WO2021258140A1 - Cd83 binding protein conjugates for treating lymphoma - Google Patents

Cd83 binding protein conjugates for treating lymphoma Download PDF

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Publication number
WO2021258140A1
WO2021258140A1 PCT/AU2021/050652 AU2021050652W WO2021258140A1 WO 2021258140 A1 WO2021258140 A1 WO 2021258140A1 AU 2021050652 W AU2021050652 W AU 2021050652W WO 2021258140 A1 WO2021258140 A1 WO 2021258140A1
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individual
lymphoma
binding protein
anthracycline
antibody
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PCT/AU2021/050652
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French (fr)
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Georgina Jane Clark
Edward Alan ABADIR
Xinsheng Ju
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Kira Biotech Pty Limited
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Priority claimed from AU2020902090A external-priority patent/AU2020902090A0/en
Application filed by Kira Biotech Pty Limited filed Critical Kira Biotech Pty Limited
Publication of WO2021258140A1 publication Critical patent/WO2021258140A1/en

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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Definitions

  • CD83 binding protein conjugates for treating lymphoma CD83 binding protein conjugates for treating lymphoma
  • the present invention relates to compositions and methods of use thereof for the treatment of lymphoma.
  • Lymphoma is a group of blood cell tumors that develop from lymphocytes.
  • the two main categories of lymphomas are Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
  • NHL Hodgkin and Reed-Sternberg cells
  • NHL includes all lymphomas except HL.
  • HRS Hodgkin and Reed-Sternberg cells
  • NHL includes all lymphomas except HL.
  • Current therapies for HL have a significant rate of success, 25% of patients experience disease relapse when they become refractory to either primary or secondary chemotherapy, and survival remains substantially lower especially in elderly patients who cannot tolerate such therapy.
  • therapies for NHL have a lower rate of success than for HL. Almost 1 in every 2 people with NHL will have the diffuse large B-cell lymphoma (DLBCL) form of the lymphoma and a further 5-10% of people with NHL will have Mantle cell lymphoma (MCL).
  • DLBCL diffuse large B-cell lymphoma
  • MCL Mantle cell lymphoma
  • the present invention provides a method of treating lymphoma in an individual who has received or is receiving chemotherapy that includes an anthracycline and/or an alkylating agent, the method comprising
  • the present invention provides a method of treating lymphoma in an individual, the method comprising
  • the present invention provides a method of treating lymphoma in an individual, the method comprising
  • the present invention provides a method of treating lymphoma in an individual, the method comprising
  • the present invention provides a method of treating lymphoma in an individual, the method comprising
  • the anthracycline may be selected from the group consisting of daunorubicin, doxorubicin (including ADRIAMYCIN®, morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride, doxorubicin HCI liposome injection (DOXIL®), dimethyl-doxorubicin (diMe-Doxo) and deoxydoxorubicin), epirubicin, idarubicin, mitoxantrone, valrubicin, aldoxorubicin, annamycin, plicamycin, piramycin, aclarubicin, zorubicin, carubicin, noglamamycin, menogaril, pitarubicin, detorubicin, esorubicin, marcellomycin, nogalamycin, rodorubicin, quelamycin, an an an agent that iso
  • the anthracycline is doxorubicin.
  • the alkylating agent may be selected from the group consisting of ethylenimines and methylamelamines such as thiotepa, altretamine, triethylenemelamine, triethylenephosphoramidetriethiylenethiophosphoraminde and trimethylolomelamine; cyclophosphamide (CY-TOXAN® or Neosar®); mitomycins such as mitomycin C; 6- diazo-5-oxo-L-norleucine; alkyl sulfonates such as busulfan, improsulfan and pipsulfan; nitrogen mustards such as bendamustine, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trof
  • the alkylating agent is cyclophosphamide.
  • the compound that activates NF-KB may be, phytohemagglutinin (PHA); phorbol esters, tetradecanoyl phorbol acetate (TPA); lipopolysaccharide (LPF); tumor necrosis factor-a (TNFa); prostratin; TRAF family member-associated NFKB activator (TANK) polypeptide; I L- 1 b , glutamate or any other compound described herein.
  • PHA phytohemagglutinin
  • TPA phorbol esters
  • TPA tetradecanoyl phorbol acetate
  • LPF lipopolysaccharide
  • TNFa tumor necrosis factor-a
  • prostratin TRAF family member-associated NFKB activator (TANK) polypeptide
  • I L- 1 b glutamate or any other compound described herein.
  • the lymphoma may be Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
  • HL Hodgkin lymphoma
  • NHL non-Hodgkin lymphoma
  • the NHL may be Mantle cell lymphoma (MCL).
  • MCL Mantle cell lymphoma
  • the NHL may be diffuse large B-cell lymphoma (DLBCL).
  • DLBCL diffuse large B-cell lymphoma
  • the NHL may be Follicular lymphoma (FL).
  • the CD83 binding protein comprises an antigen binding domain that binds to CD83.
  • the CD83 binding protein is an antibody or antigen binding fragment thereof.
  • the antibody fragment may be selected from the group consisting of a dAb, Fab, Fd, Fv, F(ab’)2, scFv or any other antibody fragment format described herein.
  • the chemotherapy may be administered simultaneously with the CD83 binding protein conjugated to a cytotoxic agent.
  • the chemotherapy may be administered sequentially to the CD83 binding protein conjugated to a cytotoxic agent.
  • the chemotherapy may be administered prior to the CD83 binding protein conjugated to a cytotoxic agent.
  • the CD83 binding protein conjugated to a cytotoxic agent may be administered prior to the chemotherapy.
  • the method or use of the invention further comprises a step of determining whether the individual or subject has a lymphoma that is CD83+ or CD83-. Preferably, this step is performed prior to any administering step.
  • the individual has a lymphoma that is CD83+.
  • the individual has a lymphoma that is CD83-.
  • the present invention also provides a CD83 binding protein conjugated to a cytotoxic agent for use in the treatment of lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent.
  • the present invention also provides a CD83 binding protein conjugated to a cytotoxic agent for use in the treatment of lymphoma in an individual, who has received, or who is receiving, an activator of NF-KB.
  • the present invention also provides a medicament comprising, consisting essentially of or consisting of a CD83 binding protein conjugated to a cytotoxic agent, wherein the medicament is used for treating lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent.
  • the present invention also provides a kit comprising, consisting essentially of or consisting of
  • the instructions describe any method of treatment described herein.
  • the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a medicament for the treatment of lymphoma in an individual who has received, or who is receiving, a chemotherapy that includes an anthracycline and/or an alkylating agent.
  • the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a medicament for the treatment of lymphoma in an individual who has received, or who is receiving, an activator of NF-KB.
  • the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a first medicament and use of an anthracycline and/or an alkylating agent in the manufacture of a second medicament, wherein the first and second medicaments are used to treat lymphoma in an individual.
  • the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a first medicament and use of an activator of NF-KB in the manufacture of a second medicament, wherein the first and second medicaments are used to treat lymphoma in an individual.
  • the cytotoxic agent conjugated to a CD83 binding protein may be any cytotoxic agent described herein.
  • the individual has received, or is receiving, a sub-therapeutic dose of the anthracycline and/or the alkylating agent.
  • a sub-therapeutic dose of the anthracycline and/or the alkylating agent is administered to the individual in a sub-therapeutic dose.
  • the sub-optimal or sub-therapeutic doses may be expressed as a % of a therapeutic or optimal dose, or a % reduction of a therapeutic or optimal dose.
  • a sub-optimal dose may be provided in 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of a therapeutic dose.
  • the sub-optimal dose may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction of a therapeutic or optimal dose.
  • the individual may have lymphoma that is resistant to an anthracycline and/or alkylating agent.
  • the individual may have received an anthracycline and/or alkylating agent to which the lymphoma responded but overtime may have developed resistance such that previously therapeutic doses no longer provide any therapeutic benefit.
  • the individual may have never responded to an anthracycline and/or alkylating agent.
  • FIG. 1 Mantle cell lymphoma (MCL) cells express of CD83.
  • MCL Mantle cell lymphoma
  • B Primary blastoid non- nodal MCL patient (MCL01 and MCL02) blood samples were stained with CD19-V450, CD5-APC and 3C12C-FITC and analysed by flow cytometry for the CD83 expression on CD19+/CD5+ cells.
  • C Quantitative real time PCR analysis of mRNAs for CD83 in MCL cell lines and one MCL primary sample.
  • FIG. 1 CD83 ADC kills MCL in xenograft mice.
  • A Schematic design of the MCL xenograft NSG mouse model.
  • B
  • CD83 + cells were treated with either a DMSO control or canonical NF-KB inhibitor BAY-11-7082 at different concentrations (0.25mM or 1.25mM) for 4 or 24 hrs.
  • Real-time PCR (qPCR) analyses of CD83 mRNA level in Mino (left) and Rec-1 (right) cells is shown. *: p ⁇ 0.05.
  • CD83+ cells were treated with either a DMSO control or BAY-11-7082 (0.25pM) for 24 hours.
  • Mino or Z138 cells were cultured in the presence of Doxorubicin (DOX, 0.2ug/ml) or Cyclophosphamide (CP, 0.5mg/ml) for 30 minutes, 2 hrs, 6 hrs and 24 hrs. Cytoplasmic and nuclear protein were isolated. Immunoblot analysis of cell lysate was performed with anti-NF-kB antibodies.
  • DOX Doxorubicin
  • CP Cyclophosphamide
  • Mino or Z138 cells were cultured in the presence of Doxorubicin (DOX, 0.02pg/ml) or Cyclophosphamide (CP, 0.2mg/ml) for 48 hrs, cell surface CD83 expression was analysed by flow cytometry. Mean fluorescent intensity ⁇ SEM of three experiments were shown. (A) and one representative data (B) was shown.
  • DOX Doxorubicin
  • CP Cyclophosphamide
  • (C) Mino or Z138 cells were cultured with serial dilution of 3C12C-MMAE (0.176, 0.088, 0.044, 0.022, 0.011, 0.0055, 0.00275 pg/ml), DOX (0.064, 0.032, 0.016, 0.008, 0.004, 0.002, 0.001 pg/ml), CP (1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 pg/ml) or the combination of 3C12C-MMAE/DOX (0.176/0.064, 0.088/0.032, 0.044/0.016, 0.022/0.008, 0.011/0.004, 0.0055/0.002, 0.00275/0.001 pg/ml), the combination of 3C12C-MMAE/CP (0.176/1.6, 0.088/0.8, 0.044/0.4, 0.022, /0.2 0.011/0.1, 0.0055/0.05, 0.00275/0.025 pg/ml for 72 hrs
  • Mino or Z138 cells were cultured with serially diluted 3C12C-MMAE (0.176, 0.088, 0.044, 0.022, 0.011, 0.0055, 0.00275 pg/mL), Ibrutinib (5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 nM) or the combination of 3C12C-MMAE/lbrutinib (0.176/5, 0.088/2.5, 0.044/1.25, 0.022/0.625, 0.011/0.312, 0.0055/0.0.156, 0.00275/0.078) for 72 hours.
  • CellTiter-Glo Luminescent cell viability assay was used to determine the killing effect. Data were from one of three independent experiments.
  • the present inventors have surprisingly identified that a CD83 binding protein conjugated to a cytotoxic agent and some chemotherapies act synergistically to kill lymphoma cells.
  • chemotherapies that include an anthracycline or an alkylating agent in combination with a CD83 binding protein conjugated to a cytotoxic agent result in synergistic cell killing of lymphoma cells.
  • the experimental data described herein shows that surprisingly sub-therapeutic doses of an anthracycline or an alkylating agent can be used to synergise with an CD83 binding protein conjugated to a cytotoxic agent result to kill lymphoma cells.
  • the invention may then find particular use in the treatment of individuals having a lymphoma that has not responded to, or become resistant to, an anthracycline or an alkylating agent.
  • the present disclosure relates to a method for treating lymphoma in an individual.
  • Lymphoma is a group of blood cell tumors that develop from lymphocytes.
  • the lymphoma may be HL or NHL.
  • the lymphoma is HL.
  • Hodgkin lymphoma is a lymphoma characterised by the presence of Hodgkin and Reed-Sternberg cells (HRS cells). HRS cells are identified typically as large bi-nucleated cells with prominent nucleoli and a CD45-, CD30 + , CD15 + immunophenotype. Typical characteristics of HRS cells include large size (20-50 micrometres) and are either multinucleated or have a bilobed nucleus with prominent eosinophilic inclusion-like nucleoli (thus resembling an "owl's eye” appearance).
  • the lymphoma is NHL. NHL is lymphoma not involving HRS cells.
  • the NHL is MCL.
  • Mantle cell lymphoma is a subtype of B-cell lymphoma, due to CD5 positive antigen-naive pre-germinal center B-cells within the mantle zone that surrounds normal germinal center follicles.
  • Mantle cell lymphoma cells generally over-express cyclin D1.
  • the NHL is diffuse large B-cell lymphoma (DLBCL).
  • DLBCL diffuse large B-cell lymphoma
  • the NHL sub-type is Follicular lymphoma (FL).
  • CD83 is a single-pass type I membrane protein and member of the immunoglobulin superfamily. Three human transcript variants encoding different isoforms of CD83 have been identified.
  • the amino acid sequence of the human CD83 (hCD83) isoforms are shown in SEQ ID NO: 1 (NP_004224.1; isoform a), SEQ ID NO: 2 (NP_001035370.1; isoform b) and SEQ ID NO: 3 (NP_001238830.1; isoform c).
  • the amino acid sequence of human CD83 comprises an amino acid sequence as shown in SEQ ID NO: 1, 2, or 3.
  • CD83 Homologs of CD83 can be found in Pan troglodytes (XP_518248.2), Macaca mulatta (XP_001093591.1), Canis lupus familiaris (XP_852647.1), Bos Taurus (NP_001040055.1), Mus musculus (NP_033986.1), Rattus norvegicus (NP_001101880.1), Papio Anubis (XP_003897130.1, XM_003897081.2) and Gallus gallus (XP_418929.1).
  • CD83 is a marker of activated dendritic cells (DC), and is also expressed on activated B cell, T cells, macrophages, neutrophils etc. There are membrane-bound forms of CD83, and soluble forms of CD83 (sCD83).
  • a CD83 binding protein is a protein which is capable of specifically binding to CD83.
  • the term “CD83 binding protein” includes a single polypeptide chain (i.e., a series of contiguous amino acids linked by peptide bonds), or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex or protein), capable of specifically binding to CD83.
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • the CD83 binding protein typically comprises an antigen binding domain.
  • An “antigen binding domain” is a region of an antibody that is capable of specifically binding to an antigen.
  • the antigen binding domain of a CD83 binding protein specifically binds to CD83.
  • An antigen binding domain typically comprises the complementarity determining region (CDR) 1 , 2 and/or 3 of the heavy chain variable region, and/or the CDR 1, CDR2 and/or CDR3 of the light chain variable region, of an antibody. More typically, the antigen binding domain comprises CDR 1, 2 and 3 of the heavy chain variable region, and CDR 1, 2 and 3 of the light chain variable region, of an antibody.
  • the antigen binding domain comprises a heavy chain variable region (VH), and/or a light chain variable region (VL), of an antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen binding domain need not be in the context of an entire antibody, for example, it can be in isolation (e.g., a domain antibody) or in another form (e.g., scFv).
  • an “antibody” refers to a protein capable of specifically binding to one or a few closely related antigens (e.g., CD83) by an antigen binding domain contained within an Fv region of the antibody.
  • An antibody comprises four chain antibodies (e.g., two light (L) chains and two heavy (H) chains), recombinant, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, human antibodies, CDR-grafted antibodies, half antibodies, and bispecific antibodies).
  • An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit.
  • Full-length antibodies comprise two heavy chains ( ⁇ 50 to 70 kDa each) covalently linked and two light chains ( ⁇ 23 kDa each).
  • a light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a k light chain or a l light chain.
  • a heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s).
  • Heavy chains of mammals are of one of the following types a, d, e, g, or m.
  • Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions.
  • the number of inter-chain disulfide bonds can vary among different types of antibodies.
  • Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus.
  • the constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain which is 330 to 440 amino acids in length.
  • the light chain variable region is aligned with the variable region of the heavy chain.
  • the antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.
  • the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody.
  • the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.
  • variable region refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs), that is, CDRI, CDR2, and CDR3, and framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs.
