WO2024223769A1 - Composés de liaison à la mutéine d'histone h3 - Google Patents

Composés de liaison à la mutéine d'histone h3 Download PDF

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WO2024223769A1
WO2024223769A1 PCT/EP2024/061448 EP2024061448W WO2024223769A1 WO 2024223769 A1 WO2024223769 A1 WO 2024223769A1 EP 2024061448 W EP2024061448 W EP 2024061448W WO 2024223769 A1 WO2024223769 A1 WO 2024223769A1
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binding compound
cancer
binding
antibody
amino acid
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PCT/EP2024/061448
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English (en)
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Edward Green
Tamara BOSCHERT
John Lindner
Katharina SAHM
Michael Platten
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Universität Heidelberg
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention relates to a binding compound binding to a mutein of Histone H3 comprising the amino acid sequence of SEQ ID NO: 1 (H3 mutein), wherein said binding compound is a human antibody or a fragment and/or a derivative thereof, and to polynucleotides, host cells, medical uses and methods related thereto.
  • Histones are generally known as highly basic intranuclear proteins essential in organizing DNA structure within the chromatin. Histones have been found to be released into the extracellular space e.g. during necrosis or apoptosis by cancer cells (Sato et al. (2023), ACS Omega 8:25059); moreover, histones have been found to be released after cell death such as in sepsis, trauma, ischemia/reperfusion injury and autoimmune disease, where they can act as cytotoxic damage-associated molecular pattern (DAMP) proteins by activating Toll-like receptors (TLRs), promoting proinflammatory cytokine pathways and altering phospholipid membrane permeability (Silk et al. (2017), Official Journal of the Cell Death Differentiation Association 8:e2812).
  • DAMP cytotoxic damage-associated molecular pattern
  • H3K27M A recurrent monoallelic point mutation at position 27 in the histone-3 gene (H3K27M) defines a distinct subtype of highly aggressive diffuse intrinsic pontine glioma (DIPG) characterized by high mortality and morbidity rates (Zhang et al., Front Immunol 11 :592612, (2020)). Mutations in both H3-3 histone A (H3-3, HGNC:4764) and variant H3.1 histone A (H3C1, HGNC:4766) can give rise to DIPG. H3K27M-specific antibodies are available commercially for diagnostic purposes, both as polyclonal antibodies and as monoclonal rabbit antibodies.
  • DIPG diffuse intrinsic pontine glioma
  • DIPG Immunosorbent et al., J Neurooncol 119, 7-15 (2014). Due to a generally low mutational burden and a lack of immune cell infiltration, DIPG are unsuitable for a generalized immune checkpoint inhibition treatment, but may benefit from targeted immunotherapies (Ross et al., Brain 144, 2594-2609 (2021)).
  • Recent DIPG-specific immunotherapies include a phase I dose-escalation clinical trial investigating the safety and efficacy of a chimeric antigen receptor (CAR) T cell therapy targeting the uniformly upregulated disialoganglioside GD2 on DIPG (Majzner et al., Nature 603, 934-941 (2022)).
  • CAR chimeric antigen receptor
  • Peptide vaccinations targeting the neoantigen H3K27M have also been developed with subsequent analysis of a mutation-specific T cell response (Ochs et al., Oncoimmunology 6, el328340 (2017); Mueller et al. J Clin Invest 130, 6325-6337 (2020)).
  • H3K27M-reactive CD8+ T cells In HLA A*02:01+ patients, vaccination with a short H3K27M peptide induced an expansion of H3K27M-reactive CD8+ T cells (Chheda et al., J Exp Med 215, 141-157 (2018)). However, cytotoxicity of these CD8+ T cells against endogenously expressing HLA A*02:01+ H3K27M tumor cells remains controversial (Chedda et al., loc. cit., Immisch et al., J Immunother Cancer 10(10):e005535, (2022).
  • the present invention relates to a binding compound binding, preferably specifically binding, to a mutein of H3 histone (H3) comprising the amino acid sequence of SEQ ID NO: 1 (KAPRKQLATKAARMSAPSTGGVKKPHR, H3 mutein).
  • the term "multitude”, as referred to herein, relates to a number of more than one, i.e. at least two, preferably at least three, more preferably at least four, even more preferably at least five. A multitude may however, also be a number of at least ten, at least 25, at least 100, or more.
  • the methods specified herein below are in vitro methods.
  • the method steps may, in principle, be performed in any arbitrary sequence deemed suitable by the skilled person, but preferably are performed in the indicated sequence; also, one or more, preferably all, method steps may be assisted or performed by automated equipment.
  • the methods may comprise steps in addition to those explicitly mentioned.
  • the term "about” relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ⁇ 20%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • the term “essentially” indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ⁇ 20%, more preferably ⁇ 10%, most preferably ⁇ 5%.
  • “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention.
  • composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like.
  • a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of non-specified component(s).
  • the degree of identity (e.g. expressed as "%identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art.
  • the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment.
  • the percentage is calculated by determining, preferably over the whole length of the polynucleotide or polypeptide, the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e.g. BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection.
  • GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity.
  • the default values of 5.00 for gap weight and 0.30 for gap weight length are used.
  • the Basic Local Alignment Search Tool (BLAST) implementation is used with default parameter values for alignment.
  • BLAST Basic Local Alignment Search Tool
  • the term "essentially identical" indicates a %identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The aforesaid applies to the term "essentially complementary” mutatis mutandis.
  • fragment of a biological macromolecule, preferably of a polynucleotide, polypeptide, or polypeptide complex, is used herein in a wide sense relating to any sub-part, preferably subdomain, of the respective biological macromolecule comprising the indicated sequence, structure and/or function.
  • the term includes sub-parts generated by actual fragmentation of a biological macromolecule, but also sub-parts derived from the respective biological macromolecule in an abstract manner, e.g. in silico.
  • an Fc or Fab fragment but also e.g. a single-chain antibody, a bispecific antibody, and a nanobody may be referred to as fragments of an immunoglobulin.
  • the compounds specified in particular the polynucleotides and polypeptides, may be comprised in larger structures, e.g. may be covalently or non-covalently linked to further sequences, carrier molecules, retardants, and other excipients.
  • polypeptides as specified may be comprised in fusion polypeptides comprising further peptides, which may serve e.g. as a tag for purification and/or detection, as a linker, or to extend the in vivo half-life of a compound.
  • detectable tag refers to a stretch of amino acids which are added to or introduced into the fusion polypeptide; preferably, the tag is added C- or N- terminally to the fusion polypeptide. Said stretch of amino acids preferably allows for detection of the polypeptide by an antibody which specifically recognizes the tag; or it preferably allows for forming a functional conformation, such as a chelator; or it preferably allows for visualization, e.g. in the case of fluorescent tags.
