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WO2025037120A1 - Composé comprenant un premier et un second anticorps ou un fragment de liaison à l'antigène et procédés de conjugaison de celui-ci - Google Patents

Composé comprenant un premier et un second anticorps ou un fragment de liaison à l'antigène et procédés de conjugaison de celui-ci Download PDF

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WO2025037120A1
WO2025037120A1 PCT/GB2024/052163 GB2024052163W WO2025037120A1 WO 2025037120 A1 WO2025037120 A1 WO 2025037120A1 GB 2024052163 W GB2024052163 W GB 2024052163W WO 2025037120 A1 WO2025037120 A1 WO 2025037120A1
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Prior art keywords
antibody
group
antigen
binding fragment
aliphatic
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Nyle Owen Saul JONES
Oliver Schon
Tiffany Jane DANIELS
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Bivictrix Ltd
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Bivictrix Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • a compound comprising a first and second antibody or antigen-binding fragment and methods of conjugating the same
  • the present invention relates to a compound, in particular to a compound comprising a first and second antibody or antigen-binding fragment, wherein the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.
  • the invention extends to methods of producing the compounds. The method also extends to the use of click chemistry to produce the compounds.
  • Therapeutic Antibody Database approximately 2,800 antibodies have been studied or are being planned for studies in human clinical trials, and approximately 80 antibodies have been approved by governmental drug regulatory agencies for clinical uses.
  • Antibodies can also serve as carriers of cytotoxic molecules or other therapeutic agents. Indeed, in recent years there has been extensive research and development in the field of antibody drug conjugates (ADC), wherein an antibody is conjugated to one or more cytotoxic drug, such as auristatin, maytansine, calicheamicin and camptothecin.
  • ADCs have been developed as anti-cancer therapeutics designed to target solid tumours as well as diffusive - or liquid - tumours of the blood, lymphoid system and bone marrow (including various types of lymphomas and leukaemia).
  • the ADCs target one or more unique antigens on the surface of the cancer cell of interest. Methods for conjugating the drugs to the antibody molecules have also been developed.
  • ADCs as therapeutics may be improved by increasing the specificity for the target cell.
  • antibodies having bi- or multi-specificity are known in the art.
  • antibodies containing two or more antigen-binding sites are known.
  • a number of methods have been reported for preparing multivalent antibodies, for example, by covalently linking three or four Fab fragments via a connecting structure.
  • antibodies have been engineered to express tandem three or four Fab repeats.
  • the invention relates to a compound comprising the formula: A 1 -S’-L-S”-
  • a 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • a 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment; and L is a linker; wherein the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.
  • the compound may consist essentially of the formula: A 1 -S’-L-S”- A 2 , wherein each of A 1 , A 2 , S’, S” and L are as defined herein.
  • the compound may consist of the formula: A 1 -S’-L-S”-A 2 , wherein each of A 1 , A 2 , S’, S” and L are as defined herein.
  • the invention relates to a method of producing a compound comprising conjugating together a first unit and a second unit via click chemistry; wherein the first unit comprises the formula: A 1 -S’-Y’, wherein A 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • Y’ is hydrogen or -L’-X’, wherein L’ is a first linker arm; and X’ is a first conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and wherein the second unit comprises the formula: A 2 -S”-Y”, wherein A 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • Y is hydrogen or -L”-X”, wherein L” is a second linker arm; and X” is a second conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when Y’ is hydrogen, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; when Y’ is -L’-X’ and X’ is an azide, nitrone, tetrazine or tetrazole group, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cycl
  • the invention relates to the use of click chemistry to conjugate together a first unit and a second unit to produce a compound; wherein the first unit comprises the formula A 1 -S’-Y’, wherein A 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • Y’ is hydrogen or -L’-X’, wherein L’ is a first linker arm; and X’ is a first conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and wherein the second unit comprises the formula: A 2 -S”-Y”, wherein A 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • Y is hydrogen or -L”-X”, wherein L” is a second linker arm; and X” is a second conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when Y’ is hydrogen, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; when Y’ is -L’-X’ and X’ is an azide, nitrone, tetrazine or tetrazole group, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cycl
  • Figure 2 shows an SDS-PAGE gel of crude samples taken after 22 hours from Bi-Fab synthesis reactions using SPAAC, CuAAC and iEDDA-based reactions. Molecular weight markers (left) indicate rough band sizing in kDa.
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.
  • the nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties of the molecules disclosed herein are also described in the examples.
  • the compound comprises a first antibody or antigen-binding fragment, A 1 , and a second antibody or antigen-binding fragment, A 2 .
  • the first and second antibody or antigenbinding fragments each capable of binding to an antigen.
  • the terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell I B-cell antigen receptor.
  • the term “antigen(s)” encompasses antigenic epitopes, e.g., fragments of antigens which are recognized by, and bind to, immune components.
  • Epitopes can be recognized by antibodies in solution, e.g., free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.
  • epitopes or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antigen-binding fragment thereof specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes.
  • Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation.
  • epitope mapping Methods for determining what epitopes are bound by a given antibody or antigenbinding fragment thereof (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antigen-binding fragment thereof.
  • Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. Suitable competition assays are mentioned elsewhere herein and also shown in the examples.
  • the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, mutagenesis mapping (e.g, site-directed mutagenesis mapping), and/or in silico modelling.
  • antibody refers to an immunoglobulin (Ig) protein that is capable of binding an antigen.
  • antibody as used herein broadly refers to any polypeptide comprising complementarity determining regions (CDRs) that confer specific binding affinity of the polypeptide for an antigen.
  • CDRs complementarity determining regions
  • the term antibody as used herein encompasses polyclonal and monoclonal antibody preparations.
  • antibody should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain.
  • antibody should also be construed as covering antibody mimetics, such as, but not limited to, cyclic peptides, for example bicyclic peptides, cysteine knots and anticalins etc.
  • the antibody may be an immunoglobulin (Ig) molecule, or antigen binding portion/fragment thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • Ig immunoglobulin
  • Such fragments are known in the art for example F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Therefore, an antibody fragment comprises an antigen binding portion.
  • the present invention extends to such antibody fragments.
  • the term "monoclonal antibody” refers to an antibody obtained from a single close of cells or cell line.
  • the individual antibodies are identical and/or bind the same epitope.
  • polyclonal antibodies which include different antibodies directed against different epitopes, each monoclonal antibody of in a preparation is directed against a single epitope.
  • the term “specific” may refer to the situation in which the antibody or antigen-binding fragment will not show any significant binding to molecules other than its specific binding partner.
  • binds means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions.
  • the ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • the binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.
  • polypeptide(s) and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid(s) or “peptide residue(s)”, as used herein denote both naturally occurring and synthetic amino acids.
  • the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.
  • a full-length antibody comprises two heavy (H) chains and two light (L) chains.
  • Each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR, VH or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR, VL or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • Immunoglobulin molecules can be subdivided into various types class and subclass.
  • the classes (isotype) of immunoglobulin include IgG, IgM, IgA, IgE, and IgD.
  • the immunoglobulin classes are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as y-chains; IgM have p-chains; IgA have a-chains; IgE have e-chains; and IgD have b-chains.
  • Antibodies may include the kappa (K) and lambda (A) light chains and the alpha (IgA), gamma (lgG1 , lgG2, lgG3, lgG4), delta (IgD), epsilon (IgE) and mu (IgM) heavy chains, or their equivalents in other species.
  • Full-length immunoglobulin “light chains” (usually of about 25 kDa or 214 amino acids long) consist of a variable region of approximately 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus.
  • Fully-length immunoglobulin “heavy chains” (usually of about 50 kDa or 446 amino acids long), likewise consist of a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • Light or heavy chain variable regions are generally composed of a “framework” region (FR) interrupted by three hypervariable regions, also called CDRs.
  • the extent of the framework region and CDRs have been precisely defined. The sequences of the framework regions of different light and heavy chains are relatively conserved within a species.
  • the framework region of an antibody i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.
  • Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3.
  • Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.
  • the antibody may be comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.
  • Such mutant, variant, or derivative antibody formats are known in the art.
  • CDR complementarity-determining region
  • CDR1 complementarity-determining region
  • CDR2 complementarity-determining region
  • CDR3 complementarity-determining region
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.
  • CDRs Different definitions of the CDRs are commonly in use. The method described by Kabat is the most commonly used and CDRs are based on sequence variability (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1 -113 of the heavy chain).
  • Another system is the ImMunoGeneTics (IMGT) numbering scheme (Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005)).
  • IMGT ImMunoGeneTics
  • a CDR is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain.
  • CDR1-IMGT loop BC
  • CDR2-IMGT loop C'C
  • CDR3-IMGT loop FG
  • antibody is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one CDR capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo. Methods of producing polyclonal and monoclonal antibodies are known in the art and described more fully below.
  • the antibody or antigen-binding fragment thereof may be chimeric, human or humanised.
  • a “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody.
  • Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody therapies, as such molecules would elicit little or no immune response in the human subject.
  • a humanised antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent 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.
  • Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody diagnostics or therapies, as such molecules would elicit little or no immune response in the human subject.
  • the term invention includes antibodies and antigen-binding fragments thereof.
  • the antigen-binding fragments may be selected from any fragment capable of binding the antigen or antigenic fragment of interest.
  • Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, F(ab')3, Fabc, Fd, single chain Fv (scFv), (scFv)2, Fv, scFv-Fc, heavy chain only antibody, diabody, tetrabody, triabody, minibody, antibody mimetic protein, single domain antibody, e.g. a VH.
  • the antigen-binding fragment may comprise or consist of any of these fragments.
  • Antigen-binding fragments derived from an antibody, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and/or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.
  • Fv fragments ( ⁇ 25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).
  • VH and VL domains respectively are capable of binding to an antigen. They are generally referred to as a “single domain antibody” or “immunoglobulin single variable domain”.
  • a single domain antibody ( ⁇ 12 to 15 kDa) has thus either the VH or VL domain.
  • Antigen-binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341 : 544-546). Ward et al.
  • dAbs for single domain antibodies
  • dAb generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen.
  • VH, VHH or VL immunoglobulin variable domain
  • human single domain antibodies are preferred over camelid derived VHH, primarily because they are not as likely to provoke an immune response when administered to a patient.
  • a "Fab molecule” fragment antigen binding as such a Fab domain refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the "Fab light chain”) of an immunoglobulin.
  • the Fab light chain and Fab heavy chain in the Fab construct are linked by a polypeptide sequence to yield a single chain Fab (scFab).
  • a “Fab” or “Fab fragment” comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains.
  • a Fab contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively.
  • the variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen binding.
  • a Fab’ comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains and the thiol group which forms the disulphide bridge between two heavy chains in a full-length antibody.
  • the antigen binding fragment may comprise a Fab.
  • the antigen binding fragment may comprise a Fab’.
  • the antigen binding fragment may comprise an scFv.
  • the first and/or second antibody or antigen binding fragment may comprise a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody.
  • the first antigen binding fragment may comprise a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody.
  • the second antibody or antigen binding fragment may comprise a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody.
  • the first and second antibody or antigen binding fragment may comprise a Fab or a Fab’.
  • the first and second antibody or antigen binding fragment may comprise a Fab. In an embodiment, the first and second antibody or antigen binding fragment may comprise or consist of a Fab. In an embodiment, the first and second antibody or antigen binding fragment may comprise or consist of a Fab. In an embodiment, the first antibody or antigen binding fragment may comprise or consist of a Fab. In an embodiment, the second antibody or antigen binding fragment may comprise or consist of a Fab. In an embodiment, the first and second antibody or antigen binding fragment may each comprise or consist of a Fab. In an embodiment, the first and second antibody or antigen binding fragment may each comprise an scFv.
  • single-chain refers to a molecule comprising amino acid monomers linearly linked by peptide bonds.
  • one of the antigen binding moieties e.g., antigen binding polypeptide construct
  • the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single- chain Fab molecule.
  • Fv fragments ( ⁇ 25kDa) consist of the two variable domains, VH and VL.
  • VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate.
  • stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).
  • one of the antigen binding moieties is a single-chain Fv molecule (scFv).
  • Single chain fragment variable (scFv) antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • the scFv is a fusion protein comprising the heavy chain variable region and the light chain variable region linked via a peptide linker
  • the antibody or antigen-binding fragment may be chimeric, human or humanised.
  • a “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody.
  • the antibody or antigen-binding fragment may comprise a monoclonal antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment may comprise a CH2 domain.
  • the CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule.
  • the CH2 domain of the antibody may be the CH2 domain of human IgG 1 , lgG2, lgG3, or lgG4, e.g., the CH2 domain of human lgG1.
  • the sequences of human IgG domains are known in the art.
  • the antibody or antigen-binding fragment may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain.
  • the immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer.
  • the hinge region, or part thereof may be a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof.
  • the hinge region, or part thereof may be an IgG 1 hinge region, or part thereof.
  • the sequence of the CH3 domain is not particularly limited.
  • the CH3 domain may be a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.
  • the antibody or antigen-binding fragment may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region.
  • the sequences of human lgG1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art.
  • the antibody or antigen-binding fragment may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.
  • the antibody or antigen-binding fragment may comprise an Fc domain.
  • the first and/or second antibody or antigen-binding fragment may comprise an Fc domain.
  • one of the first or second antibody or antigen-binding fragment may comprise an Fc domain.
  • the first and/or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody and ii) an Fc domain.
  • one of the first or second antibody or antigenbinding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody and ii) an Fc domain.