  • VH refers to the variable region of the heavy chain.
  • VL refers to the variable region of the light chain.
  • variable region domains refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding.
  • Each variable region domain typically has three CDR regions identified as CDRI, CDR2 and CDR3.
  • the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as "the Kabat numbering system”).
  • amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk). According to the numbering system of Kabat, VH FRS and CDRs are positioned as follows: residues 1 to 30 (FRI), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113 (FR4).
  • VL FRs and CDRs are positioned as follows: residues 1 to 23 (FRI), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3) and 98 to 107 (FR4).
  • the present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et aL, Nature 342: 877-883, 1989; and/or Al-Lazikani et al., J. Mol. Biol.
  • the CDRs are defined according to the Kabat numbering system.
  • FRs Framework regions
  • the term “Fv” refers to any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding domain that is capable of specifically binding to an antigen.
  • the VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains.
  • an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means.
  • Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, for example, CH2 or CH3 domain, for example, a minibody including other proteins like CAR T cell constructs.
  • An "Fab fragment” consists of a monovalent antigen-binding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means.
  • An "Fab 1 fragment” of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means.
  • F(ab')2 fragment of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • Fab2 M fragment is a recombinant fragment comprising two Fab fragments linked using, for example, a leucine zipper or a CH3 domain.
  • a “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
  • the term "binds" in reference to the interaction of a CD83 binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A”, the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled "A" bound to the antibody.
  • a protein that "specifically binds" or "binds specifically" to a particular antigen is a protein that reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with the particular antigen than it does with alternative antigens.
  • a protein that specifically binds CD83 binds CD83 with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens.
  • telomere binding protein binds to the antigen with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, for example, 20 nM or less, such as, 15 nM or less or 10 nM or less or 5 nM or less or 1 nM or less or 500 pM or less or 400 pM or less or 300 pM or less or 200 pM or less or 100 pM or less.
  • KD equilibrium constant
  • epitope As used herein, the term “epitope” (syn. "antigenic determinant”) means a region of an antigen to which a protein comprising an antigen binding domain of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or at least 5 to 10 or 2 to 5 or 1 to 3 amino acids outside of this region. In some examples, the epitope is a linear series of amino acids. An epitope may also comprise a series of discontinuous amino acids that are positioned close to one another when an antigen is folded, that is, a "conformational epitope".
  • epitope is not limited to peptides or polypeptides.
  • the term “epitope” includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.
  • the method may employ any CD83 binding protein which is tolerated by the individual and which has a high affinity for CD83.
  • CD83 binding proteins suitable for use in the method of the invention may be identified by screening libraries of antibodies or proteins comprising an antigen binding domain (e.g. comprising variable regions of antibodies) to identify CD83 binding proteins. Methods for screening libraries of proteins comprising antigen binding domains which specifically bind CD83 are described in, for example, WO2014/117220, and W02016/061617.
  • CD83 binding protein is an antibody.
  • the antibody is a polyclonal antibody.
  • Polyclonal antibodies may be prepared using methods that are known in the art. Polyclonal antibodies can be raised in an animal, preferably mammal, e.g., by one or more injections of an antigenic composition which is used to immunize the animal or mammal. Typically, the antigenic composition is administered by multiple intravenous, subcutaneous or intraperitoneal injections. The immunization protocol may be readily selected by those skilled in the art. Methods for immunization and isolation of polyclonal antibodies are described in, for example, Antibodies: a Laboratory Manual by E. Harlow and D. Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapter 5.
  • the CD83 binding protein is a monoclonal antibody or antigen binding fragment thereof.
  • Monoclonal antibodies may be prepared using methods know in the art, and described in, for example Antibodies: A Laboratory Manual by E. Harlow and D. Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-7.
  • a monoclonal antibody may be prepared, for example, by immunizing a mouse, hamster, or other appropriate host animal, with an antigen to elicit lymphocytes that produce or can produce antibodies that will specifically bind to the antigen.
  • the antigen will typically be administered by administering an antigenic composition which includes, for example, a CD83 protein, such as that described in W02016/061617.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • a suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”), which substances prevent the growth of HGPRT- deficient cells.
  • HGPRT medium hypoxanthine, aminopterin, and thymidine
  • the antibodies can be sequenced and subsequently prepared by recombinant techniques to produce chimeric antibodies, such as humanized antibodies. Chimerisation of murine antibodies and antibody fragments are known to those skilled in the art. The use of antibody components derived from chimerized monoclonal antibodies reduces potential problems associated with the immunogenicity of murine sequence.
  • variable domains from murine antibodies may be cloned using conventional techniques that are known in the art and described in, for example, Sambrook and Russell, Eds, Molecular Cloning: A Laboratory Manual, 3 rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001.
  • the variable light chain and variable heavy chain sequences for murine antibodies can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening.
  • a chimeric antibody is an antibody protein that comprises the variable region, including the complementarity determining regions (CDRs) of an antibody derived from one species, typically a mouse antibody, while the constant domains of the antibody molecule are derived from another species, such as a human.
  • CDRs complementarity determining regions
  • the CD83 binding protein is a humanised antibody.
  • a humanised antibody is a form of chimeric antibody in which the CDRs from an antibody from one species; e.g., a mouse antibody, are transferred from the heavy and light variable chains of the mouse antibody into human heavy and light variable domains (e.g., framework region sequences).
  • the constant domains of the antibody molecule are derived from those of a human antibody.
  • the CD83 binding protein may thereof be a chimeric antibody.
  • the chimeric antibody for use in the method described herein comprises the complementarity determining regions (CDRs), and typically framework regions (FR), of a murine mAb which specifically binds a CD83 protein.
  • the chimeric antibody may comprise the light and heavy chain constant regions of a human antibody.
  • the use of antibody components derived from chimerized monoclonal antibodies reduces potential problems associated with the immunogenicity of murine constant regions. Humanization of murine antibodies and antibody fragments is known to those skilled in the art, and described in, for example, US5225539; US6054297; and US7566771.
  • humanized monoclonal antibodies may be produced by transferring murine complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts.
  • Antibodies can be isolated and purified from serum and hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al. , "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, vol. 10, pages 79-104 (The Humana Press, Inc. 1992).
  • the CD83 binding protein is a fully humanised monoclonal antibody.
  • a humanised antibody is a form of chimeric antibody in which the CDRs from an antibody from one species; e.g., a mouse antibody, are transferred from the heavy and light variable chains of the mouse antibody into human heavy and light variable domains (e.g., framework region sequences).
  • the constant domains of the antibody molecule are derived from those of a human antibody.
  • Antibodies which target CD83 can be characterized by a variety of techniques that are well-known to those of skill in the art. For example, the ability of an antibody to specifically bind to CD83 can be verified using, for example, an indirect enzyme immunoassay, flow cytometry analysis, ELISA or Western blot analysis.
  • a CD83 binding protein typically comprises the variable region of the heavy and/or light chain of an antibody, which specifically binds CD83.
  • the portions of the variable heavy and/or light chain may be on separate polypeptide chains, such as Fv fragments, or in a single polypeptide chain in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
  • the CD83 binding protein is an antigen binding fragment of an antibody.
  • An antigen binding fragment of an antibody comprises the antigen binding domain of the antibody. Examples of antigen binding fragments include F(ab')2, Fab', Fab, Fv, sFv, scFv, and the like.
  • the antigen binding fragment comprises the CDR1, 2 and/or 3 region of the variable heavy chain and/or the variable light chain. More typically, the antigen binding fragment comprises the CDR1, 2 and 3 region of the variable heavy chain and/or the variable light chain. Still more typically, the antigen binding fragment comprises the CDR1, 2 and 3 regions of the variable heavy chain and the CDR1, CDR2 and CDR3 of the variable light chain.
  • Antigen binding fragments which recognize specific epitopes can be generated by known techniques. F(ab')2 fragments, for example, can be produced by pepsin digestion of the antibody molecule. These and other methods are described, for example, by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. Alternatively, Fab' expression libraries can be constructed to allow rapid and easy identification of Fab' fragments with the desired specificity.
  • the CD83 binding protein is a single chain Fv molecule (scFv).
  • a single chain Fv molecule (scFv) comprises a VL domain and a VH domain.
  • the VL and VH domains are typically covalently linked by a peptide linker (L) and fold to form an antigen binding site. While the VH and VL regions may be directly joined together, those skilled in the art will appreciate that the regions may be separated by a peptide linker consisting of one or more amino acids. Peptide linkers and their use are known in the art. Generally the peptide linker will have no specific biological activity other than to join the regions or to preserve some minimum distance or other spatial relationship between the VH and VL.
  • Single chain Fv (scFv) antibodies optionally include a peptide linker of no more than 50 amino acids, generally no more than 40 amino acids, preferably no more than 30 amino acids, and more preferably no more than 20 amino acids in length.
  • scFv antibodies are known in the art, and have been described in, for example, US5260203.
  • mRNA from B-cells from an immunized animal, or mRNA obtained from B lymphocytes purified from a panel of human donors is isolated and cDNA is prepared.
  • the cDNA is amplified using primers specific for the variable regions of heavy and light chains of immunoglobulins.
  • the PCR products are purified, and the nucleic acid sequences are joined. If a linker peptide is desired, nucleic acid sequences that encode the peptide are inserted between the heavy and light chain nucleic acid sequences.
  • the nucleic acid which encodes the scFv is inserted into a vector and expressed in the appropriate host cell.
  • the scFv that specifically bind to the desired antigen are typically found by panning of a phage display library. Panning can be performed by any of several methods. Panning can conveniently be performed using cells expressing the desired antigen on their surface or using a solid surface coated with the desired antigen. Conveniently, the surface can be a magnetic bead. The unbound phage are washed off the solid surface and the bound phage are eluted. Methods for preparing other antigen binding fragments are known in the art. For example, antigen binding fragments can also be prepared by proteolytic hydrolysis of a full-length antibody or by expression in E.
  • An antibody fragment can be obtained by pepsin or papain digestion of full- length antibodies by conventional methods.
  • an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximate 100 Kd fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce an approximate 50 Kd Fab' monovalent fragment.
  • an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the epitope that is recognized by the intact antibody.
  • the CD83 binding protein is a bispecific antibody.
  • Bispecific antibodies preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen.
  • the bispecific antibodies are bi-specific T-cell engagers.
  • Bi-specific T-cell engagers are a class of artificial bispecific monoclonal antibodies.
  • BiTEs are fusion proteins, typically comprising two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain.
  • scFvs single-chain variable fragments
  • One of the scFvs binds to tumor antigen (e.g. CD83 target described herein) and the other generally to an effector cell, such as a T cell via the CD3 receptor.
  • tumor antigen e.g. CD83 target described herein
  • Method for preparing bispecific antibodies are described in, for example, Laszlo et al. Blood. 2014 Jan 23; 123(4): 554-561; Loffler, Blood (2000), 95: 2098-103.
  • the CD83 binding protein may be a human monoclonal antibody.
  • Human monoclonal antibodies can be generated by immunizing transgenic mice carrying genes from the human immune system or can be derived from a phage human scFv library. For example, mice containing human immunoglobulin gene loci that encode unrearranged human heavy and light chain immunoglobulin sequences, may be immunized to produce human monoclonal antibodies. Examples of transgenic mice for production of human antibodies are known in the art and described in, for example, Lonberg et al. (1994) Nature 368: 856-859; Kellermann et al. (2002) Curr. Opin. Biotechnol. 13: 593-597; Tomizuka et al. (2000) PNAS 97: 722-727.
  • the CD83 binding protein is a fully human antibody.
  • Such an antibody may be produced from a human scFv and reformatted into an antibody with constant domains from a human antibody.
  • mRNA obtained from B lymphocytes purified from a panel of human donors may be used to produce human scFv as described herein.
  • Human antibodies may be prepared by adding heavy and light chain constant regions to the heavy and light chain variable regions contained in the scFv sequences.
  • the antibodies described herein may be used to isolate other CD83 binding proteins, such as antibodies, which bind the same epitope, or overlapping epitope, by assessing cross-competition for the epitope.
  • Cross-competition with the antibody or antigen binding fragments described herein can be assessed using methods known in the art, such as BIAcore analysis, flow cytometry, ELISA analysis.
  • the CD83 binding protein comprises a heavy chain variable region (VH) which comprises:
  • the CD83 binding protein comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • CDRs three complementarity determining regions
  • the CD83 binding protein comprises an antigen binding domain which comprises:
  • VH heavy chain variable region
  • a light chain variable region which comprises: (i) a CDR1 sequence comprising the amino acid sequence of SEQ ID NO:
  • the CD83 binding protein comprises an antigen binding domain which comprises:
  • a heavy chain variable region which comprises a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 5 and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 6;
  • a light chain variable region which comprises a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 8 and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 9.
  • the CD83 binding protein comprises an antigen binding domain which comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 10, and a variable light chain comprising an amino acid sequence of SEQ ID NO: 11.
  • the CD83 binding protein is monoclonal antibody 3C12C as described in WO2014/117220.
  • the CD83 binding protein comprises an antigen binding domain which comprises:
  • nucleotide sequences encoding the light and heavy chains of antibodies described herein are shown in SEQ ID Nos: 41-59.
  • NF-KB activators or agonists Compounds or agents that activate NF-KB may also be referred to herein as NF- KB activators or agonists. Compounds or agents may increase the activity of NF-KB and/or increase the expression of or amount of NF-KB in a cell.
  • Activation of NF-KB involves the nuclear translocation of a canonical or non- canonical NF-KB complex, as described herein, which can be determined using any assay known in the art including the assays described herein.
  • Translocation may be of the p50/RelA and p50/c-Rel dimers, or of p52/RelB.
  • Exemplary compounds or agents that activate NF-KB include those described herein, including cytokines, mitogens, prostaglandins, leukotrienes, bacteria, bacterial proteins, viruses, viral proteins, chemical agents, oxidising agents, microtubule depolymerising agents (such as colchicine, nocodazole, podophyllotoxin and vinblastine), genotoxins (such as etoposide) and the RelA(p65) subunit of NF-KB or agents which cause its expression, overexpression or activation (e.g. an expression vector for RelA(p65) or a transcription factor which causes its upregulation or an agent which induces its translocation to the cell nucleus).
  • cytokines include those described herein, including cytokines, mitogens, prostaglandins, leukotrienes, bacteria, bacterial proteins, viruses, viral proteins, chemical agents, oxidising agents, microtubule depolymerising agents (such as colchicine, nocodazole,
  • NF-KB is a protein complex that controls, among other things, transcription of cellular DNA; thus, it is a transcription factor.
  • activated NF-KB binds to DNA-binding sites where expression of specific genes is turned on.
  • activators of NF-KB are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of NF-KB.
  • activators of NF-KB are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes NF-KB.
  • TNF- a Tumour necrosis factor-a
  • LT Lymphotoxin
  • IL-I lnterleukin-1 a and b
  • IL- 2 lnterleukin-2
  • LIF Leukemia Inhibitory factor
  • M- CSF Macrophage colony-stimulating factor
  • GM-CSF Granulocyte/macrophage colony-stimulating factor
  • Mitogens Antigen, Allogenic Stimulation Lectins (PHA, Con A), anti-ab T cell receptor, anti-CD3, anti CD2, anti-CD28, Phorbol esters (Diacylglycerol (DAG), Calcium ionophores (ionomycin, A2837), anti-surface IgM (P39) (CD-40 ligand), and Serum (Platelet-derived growth factor) (PDGF).
  • PHA Allogenic Stimulation Lectins
  • PDAG Allogenic Stimulation Lectins
  • DAG Diacylglycerol
  • DAG Calcium ionophores
  • P39 anti-surface IgM
  • CD-40 ligand anti-surface IgM
  • Serum Platinum-derived growth factor
  • Leukotriene B4 Prostaglandin E2 (PGE2) (Insulin), Shigella flexneri, Mycobacterium tuberculosis
  • Cell wall products Lipopolysaccharide (LPS), Muramyl peptides (G(Anh)MTetra), Toxins: Staphylococcus enterotoxin A and B (SEA and SEB), and Toxic shock syndrome toxin-1 (TSST-1) (Cholera toxin).
  • Viruses and viral products Human T cell leukemia virus-1 (HTLV-1), Tax Hepatitis B virus (HBV), Hbx, MHBs, Epstein-Barr virus (EBV), EBNA-2, LMP, Cytomegalovirus (CMV) (Human immunodeficiency virus-1) (HIV-1) Human herpes virus-6 (HHV-6), Newcastle disease virus, Sendai virus, and Adenovirus 5 ds RNA.