  • Preferred detectable tags are the Myc-tag, FLAG-tag, 6-His-tag, HA-tag, GST-tag or a fluorescent protein tag, e.g. a GFP-tag. These tags are all well known in the art.
  • further peptides preferably comprised in a fusion polypeptide comprise further amino acids or other modifications which may serve as mediators of secretion, as mediators of blood-brain-barrier passage, as cell-penetrating peptides, and/or as immune stimulants.
  • Further polypeptides or peptides to which the polypeptides may be fused are signal and/or transport sequences, e.g. an IL-2 signal sequence, and linker sequences.
  • polypeptide refers to a molecule consisting of several, typically at least 20 amino acids that are covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are usually considered to be "peptides".
  • the polypeptide comprises of from 50 to 1000, more preferably of from 75 to 1000, still more preferably of from 100 to 500, most preferably of from 110 to 400 amino acids.
  • the polypeptide is comprised in a fusion polypeptide and/or a polypeptide complex
  • polypeptide complex relating to complex comprising a multitude of identical and/or non-identical polypeptides connected via one or more non-peptide bond(s), e.g. via at least one disulfide bond, ionic interaction, van der Waals interaction, and/or hydrophobic interaction.
  • a polypeptide complex may e.g. be an oligomeric protein.
  • the polypeptide is a binding compound as specified herein below.
  • the polypeptide comprises at least one sequence of at least three, preferably at least five, more preferably at least ten contiguous amino acids not present in the naturally occurring polypeptide.
  • Polypeptides as specified may be comprised in fusion polypeptides comprising further amino acid sequences; a further amino acid sequence preferably is an amino acid sequence of a functional polypeptide, i.e. a polypeptide providing at least one additional, preferably auxiliary, function, wherein said auxiliary function in particular may be immune stimulation, cell and/or surface tethering, opsonization, membrane integration, membrane transport, and the like.
  • polypeptide variant relates to any chemical molecule comprising at least one polypeptide as specified herein, having the indicated activity, but differing in structure from said specific polypeptide.
  • the polypeptide variant comprises a polypeptide having a contiguous amino acid sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, of the amino acid sequence of the polypeptide specifically indicated.
  • a polypeptide variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant is still, preferably, at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%, identical with the amino acid sequence of the specific polypeptide.
  • the degree of identity between two amino acid sequences can be determined by algorithms well known in the art and as described herein above.
  • Polypeptide variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs.
  • polypeptide variants referred to herein include fragments of the specific polypeptides or the aforementioned types of polypeptide variants as long as these fragments and/or variants have the biological activity as specified.
  • Such fragments may be or may be derived from, e.g., degradation products or splice variants of the polypeptides.
  • variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristyl ation, by including non-natural amino acids, and/or by being peptidomimetics.
  • H3 histone which may also be referred to as "H3” is in principle known to the skilled person to relate to a member of the human histone H3 multigene family.
  • H3 is H3-3A or is H3-1.
  • H3-3A has a human gene nomenclature (HGNC) database entry with accession number HGNC:4764 (version of 21 March 2023), the amino acid sequence of H3- 3A is obtainable e.g. from Genbank Acc No. NP 002098.1, the amino acid sequence of H3.1 is obtainable e.g. from Genbank Acc No. NP_066298.1.
  • HGNC human gene nomenclature
  • mutant is known to the skilled person to relate to a polypeptide with an amino acid sequence differing from that of the respective wild-type polypeptide in at least one position, i.e. is a "mutant protein".
  • H3 mutein relates to a H3 polypeptide, preferably a H3-3A or a H3-1 polypeptide, comprising a lysine to methionine amino acid exchange at position 27.
  • amino acid numbering in the H3 polypeptide customarily omits the methionine at the N-terminus of the polypeptide as described e.g. in Genbank Acc No. NP_002098.1; thus, a lysine to methionine amino acid exchange at position 27 of H3-3 A corresponds to a lysine to methionine amino acid exchange at position 28 of Genbank Acc No.
  • the H3 mutein comprises an amino acid sequence as shown in SEQ ID NO: 1, 11 (RKQLATKAARMSAPSTGGVKKPH), 12 (KAPRKQLATKAARMSAPSTGGVKKPHR), 13 (KQLATKAARMSAPATGGVKKPH), 14 (RKQLATKAARMSAPATGGVKKPH), or 15 (KAPRKQLATKAARMSAPATGGVKKPHR).
  • the H3 mutein preferably comprises, more preferably consists of, the amino acid sequence of SEQ ID NOTO or 16, preferably of SEQ ID NOTO.
  • the term "specific binding" and grammatical variations thereof are used herein to indicate that a binding compound does not bind significantly to other compounds, typically biomolecules, present in a sample or a subject.
  • the dissociation constant of the H3 mutein/binding compound complex is at least a factor of 5, preferably at least a factor of ten, more preferably at least a factor of 100, lower than the dissociation constant of a complex between any non-H3 mutein compound and the binding compound.
  • the binding compound binds to the H3 mutein; as the skilled person understands from the description herein, the binding compound binds to the H3 mutein specifically; thus, the binding compound preferably binds to the wildtype H3 polypeptide with a different dissociation constant as specified herein above.
  • the binding compound preferably binds to the amino acid sequence of the H3 mutein comprising the K27M amino acid exchange.
  • the binding polypeptide preferably binds to the epitope of the H3 mutein comprising the K27M amino acid exchange.
  • the binding polypeptide preferably also binds to a peptide comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1.
  • the term "binding compound” relates to a chemical molecule binding to the H3 mutein as specified herein above; the binding between the H3 mutein and the binding compound preferably is direct, i.e., preferably, there is a direct molecular interaction between the binding compound and the H3 mutein, more preferably between amino acids of SEQ ID NO: 1 and the binding compound, even more preferably between the methionine at position 27 of the H3 mutein and/or the methionine at position 14 of SEQ ID NO: 1 and the binding compound.
  • the binding compound binds an epitope of the H3 mutein comprising the K27M amino acid exchange.
  • the binding between the H3 mutein and the binding compound is specific, preferably as specified herein above.
  • the dissociation constant (KD) of the H3 mutein/binding compound complex is at most 100 nM, more preferably at most 10 nM, even more preferably at most 1 nM, most preferably at most 100 pM, preferably under standard conditions, more preferably under the conditions specified herein in the Examples.
  • the binding compound is a biological macromolecule, preferably comprising, more preferably being, a polypeptide or a polypeptide complex.