  • the antibody or antigen-binding fragment may comprise an Fc region also referred to as an Fc domain.
  • Fc or “Fc domain” or “Fc region” or “Fc construct” herein is used to define a C-terminal region of an immunoglobulin heavy chain.
  • the term includes native sequence Fc regions and variant Fc regions.
  • Fc region generally refers to a dimer complex comprising the C- terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody.
  • the Fc region may comprise native or variant Fc sequences.
  • the Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.
  • Fc polypeptide herein is meant one of the polypeptides that make up an Fc region.
  • An Fc polypeptide may be obtained from any suitable immunoglobulin, such as lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM.
  • an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence.
  • the antibody may comprise a CH2 domain.
  • the CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule.
  • the CH2 domain of the antibody is in one embodiment the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g the CH2 domain of human lgG1.
  • the sequences of human IgG domains are known in the art.
  • the binding molecule, antibody or antigen binding fragment thereof may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain.
  • the immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer.
  • the hinge region, or part thereof is a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof.
  • the hinge region, or part thereof is an lgG1 hinge region, or part thereof.
  • the binding molecule, antibody or antigen binding fragment thereof may comprise a CH3 domain.
  • the sequence of the CH3 domain is not particularly limited.
  • the CH3 domain is a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.
  • the binding molecule, antibody or antigen binding fragment thereof may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region.
  • the sequences of human lgG1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art.
  • An binding molecule, antibody or antigen binding fragment of the invention may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.
  • the Fc includes two Fc polypeptides each having a CH3 domain for dimerization. The N- terminal end of each Fc polypeptide is linked to the C-terminus of one of the antigen binding polypeptide constructs with or without a linker.
  • the Fc region may be any suitable Fc region for example a human Fc region.
  • An Fc region can mediate downstream effector functions via interaction with Fc-receptors found on immune cells or with C1q, the recognition molecule of the complement system.
  • the antibody or antigen-binding fragment may comprise an Fc region may be capable of interacting with an Fc receptor.
  • the antibody or antigen-binding fragment may comprise an Fc region may be capable of interacting with an Fc receptor and eliciting a downstream effector function.
  • Effector functions refer to downstream immune effector mechanisms such as but not limited to antibodydependent cytotoxicity, antibody dependent cellular phagocytosis, complement-dependent cytotoxicity, antibody-dependent intracellular neutralization and/or immunomodulatory functions.
  • Fc receptors are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRlla, FcyRllb and FcyRllc) and CD16 (FcyRllla and FcyRlllb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRIII are low to intermediate affinity (micromolar range KD).
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • NK Natural Killer
  • Complement dependent cytotoxicity and “CDC” refer to the lysing of a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
  • a molecule e.g. an antibody
  • Antibody-dependent cellular phagocytosis and "ADCP” refer to the destruction of target cells via monocyte or macrophage-mediated phagocytosis.
  • the antibody or antigen-binding fragment may comprise a human IgG Fc with effector function.
  • FcyRs on the surface of effector cells bind to the Fc region of an IgG which itself is bound to a target cell.
  • a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell.
  • the level of ADCC effector function various for IgG subtypes. Although this is dependent on the allotype and specific FcyRs in simple terms ADCC effector function is high for human IgG 1 and lgG3, and low for lgG2 and lgG4.
  • FcyRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions.
  • the binding molecule, antibody or antigen binding fragment thereof of the invention of the invention may have an Fc region with effector function, with enhanced effector function or with reduced effector function.
  • the binding molecule, antibody or antigen binding fragment thereof may include a modified Fc region.
  • the antibody or antigen-binding fragment may comprise a modified Fc.
  • the modified Fc may be modified in terms of amino acid sequence compared to a wild-type Fc region.
  • the Fc region may be modified to modulate the effect of the Fc region.
  • the Fc region may be modified to enhance effector function.
  • the antibody or antigen-binding fragment may comprise an Fc with enhanced effector function.
  • the Fc region may be modified to reduce or abolish effector function.
  • the Fc region may be modified to improve certain properties, e.g. to reduce complement mediated effector function, to reduce FcyR- mediated effector functions, to improve half-life.
  • the Fc region may be modified to enhance or reduce interaction of the Fc with one or more of the Fc receptors e.g. FcRn, C1q, TRIM21 , FcyRI, FcyRlla/b, FcyRllla).
  • the potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such ADCC and ADCP.
  • a number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D/A330L/I332E (“3M”), F243L or G236A.
  • enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, FcyRs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity.
  • Afucosylated (non- fucosylated) antibodies exhibit greatly enhanced ADCC activity through increased binding to FcyRllla.
  • Activation of ADCC and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred. Due to their lack of effector functions, lgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, lgG4 molecules can exchange half molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous lgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of lgG4 antibodies with a reduced propensity for Fab-arm exchange.
  • the CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors, such as FcyRI, FcyRlla, FcyRllb, FcyRIII and/or to complement.
  • CH2 domains of human IgG domains normally bind to Fey receptors and complement, decreased binding to Fey receptors is expected to decrease ADCC and decreased binding to complement is expected to decrease CDC activity of the antibody molecule.
  • An antibody molecule of the invention may comprise an Fc with modifications K322A/L234A/L235A or L234F/L235E/P331 S (“TM”), which almost completely abolish FcyR and C1q binding.
  • An antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) ("LALA mutation").
  • complement activation and ADCC can be decreased by mutation of Pro329 (position according to EU numbering), e.g., to either P329A or P329G.
  • the antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA- PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering).
  • an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297.
  • Modification of glycosylation on asparagine 297 of the Fc domain which is known to be required for optimal FcR interaction may confer a loss of binding to FcRs; a loss of binding to FcRs has been observed in N297 point mutations.
  • An antibody molecule of the invention may comprise an Fc with an N297A, N297G or N297Q mutation.
  • An antibody molecule of the invention with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor or following the expression of Fc domains in bacteria.
  • the Fc region may be modified to improve the half-life of the antibody or antigen binding fragment or compound.
  • the Fc region may be modified to enhance interaction with FcRn receptor and thereby improve half-life of the antibody or antigen binding fragment or compound.
  • IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days.
  • Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4.
  • the T250Q/M428L variant conferred an approximately 2- fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y/S254T/T256E variant, gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy.
  • the Fc region may be modified to reduce interaction with FcyR receptor and thereby improve half-life of the antibody or antigen binding fragment or compound.
  • the antibody or antigen-binding fragment may comprise an Fc which does not elicit downstream effector function, i.e., where its effector function has been knocked out.
  • the half-life of the compound may be increased.
  • Methods to modify or modulate the Fc effector function are known in the art and may be achieved via site directed mutagenesis of the Fc region.
  • the modulated Fc refers to a modulated activity compared to that of the wildtype Fc.
  • the antibody or antigen-binding fragment may comprise an additional moiety.
  • the additional moiety may provide further function to the molecule.
  • the further moiety may be selected from a half-life extending moiety and/or a label.
  • the additional moiety may be selected from one or more half-life extending molecules for example, one or more PEG molecules, a liposome, a serum albumin protein an antibody or antibody fragment that binds serum albumin.
  • the serum albumin may be human serum albumin.
  • the antibody or antigen-binding fragment may be conjugated or linked to the additional moiety in any suitable manner.
  • the additional moiety may be a detectable or functional label.
  • a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.
  • the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
  • the antibody or antigen-binding fragment may be modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome, or using a serum albumin protein or an antibody or antibody fragment that binds human serum albumin. Increased half-life can also be conferred by conjugating the molecule to an antibody fragment.
  • half-life refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.
  • the first and/or second antibody or antigen-binding fragment may be modified by serum albumin protein.
  • the first and/or second antibody or antigen-binding fragment may be conjugated to serum albumin protein.
  • the serum albumin protein may be human serum albumin or a modified version.
  • the first and/or second antibody or antigen-binding fragment may comprise an antibody or antibody fragment that binds human serum albumin.
  • the first and/or second antibody or antigen-binding fragment may be conjugated to a further antibody or fragment thereof that binds human serum albumin.
  • Increased half-life can also be conferred by conjugating the bispecific molecule to an antibody fragment, for example wherein the antibody fragment binds serum albumin.
  • the antibody fragment that binds serum albumin may be a F(ab')2, Fab, Fab’, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • the antigen binding fragment that binds serum albumin may be a single domain antibody for example a VH.
  • the antigen binding fragment that binds serum albumin may be an scFv.
  • Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding antibodies without such modification.
  • increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding antibodies without such modification.
  • the in vivo half-life of the antibody or antigen-binding fragment or compound of the invention a can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art.
  • Half-life can for example be expressed using parameters such as the t1/2-alpha t1/2-beta and the area under the curve (AUC). It will be appreciated by a person skilled in the art that reference to the half-life of an antibody may also refer to the half-life of the compounds of the invention (and may be used interchangeably herein).
  • the antibody or antigen-binding fragment may be produced by any suitable method.
  • the antibody or antigen-binding fragment may be produced in/by murine, mammal or other animal models, by using hybridoma technology or other methods known in the art.
  • nucleic acids encoding the antibody or antigen-binding fragment or binding molecule as described herein may be inserted into a plasmid and expressed in a suitable expression system.
  • the present invention includes methods for expressing an antibody or antigen-binding fragment or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E.
  • a bacterial host cell such as an E. coli, may include a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D- thiogalactopyranoside).
  • IPTG isopropyl-beta-D- thiogalactopyranoside
  • Transformation can be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.
  • the antibody or antigen-binding fragment may be produced by a method comprising the steps of (i) introducing one or more polynucleotides encoding light and/or heavy immunoglobulin domains of the antibody wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., E. coli, CHO or Pichia or Pichia pastoris) under conditions favourable to expression of the polynucleotide and, (iii) optionally, isolating the antibody or antigen-binding fragment from the host cell and/or medium in which the host cell is grown.
  • a host cell e.g., E. coli, CHO or Pichia or Pichia pastoris
  • an antibody or antigen-binding fragment comprising more than one immunoglobulin chain, e.g., an antibody or antigen-binding fragment that comprises two heavy immunoglobulin chains and two light immunoglobulin chains
  • co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antibody or antigen-binding fragment.
  • the methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed.
  • Such chains are useful, for example, as intermediates in the expression of an antibody that includes such a chain.
  • the first and second antibody or antigen-binding fragment may each independently comprise a full-length antibody.
  • the full-length antibody may comprise human constant regions and human light chain regions.
  • the first and second antibody or antigen-binding fragment may each independently comprise an antigen-binding fragment.
  • the first and second antibody or antigen-binding fragment may each independently bind to any suitable antigen.
  • the first and second antibody or antigen-binding fragment may each bind to the same antigen or may each bind to different antigens.
  • the first and second antibody or antigen-binding fragment may each bind to different antigens.
  • the compound is bispecific.
  • bispecific is meant that the compound is able to simultaneously bind to two different types of antigen or two different epitopes on the same antigen.
  • the first and second antibody or antigen-binding fragment may each independently comprise any suitable antibody or antigen-binding fragment.
  • the first and second antibody or antigen-binding fragment may each independently comprise an antigen-binding fragment.
  • the first and second antibody or antigen-binding fragment may each independently comprise a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody.
  • the first and second antibody or antigen-binding fragment may each independently bind to any suitable antigen.
  • the first and second antibody or antigen-binding fragment may each independently bind to CD33, CD7, B7H4, and/or CD56.
  • the first antibody or antigen-binding fragment may bind to CD33.
  • the first antibody or antigen-binding fragment may bind to CD7.
  • the first antibody or antigen-binding fragment may bind to B7H4.
  • the first antibody or antigen-binding fragment may bind to CD56.
  • the second antibody or antigen-binding fragment may bind to CD33.
  • the second antibody or antigenbinding fragment may bind to CD7.
  • the second antibody or antigen-binding fragment may bind to B7H4.
  • the second antibody or antigen-binding fragment may bind to CD56.
  • CD33 is an AML antigen that has been extensively investigated in this setting and is a well-established and validated AML target, which is still the antigen of choice for many existing and novel therapeutics. Additionally, CD33 has also been shown to be a validated ADC target antigen, displaying a sufficient antigen density and internalisation. This is evident through the marketed ADC therapeutic Mylotarg, launched in 2000, this ADC targets the CD33 antigen in AML.
  • CD7 is an antigen commonly associated with thymocytes and mature T cells and is believed to play an essential role in T cell interactions and T/B cell interactions in early lymphoid development. This antigen does not currently represent a commonly targeted antigen in therapeutic development, assumed to be due to the extensive expression of this antigen on healthy T cell populations.
  • the first and second antibody or antigen-binding fragment may each bind to different antigens.
  • the first antibody or antigen-binding fragment may bind to CD33 and the second antibody or antigen-binding fragment may bind to CD7, CD56 or B7H4.
  • the first antibody or antigen-binding fragment may bind to CD33 and the second antibody or antigen-binding fragment may bind to CD7.
  • the first antibody or antigen-binding fragment may bind to CD33 and the second antibody or antigen-binding fragment may bind to CD56.
  • the first antibody or antigen-binding fragment may bind to CD33 and the second antibody or antigen-binding fragment may bind to B7H4.
  • the first antibody or antigen-binding fragment may bind to CD7 and the second antibody or antigen-binding fragment may bind to CD33.
  • the first antibody or antigen-binding fragment may bind to CD7 and the second antibody or antigen-binding fragment may bind to CD56.
  • the first antibody or antigen-binding fragment may bind to CD7 and the second antibody or antigenbinding fragment may bind to B7H4.