  • Eukaryotic parasite Theileria parva, Physical stress: UV light, Ionizing radiations (X and y) (Photofrin plus red light) (Hypoxia) and Partial hepatectomy.
  • Oxidative stress Hydrogen peroxide, Butyl peroxide, Oxidised lipids (Antimycin A).
  • Chemical agents Calyculin A, Okadaic acid (Pervanadate) (Ceramide) (Dibutyrl c- AMP) (Forskolin), Protein synthesis inhibitors, Cycloheximide, Anisomycin and Emetine.
  • the activator of NF-KB is administered simultaneously with the CD83 binding protein conjugated to a cytotoxic agent.
  • the activator of NF-KB is administered sequentially with the CD83 binding protein conjugated to a cytotoxic agent.
  • the activator of NF-KB is administered before administration of the CD83 binding protein conjugated to a cytotoxic agent.
  • a CD83 binding protein may be conjugated to a cytotoxic agent.
  • cytotoxic agents examples include chemotherapeutic agents; pro-apoptotic agents; radioisotopes; immunotoxins.
  • a cytotoxic agent is a compound which is toxic to cells.
  • cytotoxic agents include doxorubicin, cyclophosphamide, methotrexate, mustine, vincristine, procarbzine, prednisolone, bleomycin, vinblastine, dacarbazine, cyclophosphamide, Procarbazine, Paclitaxel, Irinotecan, Gemcitabine, Fluorouracil, Cytarabine, ozogamicin, adriamycin, etoposide, melphalan, mitomycin C, chlorambucil, daunorubicin.
  • a cyototoxic agent may be monomethyl-auristatin E.
  • radioisotopes examples include phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-1221, iodine-125, iodine-131, holmium-166, lutetium-177, rhenium-186, iridium-194, gold-199, astatium-211, yttrium-90, and bismuth-212.
  • immunotoxins are described in, for example, Wayne et al.
  • Anthracyclines are a class of drugs used in cancer chemotherapy that are extracted from Streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer, and lung cancers.
  • the first anthracycline discovered was daunorubicin (trade name Daunomycin), which is produced naturally by Streptomyces peucetius, a species of actinobacteria.
  • Clinically the most relevant anthracyclines are doxorubicin, daunorubicin, epirubicin and idarubicin.
  • the drugs act mainly by intercalating with DNA and interfering with DNA metabolism and RNA production. Cytotoxicity is primarily due to inhibition of topoisomerase II after the enzyme induces a break in DNA, preventing religation of the break and leading to cell death.
  • the basic structure of anthracyclines is that of a tetracyclic molecule with an anthraquinone backbone connected to a sugar moiety by a glycosidic linkage. When taken up by a cell the four ring structure intercalates between DNA bases pairs while the sugar sits within the minor groove and interacts with adjacent base pairs.
  • An anthracycline may be selected from the group consisting of daunorubicin, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride, doxorubicin HCI liposome injection (DOXIL®), dimethyl-doxorubicin (diMe-Doxo) and deoxydoxorubicin), epirubicin, idarubicin, mitoxantrone, valrubicin, aldoxorubicin, annamycin, plicamycin, piramycin, aclarubicin, zorubicin, carubicin, noglamamycin, menogaril, pitarubicin, detorubicin, esorubicin, marcellomycin, nogalamycin, rodorubicin, quelamycin, an anthracycline
  • the anthracycline is doxorubicin.
  • An alkylating antineoplastic agent is an alkylating agent used in cancer treatment that attaches an alkyl group (CnH2n+1) to DNA.
  • the alkyl group is attached to the guanine base of DNA, at the number 7 nitrogen atom of the purine ring. Since cancer cells, in general, proliferate faster and with less error-correcting than healthy cells, cancer cells are more sensitive to DNA damage — such as being alkylated.
  • the alkylating agents may be classical alkylating agents, alkylating-like or non- classical alkylating agents.
  • An alkylating agents may be selected from the group consisting of ethylenimines and methylamelamines such as thiotepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; cyclophosphamide (CY TOXAN®); mitomycins such as mitomycin C; 6-diazo-5-oxo-L-norleucine; alkyl sulfonates such as busulfan, improsulfan and pipsulfan; nitrogen mustards such as bendamustine, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard;
  • the alkylating agent is cyclophosphamide.
  • compositions and administration are typically formulated as a pharmaceutical composition for administration to the individual or subject.
  • the pharmaceutical composition comprises a CD83 binding protein formulated with a pharmaceutically acceptable carrier.
  • compositions may be formulated, for example, by employing conventional liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).
  • compositions comprising the CD83 binding protein are typically in the form of a sterile injectable aqueous suspension.
  • This suspension may be formulated according to the known art and contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients may include suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally- occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • suspending agents for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectable formulations.
  • the pharmaceutical composition may be administered by any suitable means, typically, parenterally, such as by subcutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous solutions or suspensions); in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.
  • the CD83 binding protein may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • compositions for administration to the subject may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy.
  • the pharmaceutical compositions are prepared by uniformly and intimately bringing the compound into association with a liquid carrier.
  • the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • treating means affecting an individual or subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.
  • treatment achieves the result of reducing the number of malignant lymphocytes in the recipient subject.
  • mammals refers to any animal having lymphoma which requires treatment by the present method.
  • primates such as humans
  • a variety of other mammals can be treated using the methods of the present invention.
  • mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated.
  • the individual or subject may be one that has a lymphoma that is CD83+ or CD83 high, for example, has biochemically or clinically detectable CD83 mRNA transcripts, or CD83 protein on the surface of a lymphoma cell.
  • the individual or subject may be one that has a lymphoma that is CD83- or CD83 low, for example, has biochemically or clinically undetectable CD83 mRNA transcripts or CD83 protein on the surface of a lymphoma cell.
  • the capacity of an anthracycline and/or an alkylating agent to increase the expression of CD83 makes the present invention applicable to individuals or subjects with lymphoma that is CD83-.
  • the individual or subject is not receiving ibrutinib or any other compounds that inhibits Bruton Tyrosine Kinase (BTK) or NF-KB signalling.
  • BTK Bruton Tyrosine Kinase
  • the term "effective amount” refers to the amount of the CD83 binding protein that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • an appropriate dosage level will generally be about 0.01 to 50 mg per kg patient body weight per dose.
  • the dosage level will be about 0.1 to about 25 mg/kg per dose; more preferably about 0.5 to about 10 mg/kg per dose.
  • a suitable dosage level may be about 0.01 to 25 mg/kg per dose, about 0.05 to 10 mg/kg per dose, or about 0.1 to 5 mg/kg per dose. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 5 mg/kg per dose. Dosage may be administered once or multiple times.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • a dose escalation regime in which a CD83 binding protein or other active ingredient is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject's initially suffering adverse events.
  • multiple doses in a week may be administered.
  • increasing doses may be administered.
  • One or more CD83 binding protein conjugated to a cytotoxic agent can be administered to a subject by an appropriate route, either alone or in combination with (before, simultaneous with, or after) an anthracycline and/or an alkylating agent.
  • the CD83 binding protein conjugated to a cytotoxic agent of the present disclosure can be administered in combination with, for example, one or more agents, such as one or more anthracycline and/or an alkylating agents typically used for the treatment of lymphoma.
  • the dose of anthracycline and/or alkylating agent administered is a sub-optimal or sub-therapeutic dose.
  • a sub-optimal or sub-therapeutic dose is a dose that is unable to achieve the therapeutic goal. That goal may be for example, a reduction in tumour size or mere stasis of tumour growth.
  • a sub-optimal dose is one which does not cause significant adverse side effects in the patient.
  • a sub-optimal dose can be determined according to methods well known in the art. For example, generally an optimal or therapeutic dose for an anthracycline is 50mg/m 2 with a maximum dose of 75mg/m2. A sub optimal dose is generally less than this. This can be further determined by providing an amount of anthracycline less than 25mg/m 2 which is the mini CHOP dosage and is still clinically active, and measuring therapeutic impact and/or side effects of anthracycline administration. In one embodiment, a sub-therapeutic dose of doxorubicin may be less than 25mg/m 2 .
  • the sub-optimal dose of an anthracycline may be mg/m 2 of less than 75mg/m 2 , preferably from less than 50mg/m 2 , or even more preferably less than 25mg/m 2 .
  • the sub-optimal dose of an anthracycline may be expressed as a % of a therapeutic or optimal dose, or % reduction of a therapeutic or optimal dose.
  • a sub-optimal dose may 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of the maximum 75mg/m 2 therapeutic dose, of a 50mg/m 2 therapeutic dose, or of a 25mg/m 2 therapeutic dose.
  • an optimal dose is normally expressed as 400mg/m 2 for MCL but maxi CHOP uses 750mg/m 2 .
  • Exemplary ranges for cyclophosphamide are generally from 250mg/m 2 to 1200mg/m 2 .
  • the sub-optimal doses of an alkylating agent may be expressed as a % of a therapeutic or optimal dose, or % reduction of a therapeutic or optimal dose.
  • a sub-optimal dose may 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of 250mg/m 2 , 400mg/m 2 , 750mg/m 2 or of 1200mg/m 2 therapeutic dose.
  • the sub-optimal doses of alkylating agents or anthracyclines may be expressed as a % of a therapeutic or optimal dose, % reduction of a therapeutic or optimal dose.
  • a sub-optimal dose may be provided in 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% reduction of a therapeutic dose.
  • the sub-optimal dose may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction of a therapeutic or optimal dose.
  • Each treatment cycle typically consists of about 21 to 28 days.
  • the cycle commences on day 1 which is the day on which the anthracycline and/or alkylating agent is first given. The drug is not given for the following 20 days.
  • the cycle is completed at 20 days from day 1.
  • a second treatment cycle may be provided. This cycle will commence on the day after the 1 st treatment cycle is completed by giving the drug (i.e. anthracycline and/or alkylating agent) on the day after the 1 st treatment cycle is completed. The drug is not given for the following 20 days.
  • the second treatment cycle is completed by 20 days from the commencement of the 2 nd treatment cycle.
  • Third, fourth, fifth and more treatment cycles may be provided accordingly.
  • anthracycline and/or alkylating agent is given once every 21 or 28 days is because the therapeutic dose of the drug is so toxic that the patient cannot receive it more frequently.
  • the sub-therapeutic doses of anthracycline and/or alkylating agent which are given according to the invention do not cause such significant side effects and therefore are able to be given more frequently than otherwise is possible with a therapeutic dose.
  • a sub-therapeutic dose of anthracycline and/or alkylating agent is given every 7 to 21 days, preferably about every week or 2 weeks.
  • anthracycline and/or alkylating agent which are given according to the invention enable an enhanced or potentiated effect of these cytotoxic agents in each treatment cycle, enabling a treatment cycle to be longer than the conventional 21 or 28 day period. This would enable a patient to have more time between outpatient visits for administration of anthracycline and/or alkylating agent.
  • a therapeutic dose is given in a treatment cycle that is greater than 28 days, preferably about 30 to 90 days, preferably about 1.5 to 2 months. Fewer treatment cycles
  • cancer treatment regimes are conventionally applied over a 5 to 6 months period, involving about 5 to 6 treatment cycles.
  • an anthracycline and/or alkylating agent which are given according to the invention enable an enhanced or potentiated effect of these cytotoxic agents in each treatment cycle, thereby requiring few treatment cycles.
  • a therapeutic dose is given in few than 5 to 6 treatment cycles, preferably 2 to 5 cycles, preferably 3 or 4 cycles.
  • MCL patient samples were collected with informed consent approved by the Sydney Local Health District Human Research Ethics Committee (X15- 0464&LNR15/RPAH/615), consistent with the declaration of Helsinki.
  • Archival paraffin embedded lymph node biopsies were obtained from 18 MCL patients at initial diagnosis (Table 1) were obtained.
  • Fresh peripheral blood mononuclear cells (PBMC) were purified by Ficoll-Hypaque (GE Healthcare) density gradient one MCL patient’s sample collected at initial diagnosis.
  • Table 1 Characteristics of 18 Mantle Cell Lymphoma patients
  • a lysosomal cathepsin B cleavable, self-emolative dipeptide (ValCit) maleimide linker was prepared from auristatin E for conjugation to partially reduced human anti-CD83 antibody 3C12C as per Francisco et al. Blood. 2003; 102(4): 1458-1465.
  • MCL cell lines (Mino, Rec-1, JVM2, Z138, purchased from ATCC), HL cell line KM-H2 (gift from Prof Volker Diehl, University of Cologne, Germany) and the Erythro- Leukemia cell line HEL (Purchased from ATCC) were cultured in complete RPMI-1640 medium containing 10% human fetal calf serum, 2mM glutaMAXTM, 100U/ml penicillin, 100pg/ml streptomycin, 1mM sodium pyruvate, 10mM HEPES, 10mM b Mercaptoethanol (Thermo Fisher Scientific) at 37°C, in 5% C0 2 .
  • BAY11-7082 Sigma-Aldrich
  • Doxorubicin DOX, DBL
  • cyclophosphamide CP, Slade Health
  • Immunohistochemical staining was performed on 3pm sections prepared from formalin fixed paraffin embedded biopsy tissue of lymph node or bone marrow from MCL patients as described previously (Li et al. Haematologica. 2018 Apr;103(4):655- 665; Ju et al. J Immunol. 2016 15;197(12):4613-25).
  • the primary CD83 antibody used was the F5 clone (Santa Cruz Biotechnology).
  • Staining was performed on a Leica Bond III Autostainer (Leica Biosystems) using a Bond Polymer Refine Detection kit for visualization with 3,3'-diaminobenzidine (DAB).
  • anti-human CD5-APC (BD Biosciences, clone L17F12), anti-human CD19-PE (BD Biosciences, clone HIB19), anti human CD45-AF488 (BioLegend, clone HI30), anti-mouse CD45-PerCP Cy5.5 (BD Biosciences, clone 30-F11), anti-human CD83-FITC (Beckman and Coulter, clone HB15a) or 3C12C-FITC (made in house) (Seldon et al. Leukemia. 2016 Mar;30(3):692- 700). Data was collected using a Fortessa X-20 or Accuri C6 (BD Biosciences) flow cytometer and the data analyzed with FlowJo software (TreeStar).
  • Cells (5000 per well) were cultured for 72 hours at in 200mI complete RPMI-1640 medium containing various concentration of 3C12C-MMAE, DOX, CP or their combination. The cell viability was analysed with the Cell-Titer-Glo Luminescent Cell viability kit according to the manufacturer’s instruction (Promega) and half maximal inhibitory concentration (IC50) was calculated.
  • IC50 half maximal inhibitory concentration
  • cells were cultured in complete RPMI-1640 medium with 3C12C, 3C12C-MMAE, or irrelevant Ig- MMAE (Herceptin-MMAE conjugated with the same chemistry as 3C12C-MMAE in house) for the indicated times and then cells were fixed in 70% cold ethanol for 2 hours. Cells were stained with propidium iodide (PI) in the presence of DNase-free RNase A (Sigma-Aldrich) and analyzed by flow cytometry.
  • PI propidium iodide
  • MCL cell lines and cells from one primary MCL sample treated with or without DOX (0.2ug/ml), CP (0.2mg/ml) for different time points were lysed to enable isolation of and the nuclear and cytoplasmic fractions were isolated using NE-PER kit (ThemoFisher Scientific) according to the manufacturer's instruction.
  • the protein concentration of each fraction was measured using a BCA protein assay reagent kit (Thermo Fisher Scientific). 5-10 pg of protein were separated by SDS-PAGE with 4- 12% Bis-Tris Plus gel (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using an Iblot blotting system (Thermo Fisher Scientific).
  • TTBS Tris-buffered saline
  • the primary antibodies included: rabbit anti-p105/p50 (ab32360, Abeam), mouse anti-p100/p52 (05- 361, EMD Millipore), rabbit anti-p65 (8242S, Cell Signaling Technology), rabbit anti- RelB (ab18027, Abeam), rabbit anti-alpha-Tubulin (T6074, Sigma-Aldrich), mouse anti human actin beta (Bio-rad) and rabbit anti-human Histone (#9715, Cell Signaling Technology).