  • the binding compound may comprise additional molecules and/or substructures; e.g., in case the binding compound is an antibody, the antibody may be glycosylated. Also, in particular in case the binding compound is an antibody fragment and/or an antibody derivative, the binding compound may be comprised in a larger structure, e.g. in a dendrimer.
  • the binding compound is an antibody or a fragment and/or a derivative of said antibody, having the activity of binding to a H3 mutein as specified herein above.
  • antibody is, in principle, understood by the skilled person and preferably relates to an immunoglobulin from any of the classes IgA, IgD, IgE, IgG, or IgM, having the activity of binding, preferably specifically binding, to a H3 mutein as specified herein above.
  • Antibodies against a H3 mutein or fragments thereof can be prepared by well-known methods using a purified H3 polypeptide or a suitable fragment derived therefrom as an antigen.
  • a fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Suitable fragments may also be obtained either from the H3 mutein by proteolytic digestion, may be synthetic peptides, or may be recombinantly expressed.
  • a peptide comprising, more preferably consisting of, SEQ ID NO:1 is used as an antigen.
  • the binding compound is an IgG, an IgA, or an IgM, wherein said IgG preferably is an IgGl, IgG2, IgG3, or IgG4 and/or wherein said IgA preferably is an IgAl or IgA2.
  • the antibody is a monoclonal antibody and/or is a human, primatized, chimerized, or humanized antibody, or a fragment or derivative thereof.
  • the antibody is a fully human antibody, i.e. is an antibody derived or derivable from human immunoglobulin sequences.
  • the antibody comprises an amino acid sequence derived from a human immunoglobulin sequence, preferably by somatic hypermutation.
  • the antibody comprises a heavy chain variable domain comprising CDR sequences as shown in SEQ ID NOs:2 (GFNFSSYG), 3 (ISYDGSKK), and 4 (AKVGDGYKIDAFDI); or amino acid sequences at least 70% identical thereto.
  • the antibody comprises a light chain variable domain comprising CDR sequences as shown in SEQ ID NOs:5 (PLTKRY), 6 (KDS), and 7 (QSVDDSGTLNYV); or amino acid sequences at least 70% identical thereto. More preferably, the antibody comprises a heavy chain variable domain comprising CDR sequences as shown in SEQ ID NOs:2, 3, and 4; or amino acid sequences at least 70% identical thereto; and a light chain variable domain comprising CDR sequences as shown in SEQ ID NOs: SEQ ID NOs:5, 6, and 7; or amino acid sequences at least 70% identical thereto.
  • antibodies arise through somatic hypermutation and class switching.
  • an antibody with modified CDR3 sequences (SEQ ID NOs: 17 (AKVGDGYKADGFDM) and 18 (QSADNSGTLNYV)) related to the sequence indicated above is also able to bind the H3.3 mutein.
  • the antibody comprises a heavy chain variable domain comprising CDR sequences as shown in SEQ ID NOs:2, 3, and 17; or amino acid sequences at least 70% identical thereto.
  • the antibody comprises a light chain variable domain comprising CDR sequences as shown in SEQ ID NOs:5, 6, and 18; or amino acid sequences at least 70% identical thereto.
  • the antibody comprises a heavy chain variable domain comprising CDR sequences as shown in SEQ ID NOs:2, 3, and 17; or amino acid sequences at least 70% identical thereto; and a light chain variable domain comprising CDR sequences as shown in SEQ ID NOs: SEQ ID NOs: 5, 6, and 18; or amino acid sequences at least 70% identical thereto.
  • the antibody comprises (i) the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 70% identical thereto, and/or the amino acid sequence of SEQ ID NO:9 or an amino acid sequence at least 70% identical thereto.
  • the antibody comprises, preferably consists of, (i) the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO:9.
  • CDR complementarity determining regions
  • FWR framework regions
  • a derivative of an antibody relates to any polypeptide or polypeptide complex comprising at least one amino acid sequence of a light chain variable region and/or at least one amino acid sequence of a heavy chain variable region of an antibody.
  • a derivative of an antibody preferably is a single-chain antibody (scFv) or a single-domain antibody.
  • scFv single-chain Fv
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species.
  • HVRs hypervariable regions
  • CDRs complementarity determining regions
  • a derivative of an antibody may also comprise at least one additional amino acid sequence, e.g. a transmembrane domain or a signal peptide; thus, an antibody derivative may e.g.
  • the H3 binding domain of the antibody derivative preferably is the binding compound as specified herein, preferably is a scFv.
  • the derivative of an antibody may also comprise one or more additional polypeptide(s).
  • the antibody derivative may e.g. be a multitude-specific antibody, preferably a bispecific or a trispecific antibody, preferably further comprising a binding domain binding a tumor antigen, preferably a glioma antigen, more preferably GD2 (CAS No. 65988-71-8).
  • the derivative of an antibody may also be a mutein of an antibody, i.e. comprise an amino acid sequence differing in at least one amino acid position from an amino acid sequence as specified herein elsewhere.
  • an antibody derivative may in particular be a polypeptide variant as specified herein above of an antibody.
  • fragment of an antibody, as referred to herein, relates to a portion of an antibody comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment.
  • Pepsin treatment yields an F(ab’)2 fragment that has two antigencombining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-binding site.
  • a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • diabodies refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, WO 1993/01161. Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9 (2003): 129-134.
  • VHH single-domain antibody
  • the VHH comprises the CDRs of a heavy-chain antibody, preferably obtained from an alpaca, dromedar, camel, llama, or shark immunized with a target polypeptide.
  • a binding polypeptide may be a fragment and a derivative of an antibody; e.g. a binding polypeptide may be an Fab fragment of a somatic hypermutation derivative of an antibody described herein above having an amino acid sequence at least 70 % identical to an amino acid sequence as specified herein above.
  • Antibodies, as well as derivatives and/or fragments thereof, can be obtained by using textbook methods, e.g. as described herein above.
  • the antibody is an antibody as specified above or a polypeptide derivative thereof; more preferably, the antibody is an antibody as specified above.
  • the binding compound as specified herein may be comprised in a host cell, the term "host cell", as used herein, relating to any cell capable of receiving and/or producing a binding compound and/or receiving and integrating or stably replicating a polynucleotide and/or expressing an expressible construct, both as specified herein below.
  • the host cell is a eukaryotic cell, preferably a plant or yeast cell, e.g. a cell of a strain of baker's yeast, or is an animal cell. More preferably, the host cell is an insect cell or a mammalian cell, in particular a mouse or rat cell. Even more preferably, the host cell is a mammalian cell, most preferably is a human cell.