  • the first antibody or antigen-binding fragment may bind to CD56 and the second antibody or antigen-binding fragment may bind to CD7.
  • the first antibody or antigen-binding fragment may bind to CD56 and the second antibody or antigen-binding fragment may bind to CD33.
  • the first antibody or antigen-binding fragment may bind to CD56 and the second antibody or antigen-binding fragment may bind to B7H4.
  • the first antibody or antigen-binding fragment may bind to B7H4 and the second antibody or antigen-binding fragment may bind to CD7.
  • the first antibody or antigen-binding fragment may bind to B7H4 and the second antibody or antigen-binding fragment may bind to CD56.
  • the first antibody or antigen-binding fragment may bind to B7H4 and the second antibody or antigen-binding fragment may bind to CD33.
  • the first and second antibody or antigen-binding fragment each independently comprise a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering.
  • the first and second antibody and antigen-binding fragment each independently comprise one or more non-naturally occurring cysteine residue(s), i.e., a non-wild-type cysteine residue.
  • the first antibody or antigen-binding fragment may comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering.
  • the second antibody or antigen-binding fragment may comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering.
  • the compound comprises a first sulphur atom, S’, wherein the first sulphur atom is derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment.
  • the compound comprises a second sulphur atom, S”, wherein the second sulphur atom is derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment.
  • first and second sulphur atoms are suitably derived from a substituted cysteine residue (i.e., non-naturally occurring cysteine residue) at one of positions 153, 156, 168, 173 and/or 207, according to Kabat numbering, on the first and second antibody or antigen-binding fragment, respectively.
  • a substituted cysteine residue i.e., non-naturally occurring cysteine residue
  • the first sulphur atom, S’ may be derived from a non-naturally occurring cysteine at position 207 of the first antibody of antigen-binding fragment according to Kabat numbering.
  • the second sulphur atom, S may be derived from a non-naturally occurring cysteine at position 207 of the second antibody of antigen-binding fragment according to Kabat numbering.
  • the compound comprises a linker, L.
  • the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antibody or antigen-binding fragment, respectively.
  • the linker may be any suitable linker.
  • the linker may comprise a succinimide group.
  • the linker may comprise a succinimide group attached to the first and/or second sulphur atom.
  • the linker may comprise a succinimide group attached to each of the first and second sulphur atom.
  • the succinimide group(s) may suitably be derived from the reaction of the first and/or second sulphur atom with a maleimide group.
  • the linker may comprise one or more polyethylene glycol (PEG) groups.
  • the linker may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. polyethylene glycol groups.
  • the linker may comprise at least one moiety of the formula - (-O-CH2-CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3.
  • the linker may comprise two moieties of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3, wherein each moiety of the formula -(- O-CH2-CH2-)n- is separated by a suitable chemical moiety.
  • the linker may comprise a polycyclic group.
  • polycyclic and like terms as used herein, is meant a group comprising a plurality of ring moieties wherein at least two of said ring moieties share at least one atom, preferably at least two atoms, in common.
  • the polycyclic group comprises first and second fused rings that share at least one atom in common, preferably at least two atoms in common, more preferably two atoms in common.
  • the polycyclic group may comprise a first 6-membered heterocyclic ring fused to a second 8-membered ring, wherein the first and second rings share two atoms in common.
  • the linker may be formed via a click chemistry reaction.
  • click chemistry reaction refers to a group of reactions that are modular lining reactions, there are four key classes of click reactions including cycloadditions, nucleophilic ring openings, nonaldol carbonyl chemistry and carbon multiple bond additions.
  • the polycyclic group is formed via a click chemistry reaction, suitably wherein a first ring is caused to fuse with a second ring via a click chemistry reaction.
  • the polycyclic group may be derived from the click chemistry reaction of a cyclic alkene or cyclic alkyne group with a tetrazine group, preferably a cyclooctene group or bicyclononyne group with a tetrazine group, most preferably trans-cyclooctene or bicyclo[6.1 ,0]non-4-yne with a tetrazine group.
  • the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.
  • conjugation and “conjugate(d)” refer to chemical linkages, either covalent or non-covalent, which proximally associates one molecule of interest with a second molecule of interest.
  • each of the first and second antibody or antigen-binding fragment typically comprise at least one functional group capable of participating in a click chemistry reaction.
  • the functional group capable of participating in a click chemistry reaction may be present as part of a “linking arm” or a “click handle”, “click arm”, etc (which terms may be used interchangeably herein).
  • the first and second antibody or antigen binding fragment may each comprise a click handle, wherein each click handle comprises a complimentary conjugating group, such as those defined herein.
  • a click handle may be attached to the first and/or second antibody or antigen binding fragment, as appropriate, in any suitable manner.
  • a click handle may be attached to the first and/or second antibody or antigen-binding fragment, as appropriate, via the reaction of a thiol group of one or more of the non-naturally occurring cysteine residues as described herein and a maleimide, bromomaleimide, bromopyridazinedione, chloroacetamide, bromoacetamide, iodoacetamide, a-haloketone, unsaturated carbonyl, vinylsulfone, vinyl- pyridine, vinyl-pyridinium and/or unsaturated phosphonamidite group (Chem.
  • a click handle may comprise a maleimide, bromomaleimide bromopyridazinedione, a-haloketone, unsaturated carbonyl, vinylsulfone, vinyl- pyridine, vinyl-pyridinium and/or unsaturated phosphonamidite group and a conjugating group, wherein the conjugating group is operable to participate in a click chemistry reaction.
  • Any suitable click chemistry may be used to conjugate together the first and second antibody or antigen-binding fragments. Suitable click chemistries will be known to a person skilled in the art. It will be appreciated by a person skilled in the art that the choice of click chemistry will typically depend upon the functional groups that are to be conjugated together.
  • the click chemistry may be selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and/or an inverse electron demand Diels-Alder (iEDDA) reaction.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strained-promoted azide-alkyne click chemistry
  • iEDDA inverse electron demand Diels-Alder
  • the click chemistry may be a thiol-ene reaction.
  • the click chemistry may be a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
  • the click chemistry may be a strained- promoted azide-alkyne click chemistry (SPAAC) reaction.
  • the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction.
  • the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction.
  • iEDDA inverse electron demand Diels-Alder
  • a thiol-ene reaction (which may also be referred to as an alkene hydrothiolation reaction) is the reaction between a thiol group, i.e., R-SH, and an alkene to form a thioether.
  • the reaction results in an anti-Markovnikov addition of a thiol compound to an alkene.
  • Thiol-ene reactions may proceed through two mechanisms: free-radical additions and catalyzed Michael additions. Free-radical addition reactions can be initiated by light, heat or radical initiators, which form a thiyl radical species. The radical then propagates with an ene functional group via an anti- Markovnikov addition to form a carbon-centred radical.
  • a chain-transfer step removes a hydrogen radical from a thiol, which can subsequently participate in multiple propagation steps.
  • Michael addition reactions are catalyzed by either a base or a nucleophile, resulting in a similar anti-Markovnikov addition product as the free-radical addition reactions.
  • a thiol-ene reaction is exemplified in scheme I.
  • the reaction may be between a thiol group and a maleimide group.
  • the first and second antibody or antigen-binding fragment may be conjugated together via a thiol-maleimide reaction.
  • the first antibody or antigen-binding fragment may be conjugated to the second antibody or antigen-binding fragment via the -S’-H group present on the first antibody or antigen-binding fragment before conjugation and an alkene group, such as a cyclic alkene group, or even a maleimide group, present on the second antibody or antigen-binding fragment before conjugation or vice versa.
  • the second antibody or antigen-binding fragment (or first antibody or antigen-binding fragment if vice versa) may comprise a linker arm, L” (or L’), comprising an alkene group, such as a cyclic alkene group, or even a maleimide group.
  • the alkene group such as cyclic alkene group, or even maleimide group
  • the linker arm may itself be attached to the first or second antibody or antigen-binding fragment, as appropriate, via click chemistry, preferably via a thiolene reaction, more preferably via a thiol-maleimide reaction.
  • the -S’-H and S”-H groups as present on the first and second antibody or antigen-binding fragment before conjugation, respectively, may be reacted with a linker unit of formula X’-L 2 -X”, wherein L 2 is a linker and each of X’ are alkene groups, such as cyclic alkene groups, or even maleimide groups.
  • a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction is the reaction between an azide and an alkyne to form a 1 ,5-disubstituted 1 ,2,3-triazole.
  • the reaction between azide and alkyne is very selective.
  • the reaction is also quick and pH sensitive.
  • a CuAAC reaction is exemplified in scheme II:
  • the catalyst for the CuAAC reaction may comprise a copper(l) catalyst, such as cuprous bromide or cuprous iodide and/or a mixture of copper(ll) and a reducing agent, such as sodium ascorbate, to produce copper(l) in situ (i.e., in the reaction mixture).
  • a copper-free catalyst may be used.
  • a pentamethylcyclopentadienyl ruthenium chloride complex may be used as a catalyst to catalyse the azide-alkyne cycloaddition.
  • a strained-promoted azide-alkyne click chemistry (SPAAC) reaction is the reaction between an azide and a strained alkyne.
  • the strained alkyne may comprise a strained cyclooctyne, such as difluorooctyne (DIFO), dibenzylcyclooctyne (DIBO) and biarylazacyclooctynone (BARAC).
  • DIFO difluorooctyne
  • DIBO dibenzylcyclooctyne
  • BARAC biarylazacyclooctynone
  • DIFO difluorooctyne
  • the electron-withdrawing, propargylic, gem-fluorines act together with the ring strain to destabilize the alkyne. This destabilization increases the reaction driving force, and the desire of the cycloalkyne to relieve its ring strain.
  • An inverse electron demand Diels-Alder (iEDDA) reaction is the reaction between an electron poor diene group, such as a tetrazine group, and an electron rich dienophile group, such as a cycloalkene or cycloalkyne group, for example a cyclooctene or bicyclononyne group.
  • an electron poor diene group such as a tetrazine group
  • an electron rich dienophile group such as a cycloalkene or cycloalkyne group, for example a cyclooctene or bicyclononyne group.
  • Frontier orbital overlap between the HOMOpienophiie) and LUMOpiene is the driving force for this reaction.
  • High ring strain of dienophiles increases the HOMO energy leading to a small HOMO(Dienophiie)-LUMO(Diene) separation.
  • the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction.
  • the functional groups to be conjugated together may suitably be a tetrazine group and a cycloalkene group, such as a cyclooctene group, and/or a cycloalkyne group, such as a cyclooctyne group.
  • the functional groups to be conjugated together may be a tetrazine group and a cyclooctene group, such as transcyclooctene (TCO).
  • the functional groups to be conjugated together may be a tetrazine group and a cyclooctyne group, such as bicyclononyne (BCN).
  • BCN bicyclononyne
  • bicyclononyne includes an 8-membered ring having an unsaturated carbon-carbon triple bond, i.e., a cyclooctyne group, and a further 3 membered ring (sharing 2 carbon atoms in common with the cyclooctyne group).
  • the first and second antibody or antigen-binding fragments may be conjugated together via a click chemistry reaction between a first linker arm, L’, attached to the first antibody or antigen-binding fragment and a second linker arm, L”, attached to the second antibody or antigen-binding fragment.
  • the first linker arm, L’ may be attached to the first antibody or antigen-binding fragment via the first sulphur atom, S’.
  • the second linker arm, L may be attached to the second antibody or antigen-binding fragment via the second sulphur atom, S”.
  • the first linker arm, L’ may comprise a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group.
  • the second linker arm, L may comprise a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when the conjugating group of the first linker arm, L’, is an azide, nitrone, tetrazine or tetrazole group, then the conjugating group of the second linker arm, L”, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and when the conjugating group of the first linker arm, L’, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, then the conjugating group of the second linker arm, L”. is an azide, nitrone, tetrazine or tetrazole group.
  • the first linker arm, L’ may comprise a tetrazine group. In some embodiments, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, may comprise a tetrazine group and the second linker arm, L”, may comprise an aliphatic or cyclic alkene or an aliphatic or cyclic alkyne group, such as a cyclic alkene group or a cyclic alkyne group.
  • the first linker arm, L’ may comprise a tetrazine group.
  • the first linker arm, L’ before the first and second antibody or antigen-binding fragment are conjugated together, may comprise a tetrazine group and the second linker arm, L”, may comprise a cyclooctene group, such as trans-cyclooctene (TCO), or a cyclooctyne group, such as bicyclononyne (BCN).
  • TCO trans-cyclooctene
  • BCN bicyclononyne
  • tetrazine as used herein is intended to include 1 , 2,3,4- tetrazine, 1 , 2, 3, 5-tetrazine, and 1 , 2, 4, 5-tetrazine, as well as derivatives thereof, such as 6- methyl tetrazine.
  • tetrazole as used herein is intended to include 1/7-tetrazole, 2/7-tetrazole and 5/7-tetrazole, as well as derivatives thereof, such as 5-methyl-1 /7-tetrazole and 1-methyl-1/7-tetrazole
  • a method of producing a compound comprising conjugating together a first unit and a second unit via click chemistry; wherein the first unit comprises the formula: A 1 -S’-Y’, wherein A 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • Y’ is hydrogen or -L’-X’, wherein L’ is a first linker arm; and X’ is a first conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and wherein the second unit comprises the formula: A 2 -S”-Y”, wherein A 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • Y is hydrogen or -L”-X”, wherein L” is a second linker arm; and X” is a second conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when Y’ is hydrogen, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; when Y’ is -L’-X’ and X’ is an azide, nitrone, tetrazine or tetrazole group, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cycl
  • Y’ may be -L’-X’, wherein X’ is an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and Y” may be -L”-X”, wherein X” is an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when X’ is an azide or tetrazine group, then X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and when X’ is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, then X” is an azide or tetrazine group.