  • Secondary anti-mouse or anti-rabbit antibody-horseradish peroxidase conjugate was incubated with membranes for 1 hour at room temperature and washed with TTBS. The blots were detected with the enhanced chemiluminescent (ECL) (Bio-Rad) reagents according to the manufacturer’s instructions.
  • ECL enhanced chemiluminescent
  • BD Falcon 100pm nylon cell strainer
  • CD83 is expressed on some MCL cell lines and in lymph node/bone marrow biopsies of MCL patients
  • CD83 expression was analyzed on MCL cell lines. Mino cells and Rec-1 cells expressed the CD83 on the cell surface, whilst the Z138 and JVM2 lines did not.
  • the HL cell line KM-H2 expressed the highest level of CD83 on the cell surface ( Figure 1 A).
  • Cell surface CD83 was expressed on the CD19+/CD5+ population of PBMC from two primary MCL patients, 30.3% for MCL01 and 19.1% for MCL02 ( Figure 1B).
  • CD83 mRNA transcript level was higher in CD83 + MCL lines (Mino and Rec-1) than CD83- MCL cells (Z138 and JVM2).
  • primary MCL cells expressed more CD83 mRNA transcripts than a primary acute myeloid leukemia or healthy donor PBMC ( Figure 1C).
  • CD83 expression was analyzed on bone marrow biopsies or formalin fixed paraffin embedded lymph node biopsies of 18 MCL patients. Patient clinical characteristics are detailed in Table 1. The average age of patients was 67.2 years and male samples were predominant (16/18, 88.9%). 83.3% (15/18) of the patients were assessed to be stage IV based on Ann Arbor Stage criteria (Matasar et al. Radiol Clin North Am 2008 Mar;46(2): 175-98, vii). The Average Mantle Cell Lymphoma International Prognostic Index (Ml PI) scores of high risk or intermediate/low/risk were 55.6% and 22.2%, respectively.
  • Ml PI Mantle Cell Lymphoma International Prognostic Index
  • CD83-MMAE drug activity was tested on four MCL lines (Mino, Rec-1, Z138 and JVM2).
  • CD83 + HL cell line KM-H2 and CD83 Erythro-Leukemia cell line HEL were included as control.
  • Cells were exposed to 3C12C-MMAE for 72 hours, then the metabolic-based luciferase assay CellTitre-Glo was used to quantify viable cells.
  • 3C12C-MMAE killed MCL CD83 + cells and Mino cells efficiently. Although Mino cells expresses less CD83 on the cell surface than KM-H2 cells, the IC50 level of both cell lines is similar - IC50: 0.017ug/ml KM-H2; 0.021 ug/ml for Mino.
  • Anti-CD83 antibody drug conjugate effectively kills MCL in a xenograft model
  • 3C12C-MMAE inhibited the tumor growth and increased the survival in tumour engrafted mice compared with control antibody-MMAE conjugate (Figure 3B&3C).
  • Figure 3C the saline injection and isotype-MMAE survival overlapped whereas the 3C12C-MMAE increased survival and is shown on the graph as the far-right line.
  • CD83 upregulation correlates with NF-KB activation in MCL
  • the CD83 promoter contains NF-KB binding sites. Activation of NK-KB in normal B-cells and some B-cell malignancies induces CD83 expression.
  • the canonical NF-KB pathway is activated in some MCL cell lines and primary samples. To reveal the potential relationship between CD83 expression and NF-KB activation status in MCL, we extracted cytosol and nuclear protein from MCL cell lines and analysed NF-KB activation by western blot.
  • CD83 + MCL cells Mino and Rec-1
  • p50 and RelA in the nuclear fraction, indicating strong canonical NF-KB pathway activation.
  • p52 and RelB levels were high in cytosol and nuclear portion indicating non-canonical NF-KB pathway activation ( Figure 4A).
  • MCL01 The primary MCL PBMC cell lysate (MCL01), which was surface CD83 positive, had a similar canonical NF-KB pathway activation pattern to Mino cells ( Figure 4B).
  • CD83 mRNA level was reduced in both Mino and Rec-1 cells at concentration of 1.25mM for 18 hours exposure of BAY11-7082 (Figure 4C).
  • CD83 cell surface protein level was also reduced by canonical NF-KB inhibitors ( Figure 4D).
  • Ibrutinib a reagent for treatment of refractory and relapsed MCL, blocks activity of a specific protein called Bruton’s tyrosine kinase (BTK) leading to inhibition of NF-KB.
  • BTK tyrosine kinase
  • CD83 ADC has synergic killing effect with anthracvclines and alkylating agents
  • NF-KB activation can be induced in some malignancies by cytotoxic medications such as doxorubicin.
  • Mino and Z138 cells were cultured with drugs included in standard chemotherapy regimens and novel treatment agents (Table 2).
  • Table 2 Effect of chemotherapy reagents on CD83 expression of mantle cell lymphoma cell lines
  • CD83 + Mino cells were more sensitive to the killing with DOX and CP and upregulated NF-KB molecules after short culture with DOX or CP ( Figure 5).
  • DOX and CP upregulated CD83 on CD83 + Mino and CD83- Z138 cells whilst other chemotherapy drugs had no effect on its expression. This renders the possibility of a synergistic interaction between CD83-MMAE with some chemotherapies.
  • MCL cells were cultured with 3C12C-MMAE, DOX, CP alone or in combination.
  • the Combination Index (Cl) of less than one showed the killing effect of 3C12C-MMAE combined with DOX or CP on Mino and Z138 cells was synergistic ( Figure 6c). Whilst the Cl of Z138 with 3C12C-MMAE and DOX was close to one, in three experiments it was consistently less than one. ( Figure 6C). This synergistic activity was observed at subtherapeutic concentrations of DOX or CP.
  • CD83 expression has been reported in some lymphomas and leukemias. We showed here that CD83 is upregulated in 50% of primary MCL samples and some MCL cell lines, as well as tumor cells in MCL cell line engrafted mice.
  • Ibrutinib reduced the CD83 expression and neutralized the killing of anti-CD83 ADC on MCL. This indicates that Ibrutinib should not be combined with anti-CD83 ADC clinically.
  • Current treatment of younger patients with MCL often includes DOX and CP.

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Abstract

The present invention relates to compositions and methods of use thereof for the treatment of lymphoma. In one aspect, the present invention provides a method of treating lymphoma in an individual who has received or is receiving chemotherapy that includes an anthracycline and/or an alkylating agent, the method comprising administering a CD83 binding protein conjugated to a cytotoxic agent to the individual, thereby treating lymphoma in the individual. The invention also relates to the use of treating lymphoma with an activator of NF-κB and a CD83 binding protein conjugated to a cytotoxic agent.

Description

CD83 binding protein conjugates for treating lymphoma
Field of the invention
The present invention relates to compositions and methods of use thereof for the treatment of lymphoma.
Cross-reference to earlier application
This application claims priority from Australian provisional application no. 2020902090, the entire contents of which are hereby incorporated by reference in their entirety.
Background of the invention
Lymphoma is a group of blood cell tumors that develop from lymphocytes. The two main categories of lymphomas are Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
HL is a haematological malignancy caused by Hodgkin and Reed-Sternberg cells (HRS). NHL includes all lymphomas except HL. Currently, patients suffering from HL are treated with multi-agent chemotherapy and radiotherapy. While current therapies for HL have a significant rate of success, 25% of patients experience disease relapse when they become refractory to either primary or secondary chemotherapy, and survival remains substantially lower especially in elderly patients who cannot tolerate such therapy. Therapies for NHL have a lower rate of success than for HL. Almost 1 in every 2 people with NHL will have the diffuse large B-cell lymphoma (DLBCL) form of the lymphoma and a further 5-10% of people with NHL will have Mantle cell lymphoma (MCL).
Given the above, there is a need for new and/or improved treatments for lymphoma.
Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
Summary of the invention
In one aspect, the present invention provides a method of treating lymphoma in an individual who has received or is receiving chemotherapy that includes an anthracycline and/or an alkylating agent, the method comprising
- administering a CD83 binding protein conjugated to a cytotoxic agent to the individual; thereby treating lymphoma in the individual.
In another aspect, the present invention provides a method of treating lymphoma in an individual, the method comprising
- providing an individual who has received or is receiving chemotherapy that includes an anthracycline and/or an alkylating agent;
- administering a CD83 binding protein conjugated to a cytotoxic agent to the individual; thereby treating cancer in the individual.
In another aspect, the present invention provides a method of treating lymphoma in an individual, the method comprising
- providing an individual in whom the lymphoma is to be treated;
- administering to the individual a compound that activates NF-KB; and
- administering to the individual a CD83 binding protein conjugated to a cytotoxic agent; thereby treating cancer in the individual.
In another aspect, the present invention provides a method of treating lymphoma in an individual, the method comprising
- providing an individual in whom the lymphoma is to be treated; - administering to the individual a chemotherapy that includes an anthracycline and/or an alkylating agent; and
- administering to the individual a CD83 binding protein conjugated to a cytotoxic agent; thereby treating cancer in the individual.
In another aspect, the present invention provides a method of treating lymphoma in an individual, the method comprising
- providing an individual in whom the lymphoma is to be treated;
- administering to the individual an anthracycline and/or an alkylating agent; and
- administering to the individual a CD83 binding protein conjugated to a cytotoxic agent; thereby treating cancer in the individual.
In any aspect or embodiment, the anthracycline may be selected from the group consisting of daunorubicin, doxorubicin (including ADRIAMYCIN®, morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride, doxorubicin HCI liposome injection (DOXIL®), dimethyl-doxorubicin (diMe-Doxo) and deoxydoxorubicin), epirubicin, idarubicin, mitoxantrone, valrubicin, aldoxorubicin, annamycin, plicamycin, piramycin, aclarubicin, zorubicin, carubicin, noglamamycin, menogaril, pitarubicin, detorubicin, esorubicin, marcellomycin, nogalamycin, rodorubicin, quelamycin, an anthracycline-containing liposome such as a doxorubicin-containing liposome (e. g., 2B3-101) and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above.
Preferably, the anthracycline is doxorubicin.
In any aspect or embodiment, the alkylating agent (including monofunctional and bifunctional alkylators) may be selected from the group consisting of ethylenimines and methylamelamines such as thiotepa, altretamine, triethylenemelamine, triethylenephosphoramidetriethiylenethiophosphoraminde and trimethylolomelamine; cyclophosphamide (CY-TOXAN® or Neosar®); mitomycins such as mitomycin C; 6- diazo-5-oxo-L-norleucine; alkyl sulfonates such as busulfan, improsulfan and pipsulfan; nitrogen mustards such as bendamustine, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard; triazines such as dacarbazine and temozolomide,; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, streptozocin and ranimnustine; DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, carboplatin, oxaliplatin, methotrexate, 5-fluorouracil, and aranoside (“Ara-C”) and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above. The most clinically relevant alkylating agents were chosen for experiments. Some alkylating agents are known to be toxic in humans.
Preferably, the alkylating agent is cyclophosphamide.
In any aspect or embodiment, the compound that activates NF-KB may be, phytohemagglutinin (PHA); phorbol esters, tetradecanoyl phorbol acetate (TPA); lipopolysaccharide (LPF); tumor necrosis factor-a (TNFa); prostratin; TRAF family member-associated NFKB activator (TANK) polypeptide; I L- 1 b , glutamate or any other compound described herein.
In any aspect of the present invention, the lymphoma may be Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
In any embodiment, the NHL may be Mantle cell lymphoma (MCL).
In any embodiment, the NHL may be diffuse large B-cell lymphoma (DLBCL).
In another embodiment, the NHL may be Follicular lymphoma (FL).
In any aspect of the present invention, the CD83 binding protein comprises an antigen binding domain that binds to CD83.
In aspect of the present invention, the CD83 binding protein is an antibody or antigen binding fragment thereof. The antibody fragment may be selected from the group consisting of a dAb, Fab, Fd, Fv, F(ab’)2, scFv or any other antibody fragment format described herein.
In any aspect, the chemotherapy may be administered simultaneously with the CD83 binding protein conjugated to a cytotoxic agent.
In any aspect, the chemotherapy may be administered sequentially to the CD83 binding protein conjugated to a cytotoxic agent.
In any aspect, the chemotherapy may be administered prior to the CD83 binding protein conjugated to a cytotoxic agent.
In any aspect, the CD83 binding protein conjugated to a cytotoxic agent may be administered prior to the chemotherapy.
In any aspect, the method or use of the invention further comprises a step of determining whether the individual or subject has a lymphoma that is CD83+ or CD83-. Preferably, this step is performed prior to any administering step.
In any aspect, the individual has a lymphoma that is CD83+.
In any aspect, the individual has a lymphoma that is CD83-.
In another aspect, the present invention also provides a CD83 binding protein conjugated to a cytotoxic agent for use in the treatment of lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent.
In another aspect, the present invention also provides a CD83 binding protein conjugated to a cytotoxic agent for use in the treatment of lymphoma in an individual, who has received, or who is receiving, an activator of NF-KB.
In another aspect, the present invention also provides a medicament comprising, consisting essentially of or consisting of a CD83 binding protein conjugated to a cytotoxic agent, wherein the medicament is used for treating lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent. In another aspect, the present invention also provides a kit comprising, consisting essentially of or consisting of
(a) a CD83 binding protein conjugated to a cytotoxic agent; and
(b) an anthracycline and/or an alkylating agent; and instructions for using (a) and (b) in treating lymphoma in an individual.
Preferably, the instructions describe any method of treatment described herein.
In another aspect, the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a medicament for the treatment of lymphoma in an individual who has received, or who is receiving, a chemotherapy that includes an anthracycline and/or an alkylating agent.
In another aspect, the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a medicament for the treatment of lymphoma in an individual who has received, or who is receiving, an activator of NF-KB.
In another aspect, the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a first medicament and use of an anthracycline and/or an alkylating agent in the manufacture of a second medicament, wherein the first and second medicaments are used to treat lymphoma in an individual.
In another aspect, the present invention also provides, use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a first medicament and use of an activator of NF-KB in the manufacture of a second medicament, wherein the first and second medicaments are used to treat lymphoma in an individual.
In any aspect, the cytotoxic agent conjugated to a CD83 binding protein may be any cytotoxic agent described herein.
In any aspect, the individual has received, or is receiving, a sub-therapeutic dose of the anthracycline and/or the alkylating agent. In any aspect, the anthracycline and/or an alkylating agent is administered to the individual in a sub-therapeutic dose.
In certain embodiments, the sub-optimal or sub-therapeutic doses may be expressed as a % of a therapeutic or optimal dose, or a % reduction of a therapeutic or optimal dose. For example, a sub-optimal dose may be provided in 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of a therapeutic dose. Alternatively, the sub-optimal dose may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction of a therapeutic or optimal dose.
In any aspect, the individual may have lymphoma that is resistant to an anthracycline and/or alkylating agent. In one embodiment, the individual may have received an anthracycline and/or alkylating agent to which the lymphoma responded but overtime may have developed resistance such that previously therapeutic doses no longer provide any therapeutic benefit. Alternatively, the individual may have never responded to an anthracycline and/or alkylating agent.
As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. As used herein, “comprising” and “including” are used interchangeably.
Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Brief description of the drawings
Figure 1. Mantle cell lymphoma (MCL) cells express of CD83. (A) CD83 expression on four MCL cell lines (Mino, Rec-1, Z138 and Jvm2) was analysed by flow cytometry with human anti-CD83 antibody 3C12C-FITC (n=4). (B) Primary blastoid non- nodal MCL patient (MCL01 and MCL02) blood samples were stained with CD19-V450, CD5-APC and 3C12C-FITC and analysed by flow cytometry for the CD83 expression on CD19+/CD5+ cells. (C) Quantitative real time PCR analysis of mRNAs for CD83 in MCL cell lines and one MCL primary sample. Data were normalized by the amount of HPRT mRNA and calculated as fold change compared with CD83 level in Minami-1 and PBMC from healthy donors, respectively. (D) Formalin fixed paraffin embedded (FFPE) biopsies of 18 primary MCL patients were stained with anti-CD83 antibody. Percentage of CD83 positive cells is shown.