  • the host cell is a B cell, preferably an activated B cell, more preferably a recombinant activated B cell.
  • the host cell is an activated plasmablast, a memory B cell, or a B cell with cytotoxic activity.
  • the host cell may, however, also be different cell, such as a tissue stem cell, a hematopoietic precursor cells, and the like.
  • the binding compound is a chimeric antigen receptor (CAR), a T cell receptor fusion construct (TruC), a transmembrane signaling protein, comprises a T cell engagement element, or is a bi- or trispecific antibody
  • the host cell may also be a T cell, in particular a CD4+ T cell or a CD8+ T cell, more preferably a cytotoxic T cell.
  • subjects suffering from brain tumors may comprise B cells producing antibodies recognizing the H3 mutein as specified herein above.
  • Antibodies produced by such cells and substructures thereof can be used for producing therapeutic antibodies, bispecific antibodies, CARs, recombinant transmembrane sensors, and other derivatives making use of the binding specificity of such antibodies.
  • fully human antibodies are less likely to be immunogenic in human treatment.
  • the present invention also relates to a polynucleotide encoding a binding compound as specified herein, wherein said binding compound preferably comprises or is a polypeptide, preferably an antibody or a derivative and/or a fragment thereof.
  • polynucleotide refers to single- or double-stranded DNA molecules as well as to RNA molecules. Encompassed by the term is genomic DNA, cDNA, hnRNA, mRNA, as well as all naturally occurring or artificially modified derivatives of such molecular species.
  • the polynucleotide may be, preferably, a linear or circular molecule.
  • a polynucleotide according to the present invention may comprise additional sequences required for proper transcription and/or translation such as 5'- or 3 -UTR sequences or sequences required for splicing or RNA stability.
  • the polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. purified or at least isolated from its natural context such as its natural gene locus) or in genetically modified or exogenously (i.e. artificially) manipulated form.
  • the polynucleotide preferably, is provided in the form of a double or single stranded molecule. It will be understood that referring to any of the aforementioned polynucleotides also refers to complementary or reverse complementary strands of the specific sequences referred to.
  • polynucleotides comprising chemically modified nucleotides, including naturally occurring modified nucleotides such as glycosylated or methylated nucleotides, or artificial modified ones such as biotinylated nucleotides.
  • the polynucleotide is comprised in an expression construct allowing for expression of the polynucleotide in a host cell or a subject.
  • expression construct refers to a heterologous polynucleotide comprising the aforementioned polynucleotide encoding the binding compound as well as one or more nucleic acids being heterologous thereto which are required for expression of the polynucleotide encoding the fusion polypeptide.
  • heterologous nucleic acids may be promoter sequences, enhancer sequences and/or transcription termination sequences such as terminators.
  • the polynucleotide is usually operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic host cells or isolated fractions thereof.
  • Expression of the polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in host cells are well known in the art. Preferably, they comprise regulatory sequences ensuring initiation of transcription and/or poly- A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Examples for regulatory elements permitting expression in eukaryotic host cells are the A0X1- or the GALI- promoter in yeast or the CMV- , SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • expression systems envisaged by the invention shall permit expression in insect cells, such as polyhedrin promoter based systems.
  • inducible expression control sequences may be used, which are well known in the art.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • the expression construct and/or the polynucleotide referred to herein is a vector.
  • the vector may be incorporated into a host cell by various techniques well known in the art.
  • a plasmid vector can be introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens.
  • a plasmid vector may be introduced by heat shock or electroporation techniques.
  • the vector may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
  • Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
  • the vector may also comprise further nucleic acids required for introducing the vector into a host.
  • the vector may comprise further nucleic acids required for transformation or transfection and for propagation of the vector in the host cells.
  • the vector is an expression vector and a gene transfer or targeting vector.
  • suitable expression vectors are known in the art. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of expression constructs into targeted cell population, e.g. also in therapeutic approaches.
  • the vector may also comprise nucleic acids that allow for either heterologous or homologous integration of at least part of the vector into a genome of a host cell.
  • the vector referred to herein may also be or comprise a targeting construct which allow for random or site- directed integration of the targeting construct into genomic DNA.
  • Such targeting constructs preferably, comprise DNA of sufficient length for either homologous or heterologous recombination flanking the polynucleotide or the expression construct.
  • the polynucleotide or expression construct may also be introduced using integration systems like Cre/LoxP or CRISPR/CAS. In such cases, the polynucleotide or expression construct may comprise further nucleic acids allowing for the use of such integration systems. Suitable modifications/additions depend on the envisaged integration system and are well known to those skilled in the art.
  • the present invention further relates to a host cell comprising a binding compound as specified herein and/or a polynucleotide, preferably an expression construct and/or vector as specified herein.
  • the host cell has been described herein above.
  • the host cell produces a binding compound as specified herein, more preferably expresses the expression construct specified herein above.
  • the host cell may in particular be an activated B cell, such as an activated plasmablast producing the binding compound, in particular the antibody or derivative or fragment thereof, or a T cell producing a CAR or TruC, all as specified herein above.
  • the present invention also relates to a binding compound of the present invention, a polynucleotide of the present inventions, and/or a host cell of the present invention, for use in medicine; as well as for use in treating and/or preventing cancer in a subject.
  • treating refers to an amelioration of a disease or disorder referred to herein or the symptoms accompanied therewith to a significant extent; as used herein, the term includes prevention of deterioration of a disease, disorder, or symptoms associated therewith. Said treating may also include an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated.
  • Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %.
  • the p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.
  • the treatment shall be effective for at least 10%, at least 20% at least 50% at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.
  • treating comprises inhibiting proliferation, more preferably killing, of cancer cells.
  • treating cancer is reducing tumor and/or cancer cell burden in a subject.
  • effectiveness of treatment of e.g. cancer is dependent on a variety of factors including, e.g. cancer stage and cancer type.
  • cancer treatment further comprises at least one of chemotherapy, immunotherapy, surgery, and radiotherapy.
  • treating comprises treating a cancer producing a H3 mutein.
  • preventing and prevention refer to retaining health with respect to the diseases or disorders referred to herein for a certain period of time in a subject. It will be understood that the said period of time may be dependent on the amount of the drug compound which has been administered and individual factors of the subject discussed elsewhere in this specification. It is to be understood that prevention may not be effective in all subjects treated with the compound according to the present invention. However, the term requires that, preferably, a statistically significant portion of subjects of a cohort or population are effectively prevented from suffering from a disease or disorder referred to herein or its accompanying symptoms. Preferably, a cohort or population of subjects is envisaged in this context which normally, i.e.