  • Y’ may be hydrogen
  • Y may be -L”-X”
  • X may be an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, such a cyclic alkene group, such as maleimide.
  • the first and second antibody or antigen binding fragments may be conjugated together via a thiol-ene reaction, such as a thiol-maleimide reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be - L”-X”, wherein X” is a cycloalkene or cycloalkyne group.
  • Y’ may be -L’-X’, wherein X’ is a cycloalkene or cycloalkyne group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be - L”-X”, wherein X” is a cycloalkene group.
  • Y’ may be -L’-X’, wherein X’ is a cycloalkene, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a cycloalkyne group.
  • Y’ may be -L’-X’, wherein X’ is a cycloalkyne, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a cyclooctene or cyclooctyne group.
  • Y’ may be -L’-X’, wherein X’ is a cyclooctene or cyclooctyne group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”-X”, wherein X” is a cyclooctene.
  • Y’ may be -L’-X’, wherein X’ is a cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”-X”, wherein X” is a cyclooctyne.
  • Y’ may be -L’- X’, wherein X’ is a cyclooctyne group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • iEDDA inverse electron demand Diels-Alder
  • Y’ may be -L’-X’, wherein X’ is an azide group, and Y” may be -L”-X”, wherein X” is an alkyne or cyclooctyne group.
  • Y’ may be -L’-X’, wherein X’ is an alkyne or cyclooctyne group, and Y” may be -L”-X”, wherein X” is an azide group.
  • the first and second antibody or antigen binding fragments may be conjugated together via a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and/or a strained- promoted azide-alkyne click chemistry (SPAAC) reaction.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strained- promoted azide-alkyne click chemistry
  • the cyclic alkene conjugating groups may comprise any suitable group.
  • the cyclic alkene conjugating groups may comprise cyclooctene, such as cis or trans-cyclooctene.
  • Cyclooctene groups in accordance with the present invention include any group having an 8- memebered cyclic ring that has at least one double bond within the ring structure.
  • the cyclic alkene conjugating group may comprise trans-cyclooctene (TCO).
  • the cyclic alkyne conjugating groups may comprise any suitable group.
  • the cyclic alkyne conjugating groups may comprise a dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN) and/or dibenzocyclooctyne (DICO) group.
  • the cyclic alkyne conjugating group may comprise a bicyclononyne (BCN) group, such as bicyclo[6.1 ,0]non-4-yne.
  • the cyclic alkyne conjugating groups may comprise a cyclooctyne group, more preferably a bicyclononyne (BCN) group, most preferably bicyclo[6.1 ,0]non-4-yne.
  • BCN bicyclononyne
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group and Y” may be -L”- X”, wherein X” is a cyclooctene group.
  • Y’ may be -L’-X’, wherein X’ is a cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group and Y” may be -L”- X”, wherein X” is a trans-cyclooctene group.
  • Y’ may be -L’-X’, wherein X’ is a trans-cyclooctene group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a bicyclononyne (BCN) group.
  • Y’ may be -L’-X’, wherein X’ is a bicyclononyne (BCN) group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • Y’ may be -L’-X’, wherein X’ is a tetrazine group, and Y” may be -L”- X”, wherein X” is a bicyclo[6.1 ,0]non-4-yne group.
  • Y’ may be -L’-X’, wherein X’ is a bicyclo[6.1 ,0]non-4-yne group, and Y” may be -L”-X”, wherein X” is a tetrazine group.
  • the first and second antibody or antigen binding fragments may be conjugated together via an inverse electron demand Diels-Alder (iEDDA) reaction, as defined herein.
  • the -L’-X’ group may be attached to the first sulphur atom, S’, via any suitable method.
  • -L’-X’ may be attached to S’ via the reaction of a thiol group of one or more of the non-naturally occurring cysteine residues as described herein and a maleimide, bromomaleimide, bromopyridazinedione, chloroacetamide, bromoacetamide, iodoacetamide, a-haloketone unsaturated carbonyl, vinylsulfone, vinyl-pyridine, vinyl-pyridinium and/or unsaturated phosphonamidite group.
  • the -L’-X’ group may be attached to the first sulphur atom, S’, via a succinimide group.
  • the succinimide group may suitably be derived from the reaction of the first sulphur atom with a maleimide group.
  • the -L”-X” group may be attached to S” via any suitable method.
  • -L”-X may be attached to S” via the reaction of a thiol group of one or more of the non-naturally occurring cysteine residues as described herein and a maleimide, bromomaleimide, bromopyridazinedione, chloroacetamide, bromoacetamide, iodoacetamide, a- haloketone, unsaturated carbonyl, vinylsulfone, vinyl-pyridine, vinyl-pyridinium and/or unsaturated phosphonamidite group.
  • the -L”-X” group may be attached to the second sulphur atom, S”, via a succinimide group.
  • the succinimide group may suitably be derived from the reaction of the second sulphur atom with a maleimide group.
  • L’ may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. polyethylene glycol (PEG) groups.
  • PEG polyethylene glycol
  • L’ may comprise a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3.
  • the -L”-X group may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. polyethylene glycol (PEG) groups.
  • the second linker arm, L may comprise a moiety of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3.
  • the compound may optionally comprise one or more further moieties.
  • the compound may comprise a half-life extension moiety.
  • the inclusion of a half-life extension moiety suitably increases the half-life of the compound when compared to the same compound that does not include a half-life extension moiety (wherein the term “half-life” is as already defined herein).
  • the half-life extension moiety may comprise an antibody and/or antigen-binding fragment, a protein, and or a polypeptide.
  • the half-life extension moiety may comprise a serum albumin protein or an antibody or antigen-binding fragment that binds human serum albumin.
  • the half-life extension moiety may comprise an antibody or antigen-binding fragment that binds human serum albumin, more preferably an antigen-binding fragment that binds human serum albumin, more preferably a single domain antibody that binds human serum albumin, most preferably a VH domain that that binds human serum albumin.
  • the compound may comprise one or more further antibodies and/or antigen-binding fragments.
  • the compound may comprise a total of 3, 4, 5, etc., antibodies and/or antigen-binding fragments.
  • the compound may comprise a total of 3 antibodies and/or antigen-binding fragments.
  • the further antibodies and/or antigenbinding fragments may be any suitable antibodies and/or antigen-binding fragments.
  • the antibodies and/or antigen-binding fragments may bind to one or more of the antigens as already defined herein (i.e., CD33, CD7, B7H4, and CD56), may bind to human serum albumin or may bind to any other suitable antigen.
  • Suitable antigens include antigens such as CD33, CD7, B7H4, and CD56.
  • the further antibodies and/or antigen-binding fragments may be attached to the compound in any suitable manner for example recombinantly attached or chemically linked. Where the further antibodies and/or antigen-binding fragments are chemically linked this may be by using the click-chemistry methods as set out herein. Preferably, where the further antibodies and/or antigen-binding fragments are chemically linked, this may be via an inverse electron demand Diels-Alder (iEDDA) reaction as set out herein.
  • iEDDA inverse electron demand Diels-Alder
  • a further antibody or antigen-binding fragment may also be a half-life extension moiety and vice versa.
  • the or each further antibody or antigen-binding fragment may bind to CD33, CD7, B7H4, CD56 and/or human serum albumin, preferably human serum albumin.
  • the further moiety may be a detectable or functional label.
  • a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.
  • the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
  • the one or more further moieties may be attached to the compound at any suitable position and by any suitable means.
  • the compound comprises a further antibody and/or antigen-binding fragment, protein and/or polypeptide
  • said antibody and/or antigen-binding fragment, protein and/or polypeptide may be recombinantly attached to the first and/or second antibody and/or antigen-binding fragment, A 1 and/or A 2 .
  • nucleic acid molecule has been designed which encodes the first and/or second antibody and/or antigen-binding fragment, A 1 and/or A 2 in combination with the further moiety such that when then nucleic acid is transfected into a host organism the protein is expressed in a linked manner.
  • nucleic acids encoding the first or second antibody and/or antigen-binding fragment as described herein, A 1 and/or A 2 , and the further antibody and/or antigen-binding fragment, protein and/or polypeptide, suitably separated by a polypeptide spacer/linker sequence may be inserted into a plasmid and expressed in a suitable expression system.
  • nucleic acids encoding A 1/2 -(peptide linker) n -M may be expressed in a suitable expression system, wherein A 1/2 represents either of A 1 or A 2 , depending on where it is desired that the further moiety is attached, n is 0 or 1 , preferably 1 , and M is a further moiety selected from an antibody and/or antigen-binding fragment, protein or polypeptide.
  • suitable expression systems include bacterial host cells, such as E. coli, CHO or other host cells expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding A 1/2 -(peptide linker) n -M that is operably linked to a T7 promoter.
  • a bacterial host cell such as an E. coli
  • the compound when the compound comprises one or more further antibodies and/or antigen-binding fragments, said further antibodies and/or antigen-binding fragments may be conjugated to the first and/or second antibody and/or antigen-binding fragment, A 1 and/or A 2 , via click chemistry, such as via an inverse electron demand Diels-Alder (iEDDA) reaction.
  • the compound may suitably comprise the formula:
  • a 3 and A 4 each independently represent a further antibody or antigen-binding fragment as defined herein
  • S’ 1 represents a sulphur atom, other than S’, that is derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • S” 2 represents a sulphur atom, other than S”, that is derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • L 3 and L 4 are each linkers; m is 0 to 4; and n is 0 to 4; wherein at least one of m and n is at least 1 ; and wherein each further antibody or antigen-binding fragment is conjugated to A 1 or A
  • each further antibody or antigen-binding fragment when each further antibody or antigen-binding fragment is conjugated to A 1 or A 2 via an inverse electron demand Diels-Alder (iEDDA) reaction, each further antibody or antigen-binding fragment may have a tetrazine functional group and A 1 or A 2 (as appropriate) may have a cyclic alkene group, such as a cyclooctene group, such as trans-cyclooctene (TCO), or a cyclic alkyne group, such as a cyclooctyne group, such as bicyclononyne (BCN).
  • a cyclic alkene group such as a cyclooctene group, such as trans-cyclooctene (TCO)
  • TCO trans-cyclooctene
  • BCN bicyclononyne
  • m is 1 and n is 0 or m is 0 and m is 1 .
  • L 3 and L 4 are each linkers. Suitable linkers are as defined herein in relation to L. the linkers, L 3 and L 4 , are suitably formed by the conjugation of two linking arms using click chemistry, such as an inverse electron demand Diels-Alder (iEDDA) reaction. Suitable linking arms are as defined herein in relation to L’ and L”.
  • click chemistry such as an inverse electron demand Diels-Alder (iEDDA) reaction.
  • Suitable linking arms are as defined herein in relation to L’ and L”.
  • L 3 is attached to A 1 via a non-naturally occurring cysteine residue, S’ 1 , present in A 1 .
  • L 4 is attached to A 2 via a non-naturally occurring cysteine residue, S” 2 , present in A 2 .
  • Said non-natural occurring cysteine residues may be at position 153, 156, 168, 173 and/or 207 of the first or second antibody or antigen-binding fragment, as appropriate, according to Kabat numbering. It will be appreciated by a person skilled in the art that S’ should be at a different position to S’ 1 . It will be appreciated by a person skilled in the art that S” should be at a different position to S” 2 .
  • L 3 may be attached to A 3 via any suitable residue.
  • L 3 may be attached to A 3 via a naturally occurring amino acid residue, such as a naturally occurring lysine or cysteine residue, such as a naturally occurring cysteine residue.
  • L 4 may be attached to A 4 via any suitable residue.
  • L 4 may be attached to A 4 via a naturally occurring amino acid residue, such as a naturally occurring lysine or cysteine residue, such as a naturally occurring cysteine residue.
  • L 3 may be attached to A 3 via a non-naturally occurring cysteine residue.
  • a 3 may comprise a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that it comprises one or more non-naturally occurring cysteine residue(s).
  • L 4 may be attached to A 4 via a non-naturally occurring cysteine residue.
  • a 4 may comprise a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that it comprises one or more non-naturally occurring cysteine residue(s).
  • the compound may be of the following formula:
  • a 3 represents a third antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the third antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • a 4 represents a fourth antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the fourth antibody or antigen-bind
  • the compound may further comprise a payload.
  • the payload may be a cell killing agent.
  • the payload may be a macrophage class switching agent.
  • the payload may be an immune-modulating payload.
  • the payload may be a light activatable payload.
  • the payload may be a molecular label.
  • molecular label and like terms as used herein, is meant a group that is operable to aid the detection of the compound. Detection of the compound may be ex vivo and/or in vivo.
  • suitable molecular labels include, but are not limited to, fluorescent molecules, p-galactosidase, luciferase molecules, chemical dyes, fluorophores and/or radioisotopes.
  • the additional moiety may be a detectable or functional label.
  • a label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers.
  • the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.
  • the molecular label may be a fluorophore.
  • Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • API allophycocyanin
  • Indocicarbocyanine Cy5
  • Indocarbocyanine Cy3
  • DyLight such as 405, 488, 550, 650, 680, 755, 800.
  • the molecular label may also be a biotin tag, derived from biotin.
  • the labelling moiety may also be a radioisotope or a radioisotope containing moiety.