Figure 2. 3C12C conjugation with monomethyl auristatin E (3C12C-MMAE) kill Mantle Cell Lymphoma (MCL) lines in vitro
(A) CD83+ KM-H2, Mino, Rec-1 or CD83 Z138, JVM2, HEL cells were cultured with different concentrations of 3C12C-MMAE for 72 hours before determining viable cells by CellTiter-Glo Luminescent Cell Viability assay. Data from one representative experiment with half maximal inhibitory concentration (IC50) (left) and the mean ± SEM of three experiment data (right) is shown. (B) Mino cells were cultured in of 3C12C- MMAE (antibody concentration of 0.2pg/ml) for 18 hours, then cells were fixed for cell cycle analysis by flow cytometry. Data from one representative experiment (upper panel) and the meant SEM of three experiment data (bottom panel) is shown.
Figure 3. CD83 ADC kills MCL in xenograft mice. (A) Schematic design of the MCL xenograft NSG mouse model. (B)
Quantification of tumor volume from MCL engrafted mice treated with 3C12C-MMAE, isotype control-MMAE or saline is shown (n =6 mice per group). One of three representative experiments is shown. (C) Kaplan-Meier survival curves of mice implanted with Mino cells (n =6 mice per group). Data was analysed with log-rank test. ***: p<0.001. One of three representative experiments is shown.
Figure 4. Activation of NF-KB increases CD83 expression in MCL
(A) Western blot analysis of canonical pathway NF-KB proteins (p105/p50 and RelA) and non-canonical pathway NF-KB proteins (p100/p52 and RelB) levels in the cytosolic and nuclear extracts of CD83+ MCL cells (Mino and Rec-1) and CD83- MCL cells (Z138 and JVM2). (B). Western blot analysis of canonical N F-KB proteins (p105/p50 and RelA) and non-canonical NF-KB proteins (p100/p52 and RelB) levels in the cytosolic and nuclear extracts of CD83+ MCL cells (Mino) and primary MCL cells (MCL01). (C) CD83+ cells were treated with either a DMSO control or canonical NF-KB inhibitor BAY-11-7082 at different concentrations (0.25mM or 1.25mM) for 4 or 24 hrs. Real-time PCR (qPCR) analyses of CD83 mRNA level in Mino (left) and Rec-1 (right) cells is shown. *: p<0.05. (D) CD83+ cells were treated with either a DMSO control or BAY-11-7082 (0.25pM) for 24 hours. Cell surface CD83 expression on Mino (upper) and Rec-1 (bottom) was analysed by flow cytometry and mean fluorescence intensity (MFI) normalized to the untreated samples are shown ( n = 3). *: P < 0.05; **: P <0.01. Data from one representative experiment is shown in right panel. Filled histogram: isotype control. Open histogram with solid line: CD83 staining on cells treated with DMSO. Open histogram with dash lines: cells treated with BAY-11-7082 (0.25pM). (E) Mino cells or Rec-1 were cultured in different concentrations of Ibrutinib for 72 hours, CD83 expression was analysed by flow cytometry with human anti-CD83 antibody 3C12C- FITC, mean fluorescent intensity of one representative experiment ( n = 3) is shown.
Figure 5. Doxorubicin and Cyclophosphamide activated NF-KB in MCL lines.
Mino or Z138 cells were cultured in the presence of Doxorubicin (DOX, 0.2ug/ml) or Cyclophosphamide (CP, 0.5mg/ml) for 30 minutes, 2 hrs, 6 hrs and 24 hrs. Cytoplasmic and nuclear protein were isolated. Immunoblot analysis of cell lysate was performed with anti-NF-kB antibodies.
Figure 6. 3C12C ADC synergized with Doxorubicin/Cyclophosphamide
Mino or Z138 cells were cultured in the presence of Doxorubicin (DOX, 0.02pg/ml) or Cyclophosphamide (CP, 0.2mg/ml) for 48 hrs, cell surface CD83 expression was analysed by flow cytometry. Mean fluorescent intensity ± SEM of three experiments were shown. (A) and one representative data (B) was shown. (C) Mino or Z138 cells were cultured with serial dilution of 3C12C-MMAE (0.176, 0.088, 0.044, 0.022, 0.011, 0.0055, 0.00275 pg/ml), DOX (0.064, 0.032, 0.016, 0.008, 0.004, 0.002, 0.001 pg/ml), CP (1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 pg/ml) or the combination of 3C12C-MMAE/DOX (0.176/0.064, 0.088/0.032, 0.044/0.016, 0.022/0.008, 0.011/0.004, 0.0055/0.002, 0.00275/0.001 pg/ml), the combination of 3C12C-MMAE/CP (0.176/1.6, 0.088/0.8, 0.044/0.4, 0.022, /0.2 0.011/0.1, 0.0055/0.05, 0.00275/0.025 pg/ml for 72 hrs. CellTitre-Glo Luminescent cell viability assay was used to determine the killing effect. Data was from one of three independent experiments. Combination index (Cl) was analysed with CompuSyn. Figure 7. The combination effect of Ibrutinib and 3C12C-MMAE on the killing of Mino orZ138 cells.
Mino or Z138 cells were cultured with serially diluted 3C12C-MMAE (0.176, 0.088, 0.044, 0.022, 0.011, 0.0055, 0.00275 pg/mL), Ibrutinib (5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078 nM) or the combination of 3C12C-MMAE/lbrutinib (0.176/5, 0.088/2.5, 0.044/1.25, 0.022/0.625, 0.011/0.312, 0.0055/0.0.156, 0.00275/0.078) for 72 hours. CellTiter-Glo Luminescent cell viability assay was used to determine the killing effect. Data were from one of three independent experiments.
Detailed description of the embodiments
Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.
All of the patents and publications referred to herein are incorporated by reference in their entirety. For purposes of interpreting this specification, the following definitions will generally apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.
The present inventors have surprisingly identified that a CD83 binding protein conjugated to a cytotoxic agent and some chemotherapies act synergistically to kill lymphoma cells. Specifically, chemotherapies that include an anthracycline or an alkylating agent in combination with a CD83 binding protein conjugated to a cytotoxic agent result in synergistic cell killing of lymphoma cells.
The experimental data described herein shows that surprisingly sub-therapeutic doses of an anthracycline or an alkylating agent can be used to synergise with an CD83 binding protein conjugated to a cytotoxic agent result to kill lymphoma cells. This indicates that sub-therapeutic doses, which minimise side effects, of an anthracycline or an alkylating agent could be administered to an individual in conjunction with a CD83 binding protein conjugated to a cytotoxic agent thereby surprisingly achieving a therapeutic outcome. Further, the invention may then find particular use in the treatment of individuals having a lymphoma that has not responded to, or become resistant to, an anthracycline or an alkylating agent.
Lymphoma
The present disclosure relates to a method for treating lymphoma in an individual.
Lymphoma is a group of blood cell tumors that develop from lymphocytes. The lymphoma may be HL or NHL.
In one embodiment, the lymphoma is HL. Hodgkin lymphoma is a lymphoma characterised by the presence of Hodgkin and Reed-Sternberg cells (HRS cells). HRS cells are identified typically as large bi-nucleated cells with prominent nucleoli and a CD45-, CD30+, CD15+ immunophenotype. Typical characteristics of HRS cells include large size (20-50 micrometres) and are either multinucleated or have a bilobed nucleus with prominent eosinophilic inclusion-like nucleoli (thus resembling an "owl's eye" appearance). In another embodiment, the lymphoma is NHL. NHL is lymphoma not involving HRS cells.
In one embodiment, the NHL is MCL. Mantle cell lymphoma is a subtype of B-cell lymphoma, due to CD5 positive antigen-naive pre-germinal center B-cells within the mantle zone that surrounds normal germinal center follicles. Mantle cell lymphoma cells generally over-express cyclin D1.
In one embodiment, the NHL is diffuse large B-cell lymphoma (DLBCL).
In another embodiment, the NHL sub-type is Follicular lymphoma (FL).
CD83 binding protein
CD83 is a single-pass type I membrane protein and member of the immunoglobulin superfamily. Three human transcript variants encoding different isoforms of CD83 have been identified. For the purposes of nomenclature and not limitation, the amino acid sequence of the human CD83 (hCD83) isoforms are shown in SEQ ID NO: 1 (NP_004224.1; isoform a), SEQ ID NO: 2 (NP_001035370.1; isoform b) and SEQ ID NO: 3 (NP_001238830.1; isoform c). Accordingly, in one example, the amino acid sequence of human CD83 comprises an amino acid sequence as shown in SEQ ID NO: 1, 2, or 3. Homologs of CD83 can be found in Pan troglodytes (XP_518248.2), Macaca mulatta (XP_001093591.1), Canis lupus familiaris (XP_852647.1), Bos Taurus (NP_001040055.1), Mus musculus (NP_033986.1), Rattus norvegicus (NP_001101880.1), Papio Anubis (XP_003897130.1, XM_003897081.2) and Gallus gallus (XP_418929.1).
CD83 is a marker of activated dendritic cells (DC), and is also expressed on activated B cell, T cells, macrophages, neutrophils etc. There are membrane-bound forms of CD83, and soluble forms of CD83 (sCD83).
A CD83 binding protein is a protein which is capable of specifically binding to CD83. The term “CD83 binding protein” includes a single polypeptide chain (i.e., a series of contiguous amino acids linked by peptide bonds), or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex or protein), capable of specifically binding to CD83. For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
The CD83 binding protein typically comprises an antigen binding domain. An “antigen binding domain” is a region of an antibody that is capable of specifically binding to an antigen. The antigen binding domain of a CD83 binding protein specifically binds to CD83. An antigen binding domain typically comprises the complementarity determining region (CDR) 1 , 2 and/or 3 of the heavy chain variable region, and/or the CDR 1, CDR2 and/or CDR3 of the light chain variable region, of an antibody. More typically, the antigen binding domain comprises CDR 1, 2 and 3 of the heavy chain variable region, and CDR 1, 2 and 3 of the light chain variable region, of an antibody. Still more typically, the antigen binding domain comprises a heavy chain variable region (VH), and/or a light chain variable region (VL), of an antibody. The antigen binding domain need not be in the context of an entire antibody, for example, it can be in isolation (e.g., a domain antibody) or in another form (e.g., scFv).
An "antibody" refers to a protein capable of specifically binding to one or a few closely related antigens (e.g., CD83) by an antigen binding domain contained within an Fv region of the antibody. An antibody comprises four chain antibodies (e.g., two light (L) chains and two heavy (H) chains), recombinant, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, human antibodies, CDR-grafted antibodies, half antibodies, and bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kDa each) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a k light chain or a l light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, d, e, g, or m. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain which is 330 to 440 amino acids in length. The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In various embodiments, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.
As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs), that is, CDRI, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.
As used herein, the term "complementarity determining regions" (i.e. CDR I, CDR 2, and CDR 3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDR regions identified as CDRI, CDR2 and CDR3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as "the Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk). According to the numbering system of Kabat, VH FRS and CDRs are positioned as follows: residues 1 to 30 (FRI), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113 (FR4). According to the numbering system of Kabat, VL FRs and CDRs are positioned as follows: residues 1 to 23 (FRI), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3) and 98 to 107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et aL, Nature 342: 877-883, 1989; and/or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Plukthun J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system.
"Framework regions" (FRs) are those variable region residues other than the CDR residues.
As used herein, the term "Fv" refers to any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding domain that is capable of specifically binding to an antigen. The VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, for example, CH2 or CH3 domain, for example, a minibody including other proteins like CAR T cell constructs.
An "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. An "Fab1 fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means.
An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
An "Fab2M fragment is a recombinant fragment comprising two Fab fragments linked using, for example, a leucine zipper or a CH3 domain.
A "single chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
As used herein, the term "binds" in reference to the interaction of a CD83 binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A"), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled "A" bound to the antibody.
A protein that "specifically binds" or "binds specifically" to a particular antigen is a protein that reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with the particular antigen than it does with alternative antigens. For example, a protein that specifically binds CD83 binds CD83 with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens. In one example, "specific binding" of a CD83 binding protein to an antigen, means that the protein binds to the antigen with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, for example, 20 nM or less, such as, 15 nM or less or 10 nM or less or 5 nM or less or 1 nM or less or 500 pM or less or 400 pM or less or 300 pM or less or 200 pM or less or 100 pM or less.
As used herein, the term "epitope" (syn. "antigenic determinant") means a region of an antigen to which a protein comprising an antigen binding domain of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or at least 5 to 10 or 2 to 5 or 1 to 3 amino acids outside of this region. In some examples, the epitope is a linear series of amino acids. An epitope may also comprise a series of discontinuous amino acids that are positioned close to one another when an antigen is folded, that is, a "conformational epitope". The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.
The method may employ any CD83 binding protein which is tolerated by the individual and which has a high affinity for CD83. CD83 binding proteins suitable for use in the method of the invention may be identified by screening libraries of antibodies or proteins comprising an antigen binding domain (e.g. comprising variable regions of antibodies) to identify CD83 binding proteins. Methods for screening libraries of proteins comprising antigen binding domains which specifically bind CD83 are described in, for example, WO2014/117220, and W02016/061617.
In one embodiment, CD83 binding protein is an antibody.
In one embodiment, the antibody is a polyclonal antibody. Polyclonal antibodies may be prepared using methods that are known in the art. Polyclonal antibodies can be raised in an animal, preferably mammal, e.g., by one or more injections of an antigenic composition which is used to immunize the animal or mammal. Typically, the antigenic composition is administered by multiple intravenous, subcutaneous or intraperitoneal injections. The immunization protocol may be readily selected by those skilled in the art. Methods for immunization and isolation of polyclonal antibodies are described in, for example, Antibodies: a Laboratory Manual by E. Harlow and D. Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapter 5.
In one embodiment, the CD83 binding protein is a monoclonal antibody or antigen binding fragment thereof. Monoclonal antibodies may be prepared using methods know in the art, and described in, for example Antibodies: A Laboratory Manual by E. Harlow and D. Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-7. A monoclonal antibody may be prepared, for example, by immunizing a mouse, hamster, or other appropriate host animal, with an antigen to elicit lymphocytes that produce or can produce antibodies that will specifically bind to the antigen. The antigen will typically be administered by administering an antigenic composition which includes, for example, a CD83 protein, such as that described in W02016/061617. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells. After the initial raising of antibodies to a CD83 protein, the antibodies can be sequenced and subsequently prepared by recombinant techniques to produce chimeric antibodies, such as humanized antibodies. Chimerisation of murine antibodies and antibody fragments are known to those skilled in the art. The use of antibody components derived from chimerized monoclonal antibodies reduces potential problems associated with the immunogenicity of murine sequence.
The variable domains from murine antibodies may be cloned using conventional techniques that are known in the art and described in, for example, Sambrook and Russell, Eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001. In general, the variable light chain and variable heavy chain sequences for murine antibodies can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. A chimeric antibody is an antibody protein that comprises the variable region, including the complementarity determining regions (CDRs) of an antibody derived from one species, typically a mouse antibody, while the constant domains of the antibody molecule are derived from another species, such as a human.
In some embodiments, the CD83 binding protein is a humanised antibody. A humanised antibody is a form of chimeric antibody in which the CDRs from an antibody from one species; e.g., a mouse antibody, are transferred from the heavy and light variable chains of the mouse antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody.
The CD83 binding protein may thereof be a chimeric antibody. The chimeric antibody for use in the method described herein comprises the complementarity determining regions (CDRs), and typically framework regions (FR), of a murine mAb which specifically binds a CD83 protein. The chimeric antibody may comprise the light and heavy chain constant regions of a human antibody. The use of antibody components derived from chimerized monoclonal antibodies reduces potential problems associated with the immunogenicity of murine constant regions. Humanization of murine antibodies and antibody fragments is known to those skilled in the art, and described in, for example, US5225539; US6054297; and US7566771. For example, humanized monoclonal antibodies may be produced by transferring murine complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of human framework region sequences, in addition to human constant region sequences, further reduces the chance of inducing Human anti-mouse antibody (HAMA) reactions. Antibodies can be isolated and purified from serum and hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al. , "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, vol. 10, pages 79-104 (The Humana Press, Inc. 1992).
In some embodiments, the CD83 binding protein is a fully humanised monoclonal antibody. Whereas, a humanised antibody is a form of chimeric antibody in which the CDRs from an antibody from one species; e.g., a mouse antibody, are transferred from the heavy and light variable chains of the mouse antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody.
Antibodies which target CD83 can be characterized by a variety of techniques that are well-known to those of skill in the art. For example, the ability of an antibody to specifically bind to CD83 can be verified using, for example, an indirect enzyme immunoassay, flow cytometry analysis, ELISA or Western blot analysis.