  • preventing in particular relates to preventing cancer development, preventing metastasis formation, and/or preventing relapse, preferably relates to preventing metastasis formation and/or preventing relapse.
  • cancer relates to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells (“cancer cells”). This uncontrolled growth may be accompanied by intrusion into and destruction of surrounding tissue (infiltration) and possibly spread of cancer cells to other locations in the body (metastasis).
  • cancer is also included by the term cancer is a recurrence of a cancer (relapse).
  • the cancer is a solid cancer, a metastasis, or a relapse thereof.
  • the cancer is selected from the list consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia, adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer,
  • AML
  • the cancer is a H3 K27M positive cancer. More preferably, the cancer is cancer is a glioma, more preferably a H3 K27M positive glioma, preferably diffuse midline glioma, most preferably diffuse intrinsic pontine glioma (DIPG).
  • DIPG diffuse intrinsic pontine glioma
  • subject relates to a vertebrate animal, preferably a mammal, in particular a livestock, companion, or laboratory animal. More preferably, subject is a human. Preferably, the subject has been diagnosed to suffer from cancer as specified herein above and/or was diagnosed to be at risk of developing a relapse and/or metastases. Preferably, the subject has been assessed by a method of the present invention.
  • the present invention also relates to a use of a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein, for the manufacture of a medicament, preferably for the manufacture of a medicament for treating and/or preventing cancer.
  • the binding compound may be comprised in a pharmaceutical composition.
  • composition relates to a mixture of compounds comprising at least a binding compound as specified herein and, preferably, at least one carrier.
  • the composition may have any consistency deemed appropriate by the skilled person.
  • the composition is a solid composition, e.g. a tablet or a powder, a semisolid composition, e.g. a gel, or, more preferably, a liquid, e.g. a solution or an emulsion.
  • the composition preferably comprises a carrier.
  • the carrier(s) preferably is/are acceptable in the sense of being compatible with the other ingredients of the composition and being not deleterious to a potential recipient thereof.
  • the carrier(s) preferably is/are selected so as not to affect the biological activity of the composition.
  • the composition is sterile, more preferably a sterile solution, most preferably a sterile solution for injection.
  • the carrier is selected by the skilled person such as to achieve the consistency intended and may be, for example, a gel or, preferably, a liquid, more preferably an aqueous liquid.
  • Such carriers are distilled water, physiological saline, Ringer's solutions, dextrose solution, phosphate-buffered saline solution, and Hank's solution.
  • the carrier may include one or more solvents or other ingredients increasing solubility of the compounds comprised in the composition.
  • Further examples of liquid carriers are syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Suitable carriers comprise those mentioned above and others well known in the art.
  • the composition in particular the pharmaceutical composition, may comprise one or more further compounds; preferably, such additional compounds are selected so as to not affect the biological activity of the composition, in particular of the active compounds and/or is acceptable in the sense of being compatible with the other ingredients of the composition and being not deleterious to a potential recipient thereof.
  • the composition according to the present specification is a pharmaceutical composition; thus, preferably, the carrier is a pharmaceutically acceptable carrier.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, stabilizers and/or other compounds deemed appropriate by the skilled person, e.g. for galenic purposes.
  • the binding compound as specified herein above, in particular the antibody as specified is the "active compound" of the preparation, although “further active compounds", which are referred to under this term, may be present.
  • the active compound and the further active compound i.e. preferably the active compounds, are pharmaceutically active compounds.
  • compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound referred to herein above, preferably in admixture or otherwise associated with at least one pharmaceutically acceptable carrier or diluent.
  • the active compound(s) will usually be mixed with a carrier or the diluent.
  • the resulting formulations are to be adapted to the mode of administration, i.e. in the forms of tablets, capsules, suppositories, solutions, suspensions or the like. Dosage recommendations shall be indicated in the prescriber's or user's instructions in order to anticipate dose adjustments depending on the considered recipient.
  • the pharmaceutical composition is, preferably, administered systemically or, more preferably, topically.
  • Suitable routes of administration conventionally used for drug administration are topical, intravenous, or parenteral administration as well as inhalation.
  • administration is systemic, more preferably intravenously.
  • the pharmaceutical composition may be administered by other routes as well.
  • the cancer is a brain cancer
  • intracranial and in particular intratumoral administration may be envisaged.
  • the pharmaceutical composition may be administered topically, e.g. as a tablet, in particular as a time-delay preparation, which is preferably implanted during surgery removing a tumor.
  • the pharmaceutical composition can be administered in combination with other further active compounds either in a common pharmaceutical composition or as separated pharmaceutical compositions wherein said separated pharmaceutical compositions may be provided in form of a kit of parts.
  • the pharmaceutical composition is, preferably, administered in conventional dosage forms prepared by combining the active compound with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to obtain the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables. Similarly, the carrier or diluent may include time delay material well known in the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • a therapeutically effective dose refers to an amount of the active compound to be used in a pharmaceutical composition of the present invention which provides the effect referred to in this specification.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depend upon many factors, which may include the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment.
  • a typical dose can be, for example, in the range of 1 pg to 1000 mg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 100 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 1 mg units per kilogram of body weight per minute, respectively.
  • the pharmaceutical composition is administered once to the subject, i.e., preferably, is used as a one-time treatment.
  • the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 10 mg per kg body mass.
  • compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification.
  • the said pharmaceutical compositions may be administered more than one time, for example from two to 50 times, more preferably from five to 50 times.
  • administration is adjusted to maintain an effective concentration in the body of a subject for the time period intended, e.g. until surgical removal of one or more tumor(s) was performed.
  • the pharmaceutical preparation may be administered topically at the site of an excised tumor as a depot; in such case, the depot preferably is adjusted to maintain an effective dose until at least after additional cancer treatment, e.g. chemotherapy, was administered or, preferably, until such additional cancer treatment has been completed. Progress can be monitored by periodic assessment.
  • the present invention further relates to a method for inhibiting cancer cells comprising (a) contacting said cancer cells with a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein, and (b) thereby inhibiting said cancer cells.
  • the method for inhibiting cancer cells of the present invention preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to providing cancer cells, a binding compound, a polynucleotide, and/or a host cell for step a), and/or further treatment(s).
  • inhibiting cancer cells in the aforesaid method is treating and/or preventing cancer, so the method preferably is an in vivo method.
  • the method may also be a method for treating and/or preventing cancer in a subject, said method comprising (a) administering a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein, to said subject, and (b) thereby treating and/or preventing cancer in said subject.