  • the labelling moiety is a positron emission tomography (PET) tracer.
  • Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 C] choline, copper Cuireate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).
  • an immune-modulating payload includes any moiety that modulates the immune system, for example which stimulates the immune system and/or kills the target cell.
  • a moiety that has immuno-activating and/or antineoplastic activities can be used.
  • Such moieties may be synthetic peptides that recognise the specific target and trigger (agonist) or block (antagonist) inflammatory responses.
  • the target may be a pattern recognition receptor (PRR), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs).
  • PRR pattern recognition receptor
  • TLRs Toll-like receptors
  • NLRs NOD-like receptors
  • RLRs RIG-l-like receptors
  • CLRs C-type lectin receptors
  • CDSs cytosolic dsDNA sensors
  • Examples of payloads include agonists for the stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173).
  • STING stimulator of interferon genes protein
  • Such payloads include cyclic dinucleotides and compounds listed in see WO2021113679). Activation of the STING pathway triggers an immune response that results in generation of specific killer T-cells that shrink tumours and can provide long-lasting immunity so the tumours do not recur.
  • payloads that act on toll-like receptors (TLRs) may be used.
  • TLRs toll-like receptors
  • agonists that bind to TLR7 and/or TLR8 can be used.
  • Another example is a macrophage class switching agent.
  • a light activatable payload (IRDye® 700DX, IR700) may also be used. Light activation of the non-toxic payload results in the generation of singlet oxygen species that damage the cell membrane integrity, resulting in necrotic and immunogenic cell death of tumour cells, resulting in minimal damage to surrounding normal tissue.
  • the cell killing agent may be a cytotoxin and the skilled person will understand that a range of cytotoxins will be compatible.
  • suitable cytotoxins include, but are not limited to: i) a peptide toxin, or ii) a chemical toxin, or iii) an inhibitor of Bcl-2 or Bcl-axl, iv) an RNA Polymerase inhibitor such as a-amanitin, v) a spliceosome inhibitor, vi) a microtubuletargeting payload, or vii) a DNA-damaging payload.
  • the cytotoxin is a biologically active cytotoxic material.
  • the cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camtothecin analogues, topoisomerase inhibitors and doxorubicin.
  • the payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide.
  • the payload is preferably a cytotoxin.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • radioactive isotopes e.g., ⁇ 211 >At, ⁇ 131 >l, ⁇ 125>1 , ⁇ 90>Y, ⁇ 186>Re, ⁇ 188>Re, ⁇ 153>Sm, ⁇ 212>Bi, ⁇ 32>P, ⁇ 60>C, and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., ⁇ 211 >At, ⁇ 131 >l, ⁇ 125>1 , ⁇ 90>Y, ⁇ 186>Re, ⁇ 188>Re, ⁇ 153>Sm, ⁇ 212>Bi, ⁇ 32>P, ⁇ 60>C
  • chemotherapeutic agent and "anticancer agent” are terms that denote a chemical compound useful in the treatment of cancer, and which may be administered in combination therapy with the antibody drug conjugate compounds of the invention.
  • chemotherapeutic agents include Erlotinib (TARCEV A(R), Genentech/OSI Pharm.), Bortezomib (VELCADE(R), Millenium Pharm.), Fulvestrant (FASLODEX(R), Astrazeneca), Sutent (SUI 1248, Pfizer), Letrozole (FEMARA(R), Novartis), Imatinib mesylate (GLEEVEC(R), Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin(R), Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE(R), Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43- 9006, Bayer Labs.), and Gefitinib (IRESSA(R), Astrazene
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl, 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1 , epirubicin, GPX- 100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dact
  • cytotoxic agent anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX(R) tamoxifen
  • raloxifene droloxifene
  • 4-hydroxytamoxifen trioxifene
  • keoxifene LYI 17018, onapristone
  • FARESTON(R) toremifene aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE(R) megestrol acetate, AROMASIN(R) exemestane, formestanie, fadrozole, RIVISOR(R) vorozole, FEMARA(R) letrozole, and ARHVI
  • the cytotoxin is a biologically active cytotoxic material.
  • the cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues and doxorubicin.
  • the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.
  • the compound may further comprise (or is incorporated or associated with) a cytotoxic or cytostatic agent, i.e., a compound that kills or inhibits cells such as tumour cells.
  • a cytotoxic or cytostatic agent i.e., a compound that kills or inhibits cells such as tumour cells.
  • Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition.
  • the cytotoxic or cytostatic agent may be, for example, a peptide toxin, a small molecule toxin or a radioisotope. This is also referred to herein as drug or cytotoxic payload.
  • an “ADC” is an antibody drug conjugate.
  • the cytotoxic or cytostatic agent may be a tubulin inhibitor; or a DNA interacting agent.
  • Tubulin inhibitors modulate tubulin polymerisation.
  • DNA interacting agents target cellular DNA.
  • the cytotoxic or cytostatic agent is a tubulin inhibitor.
  • the tubulin inhibitor is selected from the group consisting of: (a) an auristatin; and (b) a maytansine derivative.
  • the cytotoxic or cytostatic agent is an auristatin.
  • Auristatins include synthetic derivatives of the naturally occurring compound Dolastatin-10.
  • Auristatins are a family of antineoplastic I cytostatic pseudopeptides. Dolastatins are structurally unique due to the incorporation of 4 unusual amino acids (Dolavaine, Dolaisoleuine, Dolaproine and Dolaphenine) identified in the natural biosynthetic product.
  • the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF.
  • the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine.
  • the cytotoxic or cytostatic agent is a maytansine.
  • Maytansines include structurally complex antimitotic polypeptides. Maytansines are potent inhibitors of microtubulin assembly which leads towards apoptosis of tumour cells.
  • the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4.
  • the drug is MMAE, MMAF or auristatin MMAF.
  • the cytotoxic or cytostatic agent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN- 38.
  • the cytotoxic or cytostatic agent is DNA interacting agent.
  • the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs).
  • the cytotoxic or cytostatic agent is a calicheamicin.
  • Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death.
  • Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,11 , 2103-2117).
  • Calicheamicin was found in the soil microorganism Micromonosporaechinospora.
  • the calicheamicin is calicheamicin gamma 1 .
  • the drug is a duocarmycin.
  • Duocarmycins are potent anti-tumour antibiotics that exert their biological effects through binding sequence-selectively in the minor groove of DNA duplex and alkylating the N3 of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, 837-844).
  • the duocarmycin is selected from the group consisting of: Duocarmycin A; Duocarmycin B1 ; Duocarmycin B2; Duocarmycin C1 ; Duocarmycin C2; Duocarmycin D; Duocarmycin SA; Cyclopropylbenzoindole (CBI) duocarmycin; Centanamycin; Rachelmycin (CC-1065); Adozelesin; Bizelesin; and Carzelesin.
  • the cytotoxic or cytostatic agent is a pyrrolobenzodiazepine.
  • Pyrrolobenzodiazepines (PBDs) are a class of naturally occurring antitumour antibiotics. Pyrrolobenzodiazepines are found in Streptomyces.
  • PBDs exert their antitumour activity by covalently binding to the DNA in the minor groove specifically at purine- guanine-purine units. They insert on to the N2 of guanine via an aminal linkage and, due to their shape, they cause minimal disruption to the DNA helix. It is believed that the formation of the DNA-PBD adduct inhibits nucleic acid synthesis and causes excision-dependent single and double stranded breaks in the DNA helix. As synthetic derivatives the joining of two PBD units together via a flexible polymethylene tether allows the PBD dimers to cross-link opposing DNA strands producing highly lethal lesions.
  • the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether.
  • the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof); Mazethramycin (and dimers thereof); Tomaymycin (and dimers thereof); Prothracarcin (and dimers thereof); Chicamycin (and dimers thereof); Neothramycin A (and dimers thereof); Neothramycin B (and dimers thereof); DC-81 (and dimers thereof); Sibiromycin (and dimers thereof); Porothramycin A (and dimers thereof); Porothramycin B (and dimers thereof); Sibanomycin (and dimers thereof); Abbeymycin (and dimers thereof); SG3199; SG2000; and SG2285.
  • the cytotoxic or cytostatic agent is a drug that targets DNA interstrand crosslinks through alkylation.
  • a drug that targets DNA interstrand crosslinks through alkylation is selected from: a DNA targeted mustard; a guanine-specific alkylating agent; and an adeninespecific alkylating agent.
  • the cytotoxic or cytostatic agent is a DNA targeted mustard.
  • the DNA targeted mustard may be selected from the group consisting of: an oligopyrrole; an oligoimidazole; a Bis-(benzimidazole) carrier; a Polybenzamide Carrier; and a 9-Anilinoacridine-4-carboxamide carrier.
  • the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.
  • the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier.
  • the drug is Hoechst 33258.
  • a guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions.
  • the cytotoxic or cytostatic agent is a guanine-specific alkylating agent selected from the group consisting of: a G-N2 alkylators; a A- N3 alkylator; a mitomycin; a carmethizole analogue; a ecteinascidin analogue.
  • the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW- 2149.
  • the a carmethizole analogue is selected from: Bis- (Hydroxymethyl)pyrrolizidine; and NSC 602668.
  • the ecteinascidin analogue is Ecteinascidin 743.
  • Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.
  • Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators.
  • the cytotoxic or cytostatic agent is a cyclopropaindolone analogue.
  • the drug is selected from: adozelesin; and carzelesin.
  • the cytotoxic or cytostatic agent is a benz[e]indolone.
  • the cytotoxic or cytostatic agent is selected from: CBI-TMI; and iso-CBI.
  • the cytotoxic or cytostatic agent is bizelesin.
  • the cytotoxic or cytostatic agent is a Marine Antitumour Drug. Marine Antitumour Drugs has been a developing field in the antitumour drug development arena (I. Bhatnagaret al, Mar. Drugs 2010, 8, P2702-2720 and T. L. Simmons et al, Mol. Cancer Ther. 2005, 4(2), P333-342). Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents.
  • the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate.
  • the EribulinMesylate is selected from: (E7389); Soblidotin (TZT 1027); Squalamine lactate; CemadotinPlinabulin (NPI-2358); Plitidepsin; Elisidepsin; Zalypsis; Tasidotin, Synthadotin; (ILX-651); Discodermolide; HT1286; LAF389; Kahalalide F; KRN7000; Bryostatin 1 ; Hemiasterlin (E7974); Marizomib; Salinosporamide A; NPI-0052); LY355703; CRYPTO 52; Depsipeptide (NSC630176); Ecteinascidin 743; Synthadotin; Kahalalide
  • cytotoxic or cytostatic agent are also encompassed by the present invention: Amatoxins (a-amanitin)-bicyclic octapeptides produced by basidiomycetes of the genus Amanita, e.g., the Green Deathcap mushroom; Tubulysins; Pseudomonas exotoxin; Cytolysins; dolabellanins; Epothilone A, B, C, D, E, F. Epothilones - constitute a class of non- taxane tubulin polymerisation agents and are obtained by natural fermentation of the myxobacteriumSorangiumcellulosum.
  • Pseudomonas exotoxin is an exotoxin produced by Pseudomonas aeruginosa which catalyzes the ADP-ribosylation and inactivation of EF2, which leads to protein synthesis inhibition and cell death
  • the drug is amatoxin.
  • the drug is tubulysin.
  • the drug is Pseudomonas exotoxin.
  • the drug is cytolysin.
  • the drug is dolabellanin.
  • the drug is epothilone.
  • the drug is selected from: Doxorubicin; Epirubicin; Esorubicin; Detorubicin; Morpholino-doxorubicin; Methotrexate; Methopterin; Bleomycin; Dichloromethotrexate; 5-Fluorouracil; Cytosine-p-D-arabinofuranoside; Taxol; Anguidine; Melphalan; Vinblastine; Phomopsin A; Ribosome-inactivating proteins (RIPs); Daunorubicin; Vinca alkaloids; Idarubicin; Melphalan; Cis-platin; Ricin; Saporin; Anthracyclines; Indolino- benzodiazepines; 6-Mercaptopurine; Actinomycin; Leurosine; Leurosideine; Carminomycin; Aminopterin; Tallysomycin; Podophyllotoxin;
  • the cell killing portion is a peptide toxin, for example an auristatin such as MMAE or MMAF.
  • the bispecific binding molecule comprises a binding portion and a cell killing portion, wherein the binding portion is a first and second antibody or fragment thereof as described herein and wherein the cell killing portion is a peptide toxin, for example an auristatin such as Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF), vcMMAE, vcMMAF, mcMMAE and mcMMAF.
  • the payload may comprise a small molecule inhibitor with an anticancer activity.
  • the small molecule inhibitor may be a Bcl-XI inhibitor, Bcl2 inhibitor, Bcl-w inhibitor, Bcr-Abl inhibitor, EGFR inhibitor VEGFR2 inhibitor, RET inhibitor, PDGFR inhibitor, FLT-3 inhibitor, KIT inhibitor, CSF-1 inhibitor, HER2 inhibitor, LCK inhibitor, B-raf inhibitor, mTOR inhibitor, c-KIT inhibitor, FGFR inhibitor, VEGFR inhibitor, HGFR inhibitor, Jak1 inhibitor, Jak2 inhibitor, VEGFR1-3 inhibitor, Src inhibitor, c.MET inhibitor, PDGFR-p inhibitor, MEK inhibitor, HSP90 inhibitor, MMP inhibitor, proteosome inhibitor, Akt inhibitor, NAMPT inhibitor.
  • the payload comprises a light-activatable payload, for example a infrared light activatable payload.
  • a light-activatable payload for example a infrared light activatable payload.