A CD83 binding protein typically comprises the variable region of the heavy and/or light chain of an antibody, which specifically binds CD83. The portions of the variable heavy and/or light chain may be on separate polypeptide chains, such as Fv fragments, or in a single polypeptide chain in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"). In one embodiment, the CD83 binding protein is an antigen binding fragment of an antibody. An antigen binding fragment of an antibody comprises the antigen binding domain of the antibody. Examples of antigen binding fragments include F(ab')2, Fab', Fab, Fv, sFv, scFv, and the like. Typically, the antigen binding fragment comprises the CDR1, 2 and/or 3 region of the variable heavy chain and/or the variable light chain. More typically, the antigen binding fragment comprises the CDR1, 2 and 3 region of the variable heavy chain and/or the variable light chain. Still more typically, the antigen binding fragment comprises the CDR1, 2 and 3 regions of the variable heavy chain and the CDR1, CDR2 and CDR3 of the variable light chain. Antigen binding fragments which recognize specific epitopes can be generated by known techniques. F(ab')2 fragments, for example, can be produced by pepsin digestion of the antibody molecule. These and other methods are described, for example, by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. Alternatively, Fab' expression libraries can be constructed to allow rapid and easy identification of Fab' fragments with the desired specificity.
In some embodiments, the CD83 binding protein is a single chain Fv molecule (scFv). A single chain Fv molecule (scFv) comprises a VL domain and a VH domain. The VL and VH domains are typically covalently linked by a peptide linker (L) and fold to form an antigen binding site. While the VH and VL regions may be directly joined together, those skilled in the art will appreciate that the regions may be separated by a peptide linker consisting of one or more amino acids. Peptide linkers and their use are known in the art. Generally the peptide linker will have no specific biological activity other than to join the regions or to preserve some minimum distance or other spatial relationship between the VH and VL. However, the constituent amino acids of the peptide linker may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. Single chain Fv (scFv) antibodies optionally include a peptide linker of no more than 50 amino acids, generally no more than 40 amino acids, preferably no more than 30 amino acids, and more preferably no more than 20 amino acids in length.
Methods of making scFv antibodies are known in the art, and have been described in, for example, US5260203. For example, mRNA from B-cells from an immunized animal, or mRNA obtained from B lymphocytes purified from a panel of human donors, is isolated and cDNA is prepared. The cDNA is amplified using primers specific for the variable regions of heavy and light chains of immunoglobulins. The PCR products are purified, and the nucleic acid sequences are joined. If a linker peptide is desired, nucleic acid sequences that encode the peptide are inserted between the heavy and light chain nucleic acid sequences. The nucleic acid which encodes the scFv is inserted into a vector and expressed in the appropriate host cell. The scFv that specifically bind to the desired antigen are typically found by panning of a phage display library. Panning can be performed by any of several methods. Panning can conveniently be performed using cells expressing the desired antigen on their surface or using a solid surface coated with the desired antigen. Conveniently, the surface can be a magnetic bead. The unbound phage are washed off the solid surface and the bound phage are eluted. Methods for preparing other antigen binding fragments are known in the art. For example, antigen binding fragments can also be prepared by proteolytic hydrolysis of a full-length antibody or by expression in E. coli or another host of the DNA coding for the fragment. An antibody fragment can be obtained by pepsin or papain digestion of full- length antibodies by conventional methods. For example, an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximate 100 Kd fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce an approximate 50 Kd Fab' monovalent fragment. Alternatively, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the epitope that is recognized by the intact antibody.
In one embodiment, the CD83 binding protein is a bispecific antibody. Bispecific antibodies, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen.
In some embodiments, the bispecific antibodies are bi-specific T-cell engagers. Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies. BiTEs are fusion proteins, typically comprising two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain. One of the scFvs binds to tumor antigen (e.g. CD83 target described herein) and the other generally to an effector cell, such as a T cell via the CD3 receptor. Method for preparing bispecific antibodies are described in, for example, Laszlo et al. Blood. 2014 Jan 23; 123(4): 554-561; Loffler, Blood (2000), 95: 2098-103.
In one embodiment, the CD83 binding protein may be a human monoclonal antibody. Human monoclonal antibodies can be generated by immunizing transgenic mice carrying genes from the human immune system or can be derived from a phage human scFv library. For example, mice containing human immunoglobulin gene loci that encode unrearranged human heavy and light chain immunoglobulin sequences, may be immunized to produce human monoclonal antibodies. Examples of transgenic mice for production of human antibodies are known in the art and described in, for example, Lonberg et al. (1994) Nature 368: 856-859; Kellermann et al. (2002) Curr. Opin. Biotechnol. 13: 593-597; Tomizuka et al. (2000) PNAS 97: 722-727.
In one embodiment, the CD83 binding protein is a fully human antibody. Such an antibody may be produced from a human scFv and reformatted into an antibody with constant domains from a human antibody. For example, mRNA obtained from B lymphocytes purified from a panel of human donors may be used to produce human scFv as described herein. Human antibodies may be prepared by adding heavy and light chain constant regions to the heavy and light chain variable regions contained in the scFv sequences.
The antibodies described herein may be used to isolate other CD83 binding proteins, such as antibodies, which bind the same epitope, or overlapping epitope, by assessing cross-competition for the epitope. Cross-competition with the antibody or antigen binding fragments described herein can be assessed using methods known in the art, such as BIAcore analysis, flow cytometry, ELISA analysis.
Examples of CD83 binding proteins suitable for use in the method of the invention include anti-CD83 antibodies HB15a (available from Beckman and Coulter), HB15e (available from STEMCELL Technologies), monoclonal antibodies 3C12, 3C12B, 3C12C, 3C12D and 3C12E as described in WO2014/117220
(US20150376277A1), and monoclonal antibodies 1F7, or derivatives thereof, as described in W02016/061617 (US20170335006A1). The amino acid and nucleotide sequences of the antibodies described in WO2014/117220 (US20150376277A1) and W02016/061617 (US20170335006A1) are herein incorporated by reference.
In one embodiment, the CD83 binding protein comprises a heavy chain variable region (VH) which comprises:
(i) a sequence which is at least 90% identical to the amino acid sequence shown in SEQ ID NO:10; or (ii) three complementarity determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO:10.
In one embodiment, the CD83 binding protein comprises:
(a) a heavy chain variable region (VH) which comprises: (i) a sequence which is at least 90% identical to the amino acid sequence shown in SEQ ID NO:10; or
(ii) three complementarity determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO:10; and
(b) a light chain variable region (VL) which comprises: (i) a sequence which is at least 90% identical to any one of the amino acid sequences shown in SEQ ID NO: 12, 13, 11, 14, or 15; or
(ii) three complementarity determining regions (CDRs) of any one of the amino acid sequences shown in SEQ ID NO: 12, 13, 11, 14, or 15; or
(iii) a consensus sequence as shown in SEQ ID NO: 40 or (iii) three CDRs, wherein the amino acid sequence of CDR1 , CDR2, or CDR3 is a consensus sequence shown in SEQ ID NO: 37, 38, or 39.
In one embodiment, the CD83 binding protein comprises an antigen binding domain which comprises:
(a) a heavy chain variable region (VH) which comprises: (i) a sequence which is at least 90% identical to the amino acid sequence shown in SEQ ID NO:10; or
(ii) three complementarity determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO: 10; and
(b) a light chain variable region which comprises: (i) a CDR1 sequence comprising the amino acid sequence of SEQ ID NO:
37, a CDR2 sequence comprising the amino acid sequence of SEQ ID
NO: 38 and a CDR3 sequence comprising the amino acid sequence of
SEQ ID NO: 39.
In one embodiment, the CD83 binding protein comprises an antigen binding domain which comprises:
(a) a heavy chain variable region which comprises a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 5 and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 6; and
(b) a light chain variable region which comprises a CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 8 and a CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 9.
In one embodiment, the CD83 binding protein comprises an antigen binding domain which comprises a variable heavy chain comprising an amino acid sequence of SEQ ID NO: 10, and a variable light chain comprising an amino acid sequence of SEQ ID NO: 11.
In one embodiment, the CD83 binding protein is monoclonal antibody 3C12C as described in WO2014/117220.
In various other embodiments, the CD83 binding protein comprises an antigen binding domain which comprises:
(i) a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO:12; or
(ii) a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO: 13; or
(iii) a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO:14; or (iv) a VH sequence as shown in SEQ ID NO: 10 and a VL sequence as shown in SEQ ID NO: 15; or
(v) a heavy chain sequence as shown in SEQ ID NO: 21 and a light chain sequence as shown in SEQ ID NO:16; or (vi) a heavy chain sequence as shown in SEQ ID NO: 21 and a light chain sequence as shown in SEQ ID NO:17; or
(vi) a heavy chain sequence as shown in SEQ ID NO:21 and a light chain sequence as shown in SEQ ID NO:18; or
(vii) a heavy chain sequence as shown in SEQ ID NO:21 and a light chain sequence as shown in SEQ ID NO:19; or
(viii) a heavy chain sequence as shown in SEQ ID NO:21 and a light chain sequence as shown in SEQ ID NO:20; or
(ix) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO:23; or (x) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO:24; or
(xi) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO:25;
(xii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO:26;
(viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO:27;
(viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 28; (viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 29; (viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 30;
(viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 31; (viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 32;
(viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 33; or
(viii) a VH sequence as shown in SEQ ID NO:22 and a VL sequence as shown in SEQ ID NO: 34; or
(vix) a heavy chain sequence as shown in SEQ ID NO: 35 and a light chain sequence as shown in SEQ ID NO: 36.
Examples of nucleotide sequences encoding the light and heavy chains of antibodies described herein are shown in SEQ ID Nos: 41-59.
A summary of the sequence listing is set out below:
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
NF-kB activators
Compounds or agents that activate NF-KB may also be referred to herein as NF- KB activators or agonists. Compounds or agents may increase the activity of NF-KB and/or increase the expression of or amount of NF-KB in a cell.
Activation of NF-KB involves the nuclear translocation of a canonical or non- canonical NF-KB complex, as described herein, which can be determined using any assay known in the art including the assays described herein. Translocation may be of the p50/RelA and p50/c-Rel dimers, or of p52/RelB. Exemplary compounds or agents that activate NF-KB include those described herein, including cytokines, mitogens, prostaglandins, leukotrienes, bacteria, bacterial proteins, viruses, viral proteins, chemical agents, oxidising agents, microtubule depolymerising agents (such as colchicine, nocodazole, podophyllotoxin and vinblastine), genotoxins (such as etoposide) and the RelA(p65) subunit of NF-KB or agents which cause its expression, overexpression or activation (e.g. an expression vector for RelA(p65) or a transcription factor which causes its upregulation or an agent which induces its translocation to the cell nucleus).
NF-KB is a protein complex that controls, among other things, transcription of cellular DNA; thus, it is a transcription factor. In some instances, activated NF-KB binds to DNA-binding sites where expression of specific genes is turned on. In certain embodiments, activators of NF-KB are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of NF-KB. In certain other embodiments, activators of NF-KB are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes NF-KB.
Further exemplary agents can be found in Siebenlist et al 1994 Annu. Rev. Cell Biol. 10 405-455. These include, Cytokines: Tumour necrosis factor-a (TNF- a), Lymphotoxin (LT) (TNF-b), lnterleukin-1 a and b (IL-I a and b), lnterleukin-2 (IL- 2), Leukemia Inhibitory factor (LIF) (lnterferon-g), (Macrophage colony-stimulating factor (M- CSF), and (Granulocyte/macrophage colony-stimulating factor) (GM-CSF). Mitogens: Antigen, Allogenic Stimulation Lectins (PHA, Con A), anti-ab T cell receptor, anti-CD3, anti CD2, anti-CD28, Phorbol esters (Diacylglycerol (DAG), Calcium ionophores (ionomycin, A2837), anti-surface IgM (P39) (CD-40 ligand), and Serum (Platelet-derived growth factor) (PDGF). Other biological mediators - Bacteria and bacterial products: Leukotriene B4 (Prostaglandin E2 (PGE2) (Insulin), Shigella flexneri, Mycobacterium tuberculosis Cell wall products: Lipopolysaccharide (LPS), Muramyl peptides (G(Anh)MTetra), Toxins: Staphylococcus enterotoxin A and B (SEA and SEB), and Toxic shock syndrome toxin-1 (TSST-1) (Cholera toxin). Viruses and viral products: Human T cell leukemia virus-1 (HTLV-1), Tax Hepatitis B virus (HBV), Hbx, MHBs, Epstein-Barr virus (EBV), EBNA-2, LMP, Cytomegalovirus (CMV) (Human immunodeficiency virus-1) (HIV-1) Human herpes virus-6 (HHV-6), Newcastle disease virus, Sendai virus, and Adenovirus 5 ds RNA. Eukaryotic parasite: Theileria parva, Physical stress: UV light, Ionizing radiations (X and y) (Photofrin plus red light) (Hypoxia) and Partial hepatectomy. Oxidative stress: Hydrogen peroxide, Butyl peroxide, Oxidised lipids (Antimycin A). Chemical agents: Calyculin A, Okadaic acid (Pervanadate) (Ceramide) (Dibutyrl c- AMP) (Forskolin), Protein synthesis inhibitors, Cycloheximide, Anisomycin and Emetine.
In any aspect, the activator of NF-KB is administered simultaneously with the CD83 binding protein conjugated to a cytotoxic agent.
In any aspect, the activator of NF-KB is administered sequentially with the CD83 binding protein conjugated to a cytotoxic agent.
In any aspect, the activator of NF-KB is administered before administration of the CD83 binding protein conjugated to a cytotoxic agent.
Cytotoxic agents
As used herein, a CD83 binding protein may be conjugated to a cytotoxic agent.
Examples of cytotoxic agents including chemotherapeutic agents; pro-apoptotic agents; radioisotopes; immunotoxins.
A cytotoxic agent is a compound which is toxic to cells. Examples of cytotoxic agents include doxorubicin, cyclophosphamide, methotrexate, mustine, vincristine, procarbzine, prednisolone, bleomycin, vinblastine, dacarbazine, cyclophosphamide, Procarbazine, Paclitaxel, Irinotecan, Gemcitabine, Fluorouracil, Cytarabine, ozogamicin, adriamycin, etoposide, melphalan, mitomycin C, chlorambucil, daunorubicin. A cyototoxic agent may be monomethyl-auristatin E.
Examples of radioisotopes include phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-1221, iodine-125, iodine-131, holmium-166, lutetium-177, rhenium-186, iridium-194, gold-199, astatium-211, yttrium-90, and bismuth-212. Examples of immunotoxins are described in, for example, Wayne et al. (2016) Blood, 123: 2470-2477, and include, for example, diphtheria toxin A, Ricin-dgA, Pseudomonas exotoxin A, Liposomes, Particles or indeed any toxin delivery.
Methods for conjugating cytotoxic agents to an antibody or antigen binding fragment are known in the art.
Anthracvclines
Anthracyclines are a class of drugs used in cancer chemotherapy that are extracted from Streptomyces bacterium. These compounds are used to treat many cancers, including leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer, and lung cancers. The first anthracycline discovered was daunorubicin (trade name Daunomycin), which is produced naturally by Streptomyces peucetius, a species of actinobacteria. Clinically the most relevant anthracyclines are doxorubicin, daunorubicin, epirubicin and idarubicin.
The drugs act mainly by intercalating with DNA and interfering with DNA metabolism and RNA production. Cytotoxicity is primarily due to inhibition of topoisomerase II after the enzyme induces a break in DNA, preventing religation of the break and leading to cell death. The basic structure of anthracyclines is that of a tetracyclic molecule with an anthraquinone backbone connected to a sugar moiety by a glycosidic linkage. When taken up by a cell the four ring structure intercalates between DNA bases pairs while the sugar sits within the minor groove and interacts with adjacent base pairs.
An anthracycline may be selected from the group consisting of daunorubicin, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride, doxorubicin HCI liposome injection (DOXIL®), dimethyl-doxorubicin (diMe-Doxo) and deoxydoxorubicin), epirubicin, idarubicin, mitoxantrone, valrubicin, aldoxorubicin, annamycin, plicamycin, piramycin, aclarubicin, zorubicin, carubicin, noglamamycin, menogaril, pitarubicin, detorubicin, esorubicin, marcellomycin, nogalamycin, rodorubicin, quelamycin, an anthracycline-containing liposome such as a doxorubicin-containing liposome (e.g., 2B3-101) and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above.
Preferably, the anthracycline is doxorubicin.