  • the method may, however, also be an in vitro method, e.g. performed on cultured cells and/or on an experimental animal which preferably is sacrificed after said treatment.
  • the present invention further relates to a method for cancer assessment in a subject, comprising (A) contacting a sample of said subject with a binding compound as specified herein and/or a host cell as specified herein; (B) determining binding of said binding compound or host cell to said sample, and (C) assessing cancer based on the determining in step (B).
  • the method for cancer assessment of the present invention preferably, is an in vitro method. Further steps optionally present may relate to e.g. providing a sample of cancer cells for step (A) and optionally sample pre-treatment.
  • sample relates to a sample of a body fluid, a sample from a tissue or an organ, or a sample of wash/rinse fluid or a swab or smear obtained from an outer or inner body surface, said sample being known or suspected to comprise cancer cells.
  • the sample may in particular be a biopsy of a tumor or of a bodily fluid suspected to comprise cells of a non-solid cancer.
  • Samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or lancets, or by surgical instrumentation.
  • samples obtained by well-known techniques including, in an embodiment, scrapes, swabs or biopsies from the urogenital tract, perianal regions, anal canal, the oral cavity, the upper aerodigestive tract and the epidermis are also included.
  • samples are obtained from body fluids known to comprise cancer cells if present in a subject, i.e., preferably, blood, saliva, or bone marrow aspirate, more preferably blood, or from tumors, including metastases and relapses.
  • sample may be further processed in order to carry out the method of the present invention.
  • cells may be obtained from the sample by methods and means known in the art.
  • sample also may relate to preparations comprising or suspected to comprise cancer cells, diluted, enriched, purified and/or cultivated from a sample.
  • determining binding of a binding compound to a sample preferably is determining binding of a binding compound to at least one sample constituent, wherein said sample constituent preferably is a H3 mutein or a cell producing the same.
  • preferred methods are methods allowing detection of binding, i.e. in particular formation of complexes between a binding compound and a H3 mutein in complex matrices. Such methods preferably are immunological methods, e.g.
  • the binding compound may e.g. be coupled to an indicator agent having at least one detectable property such as an optically detectable property.
  • the binding compound preferably is coupled to a colored or fluorescent dye for determining; the binding compound may, however also be coupled to an enzyme, as is used in ELISA and related assays.
  • the detectable property may, however also be emission of radiation, electrochemiluminescence, and the like.
  • the determining may also be accomplished by determining a structural property of the binding compound, e.g. in case the binding compound is an IgG antibody, an anti-IgG antibody may be used.
  • determining may also comprise determining a detectable change in said host cell's composition in response to said contacting; e.g. a host cell may be stimulated to secrete a cytokine, e.g. interferon-gamma, to undergo a morphological change, and/or to modulate gene expression, e.g. to produce one or more surface marker(s). Also, the host cell may undergo apoptosis or become activated to proliferate in response to said contacting. Said reactions of a host cell preferably are specific for contacting said host cell to a H3 mutein or a cell producing the same; i.e.
  • said reactions of a host cell preferably only occur in case the host cell is contacted to a H3 mutein or a cell producing the same.
  • the specific requirement of a reaction of a host cell being specific may be assay specific; thus, as referred to herein, a reaction of a host cell is specific to a H3 mutein or a cell producing the same if other compounds can be excluded as causes of said reaction, e.g. by using appropriate controls.
  • I.e., e.g. interferon-gamma production in an ELISPOT assay may be deemed specific if an increase in interferon-gamma production can be differentiated from background production not caused by contacting to a H3 mutein or a cell producing the same.
  • assessing and “assessment” are understood by the skilled person. Preferably, these terms relate to providing information on a cancer, which is preferably of medical relevance. Thus, assessing may in particular be providing information aiding in diagnosing and/or prognosing cancer, and/or is identifying a subject as being susceptible for treatment with a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein; in the latter case, step (C) preferably comprises identifying said subject as susceptible for said treatment if binding of said binding compound to said sample is detected, i.e. preferably if the sample comprises the H3 mutein, preferably comprises cancer cells producing the H3 mutein.
  • step (C) comprises comparing the binding determined in step (B) to a reference, wherein the reference may be e.g. a sample not comprising the H3 mutein; in such case, determining binding in step (B) higher than the reference is indicative of a sample comprising the H3 mutein; alternatively, a sample known to comprise the H3 mutein may be used as a reference; in such case, determining binding in step (B) higher than or equal to the reference is indicative of a sample comprising the H3 mutein.
  • the reference may be e.g. a sample not comprising the H3 mutein; in such case, determining binding in step (B) higher than the reference is indicative of a sample comprising the H3 mutein; alternatively, determining binding in step (B) higher than or equal to the reference is indicative of a sample comprising the H3 mutein.
  • the H3 mutein is not normally comprised in samples, determining binding in step (B) higher than the detection limit may already be deemed
  • the present invention also relates to a, preferably in vitro, use of a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein, for detecting a H3 mutein comprising a lysine to methionine amino acid exchange at position 27.
  • the present invention relates to a recombinant host cell produced or producible by introducing a polynucleotide as specified herein into said host cell.
  • a polynucleotide as specified herein into said host cell.
  • an expression construct and/or a vector as specified above is introduced.
  • the present invention also relates to a kit comprising a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein, comprised in a housing, wherein said kit may optionally further comprise a means of administration and/or a diluent.
  • kit refers to a collection of the aforementioned compounds, means or reagents which may or may not be packaged together.
  • the components of the kit may be comprised by separate vials (i.e. as a kit of separate parts) or provided in a single vial, e.g. as a composition as specified herein above.
  • the housing of the kit preferably allows translocation of the compounds of the kit, in particular common translocation; thus, the housing may in particular be a transportable container comprising all specified components.
  • the kit of the present invention may be used for practicing the methods referred to herein above. It is, preferably, envisaged that all components are provided in a ready- to-use manner for practicing the methods referred to above.
  • the kit preferably contains instructions for carrying out said methods.
  • the instructions can be provided by a user's manual on paper or in electronic form.
  • the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit.
  • the kit comprises further compounds, such as a reaction buffer, a hybridization solution, a lysis buffer, or the like.
  • the kit is adapted for use in a method of the present invention, more preferably is adapted to comprise all reagents required to perform said method or methods, e.g. one or more diluent(s).
  • Means of administration may include a delivery unit for the administration of the compound or composition and a storage unit for storing said compound or composition until administration.
  • the means of administration may appear as separate devices in such an embodiment and are, preferably, packaged together in said kit. Preferred means for administration are those which can be applied without the particular knowledge of a specialized technician.
  • the means for administration is a syringe, more preferably with a needle, comprising the compound or composition as specified.