  • Immunoconjugates comprising a near-infrared activatable payload enable binding molecule-mediated targeted delivery to achieve a high degree of tumor specificity, while using infrared light to activate the biophysical mechanism of the drug to accurately induce rapid death of cancer cells without harming the surrounding normal tissues.
  • Suitable light-activatable payloads include IRDye700DX.
  • a linking group is used to conjugate the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload, to the first and/or second antibody or antigen-binding fragment, as appropriate.
  • the linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment.
  • the cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease.
  • Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999).
  • Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A-expressing cells.
  • a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).
  • the cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH- sensitive linker is hydrolysable under acidic conditions.
  • an acid- labile linker that is hydrolysable in the lysosome e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like can be used.
  • linkers are cleavable under reducing conditions (e.g., a disulfide linker).
  • the cleavable linker can also be a malonate linker (Johnson et al, Anticancer Res. 15 :1387-93, 1995), a maleimidobenzoyl linker (Lau et al, Bioorg-Med-Chem. 3: 1299-1304, 1995), or a 3' -N- amide analogue (Lau et al, Bioorg-Med-Chem. 3: 1305-12, 1995).
  • the linker can be a protease cleavable linker, for example a valinecitrulline, which may be cleaved by cathepsin B in the lysosome.
  • the linker also can be a non-cleavable linker, such as a maleimidoca-proyl (me) linker or maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the binding portion.
  • a non-cleavable linker such as a maleimidoca-proyl (me) linker or maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the binding portion.
  • the payload may be conjugated to the first and/or second antibody or antigen-binding fragment by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group or an electrophilic group of an antibody or antigen-binding fragment with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody or antigen-binding fragment.
  • Conjugation methods (1) and (2) may be employed with a variety of antibody or antigen-binding fragments, drug moieties, and linkers.
  • the payload may be conjugated to the first and/or second antibody or antigen-binding fragment via click chemistry as set out herein.
  • the payload may be conjugated to the first and/or second antibody or antigen-binding fragment via an inverse electron demand Diels-Alder (iEDDA) reaction.
  • iEDDA inverse electron demand Diels-Alder
  • the linkers may be attached via nucleophilic groups on the first and/or second antibody or antigen-binding fragment, as appropriate.
  • groups include, but are not limited to: (i) N- terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the first and/or second antibody or antigen-binding fragment, as appropriate, is glycosylated.
  • Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent.
  • Additional nucleophilic groups can be introduced into the first and/or second antibody or antigen-binding fragment, as appropriate, through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Antibody-drug conjugates may also be produced by modification of the first and/or second antibody or antigen-binding fragment, as appropriate, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug.
  • the sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties.
  • the resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages.
  • reaction of the carbohydrate portion of a glycosylated first and/or second antibody or antigen-binding fragment, as appropriate, with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug.
  • proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid.
  • Such aldehyde can be reacted with a drug moiety or linker nucleophile.
  • nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • Methods for conjugating the payload to the antibody or antigen binding fragment may utilise the presence of one or more no-naturally occurring amino acids.
  • the payload and/or linker may be attached via a non-naturally occurring amino acid residue present in the antibody or antigen binding fragment.
  • a “non-naturally occurring residue” refers to a amino acid residue that has been introduced at a position in the polypeptide wherein a different amino acid residue was present in the wild-type sequence
  • Methods to introduce non-naturally occurring amino acid residues are known in the art.
  • the non-naturally occurring amino acid residue to which the payload or linker is attached is may be selected from but not limited to a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.
  • site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding molecule.
  • a non-canonical amino acid is used to attach the payload or linker.
  • a “non-canonical” amino acid refers to one of the non-proteinogenic (unnatural) amino acids i.e., an amino acid which is not introduced via the cell’s natural translation machinery.
  • non-canonical amino acids there are many examples of non-canonical amino acids in the art many of which provide a bio-orthogonal handle on which to attach a payload.
  • non-canonical amino acid is used to attach the payload linker
  • suitable techniques are known in the art to introduce such non-canonical amino acid residues such as chemical modification, tRNA suppressor technology, engineered tRNA, /tRNA synthetase pairs.
  • the payload may be attached at various positions within the bispecific molecule.
  • One or more copies of the payload may be attached to the molecule, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the payload may be attached.
  • the compounds of the invention may demonstrate low payload deconjugation e.g. ⁇ 10%.
  • the immunoconjugate or ADC may comprise a payload deconjugation of ⁇ 15%, ⁇ 14%, ⁇ 13%, ⁇ 12%, ⁇ 11%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, when assessed over 5 days.
  • the compound may comprise, consist essentially of, or consist of the formula: A 1 -S’-L-S”- A 2 , wherein each of A 1 , A 2 , S’, S” and L are as defined herein.
  • a third aspect of the invention there is provided the use of click chemistry to conjugate together a first unit and a second unit to produce a compound according to the invention. Suitable features of the third aspect of the present invention as defined herein in relation to the first and/or second aspects of the present invention.
  • composition also relates to a composition comprising a compound of the invention.
  • the composition may comprise a compound of the invention and optionally a pharmaceutically acceptable carrier, excipient and/or adjuvant.
  • the composition may comprise one or more compounds of the invention.
  • the composition may comprise only one compound of the invention.
  • the composition may comprise two or more compounds of the invention.
  • compositions may comprise the compound of the invention.
  • the compositions may comprise the immunoconjugate or the ADC comprising said compound.
  • the compositions can be administered by any convenient route including, but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.
  • delivery is of the nucleic acid encoding the drug, e.g., a nucleic acid encoding the compound of the invention is delivered.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration.
  • the compositions are administered parenterally.
  • the pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form.
  • carrier refers to a diluent, adjuvant or excipient, with which compound of the present invention is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and colouring agents can be used.
  • the compounds, compositions and/or pharmaceutically acceptable carriers may suitably be sterile.
  • Water is a preferred carrier when the compound of the present invention is to be administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be in the form of a liquid, e.g., a solution, emulsion or suspension.
  • the liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.
  • the composition When intended for oral administration, the composition may be in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition typically contains one or more inert diluents.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
  • the composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension.
  • the liquid can be useful for oral administration or for delivery by injection.
  • a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
  • compositions can take the form of one or more dosage units.
  • the amount of the compound or composition that is effective/active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.
  • target cell refers to a cell or cell-type characterized by the expression or overexpression of a target molecule, wherein one or each of the first and second antibody or antigen binding fragments suitably bind to said target molecule. Any type of cell expressing a suitable antigen may be envisaged as a target cell for treatment with the compounds of the invention.
  • the cell may be a tumour cell, for example a tumour cell from a haematological malignancy, such as an AML cell.
  • An aspect of the invention relates to the compound, immunoconjugate or antibody drug conjugate described herein for use in the treatment of a disease, such as a malignancy.
  • An aspect of the invention relates to a method of treating or preventing a disease, such as a malignancy, comprising administering a therapeutically effective amount of the compound, immunoconjugate or antibody drug conjugate described herein.
  • An aspect of the invention relates to the use of the compound, immunoconjugate or antibody drug conjugate described herein for the manufacture of a medicament for the treatment of a disease, such as a malignancy.
  • malignancy refers to any disease sate comprising the uncontrolled growth and division of abnormal cells, for example cancer.
  • the binding molecules of the invention are for use in the treatment of disease state comprising malignant cellular proliferation including but not limited to neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g.
  • lung cancer small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
  • the binding molecules are for use in haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, Acute Myeloid Leukaemia (AML) and other cancers of B or T cell origin.
  • malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular
  • Hodgkin lymphoma such as AML
  • Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
  • the malignancy may be a haematological malignancy.
  • Haematologic malignancies are forms of cancer that begin in the cells of blood-forming tissue, such as the bone marrow, or in the cells of the immune system.
  • haematologic malignancies are acute and chronic leukaemias, lymphomas, multiple myeloma, AML, non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, and myelodysplastic syndromes.
  • the malignancy is an Acute Myeloid Leukaemia (AML) or AML derived cancer.
  • the disease may be a CD7+CD33+ haematological malignancy.
  • CD7+CD33+ haematological malignancy refers to a haematological malignancy characterized by the expression of both CD7 and CD33 on the surface of the malignant cells (e.g., a haematological malignancy that over expresses CD33 and/ or CD7 on their cell surface and/or that express CD33 and/or CD7 at levels considered acceptable for therapy with the antibody or antigen binding fragments thereof that specifically binds to CD7 and CD33).
  • one of the first and second antibody or antigen binding fragments may suitably bind to CD7 and the other of the first and second antibody or antigen binding fragments may suitably bind to CD33.
  • CD7+CD33+ haematological malignancies include, but are not limited to, acute myeloid leukemia (AML), a myelodysplastic syndrome, a T-cell acute lymphoblastic leukemia, and a blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • killing of a target cell relates for example to an inhibition of protein synthesis, for example such that cell viability is reduced, or an induction of apoptosis resulting in elimination or death of target cells.
  • Assays to determine cell killing and apoptosis are well known in the art. Cytotoxicity assays assess the number of live and dead cells in a population after treatment with a pharmacological substance (e.g. an LDH cytotoxicity assay, or a live-dead cell assay). Apoptosis assays assess how cells are dying by measuring markers that are activated upon cell death (e.g.
  • PS Phosphatidylserine
  • inhibit the cell growth refers to any measurable decrease in the growth or proliferation of a target cell when contacted with the antibody or antigen binding fragments thereof according to the present invention as compared to the growth of the same cell not in contact with the antibody or antigen binding fragments thereof according to the present disclosure, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • Assays to determine cell viability or proliferation are well known in the art. Cell viability assays assess how healthy the cells are by measuring markers of cellular activity (e.g.
  • Cell proliferation assays assess the growth rate of a cell population or to detect daughter cells in a growing population (e.g. a cell cycle assay, a cell proliferation assay, a cell viability assay, or a senescence assay).
  • the compound, immunoconjugate, or antibody drug conjugate as described herein may be used in combination with any other anti-cancer therapy.
  • a compound, immunoconjugate, antibody drug conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • treatments and therapies include, but are not limited to, chemotherapy, immunotherapy, surgery and/or radiation therapy.
  • therapy or therapeutic agent includes for example an anticancer compound, such as a chemotherapy agent, biologic, cytokine, small molecule, CAR-T therapy or radiotherapy treatment.
  • Chemotherapy agents include alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors and corticosteroids.
  • the chemotherapy can include vinorelbine, cisplatin, carboplatin, gemcitabine, paclitaxel, topotecan, docetaxel, irinotecan, pemetrexed, etoposide, or any combination thereof.
  • a biologic may be an antibody therapy, for example an antibody that targets a checkpoint inhibitor, such as PD-1 (e.g. Pembrolizumab, Nivolumab or Cemiplimab), PD-L1 (e.g.
  • Atezolizumab Avelumab or Durvalumab
  • PD-L2 e.g. Relatlimab
  • LAG-3 e.g. Relatlimab
  • Tim-3 e.g. Ipilimumab
  • CTLA4 e.g. Ipilimumab
  • the small molecule therapy may be Pexidartinib.
  • the compound, immunoconjugate, antibody drug conjugate of the invention may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties.
  • the second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to the compound, immunoconjugate, antibody drug conjugate such that they do not adversely affect each other.
  • the skilled person will be able to determine appropriate dosage regimens of the compound or conjugates described herein.
  • the amount of the compound, conjugate or composition described herein that is effective/active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques.
  • in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.
  • the compound, conjugate or composition may be provided at a dose of 0.1 mg/kg to 100mg/kg, 0.5 mg/kg to 100mg/kg, 1 mg/kg to 100mg/kg, 2 mg/kg to 100mg/kg, 5 mg/kg to 100mg/kg, 10 mg/kg to lOOmg/kg, 20 mg/kg to 100mg/kg, 0.1 mg/kg to 80mg/kg, 0.5 mg/kg to 80mg/kg, 1 mg/kg to 80mg/kg, 2 mg/kg to 80mg/kg, 5 mg/kg to 80mg/kg, 10 mg/kg to 80mg/kg, 20 mg/kg to 80mg/kg, 0.1 mg/kg to 60mg/kg, 0.5 mg/kg to 60mg/kg, 1 mg/kg to 60mg/kg, 2 mg/kg to 60mg/kg, 5 mg/kg to 60mg/kg, 10 mg/kg to 60mg/kg, 20 mg/kg to 80mg/kg,
  • the compound, conjugate or composition may be administered with a specific treatment schedule.
  • the treatment cycle may be 1 day to 42 days 1 day to 35 days, 1 day to 28 days, 1 day to 21 days, 1 day to 14 days, 1 day to 7 days.
  • the treatment cycle may comprise 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses.
  • the treatment cycle may comprise 1 dose every 7 days, 2 doses every 7 days, 3 doses every 7 days.
  • the treatment cycle may comprise 1 dose every 14 days, 2 doses every 14 days, 3 doses every 14 days, 4 doses every 14 days, 5 doses every 14 days, 6 doses every 14 days.
  • treating include both preventative and curative treatment of a condition, disease or disorder. These terms also include slowing, interrupting, controlling or stopping the progression of a condition, disease or disorder and preventing, curing, slowing, interrupting, controlling or stopping the symptoms of a condition, disease or disorder.
  • “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disease.
  • treatment can include a postponement of development of the symptoms associated with a disease or disease, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease.
  • the terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., canine patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
  • the compound, immunoconjugate or antibody drug conjugate is used in therapy it will be administered to the subject at a therapeutically effective dose.
  • therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • the therapeutically effective amount of the compound or composition may be administered orally, topically, by inhalation, insufflation or parenterally.
  • the compound may be formulated, for example in a composition as described herein.
  • the compound be formulated for a particular administration route.
  • formulations suitable for oral administration include tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs.
  • Suitable formulations for topical use include, for example, creams, ointments, gels, or aqueous or oily solutions or suspensions.
  • Suitable formulations for inhalation include, for example, as a fine powder or a liquid aerosol.
  • Suitable formulations for administration by insufflation include, for example, a fine powder.
  • Suitable formulations for parenteral administration include, for example, a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing.
  • the therapeutically effective amount of the compound or composition as described herein will necessarily vary depending on the subject to be treated, the route of administration and the nature and severity of the disease to be treated.
  • the term “effective amount” means an amount of compound, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.
  • the term “effective amount” of a composition, as used herein, is intended to denote a non-lethal but sufficient amount of the composition to provide the desired effect.
  • the effective amount is the one which eliminates or diminishes the symptoms associated with the disorder.
  • An effective amount may be determined by one of ordinary skill in the art, using routine experimentation.
  • in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.
  • the amount is at least about 0.01% of the compound of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition.
  • Preferred oral compositions can comprise from about 4% to about 50% of the compound of the present invention by weight of the composition.
  • compositions of the present invention can be prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the antibody of the present invention.
  • the composition can comprise from typically about 0.1 mg/kg to about 250 mg/kg of the subject’s body weight, for example, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, for example about 1 mg/kg to about 10 mg/kg of the animal's body weight.
  • the composition is administered at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg.
  • the dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.
  • Non-Therapeutic Methods [268] The compounds may also be used in various methods, for example in non-therapeutic methods.
  • the compounds may be used in a variety of diagnostic tests that comprise the detection of a target cell or molecule, for example in immunoassays for the detection of cancer.
  • the immunoassays per se are well-known and any of the well-known immunoassays may be employed. That is, classifying the known immunoassays according to the reaction type, known immunoassays include sandwich immunoassays, competition immunoassays, agglutination immunoassays, Western blot and the like. Classifying the known immunoassays according to the label employed, known immunoassays include fluorescence immunoassays, enzyme immunoassays, radio immunoassays, biotin immunoassays and the like.
  • any of these immunoassays may be employed. Further, diagnosis may be attained by immunohistostaining. In cases where a labelled compound is used in the immunoassay, the methods per se for labelling are well-known, and any of the well-known methods may be employed.
  • detecting is used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect.
  • the compound for use in the method of detecting a target cell or molecule, may further comprise an additional moiety that allows identification of the a target cell or molecule.
  • the compound may comprise a label, for example a fluorescent molecule, p-galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope.
  • the method may the comprise a further step of detecting the fluorescent molecule, p-galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope.
  • An aspect of the invention relates to an in vitro, ex vivo or in vivo method of delivering a payload to a target cell within a biological sample, comprising: i) obtaining a biological sample; and ii) contacting said biological sample with a compound as described herein, wherein the compound is conjugated to a payload.
  • the payload may be any payload as described herein, including but not limited to a cell killing agent, an immune-modulating payload, a macrophage class switching agent, a light activatable payload, or a detectable label.
  • the biological sample may be a biological tissue sample, a biological fluid.
  • a biological fluid may be, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like. it
  • An aspect of the invention relates to a kit comprising the compound described herein and optionally instructions for use.
  • the kit may contain materials useful for the treatment of the disease described above is provided.
  • the kit may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds the compound, immunoconjugate or antibody drug conjugate of the invention which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is suitably an ADC.
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the kit may further contain a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • alk or “alkyl”, as used herein unless otherwise defined, relates to saturated hydrocarbon radicals being straight, branched, cyclic or polycyclic moieties or combinations thereof and contain 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 carbon atoms, or even 1 to 4 carbon atoms.
  • radicals may be optionally substituted with a chloro, bromo, iodo, cyano, nitro, OR 19 , OC(O)R 20 , C(O)R 21 , C(O)OR 22 , NR 23 R 24 , C(O)NR 25 R 26 , SR 27 , C(O)SR 27 , C(S)NR 25 R 26 , aryl or Het, wherein R 19 to R 27 each independently represent hydrogen, aryl or alkyl, and/or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups.
  • radicals may be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tertbutyl, 2-methylbutyl, pentyl, iso-amyl, hexyl, cyclohexyl, 3-methylpentyl, octyl and the like.
  • alkylene as used herein, relates to a bivalent radical alkyl group as defined above. For example, an alkyl group such as methyl which would be represented as -CH3, becomes methylene, -CH2-, when represented as an alkylene. Other alkylene groups should be understood accordingly.
  • alkenyl relates to hydrocarbon radicals having, such as up to 4, double bonds, being straight, branched, cyclic or polycyclic moieties or combinations thereof and containing from 2 to 18 carbon atoms, such as 2 to 10 carbon atoms, such as from 2 to 8 carbon atoms, such as 2 to 6 carbon atoms, or even 2 to 4 carbon atoms.
  • radicals may be optionally substituted with a hydroxyl, chloro, bromo, iodo, cyano, nitro, OR 19 , OC(O)R 20 , C(O)R 21 , C(O)OR 22 , NR 23 R 24 , C(O)NR 25 R 2e , SR 27 , C(O)SR 27 , C(S)NR 25 R 2e , or aryl, wherein R 19 to R 27 each independently represent hydrogen, aryl or alkyl, and/or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups.
  • radicals may be independently selected from alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1 -propenyl, 2-butenyl, 2- methyl-2-butenyl, isoprenyl, farnesyl, geranyl, geranylgeranyl and the like.
  • alkynyl relates to hydrocarbon radicals having, such as up to 4, triple bonds, being straight, branched, cyclic or polycyclic moieties or combinations thereof and having from 2 to 18 carbon atoms, such as 2 to 10 carbon atoms, such as from 2 to 8 carbon atoms, such as from 2 to 6 carbon atoms, or even from 2 to 4 carbon atoms.
  • radicals may be optionally substituted with a hydroxy, chloro, bromo, iodo, cyano, nitro, OR 19 , OC(O)R 20 , C(O)R 21 , C(O)OR 22 , NR 23 R 24 , C(O)NR 25 R 2e , SR 27 , C(O)SR 27 , C(S)NR 25 R 2e , or aryl, wherein R 19 to R 27 each independently represent hydrogen, aryl or lower alkyl, and/or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsiloxane groups.
  • alkynyl radicals examples include ethynyl, propynyl, propargyl, butynyl, pentynyl, hexynyl and the like.
  • alkynylene as used herein, relates to a bivalent radical alkynyl group as defined above.
  • an alkynyl group such as ethynyl which would be represented as -CECH
  • -CEC- when represented as an alkynylene.
  • Other alkynylene groups should be understood accordingly.
  • aryl as used herein, relates to an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, and includes any monocyclic, bicyclic or polycyclic carbon ring of up to 7 members in each ring, wherein a ring is aromatic.
  • radicals may be optionally substituted with a hydroxy, chloro, bromo, iodo, cyano, nitro, OR 19 , OC(O)R 20 , C(O)R 21 , C(O)OR 22 , NR 23 R 24 , C(O)NR 25 R 26 , SR 27 , C(O)SR 27 , C(S)NR 25 R 26 , or aryl, wherein R 19 to R 27 each independently represent hydrogen, aryl or lower alkyl, and/or be interrupted by oxygen or sulphur atoms, or by silano or dialkylsilcon groups.
  • radicals may be independently selected from phenyl, p-tolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl, 3-methyl-4- methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl, 3-aminophenyl, 3- acetamidophenyl, 4-acetamidophenyl, 2-methyl-3-acetamidophenyl, 2-methyl-3-aminophenyl, 3- methyl-4-aminophenyl, 2-amino-3-methylphenyl, 2,4-dimethyl-3-aminophenyl, 4-hydroxyphenyl, 3-methyl-4-hydroxyphenyl, 1 -naphthyl, 2-naphthyl, 3-amino-1 -naphthyl, 2-methyl-3-amino-1- naphthyl, 6-amino-2-naphthyl, 4,6-dimethoxy-2-n
  • arylene relates to a bivalent radical aryl group as defined above.
  • an aryl group such as phenyl which would be represented as -Ph, becomes phenylene, -Ph-, when represented as an arylene.
  • Other arylene groups should be understood accordingly.
  • cycloalkyl refers to any of the above mentioned alkyl groups which comprise one or more cyclic group(s).
  • cycloalkylene as used herein, relates to a bivalent radical cycloalkyl group as defined above.
  • a cycloalkyl group such as cyclohexyl which would be represented as -CeHn , becomes cyclohexylene, -CeHw-, when represented as a cycloalkylene.
  • Other cycloalkylene groups should be understood accordingly.
  • the bivalent radical may be attached to two (organic) groups via any suitable atoms on the ring.
  • the groups may be attached on adjacent atoms or otherwise (for example, at the meta, ortho or para position, as appropriate).
  • hetero refers to sulphur, phosphorus, oxygen and/or nitrogen atoms.
  • heteroalkyl refers to sulphur, phosphorus, oxygen and/or nitrogen atoms.
  • heteroalkenyl and “heteroakynyl” etc. groups are those that contain at least one sulphur, phosphorus, oxygen and/or nitrogen atom in the alkyl, alkenyl, alkynyl etc. chain.
  • Heterocyclic groups are those that contain at least one sulphur, phosphorus, oxygen and/or nitrogen atom in an alkyl or aryl ring.
  • the ring that includes the heteroatom can be aromatic or nonaromatic.
  • heterocyclic groups include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiopene), furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl.
  • the heterocyclic groups may be substituted with groups including, but not limited to, -alkyl, -O-(alkyl), aryl, -C(O)R', -OC(O)R', - C(O)OR', -C(O)NH 2 , -C(O)NHR', -C(O)N(R')2 , -NHC(O)R', -S(O) 2 R', -S(O)R', -OH, -halogen, - N3 , -NH 2 , -NH(R'), -N(R') 2 and -CN; wherein alkyl is preferably Ci-Cs alkyl and each R' is independently selected from -H, -alkyl, such as Ci-Cs alkyl and aryl.
  • alkyl, alkenyl, alkynyl, aryl or aralkyl in composite groups herein should be interpreted accordingly, for example the reference to alkyl in aminoalkyl or alk in alkoxyl should be interpreted as alk or alkyl above etc.
  • the term "and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.
  • a compound comprising the formula: A 1 -S’-L-S”-A 2 , wherein A 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • a 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • L is a linker; wherein the first and second antibody or antigen-binding fragment are conjugated together via click chemistry.
  • first and second antibody or antigen-binding fragment each independently comprise a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody.
  • first and/or second antibody or antigen-binding fragment comprises a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering, for example wherein the first and second antibody or antigen-binding fragment each comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering.
  • linker, L is formed via the conjugation of a first linker arm, L’, attached to the first antibody or antigen-binding fragment via S’ and a second linker arm, L”, attached to the second antibody or antigen-binding fragment via S”.
  • the first linker arm, L’ comprises a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and the second linker arm, L”, comprises a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when the conjugating group of the first linker arm, L’, is an azide, nitrone, tetrazine or tetrazole group, then the conjugating group of the second linker arm, L”, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and when the conjugating group of the first linker arm, L’, comprises a conjugating group selected from an azide, nitrone, te
  • the first linker arm. L’ comprises an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and the second linker arm, L”, comprises a conjugating group selected from an azide, tetrazine, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when the conjugating group of the first linker arm, L’, is an azide or tetrazine group, then the conjugating group of the second linker arm, L”, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and when the conjugating group of the first linker arm, L’, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, then the conjugating group of the first linker arm, L’, is an aliphatic or cyclic alkene or aliphatic or
  • the first linker arm, L’ comprises a tetrazine conjugating group and the second linker arm, L”, comprises a conjugating group selected from a cyclooctene or cyclooctyne group; and/or wherein, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, comprises an azide conjugating group and the second linker arm, L”, comprises a conjugating group selected from an alkene, alkyne, cyclooctyne or cyclooctene group, for example an alkyne or cyclooctene group.
  • the first linker arm, L’ comprises a tetrazine conjugating group and the second linker arm, L”, comprises a cyclooctyne group, such as a bicyclononyne group, such as bicyclo[6.1 ,0]non-4-yne.
  • the click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is an inverse electron demand Diels-Alder (iEDDA) reaction.
  • click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and/or an inverse electron demand Diels-Alder (iEDDA) reaction, for example an inverse electron demand Diels- Alder (iEDDA).
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strained-promoted azide-alkyne click chemistry
  • iEDDA inverse electron demand Diels-Alder
  • [315] 23 The compound according to any one of aspects 1-22, wherein the compound comprises one or more further moieties, for example a half-life extension moiety, a further antibody or antigen-binding fragment and/or a payload.
  • the compound comprises one or more further moieties, for example a half-life extension moiety, a further antibody or antigen-binding fragment and/or a payload.
  • [316] 24 The compound according to aspect 23, wherein the half-life extension moiety comprises a serum albumin protein or an antibody or antigen-binding fragment that binds to a serum albumin protein.
  • [317] 25 The compound according to any one of aspects 23 or 24, wherein the payload comprises a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.
  • the payload comprises a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.