Alkylating agents
An alkylating antineoplastic agent is an alkylating agent used in cancer treatment that attaches an alkyl group (CnH2n+1) to DNA. The alkyl group is attached to the guanine base of DNA, at the number 7 nitrogen atom of the purine ring. Since cancer cells, in general, proliferate faster and with less error-correcting than healthy cells, cancer cells are more sensitive to DNA damage — such as being alkylated.
The alkylating agents may be classical alkylating agents, alkylating-like or non- classical alkylating agents.
An alkylating agents (including monofunctional and bifunctional alkylators) may be selected from the group consisting of ethylenimines and methylamelamines such as thiotepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; cyclophosphamide (CY TOXAN®); mitomycins such as mitomycin C; 6-diazo-5-oxo-L-norleucine; alkyl sulfonates such as busulfan, improsulfan and pipsulfan; nitrogen mustards such as bendamustine, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard; triazines such as dacarbazine and temozolomide,; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, streptozocin and ranimnustine; DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, carboplatin, oxaliplatin, methotrexate, 5-fluorouracil, aranoside (“Ara-C”), and procarbazine and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above.
Preferably, the alkylating agent is cyclophosphamide.
Pharmaceutical compositions and administration The CD83 binding proteins described herein are typically formulated as a pharmaceutical composition for administration to the individual or subject. Typically, the pharmaceutical composition comprises a CD83 binding protein formulated with a pharmaceutically acceptable carrier.
A "pharmaceutically acceptable carrier" means that it is compatible with the other ingredients of the composition and is not deleterious to an individual or subject. The compositions may be formulated, for example, by employing conventional liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).
Pharmaceutical compositions comprising the CD83 binding protein are typically in the form of a sterile injectable aqueous suspension. This suspension may be formulated according to the known art and contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally- occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable formulations.
The pharmaceutical composition may be administered by any suitable means, typically, parenterally, such as by subcutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous solutions or suspensions); in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. The CD83 binding protein may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
The pharmaceutical compositions for administration to the subject may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the compound into association with a liquid carrier. In the pharmaceutical composition the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
Generally, the term "treating" means affecting an individual or subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease. In one embodiment, treatment achieves the result of reducing the number of malignant lymphocytes in the recipient subject.
The term "individual" refers to any animal having lymphoma which requires treatment by the present method. In addition to primates, such as humans, a variety of other mammals can be treated using the methods of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated.
In any aspect of the invention, the individual or subject may be one that has a lymphoma that is CD83+ or CD83 high, for example, has biochemically or clinically detectable CD83 mRNA transcripts, or CD83 protein on the surface of a lymphoma cell.
In any aspect of the invention, the individual or subject may be one that has a lymphoma that is CD83- or CD83 low, for example, has biochemically or clinically undetectable CD83 mRNA transcripts or CD83 protein on the surface of a lymphoma cell. The capacity of an anthracycline and/or an alkylating agent to increase the expression of CD83 makes the present invention applicable to individuals or subjects with lymphoma that is CD83-.
In any aspect of the present invention, the individual or subject is not receiving ibrutinib or any other compounds that inhibits Bruton Tyrosine Kinase (BTK) or NF-KB signalling.
The term "effective amount" refers to the amount of the CD83 binding protein that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
In the treatment or prevention of lymphoma, an appropriate dosage level will generally be about 0.01 to 50 mg per kg patient body weight per dose. Preferably, the dosage level will be about 0.1 to about 25 mg/kg per dose; more preferably about 0.5 to about 10 mg/kg per dose. A suitable dosage level may be about 0.01 to 25 mg/kg per dose, about 0.05 to 10 mg/kg per dose, or about 0.1 to 5 mg/kg per dose. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 5 mg/kg per dose. Dosage may be administered once or multiple times. It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
In some examples, a dose escalation regime is used, in which a CD83 binding protein or other active ingredient is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject's initially suffering adverse events.
In the case of a subject that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.
One or more CD83 binding protein conjugated to a cytotoxic agent can be administered to a subject by an appropriate route, either alone or in combination with (before, simultaneous with, or after) an anthracycline and/or an alkylating agent. For example, the CD83 binding protein conjugated to a cytotoxic agent of the present disclosure can be administered in combination with, for example, one or more agents, such as one or more anthracycline and/or an alkylating agents typically used for the treatment of lymphoma.
Sub-optimal or therapeutic doses
In one embodiment, the dose of anthracycline and/or alkylating agent administered is a sub-optimal or sub-therapeutic dose.
A sub-optimal or sub-therapeutic dose is a dose that is unable to achieve the therapeutic goal. That goal may be for example, a reduction in tumour size or mere stasis of tumour growth.
Preferably a sub-optimal dose is one which does not cause significant adverse side effects in the patient. A sub-optimal dose can be determined according to methods well known in the art. For example, generally an optimal or therapeutic dose for an anthracycline is 50mg/m2 with a maximum dose of 75mg/m2. A sub optimal dose is generally less than this. This can be further determined by providing an amount of anthracycline less than 25mg/m2 which is the mini CHOP dosage and is still clinically active, and measuring therapeutic impact and/or side effects of anthracycline administration. In one embodiment, a sub-therapeutic dose of doxorubicin may be less than 25mg/m2.
In one embodiment, the sub-optimal dose of an anthracycline may be mg/m2 of less than 75mg/m2, preferably from less than 50mg/m2, or even more preferably less than 25mg/m2.
In certain embodiments, the sub-optimal dose of an anthracycline may be expressed as a % of a therapeutic or optimal dose, or % reduction of a therapeutic or optimal dose. For example, a sub-optimal dose may 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of the maximum 75mg/m2 therapeutic dose, of a 50mg/m2 therapeutic dose, or of a 25mg/m2 therapeutic dose.
Regarding an alkylating agent, an optimal dose is normally expressed as 400mg/m2 for MCL but maxi CHOP uses 750mg/m2. Exemplary ranges for cyclophosphamide are generally from 250mg/m2 to 1200mg/m2.
In certain embodiments, the sub-optimal doses of an alkylating agent may be expressed as a % of a therapeutic or optimal dose, or % reduction of a therapeutic or optimal dose. For example, a sub-optimal dose may 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% of 250mg/m2, 400mg/m2, 750mg/m2 or of 1200mg/m2 therapeutic dose.
In any embodiment, the sub-optimal doses of alkylating agents or anthracyclines may be expressed as a % of a therapeutic or optimal dose, % reduction of a therapeutic or optimal dose. For example, a sub-optimal dose may be provided in 90%, or 80%, or 70%, or 60% or 50%, or 40%, or 30%, or 20%, or 10% reduction of a therapeutic dose. Alternatively, the sub-optimal dose may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction of a therapeutic or optimal dose.
Frequent administration of a sub-optimal or sub-therapeutic dose Cancer chemotherapy may be administered in treatment cycles. Each treatment cycle typically consists of about 21 to 28 days. For example, in a 21 day treatment cycle, the cycle commences on day 1 which is the day on which the anthracycline and/or alkylating agent is first given. The drug is not given for the following 20 days. The cycle is completed at 20 days from day 1. A second treatment cycle may be provided. This cycle will commence on the day after the 1st treatment cycle is completed by giving the drug (i.e. anthracycline and/or alkylating agent) on the day after the 1st treatment cycle is completed. The drug is not given for the following 20 days. The second treatment cycle is completed by 20 days from the commencement of the 2nd treatment cycle. Third, fourth, fifth and more treatment cycles may be provided accordingly.
Generally, the reason why an anthracycline and/or alkylating agent is given once every 21 or 28 days is because the therapeutic dose of the drug is so toxic that the patient cannot receive it more frequently. The sub-therapeutic doses of anthracycline and/or alkylating agent which are given according to the invention do not cause such significant side effects and therefore are able to be given more frequently than otherwise is possible with a therapeutic dose.
In one embodiment a sub-therapeutic dose of anthracycline and/or alkylating agent is given every 7 to 21 days, preferably about every week or 2 weeks.
Administration of therapeutic dose less frequently
It is generally understood that the goal of cancer therapy is not achieved where a therapeutic dose is not given at least once a month. The reason for this is that after about 21 to 28 days, the anthracycline and/or alkylating agent is washed out of the patient.
The therapeutic doses of anthracycline and/or alkylating agent which are given according to the invention enable an enhanced or potentiated effect of these cytotoxic agents in each treatment cycle, enabling a treatment cycle to be longer than the conventional 21 or 28 day period. This would enable a patient to have more time between outpatient visits for administration of anthracycline and/or alkylating agent.
In one embodiment, a therapeutic dose is given in a treatment cycle that is greater than 28 days, preferably about 30 to 90 days, preferably about 1.5 to 2 months. Fewer treatment cycles
As mentioned above, cancer treatment regimes are conventionally applied over a 5 to 6 months period, involving about 5 to 6 treatment cycles.
The therapeutic doses of an anthracycline and/or alkylating agent which are given according to the invention enable an enhanced or potentiated effect of these cytotoxic agents in each treatment cycle, thereby requiring few treatment cycles.
In one embodiment a therapeutic dose is given in few than 5 to 6 treatment cycles, preferably 2 to 5 cycles, preferably 3 or 4 cycles.
It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
Examples
Example 1 Materials and Methods
Patient sample collection
MCL patient samples were collected with informed consent approved by the Sydney Local Health District Human Research Ethics Committee (X15- 0464&LNR15/RPAH/615), consistent with the declaration of Helsinki. Archival paraffin embedded lymph node biopsies were obtained from 18 MCL patients at initial diagnosis (Table 1) were obtained. Fresh peripheral blood mononuclear cells (PBMC) were purified by Ficoll-Hypaque (GE Healthcare) density gradient one MCL patient’s sample collected at initial diagnosis.
Table 1: Characteristics of 18 Mantle Cell Lymphoma patients
Figure imgf000042_0001
Figure imgf000043_0001
Human anti-CD83 antibody and antibody drug conjugation
To produce 3C12C-MMAE, a lysosomal cathepsin B cleavable, self-emolative dipeptide (ValCit) maleimide linker was prepared from auristatin E for conjugation to partially reduced human anti-CD83 antibody 3C12C as per Francisco et al. Blood. 2003; 102(4): 1458-1465.
Cell culture
MCL cell lines (Mino, Rec-1, JVM2, Z138, purchased from ATCC), HL cell line KM-H2 (gift from Prof Volker Diehl, University of Cologne, Germany) and the Erythro- Leukemia cell line HEL (Purchased from ATCC) were cultured in complete RPMI-1640 medium containing 10% human fetal calf serum, 2mM glutaMAX™, 100U/ml penicillin, 100pg/ml streptomycin, 1mM sodium pyruvate, 10mM HEPES, 10mM b Mercaptoethanol (Thermo Fisher Scientific) at 37°C, in 5% C02. BAY11-7082 (Sigma-Aldrich), Doxorubicin (DOX, DBL), cyclophosphamide (CP, Slade Health) were added to cell cultures as described.
Immunohistochemical staining Immunohistochemical staining was performed on 3pm sections prepared from formalin fixed paraffin embedded biopsy tissue of lymph node or bone marrow from MCL patients as described previously (Li et al. Haematologica. 2018 Apr;103(4):655- 665; Ju et al. J Immunol. 2016 15;197(12):4613-25). The primary CD83 antibody used was the F5 clone (Santa Cruz Biotechnology). Staining was performed on a Leica Bond III Autostainer (Leica Biosystems) using a Bond Polymer Refine Detection kit for visualization with 3,3'-diaminobenzidine (DAB).
Flow cytometry
The following antibodies were used: anti-human CD5-APC (BD Biosciences, clone L17F12), anti-human CD19-PE (BD Biosciences, clone HIB19), anti human CD45-AF488 (BioLegend, clone HI30), anti-mouse CD45-PerCP Cy5.5 (BD Biosciences, clone 30-F11), anti-human CD83-FITC (Beckman and Coulter, clone HB15a) or 3C12C-FITC (made in house) (Seldon et al. Leukemia. 2016 Mar;30(3):692- 700). Data was collected using a Fortessa X-20 or Accuri C6 (BD Biosciences) flow cytometer and the data analyzed with FlowJo software (TreeStar).
Cell viability assays and cell cycle measurement
Cells (5000 per well) were cultured for 72 hours at in 200mI complete RPMI-1640 medium containing various concentration of 3C12C-MMAE, DOX, CP or their combination. The cell viability was analysed with the Cell-Titer-Glo Luminescent Cell viability kit according to the manufacturer’s instruction (Promega) and half maximal inhibitory concentration (IC50) was calculated. For cell cycle measurement, cells were cultured in complete RPMI-1640 medium with 3C12C, 3C12C-MMAE, or irrelevant Ig- MMAE (Herceptin-MMAE conjugated with the same chemistry as 3C12C-MMAE in house) for the indicated times and then cells were fixed in 70% cold ethanol for 2 hours. Cells were stained with propidium iodide (PI) in the presence of DNase-free RNase A (Sigma-Aldrich) and analyzed by flow cytometry.
Western blot
MCL cell lines and cells from one primary MCL sample treated with or without DOX (0.2ug/ml), CP (0.2mg/ml) for different time points were lysed to enable isolation of and the nuclear and cytoplasmic fractions were isolated using NE-PER kit (ThemoFisher Scientific) according to the manufacturer's instruction. The protein concentration of each fraction was measured using a BCA protein assay reagent kit (Thermo Fisher Scientific). 5-10 pg of protein were separated by SDS-PAGE with 4- 12% Bis-Tris Plus gel (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes using an Iblot blotting system (Thermo Fisher Scientific). Following blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline (TTBS) for 2 hours at room temperature and washing with Tween 20 with TTBS, the membranes were incubated overnight at 4 °C with primary antibody in TTBS containing 5% BSA. The primary antibodies included: rabbit anti-p105/p50 (ab32360, Abeam), mouse anti-p100/p52 (05- 361, EMD Millipore), rabbit anti-p65 (8242S, Cell Signaling Technology), rabbit anti- RelB (ab18027, Abeam), rabbit anti-alpha-Tubulin (T6074, Sigma-Aldrich), mouse anti human actin beta (Bio-rad) and rabbit anti-human Histone (#9715, Cell Signaling Technology). Secondary anti-mouse or anti-rabbit antibody-horseradish peroxidase conjugate (1:3000 dilution) was incubated with membranes for 1 hour at room temperature and washed with TTBS. The blots were detected with the enhanced chemiluminescent (ECL) (Bio-Rad) reagents according to the manufacturer’s instructions.
MCL xenograft mouse tumor model
Five to six week female NOD/SCID/IL2Ry-Null mice were purchased from Australian Bioresources and housed under specific pathogen-free conditions. Experimental procedures were approved by the Sydney Local Health District (SLHD) animal welfare committee. Each mouse received 5x106 Mino cells subcutaneously on right flank area. On day 18 post cell injection, mice were treated with a single dose of 3C12C-MMAE (2.5mg/kg), Herceptin-MMAE (2.5mg/kg) or saline only (i.p.). Mice were monitored for tumor growth and the tumor volume was measured by digital caliper (tumor volume=1/2(length c width2)). The engrafted tumors were harvested, passed through a 100pm nylon cell strainer (BD Falcon) to prepare a single cell suspension, washed with PBS and analyzed by flow cytometry.
Statistical Analysis
Statistical analyses were performed using Prism 6.0 (GraphPad Software). Standard error of the mean is shown unless otherwise stated. Unpaired two-tailed student t-test or log-rank (mantel-cox) test were used as described. Differences with p<0.05 were considered significant. *: p<0.05; **: p<0.01; ***: p<0.001. The combination index (Cl) was analysed with CompuSyn (ComboSyn, Inc) (Chou et al. Cancer Res. 2010 Jan 15;70(2):440-6).
Example 2
CD83 is expressed on some MCL cell lines and in lymph node/bone marrow biopsies of MCL patients
CD83 expression was analyzed on MCL cell lines. Mino cells and Rec-1 cells expressed the CD83 on the cell surface, whilst the Z138 and JVM2 lines did not. The HL cell line KM-H2 expressed the highest level of CD83 on the cell surface (Figure 1 A). Cell surface CD83 was expressed on the CD19+/CD5+ population of PBMC from two primary MCL patients, 30.3% for MCL01 and 19.1% for MCL02 (Figure 1B).
