;
    2) amino acid sequences as shown in SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 44, respectively,
    3) amino acid sequences as shown in SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; or
    4) amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.
  12. The bispecific antibody of claim 10 or claim 11, wherein VHH1 and VHH2 are independently from each other comprise:
    1) VHH180 or VHH194; or
    2) humanized VHH180 (VHH180_1, VHH180_2, VHH180_3, VHH180_4, VHH180_5 or VHH180_6) or humanized VHH194 (VHH194_1, VHH194_2, VHH194_3, VHH194_4, VHH194_5 or VHH194_6) ,
    provided that VHH1 differs from VHH2.
  13. The bispecific antibody of any one of claims 10-12, wherein the VHH1 and VHH2 in the symmetrical bispecific antibody are linked by a linker selected from (G 4S) n, where n is an integer number, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9; GGGSG; GGSGGS; GGSGG; GSGGG; SGGGG; GGGTS; GTSPGG; GNGGGS; G4S-GGSGG-G4S-SGGGG; GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; KESGSVSGSSE QLAQFRSLD; GGRRGGSLRQQ.
  14. The bispecific antibody of any one of claims 10-13, wherein the heavy constant region is derived from IgG1, IgG2, IgG3 or IgG4, preferably derived from IgG1 isotype, and/or, the light chain constant region is derived from κ light chain or λ light chain; preferably, wherein Fc region of the heavy constant region
    1) comprises amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 23, or IgG Fc consists of SEQ ID NO: 23;
    2) comprises mutations, for example the mutations are amino acids substitution, deletion, and addition, preferably the mutations can improve ADCC, ADCP effect, for example, the mutation are one or more amino acids substitution, such as S239D/I332E (EU numbering) substitution; knob mutation (s) or hole mutation (s) ; or
    3) comprises amino acid sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 34 or 35, or IgG Fc consists of SEQ ID NO: 34 or 35.
  15. The bispecific antibody of any one of claims 10-14, wherein the bispecific antibody is selected from:
    1) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprises or consists of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;
    2) an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 36 and another chain comprises or consists of SEQ ID NO: 37;
    3) an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 38 and the chain 2 comprises or consists of SEQ ID NO: 30;
    4) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprises or consists of SEQ ID NO: 31 or SEQ ID NO: 32;
    5) an asymmetric bispecific antibody comprising two different chains, wherein one chain comprises or consists of SEQ ID NO: 28 and another chain comprises or consists of SEQ ID NO: 29;
    6) an IgG-like bispecific having two chain 1 and two chain 2, wherein the chain 1 comprises or consists of SEQ ID NO: 33 and the chain 2 comprises or consists of SEQ ID NO: 30;
    7) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprises or consists of SEQ ID NO: 51; or
    8) a symmetrical bispecific antibody comprising two identical chains, wherein the chains comprises or consists of SEQ ID NO: 52.
  16. An isolated nucleic acid encoding the anti-CD38 VHH nanobody of any one of claims 1-6, the heavy chain antibody of any one of claims 7-9, or the bispecific antibody of any one of claims 10-15.
  17. A vector comprising the nucleic acid of claim 16.
  18. A host cell comprising the nucleic acid of claim 16 or the vector of claim 17.
  19. A method of preparing the anti-CD38 VHH nanobody of any one of claims 1-6, the heavy chain antibody of any one of claims 7-9, or the bispecific antibody of any one of claims 10-15, comprising culturing the host cell of claim 18, under conditions suitable for expression of the nucleic acid of claim 16.
  20. A pharmaceutical composition comprising the anti-CD38 VHH nanobody of any one of claims 1-6, the heavy chain antibody of any one of claims 7-9, or the bispecific antibody of any one of claims 10-15, and optionally a pharmaceutically acceptable adjuvant.
  21. A method of treating cancer in a subject comprising administering to said subject the anti-CD38 VHH nanobody of any one of claims 1-6, the heavy chain antibody of any one of claims 7-9, or the bispecific antibody of any one of claims 10-15, or the pharmaceutical composition of claim 20.
  22. A use of the anti-CD38 VHH nanobody of any one of claims 1-6, the heavy chain antibody of any one of claims 7-9, or the bispecific antibody of any one of claims 10-15, or the pharmaceutical composition of claim 20 in the manufacture of a medicament for treating cancer in a subject.
  23. The method of claim 21 or the use of claim 22, wherein the cancer is that with abnormal level of CD38 (e.g., the high protein or nucleic acid level of CD38) , preferably, the cancer is hematological tumors, malignant plasma cell tumors, multiple myeloma, relapsed/refractory multiple myeloma.
PCT/CN2022/135904 2021-12-03 2022-12-01 Antibodies binding cd38 and uses thereof WO2023098813A1 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005103083A2 (en) * 2004-02-06 2005-11-03 Morphosys Ag Anti-cd38 human antibodies and uses therefor
US20100285004A1 (en) * 2004-02-06 2010-11-11 Michael Tesar Anti-CD38 human antibodies and uses thereof
CN103421115A (en) * 2013-09-02 2013-12-04 东南大学 CD38 nanometer antibody and application
CN109232739A (en) * 2017-07-11 2019-01-18 北京大学深圳研究生院 A kind of 8 nano antibody of AntiCD3 McAb, encoding gene and application
CN111808193A (en) * 2020-06-30 2020-10-23 南京安锐生物科技有限公司 Nanobody capable of binding human CD38 and application thereof
US20210024645A1 (en) * 2018-02-12 2021-01-28 Hangzhou Sumgen Biotech Co., Ltd. Cd38 protein antibody and application thereof
US20210324102A1 (en) * 2018-09-19 2021-10-21 Acroimmune Biotech Co., Ltd. Monoclonal antibody specifically binding human and monkey cd38 antigens, preparation method and use thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005103083A2 (en) * 2004-02-06 2005-11-03 Morphosys Ag Anti-cd38 human antibodies and uses therefor
US20100285004A1 (en) * 2004-02-06 2010-11-11 Michael Tesar Anti-CD38 human antibodies and uses thereof
CN103421115A (en) * 2013-09-02 2013-12-04 东南大学 CD38 nanometer antibody and application
CN109232739A (en) * 2017-07-11 2019-01-18 北京大学深圳研究生院 A kind of 8 nano antibody of AntiCD3 McAb, encoding gene and application
US20210024645A1 (en) * 2018-02-12 2021-01-28 Hangzhou Sumgen Biotech Co., Ltd. Cd38 protein antibody and application thereof
US20210324102A1 (en) * 2018-09-19 2021-10-21 Acroimmune Biotech Co., Ltd. Monoclonal antibody specifically binding human and monkey cd38 antigens, preparation method and use thereof
CN111808193A (en) * 2020-06-30 2020-10-23 南京安锐生物科技有限公司 Nanobody capable of binding human CD38 and application thereof

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