  • the means for administration is an intravenous infusion (IV) equipment comprising the compound or composition.
  • the means for administration is an inhaler comprising the compound of the present invention, wherein, more preferably, said compound is formulated for administration as an aerosol.
  • the present invention relates to a device comprising a binding compound as specified herein, a polynucleotide as specified herein, and/or a host cell as specified herein.
  • the term "device” includes any and all contraptions comprising the components specified.
  • the device is a means of administration as specified herein above.
  • the device is adapted to perform a method as specified herein, in particular an assessment method.
  • the device may also be a diagnostic device.
  • the device preferably comprises (i) an analysis unit comprising a means for determining binding of a binding compound to a sample, and, operatively connected thereto (ii) an evaluation unit comprising tangibly embedded executable instructions for performing a method as specified herein.
  • an analysis unit comprising a means for determining binding of a binding compound to a sample, and means for carrying out the determination are known to the skilled person.
  • means for determining binding of a binding compound to a sample have been described herein above and include in particular means capable for determining an amount of binding compound bound to a sample, such as an ELISA reader, as well as means for determining effects of binding of a binding compound to a sample, such as an optical unit detecting a signal of a reporter gene assay.
  • An evaluation means is any means capable of providing the analysis as specified; preferably, the evaluation means is a data processing means, such as a microprocessor, a handheld device such as a mobile phone, or a computer. How to link the means in an operating manner will depend on the type of means included into the device. In an embodiment, the means are comprised by a single device.
  • Said device may accordingly include (i) an analyzing unit for the measurement of binding of a binding compound to a sample and a (ii) computer unit for processing the resulting data for the evaluation.
  • the instructions and interpretations are comprised in an executable program code comprised in the device, such that, as a result of determination, an assessment of cancer can be provided.
  • Typical devices are those which can be applied without the particular knowledge of a specialized technician, e.g., electronic devices which merely require loading with a sample.
  • the results may be given as output of raw data which need interpretation by a technician.
  • the output of the device is, however, processed, i.e. evaluated, raw data, the interpretation of which does not require a technician.
  • the device further comprises a memory unit, preferably comprising a database comprising at least one reference value for a binding of a binding compound to a sample.
  • Embodiment 1 A binding compound binding to a mutein of H3 histone A (H3) comprising the amino acid sequence of SEQ ID NO: 1 (H3 mutein), preferably comprising the amino acid sequence of SEQ ID NO: 12.
  • Embodiment 2 The binding compound of embodiment 1, wherein said binding compound binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 11, 12, 13, 14, or 15.
  • Embodiment 3 The binding compound of embodiment 1 or 2, wherein said binding compound comprises or is a polypeptide.
  • Embodiment 4 The binding compound of any one of embodiments 1 to 3, wherein said binding compound is an antibody or a fragment and/or a derivative thereof.
  • Embodiment 5 The binding compound of any one of embodiments 1 to 4, wherein said binding compound is an antibody, preferably an IgG, an IgA, or an IgM.
  • Embodiment 6 The binding compound of any one of embodiments 1 to 5, wherein said binding compound is a monoclonal antibody or a fragment or derivative thereof.
  • Embodiment ? The binding compound of embodiment 5 or 6, wherein said IgG is an
  • Embodiment 8 The binding compound of any one of embodiments 4 to 7, wherein said derivative of an antibody is a B cell receptor, an single-chain antibody, a single-domain antibody.
  • Embodiment 9 The binding compound of any one of embodiments 4 to 7, wherein said fragment of an antibody is an Fab, Fab', F(ab')2, or Fv fragment.
  • Embodiment 10 The binding compound of any one of embodiments 1 to 9, wherein said binding compound is a single-chain antibody.
  • Embodiment 11 The binding compound of any one of embodiments 4 to 10, wherein said antibody comprises a heavy chain variable domain comprising CDR sequences as shown in SEQ ID NO s: 2, 3, and 4; or amino acid sequences at least 70% identical thereto.
  • Embodiment 12 The binding compound of any one of embodiments 4 to 11, wherein said antibody comprises a light chain variable domain comprising CDR sequences as shown in SEQ ID NOs: SEQ ID NOs:5, 6, and 7; or amino acid sequences at least 70% identical thereto.
  • Embodiment 13 The binding compound of any one of embodiments 1 to 12, wherein said binding compound, preferably being an antibody, comprises (i) the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence at least 70% identical thereto, and/or the amino acid sequence of SEQ ID NO:9 or an amino acid sequence at least 70% identical thereto.
  • Embodiment 14 The binding compound of any one of embodiments 1 to 13, wherein said binding compound is a high-affinity compound, preferably having a KD of at most 100 nM, preferably at most 10 nM, more preferably at most 1 nM, most preferably at most 100 pM.
  • Embodiment 15 The binding compound of any one of embodiments 1 to 14, wherein said binding compound is a fully human antibody.
  • Embodiment 16 The binding compound of any one of embodiments 1 to 15, wherein said binding compound is a multitude-specific antibody, preferably a bispecific or a trispecific antibody, further comprising a binding domain binding a tumor antigen, preferably a glioma antigen, more preferably GD2.
  • said binding compound is a multitude-specific antibody, preferably a bispecific or a trispecific antibody, further comprising a binding domain binding a tumor antigen, preferably a glioma antigen, more preferably GD2.
  • Embodiment 17 The binding compound of any one of embodiments 1 to 16, wherein said binding compound further comprises a T-cell engagement element.
  • Embodiment 18 The binding compound of any one of embodiments 1 to 17, wherein said binding compound is a transmembrane signaling protein.
  • Embodiment 19 The binding compound of any one of embodiments 1 to 18, wherein said binding compound is a chimeric antigen receptor (CAR) or a T cell receptor fusion construct (TruC).
  • Embodiment 20 The binding compound of any one of embodiments 1 to 14, wherein said binding compound is a transmembrane sensor for said H3 mutein.
  • Embodiment 21 The binding compound of any one of embodiments 1 to 20, wherein said binding compound comprises at least one transmembrane domain and preferably is comprised in a cell membrane of a host cell expressing said binding compound, preferably an activated B cell, more preferably a recombinant activated B cell.
  • Embodiment 22 A polynucleotide encoding a binding compound according to any one of embodiments 1 to 21.
  • Embodiment 23 The polynucleotide of embodiment 22, wherein said polynucleotide is an expression construct.
  • Embodiment 24 A host cell comprising a binding compound according to any one of embodiments 1 to 21 and/or a polynucleotide according to embodiment 22 or 23.