  • [318] 26 A method of producing a compound comprising conjugating together a first unit and a second unit via click chemistry; wherein the first unit comprises the formula: A 1 -S’-Y’, wherein A 1 represents a first antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the first antibody or antigen-binding fragment comprises one or more non- naturally occurring cysteine residue(s);
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • Y’ is hydrogen or -L’-X’, wherein L’ is a first linker arm; and X’ is a first conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and wherein the second unit comprises the formula: A 2 -S”-Y”, wherein A 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • Y is hydrogen or -L”-X”, wherein L” is a second linker arm; and X” is a second conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when Y’ is hydrogen, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; when Y’ is -L’-X’ and X’ is an azide, nitrone, tetrazine or tetrazole group, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cycl
  • [320] 28 The method according to any one of aspects 26 or 27, wherein the first and second antibody or antigen-binding fragment each independently comprise a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody.
  • [321] 29 The method according to aspect 28, wherein the first and/or second antibody or antigen-binding fragment further comprise an Fc domain, for example wherein one of the first and second antibody or antigen-binding fragment further comprise an Fc domain.
  • the first and/or second antibody or antigen-binding fragment comprises a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering, for example wherein the first and second antibody or antigen-binding fragment each comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering.
  • [326] 34 The method according to any one of aspects 26-33, wherein Y’ is hydrogen, Y” is -L”- X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; or wherein Y’ is -L’- X’ and X’ is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group and Y” is hydrogen.
  • [328] 36 The method according to any one of aspects 26-35, wherein Y’ is -L’-X’ and X’ is a tetrazine group and Y” is -L”-X” and X” is a cyclooctene or cyclooctyne group; and/or wherein Y’ is -L’-X’ and X’ is an azide group and Y” is -L”-X” and X” is an alkene, alkyne, cyclooctyne or cyclooctene group, for example an alkyne or cyclooctene group.
  • [329] 37 The method according to aspect 36, wherein Y’ is -L’-X’ and X’ is a tetrazine group, and Y” is -L”-X”, wherein X” is a trans-cyclooctene (TCO) group; and/or wherein Y’ is -L’-X’ and X’ is a trans-cyclooctene (TCO) group, and Y” is -L”-X” and X” is a tetrazine group.
  • TCO trans-cyclooctene
  • [330] 38 The method according to aspect 37, wherein the click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is an inverse electron demand Diels-Alder (iEDDA) reaction.
  • iEDDA inverse electron demand Diels-Alder
  • [331] 39 The method according to aspect 38, wherein Y’ is -L’-X’ and X’ is a tetrazine group, and Y” is -L”-X”, wherein X” is a bicyclononyne group, such as bicyclo[6.1 ,0]non-4-yne; and/or wherein Y’ is -L’-X’ and X’ is a trans-cyclooctene (TCO) group, and Y” is -L”-X” and X” is a bicyclononyne group, such as bicyclo[6.1 ,0]non-4-yne.
  • TCO trans-cyclooctene
  • [332] 40 The compound according to aspect 39, wherein the click chemistry used to conjugate together the first and second antibody or antigen-binding fragments is an inverse electron demand Diels-Alder (iEDDA) reaction.
  • iEDDA inverse electron demand Diels-Alder
  • the click chemistry used to conjugate together the first and second units is selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and/or an inverse electron demand Diels-Alder (iEDDA) reaction, for example via an inverse electron demand Diels-Alder (iEDDA) reaction.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strained-promoted azide-alkyne click chemistry
  • iEDDA inverse electron demand Diels-Alder
  • the compound further comprises one or more further moieties, for example a half-life extension moiety, a further antibody or antigen-binding fragment and/or a payload.
  • the half-life extension moiety comprises a serum albumin protein, an antibody or antigen-binding fragment that binds to a serum albumin protein, an Fc domain and/or a modified Fc domain.
  • the payload comprises a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.
  • S’ is a sulphur atom derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment
  • Y’ is hydrogen or -L’-X’, wherein L’ is a first linker arm; and X’ is a first conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and wherein the second unit comprises the formula: A 2 -S”-Y”, wherein A 2 represents a second antibody or antigen-binding fragment comprising a substitution to a cysteine residue at one or more of positions 153, 156, 168, 173 and/or 207 according to Kabat numbering, such that the second antibody or antigen-binding fragment comprises one or more non-naturally occurring cysteine residue(s);
  • S is a sulphur atom derived from a non-naturally occurring cysteine residue of the second antibody or antigen-binding fragment
  • Y is hydrogen or -L”-X”, wherein L” is a second linker arm; and X” is a second conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, with the proviso that when Y’ is hydrogen, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; when Y’ is -L’-X’ and X’ is an azide, nitrone, tetrazine or tetrazole group, then Y” is -L”-X” and X” is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group; and when Y’ is -L’-X’ and X’ is an aliphatic or cycl
  • first and second antibody or antigen-binding fragment each independently comprise a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody.
  • first and/or second antibody or antigenbinding fragment further comprise an Fc domain, for example wherein one of the first and second antibody or antigen-binding fragment further comprise an Fc domain.
  • first and/or second antibody or antigen-binding fragment comprises a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering, for example wherein the first and second antibody or antigen-binding fragment each comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering.
  • click chemistry used to conjugate together the first and second units is selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and/or an inverse electron demand Diels-Alder (iEDDA) reaction, for example via an inverse electron demand Diels-Alder (iEDDA) reaction.
  • CuAAC copper catalyzed azide-alkyne cycloaddition
  • SPAAC strained-promoted azide-alkyne click chemistry
  • iEDDA inverse electron demand Diels-Alder
  • the half-life extension moiety comprises a serum albumin protein, an antibody or antigen-binding fragment that binds to a serum albumin protein, an Fc domain and/or a modified Fc domain.
  • the payload comprises a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.
  • Step 1 Fab (1 eq.) was added to a reaction vessel followed by 0.5 M pH 8.0 Tris buffer adjust solution and PBS to bring the reaction mixture to 1 mg/ml (relative to the Fab). Tris(2- carboxyethyl)phosphine hydrochloride (TCEP-HCI; 15 eq. @ 10 mg/mL in water), a reducing agent, was introduced and the reaction mixture was gently shaken before being incubated at 37°C or room temperature for 90 minutes.
  • TCEP-HCI Tris(2- carboxyethyl)phosphine hydrochloride
  • Step 2 After 90 minutes (L)-Dehydroascorbic acid (DHAA; 20 eq. @ 20 mg/mL in DMSO) was introduced. The reaction mixture was gently shaken before being further incubated at 37°C or room temperature for 1 hour.
  • DHAA L-Dehydroascorbic acid
  • Step 3 After 1 hour one of the following functionalisation reagents was introduced (10 eq.
  • reaction mixture Upon addition of the functionalisation reagent, the reaction mixture was gently shaken, then incubated at 37°C or room temperature for 30 minutes or 2 hours. The reaction mixture was then diluted (2x volume) and reduced back to the original volume by centrifugal ultrafiltration (molecular weight cut-off: 30 kDa). The dilution and filtration process was repeated until all traces of functionalisation reagent and DMSO were removed. The solution was then concentrated down to around 1 mg/mL.
  • Step 4 The appropriate functionalised Fabs were combined at a 1 :1 ratio and gently shaken. They were then incubated at 37°C or room temperature overnight. The conjugation pairs were as shown in Table 1 below:
  • the iEDDA reaction can be performed with a range of reaction partners.
  • Methyl-tetrazine is a commonly used tetrazine that has better stability than unsubstituted tetrazines whilst retaining high levels of reactivity (1272-1279.; Bernardes, Oliveria & Guo, Chem. Soc. Rev. 2017, 46, 4895-4950). It is often reacted with TCO or BCN in iEDDA reactions.
  • Maleimide functionalised me-Tz, TCO and BCN are all commercially available.
  • Fabs were functionalised using Mal-PEG 3 -Tz-me, Mal-PEG 3 -TCO and Mal-PEG 3 -BCN and used immediately for Bi-Fab formation.
  • Bi-Fab formation was performed at a larger scale (5 mg Fab starting material) using the Tz-BCN iEDDA reaction to dimerise Fabs. Standard conditions from the trial (37°C Fab functionalisation; room temperature Bi-Fab synthesis; concentration - 1 mg / ml) were used on a larger scale to allow for purification by pSEC. This achieved a 30% yield (3.0 mg) of Bi-Fab, doubling that of the initial SPAAC DBCO-N3 reaction conditions ( ⁇ 15 % Bi-Fab yield).
  • Example 2 (anti-CD33)-mal-PEG11-mal-(anti-CD33) homodimer conjugated to MMAF (‘(anti-CD33)-mal-PEG11 -mal-(anti-CD33) homodimer ADC’)
  • a homodimer of two anti-CD33 Fabs linked by a bis-mal-PEG11 linker was first prepared according to the following method.
  • reaction mixture was gently shaken before being further incubated at 37°C or room temperature for 1 hour. After this time, Bis-Mal-Peg11 (0.5 eq. @ 10 mg/mL in DMSO) was introduced and the reaction mixture was gently shaken before being incubated at room temperature for at least 1 hour. Typically, the reaction was left overnight to ensure maximum conversion.
  • 10OpI of assay media was pipetted in the blank control and 50pl in the cell-only control wells; the plates were incubated at 37°C, 5% CO2 for 96 hours. After 96 hours incubation, 10pl of WST-1 reagent was added per well and after 3 hours incubation at 37°C, 5% CO2 the absorbance was read at 440nm and 620nm. The data for each reading was plotted in GraphPad PRISM.
  • the (anti-CD33)-mal-PEG11-mal-(anti-CD33) homodimer ADC induces cell kill in CD33+ cells (KASUMI-3, HNT-34, MV4-11 , SHI-1) and not in CD33- cells (JURKAT and DND-39). High levels of cell kill are observed in KASUMI-3, MV4-11 and SHI-1 cells which express a similar level of CD33, whereas for CD33 low expressing HNT-34 cells the level of cell kill is lower and is at high concentrations only.
  • An anti-CD33 Fab having an I207C mutation in accordance with SEQ ID NO. 1 (1 eq.) was prepared. Fabs were modified to permit homodimer formation via by bioorthogonal reactive partners (tetrazine and bicyclononyne (BCN)). The Fab (1 eq) was added to a reaction vessel followed by Tris buffer (0.5 M pH 8.0; 5 v/v %) and enough PBS pH 7.4 to bring the reaction mixture to 1 mg/ml (relative to the Fab). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP- HCI; 15 eq.
  • Tris(2-carboxyethyl)phosphine hydrochloride TCEP- HCI; 15 eq.
  • the reaction mixture was then diluted (2x volume) and reduced back to the original volume by centrifugal ultrafiltration (MWCO 30 kDa). The dilution and filtration process was repeated until all traces of Mal-PEGs-Tz-me and DMSO were removed. The the solution was then concentrated using a centrifugal ultrafiltration tube (MWCO 50 kDa) to around 1 mg/mL.
  • MWCO 30 kDa centrifugal ultrafiltration tube
  • MMAF Payload conjugation
  • Bi-Fab-Fc (1 eq.) was added to a reaction vessel followed by Tris buffer (0.5 M pH 8.0; 5 v/v %) and enough PBS pH 7.4 to bring the reaction mixture to 1 mg/ml (relative to the Bi-Fab- Fc).
  • Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCI; 15 eq. @ 10 mg/mL in water) was then introduced, and the reaction mixture was gently shaken before being incubated at room temperature until the desired level of reduction was thought to have occurred (based on desired target average drug to antibody ratio (DAR)). Then, linker payload was introduced (DBM- MMAF/mcMMAF @ 10 mg/mL). When the reaction was deemed complete excess reagents were removed by centrifugal ultafiltration (MWCO 10 kDa). The ADC was then sterile filtered (0.22 pm) prior to storage.
  • VH CDR1 (SEQ ID NO. 2)
  • VH CDR3 (SEQ ID NO. 4)
  • Anti-CD33 VL (SEQ ID NO. 5)
  • VL CDR1 (SEQ ID NO. 6)
  • VL CDR2 (SEQ ID NO. 7)
  • VL CDR3 (SEQ ID NO. 8)

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Abstract

L'invention concerne un composé comprenant un premier et un second anticorps ou un fragment de liaison à l'antigène et des procédés de conjugaison du même composé comprenant la formule: A 1-S'-L-S''-A 2 dans laquelle A1 représente un premier anticorps ou un fragment de liaison à l'antigène comprenant une substitution à résidu cystéine au niveau d'une ou plusieurs des positions 153, 156, 168, 173 et/ou 207 selon la numérotation de Kabat, de sorte que le premier anticorps ou le fragment de liaison à l'antigène comprend un ou plusieurs résidu(s) cystéine non naturels; A2 représente un second anticorps ou fragment de liaison à l'antigène comprenant une substitution à un résidu cystéine au niveau d'une ou plusieurs des positions 153, 156, 168, 173 et/ou 207 selon la numérotation de Kabat, de sorte que le second anticorps ou fragment de liaison à l'antigène comprend un ou plusieurs résidus cystéine non naturels; S' est un atome de soufre dérivé d'un résidu cystéine non naturel du premier anticorps ou fragment de liaison à l'antigène; S''est un atome de soufre dérivé d'un résidu cystéine non naturel du second anticorps ou fragment de liaison à l'antigène; et L est un lieur; le premier et le second anticorps ou le fragment de liaison à l'antigène étant conjugués ensemble par chimie click.
PCT/GB2024/052163 2023-08-17 2024-08-16 Composé comprenant un premier et un second anticorps ou un fragment de liaison à l'antigène et procédés de conjugaison de celui-ci Pending WO2025037120A1 (fr)

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