Accordingly, CD83 mRNA transcript level was higher in CD83+ MCL lines (Mino and Rec-1) than CD83- MCL cells (Z138 and JVM2). Similarly, primary MCL cells expressed more CD83 mRNA transcripts than a primary acute myeloid leukemia or healthy donor PBMC (Figure 1C).
CD83 expression was analyzed on bone marrow biopsies or formalin fixed paraffin embedded lymph node biopsies of 18 MCL patients. Patient clinical characteristics are detailed in Table 1. The average age of patients was 67.2 years and male samples were predominant (16/18, 88.9%). 83.3% (15/18) of the patients were assessed to be stage IV based on Ann Arbor Stage criteria (Matasar et al. Radiol Clin North Am 2008 Mar;46(2): 175-98, vii). The Average Mantle Cell Lymphoma International Prognostic Index (Ml PI) scores of high risk or intermediate/low/risk were 55.6% and 22.2%, respectively. Among the 18 patients, 3/18 (16.67 %) biopsies expressed high levels of CD83 (>50% positive) on the MCL cells, 8/18 (44.44%) expressed a moderate level (10-50% positive), and 6/18 (33.33%) expressed low levels of CD83 (<10% positive) and 1/18 (5.55%) had no CD83 detection (Figure 1D). The intensity of CD83 staining on biopsies was analysed; CD83 staining was very strong (+++) in 9/18 (50%), medium level (++) in 2/18 (11.1%), weak level (+) in 6/18 (33.3%), no CD83 staining on 1/18 (5.55%). There is no correlation of CD83 expression level (cut-off 10% as low/high) with Ml PI score or clinical stage. Anti-CD83 antibody drug conjugate kills CD83+ MCL cells in vitro
The CD83-MMAE drug activity was tested on four MCL lines (Mino, Rec-1, Z138 and JVM2). CD83+ HL cell line KM-H2 and CD83 Erythro-Leukemia cell line HEL were included as control. Cells were exposed to 3C12C-MMAE for 72 hours, then the metabolic-based luciferase assay CellTitre-Glo was used to quantify viable cells. 3C12C-MMAE killed MCL CD83+ cells and Mino cells efficiently. Although Mino cells expresses less CD83 on the cell surface than KM-H2 cells, the IC50 level of both cell lines is similar - IC50: 0.017ug/ml KM-H2; 0.021 ug/ml for Mino. Both the CD83+ Mino and Rec-1 cells were susceptible to naked 3C12C via NK mediated ADCC in dose dependent manner. Rec-1 had a higher IC50 level when exposed to 3C12C-MMAE compared with Mino cells. Since MMAE conjugated antibody has been reported to induce G2/M phase arrest, we tested the effect of 3C12C-MMAE on the cell cycle of CD83+ Mino cells. About 45% Mino cells were arrested in G2/M phase after 18 hour exposure to 3C12C-MMAE and this was accompanied with a concomitant decrease in G0/G1 phase cells. The control ADC Herceptin-MMAE did not change the cell cycle of Mino cells (Figure 2B).
Anti-CD83 antibody drug conjugate effectively kills MCL in a xenograft model
We established a xenograft mouse model in which NSG mice were subcutaneously injected with Mino cells for evaluating the efficacy of CD83 ADC in vivo (Figure 3A). On day 18 post injection of Mino cells when tumors were papable, 3C12C- MMAE or control vehicle was injected (i.p.). Mice were euthanized at day 6 post injection of control vehicle and engrafted tumor cells were harvested for flow cytometry analysis. Engrafted tumors were CD5+ CD19+ and expressed CD83 at similar level to the in vitro cultured Mino cells (data not shown). 3C12C-MMAE inhibited the tumor growth and increased the survival in tumour engrafted mice compared with control antibody-MMAE conjugate (Figure 3B&3C). In Figure 3C the saline injection and isotype-MMAE survival overlapped whereas the 3C12C-MMAE increased survival and is shown on the graph as the far-right line.
CD83 upregulation correlates with NF-KB activation in MCL The CD83 promoter contains NF-KB binding sites. Activation of NK-KB in normal B-cells and some B-cell malignancies induces CD83 expression. The canonical NF-KB pathway is activated in some MCL cell lines and primary samples. To reveal the potential relationship between CD83 expression and NF-KB activation status in MCL, we extracted cytosol and nuclear protein from MCL cell lines and analysed NF-KB activation by western blot. Although the activation of NF-KB in both CD83+ and CD83- MCL lines was detected, CD83+ MCL cells, Mino and Rec-1, showed elevated p50 and RelA in the nuclear fraction, indicating strong canonical NF-KB pathway activation. Alternatively, in CD83- cell lines, p52 and RelB levels were high in cytosol and nuclear portion indicating non-canonical NF-KB pathway activation (Figure 4A). The primary MCL PBMC cell lysate (MCL01), which was surface CD83 positive, had a similar canonical NF-KB pathway activation pattern to Mino cells (Figure 4B). We then exposed CD83+ cells to the canonical NF-KB pathway inhibitor, BAY11-7082. CD83 mRNA level was reduced in both Mino and Rec-1 cells at concentration of 1.25mM for 18 hours exposure of BAY11-7082 (Figure 4C). CD83 cell surface protein level was also reduced by canonical NF-KB inhibitors (Figure 4D). Ibrutinib, a reagent for treatment of refractory and relapsed MCL, blocks activity of a specific protein called Bruton’s tyrosine kinase (BTK) leading to inhibition of NF-KB. Our data showed it downregulated CD83 expression on MCL cell lines (Mino and Rec-1) and neutralized the killing effect of 3C12C-MMAE on Mino (Figure 7)
CD83 ADC has synergic killing effect with anthracvclines and alkylating agents
NF-KB activation can be induced in some malignancies by cytotoxic medications such as doxorubicin. Mino and Z138 cells were cultured with drugs included in standard chemotherapy regimens and novel treatment agents (Table 2).
Table 2: Effect of chemotherapy reagents on CD83 expression of mantle cell lymphoma cell lines
Figure imgf000048_0001
Figure imgf000049_0001
Both canonical (p105/p50, RelA) and non-canonical NF-KB (P100/p52 and RelB) was increased in CD83+ Mino cells after treatment with DOX or CP especially in the nuclear fractions. CD83- Z138 cells were more sensitive to the killing with DOX and CP and upregulated NF-KB molecules after short culture with DOX or CP (Figure 5). Furthermore, DOX and CP upregulated CD83 on CD83+ Mino and CD83- Z138 cells (Figure 6A-B) whilst other chemotherapy drugs had no effect on its expression. This renders the possibility of a synergistic interaction between CD83-MMAE with some chemotherapies. MCL cells were cultured with 3C12C-MMAE, DOX, CP alone or in combination. The Combination Index (Cl) of less than one showed the killing effect of 3C12C-MMAE combined with DOX or CP on Mino and Z138 cells was synergistic (Figure 6c). Whilst the Cl of Z138 with 3C12C-MMAE and DOX was close to one, in three experiments it was consistently less than one. (Figure 6C). This synergistic activity was observed at subtherapeutic concentrations of DOX or CP. CD83 expression has been reported in some lymphomas and leukemias. We showed here that CD83 is upregulated in 50% of primary MCL samples and some MCL cell lines, as well as tumor cells in MCL cell line engrafted mice. Interestingly, the immunohistochemical staining of primary MCL cells showed a pattern of heterogeneous expression, which reflects the heterogeneity of subclonal populations within the MCL tumor tissue. We observed CD83 upregulation by DOX and CP in MCL which, without being bound by any theory or mode of action, results from NF-KB activation induced by the DNA damage-independent stress from chemotherapy. We tested anti-CD83 as a single ADC agent in the treatment of CD83+ MCL in vitro and in a xenogeneic mouse model. Interestingly, though CD83 expression in Mino cells is not as high as that on the classical Hodgkin lymphoma cells, KM-H2, they have a similar sensitivity to the anti-CD83 ADC. This could be the hyper sensitivity of Mino cells to toxin MMAE and/or fast internalization of anti-CD83 Ab by MCL cells. An important factor that effects naked therapeutic antibody efficacy is the expression level of targeted antigen. ADCs have shown to be more effective than naked antibodies over a wider range of antigen expression levels. An anti-CD83 ADC has the potential to be effective in a substantial proportion of MCL. Even if 40% of biopsies express minimal or no CD83, this killing effect of anti-CD83 ADC will be increased by concurrent administration of chemotherapy drugs that increase CD83 expression in MCL (see below).
Ibrutinib reduced the CD83 expression and neutralized the killing of anti-CD83 ADC on MCL. This indicates that Ibrutinib should not be combined with anti-CD83 ADC clinically. Current treatment of younger patients with MCL often includes DOX and CP.
Given these drugs were capable of inducing CD83 expression in MCL, and our data showing the synergistic effect of anti-CD83 ADC with a conventional chemotherapy (DOX and CP), these patients may benefit from combination regimens of chemotherapy and anti-CD83 ADC.

Claims

1. A method of treating lymphoma in an individual who has received or is receiving chemotherapy that includes an anthracycline and/or an alkylating agent, the method comprising
- administering a CD83 binding protein conjugated to a cytotoxic agent to the individual; thereby treating lymphoma in the individual.
2. A method of claim 1 , wherein the individual has received or is receiving a sub- therapeutic dose of an anthracycline and/or an alkylating agent.
3. A method of treating lymphoma in an individual, the method comprising
- providing an individual in whom the lymphoma is to be treated;
- administering to the individual a chemotherapy that includes an anthracycline and/or an alkylating agent; and
- administering to the individual a CD83 binding protein conjugated to a cytotoxic agent; thereby treating cancer in the individual.
4. A method according to any one of claims 1 to 3, wherein the anthracycline is selected from the group consisting of daunorubicin, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin hydrochloride, doxorubicin HCI liposome injection (DOXIL®), dimethyl-doxorubicin (diMe-Doxo), and deoxydoxorubicin), epirubicin, idarubicin, mitoxantrone, valrubicin, aldoxorubicin, annamycin, plicamycin, piramycin, aclarubicin, zorubicin, carubicin, noglamamycin, menogaril, pitarubicin, detorubicin, esorubicin, marcellomycin, nogalamycin, rodorubicin, quelamycin, an anthracycline-containing liposome such as a doxorubicin-containing liposome ( e. g., 2B3-101) and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above.
5. A method according to any one of claims 1 to 4, wherein the anthracycline is doxorubicin.
6. A method according to any one of claims 1 to 5, wherein the alkylating agent is selected from the group consisting of ethylenimines and methylamelamines such as thiotepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoraminde and trimethylolomelamine; cyclophosphamide (CY TOXAN®); mitomycins such as mitomycin C; 6-diazo-5-oxo-L-norleucine; alkyl sulfonates such as busulfan, improsulfan and pipsulfan; nitrogen mustards such as bendamustine, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard; triazines such as dacarbazine and temozolomide,; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, streptozocin and ranimnustine; DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, carboplatin, oxaliplatin, methotrexate, 5-fluorouracil, and aranoside (“Ara-C”) and pharmaceutically acceptable prodrugs, salts, acids and derivatives or equivalents of any of the above.
7. A method according to any one of claims 1 to 6, wherein the alkylating agent is cyclophosphamide.
8. A method according to any one of claims 3 to 7, wherein the chemotherapy that includes an anthracycline and/or an alkylating agent is administered in sub-therapeutic doses.
9. A method of treating lymphoma in an individual, the method comprising
- providing an individual in whom the lymphoma is to be treated;
- administering to the individual a compound that activates NF-KB; and
- administering to the individual a CD83 binding protein conjugated to a cytotoxic agent; thereby treating cancer in the individual.
10. A method according to any one of claims 1 to 9, wherein the lymphoma is Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
11. A method according to claim 10, wherein the NHL is Mantle cell lymphoma (MCL).
12. A method according to claim 10, wherein the NHL is diffuse large B-cell lymphoma (DLBCL).
13. A method according to claim 10, wherein the NHL is Follicular lymphoma (FL).
14. A method according to any one of claims 1 to 13, wherein the CD83 binding protein comprises an antigen binding domain that binds to CD83.
15. A method according to any one of claims 1 to 14, wherein the CD83 binding protein is an antibody or antigen binding fragment thereof.
16. A method according to claim 15, wherein the antibody fragment is selected from the group consisting of a dAb, Fab, Fd, Fv, F(ab’)2, and scFv.
17. A method according to claim 3, wherein the chemotherapy is administered simultaneously with the CD83 binding protein conjugated to a cytotoxic agent.
18. A method according to claim 3, wherein the chemotherapy is administered sequentially to the CD83 binding protein conjugated to a cytotoxic agent.
19. A method according to claim 3, wherein the chemotherapy is administered prior to the CD83 binding protein conjugated to a cytotoxic agent.
20. A method according to claim 3, wherein the CD83 binding protein conjugated to a cytotoxic agent is administered prior to the chemotherapy.
21. A method according to any one of claims 1 to 20, wherein the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, pro-apoptotic agents, radioisotopes, and immunotoxins.
22. A method according to any one of claims 1 to 21 , wherein the cytotoxic agent is selected from the group consisting of doxorubicin, cyclophosphamide, methotrexate, mustine, vincristine, prednisolone, bleomycin, vinblastine, dacarbazine, cyclophosphamide, Procarbazine, Paclitaxel, Irinotecan, Gemcitabine, Fluorouracil, Cytarabine, ozogamicin, adriamycin, etoposide, melphalan, mitomycin C, chloramuil, daunorubicin.
23. A method according to claim 21, wherein the radioisotopes is selected from the group consisting of phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109, silver-111, tin-1221, iodine-125, iodine-131, holmium-166, lutetium-177, rhenium-186, iridium-194, gold-199, astatium-211, yttrium-90, and bismuth-212.
24. A method according to any one of claims 1 to 23, further comprising the step of determining whether the individual has a lymphoma that is CD83+ or CD83-.
25. A method according to any one of claims 1 to 23, wherein the individual has a lymphoma that is CD83+.
26. A method according to any one of claims 1 to 23, wherein the individual has a lymphoma that is CD83-.
27. A CD83 binding protein conjugated to a cytotoxic agent for use in the treatment of lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent.
28. A medicament comprising, consisting essentially of or consisting of a CD83 binding protein conjugated to a cytotoxic agent, wherein the medicament is used for treating lymphoma in an individual, who has received, or who is receiving, at least one chemotherapy including an anthracycline and/or an alkylating agent.
29. A kit comprising, consisting essentially of or consisting of
(a) a CD83 binding protein conjugated to a cytotoxic agent; and
(b) an anthracycline and/or an alkylating agent; and instructions for using (a) and (b) in treating lymphoma in an individual.
30. A kit according to claim 29, wherein the instructions describe the method of any one of claims 1 to 26.
31. Use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a medicament for the treatment of lymphoma in an individual who has received, or who is receiving, a chemotherapy that includes an anthracycline and/or an alkylating agent.
32. Use of a CD83 binding protein conjugated to a cytotoxic agent in the manufacture of a first medicament and use of an anthracycline and/or an alkylating agent in the manufacture of a second medicament, wherein the first and second medicaments are used to treat lymphoma in an individual.
PCT/AU2021/050652 2020-06-23 2021-06-23 Cd83 binding protein conjugates for treating lymphoma WO2021258140A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019051541A1 (en) * 2017-09-13 2019-03-21 Dendrocyte Biotech Pty Ltd Treatment method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019051541A1 (en) * 2017-09-13 2019-03-21 Dendrocyte Biotech Pty Ltd Treatment method

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KANENO RAMON; SHURIN GALINA V; TOURKOVA IRINA L; SHURIN MICHAEL R: "Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, vol. 7, no. 1, 10 July 2009 (2009-07-10), pages 58, XP021059145, ISSN: 1479-5876, DOI: 10.1186/1479-5876-7-58 *
LI, Z. ET AL.: "CD 83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma", HAEMATOLOGICA, vol. 103, no. 4, 2018, pages 655 - 665, XP055583152, DOI: 10.3324/haematol.2017.178384 *
ZIDUO LI, XINSHENG JU, PABLO A. SILVEIRA, EDWARD ABADIR, WEI-HSUN HSU, DEREK N. J. HART, GEORGINA J. CLARK: "CD83: Activation Marker for Antigen Presenting Cells and Its Therapeutic Potential", FRONTIERS IN IMMUNOLOGY, vol. 10, 1 January 2019 (2019-01-01), pages 1312, XP055731281, DOI: 10.3389/fimmu.2019.01312 *

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