  • Embodiment 25 The host cell of embodiment 24, wherein said host cell is a B cell.
  • Embodiment 26 The host cell of embodiment 24 or 25, wherein said host cell is an activated B cell, more preferably an activated plasmablast.
  • Embodiment 27 The host cell of embodiment 24 or 25, wherein said host cell is a memory
  • Embodiment 28 The host cell of embodiment 24 or 25, wherein said host cell is a B cell with cytotoxic activity.
  • Embodiment 29 A binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, for use in medicine.
  • Embodiment 30 Use of a binding compound according to any one of embodiments 1 to
  • a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, for the manufacture of a medicament for the manufacture of a medicament.
  • Embodiment 31 A binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, for use in treating and/or preventing cancer in a subject.
  • Embodiment 32 Use of a binding compound according to any one of embodiments 1 to
  • Embodiment 33 A method for inhibiting cancer cells comprising (a) contacting said cancer cells with a binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, and
  • Embodiment 34 The method of embodiment 33, wherein said inhibiting cancer cells is treating and/or preventing cancer.
  • Embodiment 35 The method of embodiment 33 or 34, wherein said method is an in vitro method.
  • Embodiment 36 A method for treating and/or preventing cancer in a subject, said method comprising
  • Embodiment 37 The subject matter of any one of embodiments 31 to 36, wherein said cancer is a glioma.
  • Embodiment 38 The subject matter of any one of embodiments 31 to 37, wherein said cancer is a H3 K27M positive cancer.
  • Embodiment 39 The subject matter of any one of embodiments 31 to 38, wherein said cancer is a H3 K27M positive glioma, preferably diffuse midline glioma.
  • Embodiment 40 A method for cancer assessment in a subject, comprising
  • step (C) assessing cancer based on the determining in step (B).
  • Embodiment 41 The method of embodiment 40, wherein said cancer assessment is diagnosing cancer.
  • Embodiment 42 The method of embodiment 40, wherein said cancer assessment is prognosing cancer.
  • Embodiment 43 The method of embodiment 40, wherein said cancer assessment is identifying said subject as being susceptible for treatment with a binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, wherein step (C) is identifying said subject as susceptible for said treatment if binding of said binding compound to said sample is detected.
  • Embodiment 44 Use of a binding compound according to any one of embodiments 1 to
  • Embodiment 45 The use of embodiment 4, wherein said use is an in vitro use.
  • Embodiment 46 A recombinant host cell produced or producible by introducing a polynucleotide according to embodiment 22 or 23 into said host cell.
  • Embodiment 47 A device comprising a binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28.
  • Embodiment 48 A kit comprising a binding compound according to any one of embodiments 1 to 21, a polynucleotide according to embodiment 22 or 23, or a host cell according to any one of embodiments 24 to 28, comprised in a housing.
  • Embodiment 49 The kit of embodiment 48, further comprising a means of administration.
  • Embodiment 50 The kit of embodiment 48 or 49, further comprising a diluent.
  • Fig. 1 Dose response curves of a recombinant antibody (Embodiment 4) were assessed using a direct ELISA pre-coated with either H3.3wt or H3.3K27M peptide. OD was measured at 450 and plotted over antibody concentration in nM. Figure shows high specificity for the mutein.
  • b Affinity to the H3.3K27M peptide was determined in a competitive ELISA in which the recombinant antibody was incubated with the respective peptide overnight and then added to H3.3K27M precoated ELISA plates. Dose response curves show the OD450 over peptide concentration in nM.
  • b Affinity to the H3.3 and H3.3K27M full length protein was determined in a competitive ELISA in which the recombinant antibody was incubated with the respective protein overnight and then added to H3.3K27M precoated ELISA plates. Dose response curves show the OD450 over peptide concentration in nM.
  • Fig. 2 comparison of the BCR sequence herein described. Different residues in the CDR3 regions highlighted. SEQ ID No. 4 was found as a class switched IGHG, whilst the related antibody was found to be IGHA. When expressed as recombinant IGHG antibodies, both antibodies have similar affinity for the H3.3K27M peptide.
  • a single cell sequencing library was generated from a cerebral spinal fluid sample from a H3.3K27M (SEQ ID NO: 1 : KAPRKQLATKAARMSAPSTGGVKKPHR) vaccinated patient using a commercially available kit from lOx Genomics following the manufacturer’s protocol.
  • the constructed gene expression library was sequenced on a NovaSeq6000 platform (Illumina) respectively.
  • Raw sequencing data was processed using the cellranger pipeline (lOx Genomics, version 7.0) applying default settings.
  • Antibody secreting B cells within the sample were identified on the basis of their expression of canonical B cell markers.
  • Antibody expression vectors were prepared as endotoxin-free midipreps using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research)
  • HEK cells maintained in DMEM medium supplemented with 2 % FCS were transfected with antibody expression vectors using FuGENE HD transfection reagent (Promega) in a 1 :3 ratio.
  • Antibody-containing supernatant was harvested after 5 days and filtered through a 0.45 pM syringe filter.
  • Antibody purification was performed using Mab Select (Cytiva) according to the manufacturer's guidelines. IgG ELISA
  • Indirect ELISA was performed in maxisorp plates (Nunc) pre-coated with human H3.3K27M and H3.3wt (pl4-40) (180 ng per well in PBS). Purified antibodies were added in serial dilutions.
  • 96-well polypropylene plates Gibco-coated polypropylene plates (Greiner) were blocked with 100 % FCS for 2 h at room temperature. 125 ng of antibody was diluted with H3.3K27M and H3.3wt peptide and protein, respectively and incubated overnight at 4°C. Peptide and proteins were 1 :2 serial diluted starting from 144 ng. The following morning the mixture was added to a H3.3K27M (pl4-40, 180 ng) pre-coated MaxiSorp plate.
  • the BCR identified, cloned and synthesized as herein described binds the H3.3 mutein both when present as a short peptide, or as part of the full length H3.3 protein.
  • antibodies arise through somatic hypermutation and class switching.
  • sequences related to the BCR herein claimed are also able to bind the H3.3 mutein.

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Abstract

La présente invention concerne un composé de liaison se liant à une mutéine d'histone H3A (H3, HGNC:4764) comprenant la séquence d'acides aminés de SEQ ID NO : 1 (mutéine H3), ledit composé de liaison étant un anticorps humain ou un fragment et/ou un dérivé de celui-ci, et des polynucléotides, des cellules hôtes, des utilisations médicales et des procédés associés.
PCT/EP2024/061448 2023-04-26 2024-04-25 Composés de liaison à la mutéine d'histone h3 WO2024223769A1 (fr)

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